FIELD OF THE INVENTION

The present invention relates to novel (variant) lipolytic enzymes and to one or more polynucleotides encoding one or more novel lipolytic enzymes. The invention also relates to methods of producing lipolytic enzymes, and uses thereof. The present invention further relates to the preparation of an improved foodstuff, in particular to the preparation of improved bakery products. Specifically, the invention provides lipolytic enzymes, which enzymes are capable of conferring improved characteristics to food products, including bakery products.

TECHNICAL BACKGROUND

The beneficial use of lipolytic enzymes (E.C. 3.1.1.x) in food and/or feed industrial applications has been known for many years.

For instance, in EP 0 585 988 it is claimed that lipase addition to dough resulted in an improvement in the antistaling effect. It is suggested that a lipase obtained from Rhizopus arrhizus when added to dough can improve the quality of the resultant bread when used in combination with shortening/fat. WO94/04035 teaches that an improved bread softness can be obtained by adding a lipase to dough without the addition of any additional fat/oil to the dough. Castello, P. ESEGP 89-10 Dec. 1999 Helsinki, shows that exogenous lipases can modify bread volume.

The substrate for lipases in wheat flour is 1.5-3% endogenous wheat lipids, which are a complex mixture of polar and non-polar lipids. The polar lipids can be divided into glycolipids and phospholipids. These lipids are built up of glycerol esterified with two fatty acids and a polar group. The polar group contributes to surface activity of these lipids.

Enzymatic cleavage of one of the fatty acids in these lipids leads to lipids with a much higher surface activity. It is well known that emulsifiers, such as DATEM, with high surface activity are very functional when added to dough. Lipolytic enzymes hydrolyse one or more of the fatty acids from lipids present in the food which can result in the formation of powerful emulsifier molecules within the foodstuff which provide commercially valuable functionality. The molecules which contribute the most significant emulsifier characteristics are the partial hydrolysis products, such as lyso- phospholipids, lyso-glycolipids and mono-glyceride molecules. The polar lipid hydrolysis products, namely lyso-phospholipids and lyso-glycolipids, are particularly advantageous. In bread making, such in situ derived emulsifiers can give equivalent functionality as added emulsifiers, such as DATEM.

However, the activity of lipolytic enzymes has also been found to result in accumulation of free fatty acids, which can lead to detrimental functionality in the foodstuff. This inherent activity of lipolytic enzymes limits their functionality.

The negative effect on bread volume is often explained by overdosing. Overdosing can lead to a decrease in gluten elasticity which results in a dough which is too stiff and thus results in reduced volumes. In addition, or alternatively, such lipases can degrade shortening, oil or milk fat added to the dough, resulting in off-flavour in the dough and baked product. Overdosing and off-flavour have been attributed to the accumulation of free fatty acids in the dough, particularly short chain fatty acids.

The presence of high levels of free fatty acids (FFA) in raw materials or food products is generally recognised as a quality defect and food processors and customers will usually include a maximum FFA level in the food specifications. The resulting effects of excess FFA levels can be in organoleptic and/or functional defects.

In WO2005/087918 novel fungal lipolytic enzymes were identified from Fusarium species, such as Fusarium heterosporum CBS 782.83 which were shown to have a superior quality in certain applications. These enzymes were expressed in Hansenula polymorpha and were found to hydrolyse primarily fatty acids in the sn-1 position of galactolipids and phospholipids in dough.

The problem with some fungal lipolytic enzymes is that expression of the enzyme may be limited and therefore may be costly to produce. For example expression of the enzyme in high amounts suitable for commercial scale activities may be limited. The industry is interested in finding novel lipolytic enzymes which show enhanced expression, particularly if this can be achieved without compromising functionality and/or activity.

SUMMARY OF THE INVENTION

It has surprisingly been found that the new variant lipolytic enzymes of the present invention show increased expression compared with the wild type enzyme(s) from which they were prepared. Notably the increased expression is achieved without compromising the enzymes functionality and/or activity and/or application performance. In addition the new variant lipolytic enzymes may show improved functionality and/or activity compared with the wild type enzyme(s).

The inventors have found that by modifying (substituting or inserting) one or more surface amino acids located in an external loop(s) distal to the active site (catalytic triad) of the lipolytic enzyme to replace the surface amino acid with an amino acid which is more hydrophilic (compared with the original amino acid) or to introduce hydrophilic amino acids or to introduce a glycosylation site then it is possible to substantially and surprisingly increase the expression and/or functionality and/or activity of the variant enzyme compared with the wild type enzyme. Preferably, the surface amino acid is replaced with an Asn, Ser and/or Thr (or a combination thereof) for the purpose of introducing one or more glycosylation sites. Alternatively or in addition, an external loop(s) distal to the active site can be modified by inserting one or more amino acids selected from Asn, Ser and/or Thr to introduce one or more glycosylation sites. Lipolytic enzymes typically work in an interphase between fat and water (i.e. between a hydrophilic environment and a hydrophobic environment) and hence the performance of lipolytic enzymes in certain applications is very dependent upon this interphase as well as water activity. Without wishing to be bound by theory, by changing the hydrophilicity of the surface of the lipolytic enzyme at a position which is remote the active site of the enzyme (i.e. in the loops distal the active site of the enzyme) it is possible to control the orientation of the enzyme within the fat/water interphase, such that the active site is orientated towards the substrate for the enzyme, i.e. the fat. Thus it is possible to modify the lipolytic enzyme to optimally orientate the enzyme in the interphase to increase the activity of the enzyme. In addition, or alternatively, by introducing glycosylation sites it may be possible to enhance the folding and expression and/or secretion of the enzyme from a host organism, thus enhancing expression of the variant enzymes.

In addition to or alternatively, the present inventors have also found a number of specific modifications which surprisingly increase expression and/or functionality and/or activity of the lipolytic enzyme substantially. These may include addition of glycosylation sites and/or stabilisation of the C-terminus region of the enzyme. Pn some embodiments the specific modifications increase expression without comprising the functionality and/or activity of the lipolytic enzyme. Hence some of the specific modifications increase expression without compromising application performance of the variant enzymes compared with the wild-type

(KXMl) enzyme the propresequence of which is shown herein as SEQ ID No. 2 (the mature form of which is amino acids 31-305 of SEQ ID No. 2).

Therefore in one aspect of the present invention there is provided a method for preparing a variant lipolytic enzyme comprising expressing in a host organism a nucleotide sequence which has at least 90% identity with a nucleotide sequence encoding a fungal lipolytic enzyme or which differs from a nucleotide sequence encoding a fungal lipolytic enzyme by one or several nucleic acid additions, deletions or substitutions and comprises at least one modification at a position which corresponds in the encoded amino acid sequence to a) the introduction of at least one glycosylation site (or one additional glycosylation site) in the amino acid sequence compared with the original fungal lipolytic enzyme; b) the introduction of at least one amino acid at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme which is more hydrophilic (compared with the original amino acid); or c) a substitution or insertion at one or more of positions 33, 63, 78, 190, 305, 306 or 320 or a deletion at one or more positions 311-312 or 307-319, wherein each amino acid position corresponds to the position of the amino acid sequence when aligned with SEQ ID No. 2; wherein when the nucleotide sequence has at least 90% identity with a nucleotide sequence encoding the fungal lipolytic enzyme shown in SEQ ID No. 22 or SEQ ID No. 23, or differs by one or several nucleic acid additions, deletions or substitutions from a nucleotide sequence encoding the fungal lipolytic enzyme shown in SEQ ID No. 22 or SEQ ID No. 23, the modification is not a substitution at position 63 and the deletion is not at position 311-312 (wherein the amino acid position numbering is that shown in respect of SEQ ID No. 2 when aligned). The method may introduce at least one amino acid at a surface position on the polypeptide by substituting or inserting one or more amino acids at the surface position wherein the substitution or insertion is with an amino acid which is more hydrophilic (compared with the original amino acid).

In another embodiment the present invention provides a method of producing a variant lipolytic enzyme comprising expressing in a host organism a nucleotide sequence which has at least 90% identity with SEQ ID No. 1 or which differs from SEQ ID No. 1 by one or several nucleic acid additions, deletions or substitutions, and comprises at least one modification at a position which corresponds in the encoded amino acid sequence to a) the introduction of at least one glycosylation site in the amino acid sequence; b) the introduction of at least one hydrophilic amino acid at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme; or c) a substitution or insertion at one or more of positions 33, 63, 78, 190, 305, 306 or 320 or a deletion at one or more positions 311-312 or 307-319, wherein each amino acid position corresponds to the position of the amino acid sequence of SEQ ID No. 2.

The present invention further provides a method of producing a lipolytic enzyme comprising expressing in a host organism a nucleotide sequence comprising SEQ ID No. 8, SEQ ID No.

6, SEQ ID No. 4, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID

No. 18, SEQ ID No. 20 or SEQ ID No. 26; or a nucleotide sequence having at least 98%

(preferably at least 99%, suitably at least 99.5% such as at least 99.8%) identity therewith; or a nucleic acid which differs by one or several nucleotide additions, deletions or substitutions from or which is related to the nucleotide sequence of SEQ ID No. 8, SEQ ID No. 6, SEQ ID

No. 4, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18,

SEQ BD No. 20 or SEQ ID No. 26 by the degeneration of the genetic code.

There is also provided a method of preparing a lipolytic enzyme the method comprising transforming a host cell with a recombinant nucleic acid coding for a polypeptide having hydrolytic activity towards an ester bond in a polar lipid, which nucleic acid comprises a nucleotide sequence comprising SEQ ID No. 8, SEQ ID No. 6, SEQ ID No. 4, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 26; or a nucleotide sequence having at least 98% (preferably at least 99%, suitably at least 99.5% such as at least 99.8%) identity with SEQ TD No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 26; or a nucleic acid which differs by one or several nucleotide additions, deletions or substitutions from or which is related to the nucleotide sequence of SEQ ID No. 8, SEQ ID No. 6, SEQ ID No. 4, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 26 by the degeneration of the genetic code, the host cell being capable of expressing the nucleotide sequence coding for the polypeptide, cultivating the transformed host cell under conditions where the nucleic acid is expressed and harvesting the lipolytic enzyme.

The present invention provides an enhanced expression of the nucleic acids according to the present invention and thus an improved method of production of variant polypeptides.

In a further aspect the present invention provides a polypeptide (prepro-polypeptide or mature lipolytic enzyme) obtained by the method according to the present invention.

In a yet further aspect there is provided a nucleic acid comprising a nucleotide sequence encoding a lipolytic enzyme and which nucleotide sequence comprises at least one modification at a position which corresponds in the encoded amino acid sequence to a) the introduction of at least one glycosylation site in the amino acid sequence; b) the introduction of at least one hydrophilic amino acid at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme; or c) a substitution or insertion at one or more of positions 33, 63, 78, 190, 305, 306 or 320 or a deletion at one or more positions 311-312 or 307-319, wherein each amino acid position corresponds to the position of the amino acid sequence of SEQ ID No. 2, wherein when the nucleotide sequence encodes the fungal lipolytic enzyme shown as SEQ ID No. 22 or SEQ ID No. 23 the modification is not a substitution at position 63 and the deletion is not at position 31 1-312 (wherein the amino acid position numbering is that shown in respect of SEQ ID No. 2 when aligned).

In a further aspect the present invention provides a nucleic acid comprising a nucleotide sequence which has at least 90% identity with SEQ ID No. 1 , or which differs from SEQ ID No. 1 by one or several nucleotide additions, deletions or substitutions, and which nucleotide sequence comprises at least one modification at a position which corresponds in the encoded amino acid sequence to a) the introduction of at least one glycosylation site in the amino acid sequence; b) the introduction of at least one hydrophilic amino acid at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme; or c) a substitution or insertion at one or more of positions 33, 63, 78, 190, 305, 306 or 320 or a deletion at one or more positions 31 1 -312 or 307-319, wherein each amino acid position corresponds to the position of the amino acid sequence of SEQ ID No. 2

The present invention further provides a nucleotide sequence encoding a polypeptide having hydrolytic activity towards an ester bond in a polar lipid which nucleotide sequence comprises SEQ ID No. 8, SEQ ID No. 6, SEQ ID No. 4, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 26; or a nucleotide sequence having at least 98% (preferably at least 99%, preferably at least 99.5%, such as at least 99.8%) identity with SEQ ID No. 8, SEQ ID No. 6, SEQ ID No. 4, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 26; or a nucleic acid which differs by one or several nucleotide additions, deletions or substitutions from or which is related to the nucleotide sequence of SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 26 by the degeneration of the genetic code.

In one embodiment the preferred nucleotide sequence is that shown in SEQ ID No. 8 (mutant 5) or a nucleotide sequence which is related to SEQ ID No. 8 by the degeneration of the genetic code.

In a further aspect the present invention provides a variant polypeptide encoded by the nucleic acid or nucleotide sequence according to the present invention.

Another aspect of the present invention provides a variant polypeptide which has hydrolytic activity towards an ester bond in a polar lipid and comprises an amino acid sequence which has at least 90% identity with amino acids 33-296 of SEQ ID No. 2, or differs by one or several amino acid additions, deletions or substitutions from amino acids 33-296 of SEQ ID No. 2, and which has been modified compared with the sequence shown in SEQ ID No. 2 to a) introduce at least one glycosylation site in the amino acid sequence; b) introduce at least one hydrophilic amino acid at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme; or c) substitute or insert an amino acid at at least one or more of positions 33, 63, 78 or 190 wherein each amino acid position corresponds to the position of the amino acid sequence shown in SEQ ID No. 2.

In another aspect the present invention provides a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid and comprises an amino acid sequence shown as amino acids 33-296 (or amino acids 31-305) of SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No.

25.

In a yet further aspect the present invention provides a prepro-polypeptide which when post- translationally processed in a host organism produces a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, wherein the prepropolypeptide comprises an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 7, SEQ ID No. 5, SEQ ID No. 11 , SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 25.

In one aspect the present invention further provides a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, which polypeptide is obtainable from a prepropolypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 7, SEQ ID No. 5, SEQ ID No. 1 1, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19 SEQ ID No. 21 or SEQ ID No. 25.

Depending on the host organism prepro-sequences often go through post-translational modification. With the present enzymes it is relatively common for the organism to remove the N-terminal region of the prepro sequence, i.e. remove all or part of the amino acids 1-30 of SEQ ID No. 9, SEQ ID No. 7, SEQ ID No. 5, SEQ ID No. 1 1, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 25. In some embodiments the host organism may remove slightly more amino acids than those shown as amino acids 1-30 of SEQ ID No. 9, SEQ ID No. 7, SEQ ID No. 5, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 25, such as removing amino acids 1-31 or 1-32 or 1-33 for instance. In some instances the host organism may introduce an alternative N-terminal sequence which may encompass all or part of the amino acids shown as amino acids 1-30 or may comprise a completely different N- terminal sequence (such as EAEA or EA for instance). In some cases the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its N- terminus end. In some embodiments the post-translational modification may mean modification in the C-terminal region of the prepro sequence. For example, all or part of the amino acids 306-348 may be removed from SEQ ID No. 9, SEQ ID No. 7, SEQ ID No. 5, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ TD No. 25 in the mature form. In some embodiments the host organism may remove slightly more amino acids than those shown as amino acids 306-348 of SEQ ID No. 9, SEQ ID No. 7, SEQ ID No. 5, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 25, such as removing amino acids 305- 348 or 304-348 or 303-348 for instance. In some cases the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its C-terminus end. It is envisaged that the present invention encompasses all mature forms of the protein obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 1 1, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 25, particularly those obtained from the host organism Trichoderma reesei.

The present invention yet further provides the use of a nucleic acid according to the present invention to enhance expression of a lipolytic enzyme from a host organism. Suitably the host organism may be a fungi, preferably Trichoderma spp., preferably Trichoderma reesei. Suitably the expression is enhanced between about 2-fold upto about 25-fold compared with the wild type nucleic acid (i.e. the nucleic acid without any modifications in accordance with the present invention).

The present invention further provides a method of making a foodstuff comprising adding a polypeptide according to the present invention to one or more ingredients of the foodstuff. In another aspect the present invention provides a method of making a baked product comprising adding a polypeptide according to the present invention to a dough and baking the dough to make the baked product.

The present invention further provides a method of preparing a lyso-phospholipid comprising treating a phospholipid with a polypeptide according to the present invention to produce the lyso-phospholipid.

In a yet further embodiment the present invention provides a method of preparing a lyso- glycolipid comprising treating a glycolipid with a polypeptide according to the present invention to produce a lyso-glycolipid.

The present invention further provides a process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a polypeptide according to the present invention so as to hydrolyse a major part of the polar lipids present therein.

In another aspect the present invention provides a foodstuff or a baked product obtained by the method of the present invention.

Aspects of the present invention are presented in the claims and in the following commentary.

Other aspects concerning the nucleotide sequences which can be used in the present invention include: a construct comprising the sequences of the present invention; a vector comprising the sequences for use in the present invention; a plasmid comprising the sequences for use in the present invention; a transformed cell comprising the sequences for use in the present invention; a transformed tissue comprising the sequences for use in the present invention; a transformed organ comprising the sequences for use in the present invention; a transformed host comprising the sequences for use in the present invention; a transformed organism comprising the sequences for use in the present invention. The present invention also encompasses methods of expressing the nucleotide sequence for use in the present invention using the same, such as expression in a host cell; including methods for transferring same. The present invention further encompasses methods of isolating the nucleotide sequence, such as isolating from a host cell. Other aspects concerning the amino acid sequence for use in the present invention include: a construct encoding the amino acid sequences for use in the present invention; a vector encoding the amino acid sequences for use in the present invention; a plasmid encoding the amino acid sequences for use in the present invention; a transformed cell expressing the amino acid sequences for use in the present invention; a transformed tissue expressing the amino acid sequences for use in the present invention; a transformed organ expressing the amino acid sequences for use in the present invention; a transformed host expressing the amino acid sequences for use in the present invention; a transformed organism expressing the amino acid sequences for use in the present invention. The present invention also encompasses methods of purifying the amino acid sequence for use in the present invention using the same, such as expression in a host cell; including methods of transferring same, and then purifying said sequence.

For the ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

DETAILED DISCLOSURE OF INVENTION

All reference to amino acid positions as used herein is made by reference to the amino acid sequence SEQ ID No. 2. In other words, when the numbering of an amino acid position is considered this can be determined by alignment of the amino acid sequence with SEQ ID No. 2 and by referring to the position numbering of the aligned sequences using SEQ ID No. 2 as the reference sequence (see for example Figure 22 which shows an alignment of SEQ ID No. 2 (designated therein as KLMl) with other sequences taught herein).

Suitably, the host organism used in accordance with the present invention may be a fungi, preferably from the genus Trichoderma, more preferably from the species Tήchoderma reesei.

In one embodiment the fungal lipolytic enzyme before modification does not comprise any glycosylation sites. In other words, the methods of the present invention may be used to introduce at least one glycosylation site into a lipolytic enzyme which originally or naturally does not contain any glycosylation sites

Suitably the variant polypeptide of the present invention may comprise at least two, suitably at least three, glycosylation sites.

Preferably the nucleotide sequence of the present invention or used in the methods of the present invention has at least 90% identity (preferably at least 95%, more preferably at least 98%, suitably at least 99%, such as at least 99.5% identity) with SEQ ID No. 1 or with SEQ ID No. 24, or with a nucleotide sequence shown in positions 23-106 of SEQ ID No. 24, or with a nucleotide sequence shown in positions 113-1063 of SEQ ID No. 24 or with a nucleotide sequence shown in positions 113-929 of SEQ ID No 24 except that it comprises at least one modification compared with SEQ ID No. 1 or with SEQ ID No. 24, or with a nucleotide sequence shown in positions 23-106 of SEQ ID No. 24, or with a nucleotide sequence shown in positions 113-1063 of SEQ ID No. 24 or with a nucleotide sequence shown in positions 113-929 of SEQ ID No. 24 respectively or with a nucleotide sequence which differs from one of the recited sequences by one or several nucleotide additions, deletions or substitutions.

In one embodiment when the nucleotide sequence has at least 90% identity with nucleotide sequence encoding the fungal lipolytic enzyme shown in SEQ ID No. 22 or SEQ ID No. 23, or which differs from a nucleotide sequence encoding the fungal lipolytic enzyme shown in SEQ ID No. 22 or SEQ ID No. 23 by one or several nucleotide additions, deletions or substitutions, then the modification is not a substitution at position 63 (e.g. it is not the substation K63N) and the deletion is not at position 31 1-312. The nucleotide sequence encoding the fungal lipolytic enzyme shown in SEQ ID No. 22 or SEQ ID No. 23 is shown herein as SEQ ID No 24 or a portion thereof (such as a nucleotide sequence shown in positions 23-106 of SEQ ID No. 24, or a nucleotide sequence shown in positions 1 13-1063 of SEQ ID No. 24 or a nucleotide sequence shown in positions 113-929 of SEQ ID No. 24.

In one embodiment preferably the modification in accordance with the present invention corresponds with the introduction of at least one glycosylation site at a surface position on the polypeptide and at a location in an external loop distal to the active site of the enzyme. Preferably the nucleotide sequence is modified such that one or more amino acids located at a surface position on the polypeptide and at a location in an external loop which is distal to the active site of the enzyme is substituted with an amino acid which is more hydrophilic than the original amino acid.

Alternatively, the nucleotide sequence may be modified such that one or more hydrophilic amino acids are inserted at a surface position on the polypeptide and at a location in the external loop distal to the active site of the enzyme.

In one preferable embodiment the nucleotide sequence is modified such that in the encoded amino acid one or more amino acids are substituted or inserted to provide one or more consensus sequences Asn-Xxx-Ser or Asn-Xxx-Thr, where Xxx could be any amino acid except Pro.

In one embodiment the nucleotide sequence is modified such that in the encoded amino acid sequence one or more Asn, Ser or Thr are introduced. In other words the nucleotide sequence according to the present invention comprises a modification corresponding with the introduction of one or more of Asn, Ser or Thr into the encoded protein.

Suitably in the method or nucleic acid of the present invention least two, suitably at least three, glycosylation sites may be introduced.

The nucleotide sequence of the present invention and in the methods of the present invention may be further modified to enhance C-terminal processing of the protein compared with the original lipolytic enzyme, for example compared with a lipolytic enzyme comprising SEQ ID No. 2 or amino acids 33-296 (or 31-305) thereof.

Suitably, the nucleotide sequence or the polypeptide may include C-terminal processing, preferably to render the polypeptide more stable.

In the present case C-terminus of the polypeptide is considered to be from amino acid position 306 onwards, wherein said position corresponds to the position in the amino acid sequence of SEQ ID No. 2 when aligned.

Suitably the C-terminal processing as taught in the present invention comprises one or more of the following: a substitution or insertion at positions 306 or 320 or a deletion at one or more KEX2 positions in the C-terminus, wherein each position corresponds to the position of the amino acid sequence of SEQ ID No. 2. Suitably the C-terminal processing may comprise removal of at least one of the C-terminal KEX2 sites. Without wishing to be bound by theory it is thought that removal of the KEX2 site causes cessation or a decrease in the rate of proteolytic processing and improved stability of the enzyme without compromising its activity. One KEX2 site may be found at position 306 (when aligned with SEQ ID No. 2).

Another KEX2 site may be found at positions 31 1-312 (when aligned with SEQ ID No. 2).

Preferably, nucleotide sequence according to the present invention or for use in the present invention is modified such that there is a substitution at one or more of positions 33, 63, 78, 190 and 305, wherein the amino acid is substituted with N.

Suitably the nucleotide sequence according to the present invention or for use in the present invention may be modified such that there is a substitution at one or more of positions 63, 78, 190 and 305, wherein the amino acid is substituted with N.

In one embodiment the nucleotide sequence according to the present invention or for use in the present invention may be modified such that a glycosylation site is introduced at positions 190, 191 and 192 (such that the glycosylation site comprises the consensus sequence Asn- Xxx-Ser or Asn-Xxx-Thr, where Xxx is any amino acid except Pro).

In another embodiment the nucleotide sequence according to the present invention or for use in the present invention may be modified such that a glycosylation site is introduced at positions 33, 34 and 35 (such that the glycosylation site comprises the consensus sequence Asn-Xxx-Ser or Asn-Xxx-Thr, where Xxx is any amino acid except Pro).

In another embodiment the nucleotide sequence according to the present invention or for use in the present invention may be modified such that a glycosylation site is introduced at positions 63, 64 and 65 (such that the glycosylation site comprises the consensus sequence Asn-Xxx-Ser or Asn-Xxx-Thr, where Xxx is any amino acid except Pro).

In another embodiment the nucleotide sequence according to the present invention or for use in the present invention may be modified such that a glycosylation site is introduced at positions 78, 79 and 80 (such that the glycosylation site comprises the consensus sequence Asn-Xxx-Ser or Asn-Xxx-Thr, where Xxx is any amino acid except Pro).

The glycosyolation site may be introduced by modifying (e.g. inserting or substituting) a single amino acid. Alternatively more than one amino acid (e.g. 2 or 3 amino acids) may be modified (e.g. inserted or substituted) in order to introduce the glycosylation site.

Suitably, the nucleotide sequence according to the present invention or for use in the present invention may be modified such that there is a substitution at position 190, wherein the amino acid is substituted with N.

In one embodiment preferably the nucleotide sequence according to the present invention comprises the whole or part of the nucleotide sequence shown here as SEQ ID No. 8 (or a nucleotide sequence which is related to SEQ ID No. 8 by the degeneration of the genetic code). The present invention yet further provides a polypeptide encoded by this nucleotide sequence.

In one embodiment the variant polypeptide of the present invention or for use in the present invention comprises an amino acid sequence as shown in amino acids 33-296 (or 31-304 or 31 -305) of SEQ ID No. 9.

Suitably, the nucleotide sequence according to the present invention or for use in the present invention may be modified such that there is a substitution at position 306, wherein the amino acid is substituted with any amino acid other than K or R or A, preferably the substitution at position 306 is with amino acid S.

Suitably, the nucleotide sequence according to the present invention or for use in the present invention may be modified such that there is a substitution at position 320, wherein the amino acid is substituted with any amino acid other than T, preferably the substitution at position 320 is with amino acid E. The variant polypeptide according to the present invention or encoded by the nucleic acid of the present invention or produced by a method of the present invention preferably has phospholipase activity or galactolipase activity.

In some embodiments preferably the modifications made to the variant polypeptide or the nucleic acid encoding same are modifications which add at least one glycosylation site (or multiple glycosylation sites) to the variant polypeptide and/or relate to C-terminal processing, such as to stabilise the C-terminus of the variant polypeptide.

Suitably the polypeptide may comprise a substitution at one or more of positions 33, 63, 78, 190, suitably 63, 78 or 190. Suitably the substitution maybe with N.

In one embodiment the variant polypeptide according to the present invention may comprises at least one substitution at position 306. Preferably the substitution at position 306 is with any amino acid other than K or R. Preferably the substitution at position 306 is with any amino acid other than K or R or A. In one embodiment the substitution at position 306 is preferably with a non-charged and/or hydrophilic amino acid. Suitably the substation at position 306 may be with amino acid S.

In one embodiment, the nucleotide sequence according to the present invention or for use in the present invention may be modified such that positions which are modified in the encoded amino acid sequences are as follows:

As one skilled in the art will readily appreciate when the backbone is not KLM lwt (shown herein as SEQ ID No. 2) then the amino acid shown in the table as the starting amino acid may vary in the alternative backbone at the same position when aligned with SEQ ID No. 2.

For the avoidance of doubt the symbol "Δ" as used herein means deletion of those amino acids listed after the symbol.

In some embodiments, the introduction of at least one amino acid at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme which is more hydrophilic (compared with the original amino acid) is preferably by the introduction of at least one glycosylation site (or one additional glycosylation site) at a surface position on the polypeptide and at a location in an external loop distal to the active site (catalytic triad) of the enzyme. In other words adding a glycosylation site renders the enzyme more hydrophilic in the region where the glycosylation site has been added.

In another aspect the present invention provides the use of a variant polypeptide enzyme according to the present invention in the manufacture of a foodstuff, such as for instance a dough, a baked product, an egg, an egg-based product, a noodle product, a cheese product, a tortilla product, an animal feed product, a vegetable oil or an edible oil. Advantageously, the addition of an enzyme of the present invention to the foodstuff may lead to improved emulsification with lower accumulation of free fatty acids.

In a further aspect the present invention provides the use of variant polypeptide enzyme according to the present invention in the manufacture of a dough and/or a baked product, comprising adding said lipolytic enzyme to a dough, and (optionally) baking the dough to make a baked product for one or more of the following: reducing stickiness of the dough; improving machinability of the dough; reducing blistering during baking of the baked product; improving bread volume and/or softness; prolonging shelf life of the baked product and/or dough; improving antistaling effect of the baked product and/or dough; improving crumb structure of the baked product; reducing pore heterogeneity of the baked product; improving pore homogeneity of the baked product; reducing mean pore size of the baked product; improving flavour and/or odour of the baked product, improving the colour of the crust of the baked product. In a farther aspect of the present invention provides the use of a variant polypeptide enzyme according to the present invention in the manufacture of egg-based products for improving texture, reducing mean particle size, reducing mean particle distribution, improving heat stability, improving microwave performance and/or stability.

In another aspect of the present invention, there is provided a method of treating egg or egg- based product, which method comprises adding a variant polypeptide enzyme according to the present invention to an egg or egg-based product.

In another aspect of the invention, there is provided a method of making noodles, or a noodle dough or a noodle-based product, which method comprises adding a variant polypeptide enzyme according to the present invention to the noodle, noodle dough or noodle-based product.

In one aspect of the present invention, there is provided a use of a variant polypeptide enzyme according to the present invention in the manufacture of a noodle or a noodle-based product for one or more of improving colour/yellowness, stabilising colour characteristics, reducing brightness, reducing fat content, improving texture and bite (chewiness), reducing water activity, reducing breakage, increasing core firmness and improving shape retention during processing

In another aspect of the invention, there is provided a method of making a tortilla or tortilla dough, which method comprises adding a variant polypeptide enzyme according to the present invention to the tortilla or tortilla dough.

A further aspect of the present invention provides the use of a variant polypeptide enzyme according to the present invention in the manufacture of a tortilla or a tortilla dough for improving the reliability of a tortilla, increasing pliability of a tortilla, improving antistaling properties of the tortilla and/or tortilla dough, improving softness and/or reducing off-flavour in the tortilla and/or tortilla dough. The functionality of the lipolytic enzyme in tortilla and/or noodles may be improved by combination with emulsifiers such as DATEM.

In another aspect of the invention, there is provided a method of treating milk, cheese milk, cheese or a cheese-based product, which method comprises adding a variant polypeptide enzyme according to the present invention to the cheese or cheese-based product.

The present invention yet further provides use of a variant polypeptide enzyme according to the present invention in the manufacture of a cheese or a cheese-based product for one or more of improving flavour, texture and/or stability, decreasing in the oiling-off effect in cheese and/or to increase cheese yield in cheese production.

In another aspect of the invention, there is provided a method of treating animal feed, which method comprises adding a variant polypeptide enzyme according to the present invention to the animal feed.

The present invention further provides the use of a variant polypeptide enzyme according to the present invention in the manufacture of animal feed for enhancing one or more of: feed utilisation and/or conversion efficiency, body weight gain, digestibility nitrogen uptake, metabolisability of dry matter and palatability.

In a further aspect of the present invention provides the use of a variant polypeptide enzyme according to the present invention in a process of preparing a lyso-phospholipid, for example lysolecithin by treatment of a phospholipid (e.g. lecithin) with the enzyme to produce the partial hydrolysis product, i.e. the lyso-phospholipid.

In another aspect of the present invention there is provided a process of preparing a lyso- phospholipid, for example lysolecithin, which process comprises treating a phospholipid (e.g. lecithin) with a variant polypeptide enzyme according to the present invention.

In a further aspect of the present invention provides the use of a variant polypeptide enzyme according to the present invention in a process of preparing a lyso-glycolipid, (for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG)) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the lipolytic enzyme according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.

In a yet further aspect there is provided a process of preparing a lyso-glycolipid (for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG)), which process comprising treating a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with a variant polypeptide enzyme according to the present invention.

The present invention also provides a process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant polypeptide enzyme according to the present invention so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).

For the avoidance of doubt, a person of ordinary skill in the art would be aware of methodology suitable for carrying out the enzymatic treatment of edible oils (for instance see EP 0 869 167). Known method may suitably be used when carrying out the present invention, with the proviso that the known enzyme is replaced with the enzyme according to the present invention.

In a further aspect the present invention provides the use of a variant polypeptide enzyme according to the present invention in the manufacture of a vegetable oil or edible oil for reducing the amount phospholipid in the vegetable oil or edible oil whilst maintaining the triglyceride content of the oil and/or preventing or reducing the accumulation of free fatty acids.

In a yet further aspect the present invention provides the use of a variant polypeptide enzyme according to the present invention in a process comprising treatment of a phospholipid so as to hydrolyse fatty acyl groups.

In another aspect the present invention provides the use of a variant polypeptide enzyme according to the present invention in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with the fungal lipolytic enzyme according to the present invention so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.

Preferably, the variant lipolytic enzyme according to the present invention hydrolyses polar lipids (e.g. glycolipids and/or phospholipids). In other words the variant lipolytic enzyme according to the present invention preferably has phospholipase activity (e.g. phospholipase A2 (E.G. 3.1.1.4) activity and/or phospholipase Al (E.G. 3.1.1.32) activity) and/or galactolipase or glycolipase (E.G. 3.1.1.26) activity. The variant lipolytic enzyme according to the present invention may additionally hydrolyse triglycerides. In other words the variant lipolytic enzyme according to the present invention may additional have triglyceride lipase activity (E.G. 3.1.1.3).

The term "glycolipase activity" as used herein encompasses "galactolipase activity". The terms glycolipids and galactolipids may be used interchangeably herein and include hydrolysis of DGDG and MGDG, which are hydrolysed to DGMG or MGMG, respectively.

The term "polar lipids" as used herein means phospholipids and/or glycolipids. Preferably, the term "polar lipids" as used herein means both phospholipids and glycolipids.

Suitably the variant polypeptide according to the present invention may have phospholipase activity (e.g. phospholipase A2 (E.G. 3.1.1.4) activity and/or phospholipase Al (E.G. 3.1.1.32) activity) and/or galactolipase or glycolipase (E.G. 3.1.1.26) activity.

The glycolipase activity, phospholipase activity and triacylglyceride lipase activity of an enzyme can be determined using the assays presented hereinbelow.

Determination of galactolipase activity (glycolipase activity assay):

Substrate: 0.6% digalactosyldiglyceride (Sigma D 4651), 0.4% Triton-X 100 (Sigma X-100) and 5 mM CaCb was dissolved in 0.05M HEPES buffer pH 7. Assay procedure: 400 μL substrate was added to an 1.5 mL Eppendorf tube and placed in an Eppendorf Thermomixer at 37°C for 5 minutes. At time t= 0 min, 50 μL enzyme solution was added. Also a blank with water instead of enzyme was analyzed. The sample was mixed at 10* 100 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t=l 0 min the Eppendorf tube was placed in another thermomixer at 99 ⁰ C for 10 minutes to stop the reaction.

Free fatty acid in the samples was analyzed by using the NEFA C kit from WAKO

GmbH.

Enzyme activity GLU at pH 7 was calculated as micromoles of fatty acid produced per minute under assay conditions.

Determination of phospholipase activity (pfaøspholipase activity assay):

Phospholipase activity was measured using two different methods which give comparable results. Either of these methods can be used to determine phospholipase activity in accordance with the present invention. Preferably, the PLU assay is used for determining the phospholipase activity of any enzyme.

"PLU assay" for determination of phospholipase activity

Substrate:

0.6% L-α Phosphatidylcholine 95% Plant (Avanti #441601), 0.4% Triton-X 100 (Sigma X-

100) and 5 mM CaCl ₂ was dissolved in 0.05M HEPES buffer pH 7.

Assay procedure: 400 μL substrate was added to an 1.5 mL Eppendorf tube and placed in an Eppendorf

Thermomixer at 37 ⁰ C for 5 minutes. At time t= 0 min, 50 μL enzyme solution was added.

Also a blank with water instead of enzyme was analyzed. The sample was mixed at 10* 100 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t=10 min the Eppendorf tube was placed in another thermomixer at 99°C for 10 minutes to stop the reaction. Free fatty acid in the samples was analyzed by using the NEFA C kit from WAKO GmbH.

Enzyme activity PLU-7 at pH 7 was calculated as micromoles of fatty acid produced per minute under assay conditions "TIPU assay" for determination of phospholipase activity

1 TIPU (Titration Phospholipase Unit) is defined as the amount of enzyme, which liberates 1 μmol free fatty acid per minute at the assay conditions.

Phospholipase Al and A2 catalyse the conversion of lecithin to lyso-lecithin with release of the free fatty acid from position 1 and 2, respectively. Phospholipase activity can be determined by continuous titration of the fatty acids liberated from lecithin during enzymation, since the consumption of alkali equals the amount of fatty acid liberated.

Substrate:

4% lecithin, 4% Triton-X 100, and 6 mM CaC12: 12 g lecithin powder (Avanti Polar Lipids #44160) and 12 g Triton-X 100 (Merck 108643) was dispersed in approx. 200 ml demineralised water during magnetic stirring. 3.0 ml 0.6 M CaC12 (p. a. Merck 1.02382) was added. The volume was adjusted to 300 mL with demineralised water and the emulsion was homogenised using an Ultra Thurax. The substrate was prepared freshly every day.

Assay procedure:

An enzyme solution was prepared to give a slope on the titration curve between 0.06 and 0.18 ml/min with an addition of 300 μL enzyme.

A control sample of known activity is included.

The samples were dissolved in demineralised water and stirred for 15 min. at 300 rpm.

25.00 ml substrate was thermostatted to 37.0 C for 10-15 minutes before pH was adjusted to

7.0 with 0,05 M NaOH. 300 μL enzyme solution was added to the substrate and the continuous titration with 0.05 M NaOH was carried out using a pH-Stat titrator (Phm 290,

Mettler Toledo). Two activity determinations are made on each scaling.

After 8 minutes the titration is stopped and the slope of the titration curve is calculated between 5 and 7 minutes. The detection limit is 3 TIPU/ml enzyme solution.

Calculations:

The phospholipase activity (TIPU/g enzyme) was calculated in the following way:

Where: α is the slope of the titration curve between 5 and 7 minutes of reaction time (ml/min) N is the normality of the NaOH used (mol/1) Vl is the volume in which the enzyme is dissolved (ml) m is the amount of enzyme added to Vl (g) V2 is the volume of enzyme solution added to the substrate (ml)

Determination of triacylglyceride lipase activity: assay based on triglyceride (tributyrin) as substrate (LIPU):

Lipase activity based on tributyrin is measured according to Food Chemical Codex, Forth Edition, National Academy Press, 1996, p 803, with the modifications that the sample is dissolved in deionized water instead of glycine buffer, and the pH stat set point is 5.5 instead of 7.

1 LIPU is defined as the quantity of enzyme which can liberate 1 mol butyric acid per minute under assay conditions.

The term "variant" as used herein means a protein which is not found in nature. Typically, the variant polypeptide may be produced by modifying a naturally occurring polypeptide (or nucleotide sequence encoding same). The variant polypeptide therefore comprises one or more amino acid alterations (i.e. amino acid deletions, additions or substitutions) when compared with the natural or wild-type sequence.

Preferably, the variant polypeptide according to the present invention is obtained from a fungal lipolytic enzyme obtainable (preferably obtained) from a filamentous fungus. More preferably, the fungal lipolytic enzyme is obtainable (preferably obtained) from Fusarium spp. Preferably, the fungal lipolytic enzyme according to the present invention may be obtainable (preferably obtained) from Fusarium heterosporum or Fusarium oxysporum. Suitably, the fungal lipolytic enzyme according to the present invention may be obtainable (preferably obtained) from Fusaήum heterosporwn (CBS 782.83) or Fusarium oxysporum (taught in WO98/26057 or US 7,465,570).

In one embodiment preferably the modification is not K63N, particularly when the backbone is from Fusarium oxysporum.

Thus in one aspect, preferably the lipolytic enzyme and the variant polypeptide according to the present invention is a fungal lipolytic enzyme, preferably a filamentous fungal lipolytic enzyme.

Preferably, the fungal lipolytic enzyme or variant polypeptide according to the present invention is not a fusion protein comprising an amino acid sequence from a Thermomyces protein or part thereof fused with an amino acid sequence from a Fusarium protein or part thereof. In particular, preferably the fungal lipolytic enzyme according to the present invention is not a fusion protein comprising an amino acid sequence from a Thermomyces lanuginosa protein or a part thereof fused with an amino acid sequence from a Fusaήum oxysporum protein or part thereof.

Preferably, the fungal lipolytic enzyme according to the present invention is not obtained from Thermomyces lanuginosa and/or is not a variant of an enzyme obtained from Thermomyces lanuginosa.

The variant polypeptides of the present invention were tested in baking tests and compared with the lipolytic enzyme from Fusarium heterosporwn CBS 782.83 (SEQ ID No. 2 - which is the prepro-sequence with the mature sequence being amino acids 33-296 (suitably 31-304 or 31-305 depending on the host organism). This enzyme is also designated herein as KLMl and constitutes the "wild-type" enzyme or backbone enzyme in respect of the variant polypeptides taught herein) with very good results.

The baking effects of the variant polypeptides were found to be superior to the fungal lipolytic enzyme from F. heterosporum CBS 782.83 (an enzyme comprising the amino acids sequence shown as amino acids 33-296 (suitably 31-304 or 31-305) of SEQ ID No. 2; KLMl). Suitably, the term "foodstuff as used herein means a substance which is suitable for human and/or animal consumption.

Suitably, the term "foodstuff' as used herein may mean a foodstuff in a form which is ready for consumption. Alternatively or in addition, however, the term foodstuff as used herein may mean one or more food materials which are used in the preparation of a foodstuff. By way of example only, the term foodstuff encompasses both baked goods produced from dough as well as the dough used in the preparation of said baked goods.

In a preferred aspect the present invention provides a foodstuff as defined above wherein the foodstuff is selected from one or more of the following: eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams; edible oils and fats, aerated and non-aerated whipped products, oil-in-water emulsions, water-in-oil emulsions, margarine, shortening and spreads including low fat and very low fat spreads; dressings, mayonnaise, dips, cream based sauces, cream based soups, beverages, spice emulsions and sauces.

In one aspect the foodstuff in accordance with the present invention may be a dough product or a baked product, such as a bread, a fried product, a snack, cakes, pies, brownies, cookies, noodles, instant noodles, tortillas, snack items such as crackers, graham crackers, pretzels, and potato chips, and pasta.

In another aspect, the foodstuff in accordance with the present invention may be an animal feed. In one aspect preferably the foodstuff is selected from one or more of the following: eggs, egg-based products, including mayonnaise, salad dressings, sauces, ice cream, egg powder, modified egg yolk and products made therefrom.

In some of the applications mentioned herein, particularly the food applications, such as the bakery applications, the lipolytic enzyme according to the present invention may be used with one or more conventional emulsifiers, including for example monoglycerides, diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, sodium stearoyl lactylate (SSL) and lecithins.

In addition or alternatively, the enzyme according to the present invention may be used with one or more other suitable food grade enzymes. Thus, it is within the scope of the present invention that, in addition to the lipolytic enzyme of the present invention, at least one further enzyme may be added to the baked product and/or the dough. Such further enzymes include starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phospholipases and hexose oxidase, proteases, and acyltransferases (such as those described in WO04/064987 for instance).

It is particularly preferred that the lipolytic enzyme of the invention is used in combination with alpha amylases in producing food products. In particular, the amylase may be a non~ maltogenic amylase, such as a polypeptide having non-maltogenic exoamylase activity, in particular, glucan 1 ,4-alpha-maltotetrahydrolase (EC 3.2.1.60) activity (as disclosed in WO05/003339). A suitable non-maltogenic amylase is commercially available as Powersoft™ (available from Danisco A/S, Denmark). Maltogenic amylases such as Novamyl™ (Novozymes A/S, Denmark) may also be used. In one embodiment, the combined use of alpha amylases and the lipolytic enzyme of the invention may be used in a dough, and/or the production of a baked product, such as bread, cakes, doughnuts, cake doughnuts or bagels. The combination of alpha amylases and the lipolytic enzyme of the invention is also considered as preferable for use in methods of production of tortillas, such as wheat and/or maize tortillas. In another preferred embodiment, the lipolytic enzyme according to the present invention may be used in combination with a xylanase in producing food products. GRINDAMYL™ and POWERBake 7000 are examples of commercially available xylanase enzymes available from Danisco A/S. Other examples of xylanase enzymes may be found in WO03/020923 and WO01/42433

Preferably, the lipolytic enzyme according to the present invention may be used in combination with a xylanase and an alpha amylase. Suitably the alpha amylase may be a maltogenic, or a non-maltogenic alpha amylase (such as GRINDAMYL™ or POWERSoft, commercially available from Danisco A/S), or a combination thereof.

The lipolytic enzyme of the invention can also preferably be used in combination with an oxidising enzyme, such as a maltose oxidising enzyme (MOX), for example hexose oxidase (HOX). Suitable methods are described in WO03/099016. Commercially available maltose oxidising enzymes GRINDAMYL™ and SUREBake are available from Danisco A/S.

Optionally an alpha-amylase, such as a non-maltogenic exoamylase and/or a maltogenic amylases, and/or a maltose oxidising enzyme (MOX) in combination with the enzyme according to the present invention may be used in methods of preparing a dough, a baked product, tortilla, cake, instant noodle/fried snack food, or a dairy product such as cheese.

The lipolytic enzyme according to the present invention is typically included in the foodstuff or other composition by methods known in the art. Such methods include adding the lipolytic enzyme directly to the foodstuff or composition, addition of the lipolytic enzyme in combination with a stabilizer and/or carrier, and addition of a mixture comprising the lipolytic enzyme and a stabilizer and/or carrier.

Suitable stabilizers for use with the present invention include but is not limited to inorganic salts (such as NaCl, ammonium sulphate), sorbitol, emulsifiers and detergents (such as Tween 20, Tween 80, Panodan ABlOO without triglycerides, polyglycerolester, sorbitanmonoleate), oil (such as rape seed oil, sunflower seed oil and soy oil), pectin, trehalose and glycerol. Suitable carriers for use with the present invention include but is not limited to starch, ground wheat, wheat flour, NaCl and citrate.

Further preferable aspects are presented in the accompanying claims and the in the following description and examples.

ADVANTAGES

One advantage of the methods of the present invention, the nucleic acids of the present invention and the variant polypeptides of the present invention is that the expression of the nucleic acids in a commercial host species, e.g. Trichoderma reesei, is significantly improved compared with the wild-type enzyme (e.g. KLM 1; encoded by nucleotide sequence SEQ ID No. 1). This has the advantage that it is much cheaper to produce the variants. The increase in production is significant with the wild type in T. reesei being inefficiently produced, whereas the variants typically have an improved expression level. Typically the variants are expressed at levels between about 2 to about 25 times, preferably between about 6 times to about 25 times, higher than the wild type enzyme.

The variant enzymes of the present invention have surprisingly been found to have superior functionality when used in baking applications. The use of the variant lipolytic enzymes according to the present invention advantageously results in significantly improved properties to the dough and/or baked products compared with other lipolytic enzymes from fungi, particularly LipopanF™ and/or the wild type enzyme from Fusarium heterosporum

(comprising the amino acid sequence shown herein as amino acids 33-296 (suitably 31-304 or 31-305) of SEQ ID No. 2 and taught in WO2005/087918).

Another advantage of the variant polypeptides of the present invention is their enhanced activity and/or functionality compared with the wild-type enzyme (e.g. KLMl, comprising the amino acid sequence shown herein as amino acids 33-296 (suitably 31-304 or 31-305) of SEQ ID No.2). This can lead to the "cost-in-use" of the enzyme being reduced. For example the proportion of the units that would be needed with the variant polypeptide in order to achieve the same results/effects compared with the wild-type enzyme would be significantly reduced. For instance, it is envisaged that the variant polypeptide can be dosed at about 25%-75%, preferably about 25%-50%, preferably about 25%, of the level of the wild-type enzyme(s).

The term "modifying" (or "modification") as used herein means substituting or inserting (or substitution or insertion).

TECFTNICAL EFFECTS

For baked products, such as bread, steam buns and US white pan bread, for example, the addition of a lipolytic enzyme of the present invention may result in one or more of the following: improved bread volume and softness, prolonged shelf life and/or an antistaling effect, improved crumb structure, reduced pore heterogeneity, reduced mean pore size, improved flavour and/or odour, and improved colour of the crust.

Advantageously, the enzyme according to the present invention may be used to replace emulsifiers in foodstuffs, such as dough and/or baked products.

The lipolytic enzyme according to the present invention may have synergy with emulsifiers such as DATEM, SSL, CSL, monoglyceride, polysorbates and Tween. Thus, the lipolytic enzyme according to the present invention may be used in combination with one or more emulsifiers. Advantageously, the use of the lipolytic enzyme according to the present invention in combination with one or more emulsifiers may reduce the overall amount of emulsifier used compared with the amount needed when no enzyme according to the present invention is used.

The lipolytic enzyme according to the present invention may also have synergy with hydrocolloids, Guar, xanthum and pectin, and with maltose oxidising enzymes such as hexose oxidase.

For doughnuts, cake doughnuts, bagels, snack cakes and muffins, for example, the use of a lipolytic enzyme of the present invention may result in a synergistic effect when used in combination with one or more of alpha-amylases, maltogenic alpha-amylase and non- maltogenic alpha-amylase. For cakes, sponge cakes and palm cakes, for example, the use of the lipolytic enzyme of the present invention may result in a synergistic effect when used in combination with one or more of hydrocolloids such as Guar, and/or one or more emulsifiers such as DATEM.

For biscuits, for example, use of a lipolytic enzyme according to the present invention confers improved reliability and handling properties, particularly when cold (cold reliability).

Advantageously, in mayonnaise and other egg-based products, for example, use of a lipolytic enzyme according to the present invention may lead to improved texture, reduced mean particle size, and/or reduced mean particle distribution, improved heat stability, improved microwave performance and/or stability.

In cakes, use of the present invention advantageously leads to improved softness, volume, improved keeping properties and shelf life.

For noodles or noodle-products, e.g. instant noodles, for example, the lipolytic enzyme of the present invention may confer one or more of the following characteristics: improved colour/yellowness, more stable colour characteristics, reduced brightness, reduced fat content, improved texture and bite (chewiness), reduced water activity, reduced breakage, increased core firmness and improved shape retention during processing.

Preferably, the lipolytic enzyme of the present invention may be used to reduce the fat content of a noodle or a noodle product, for instance an instant noodle.

In tortilla, for example, use of the enzyme according to the present invention may result in one or more of the following: reduced reliability of the tortilla, for instance by increasing pliability, improved antistaling properties, improving softness and/or reducing off flavour.

Advantageously, improved reliability and/or pliability may lead to a reduced likelihood of the tortilla splitting when rolled. In cheese and/or cheese-based products, for example, the use of the enzyme according to the present invention may result in one or more of the following: an improved flavour, texture and/or stability, a decrease in the oiling-off effect in cheese and/or an increase in cheese yield.

The term "oiling off effect" as used herein refers to the free oil released when cheese is melted.

The lipolytic enzyme according to the present invention may be used to produce a low fat cheese. Advantageously, the enzyme of the present invention may stabilise fat in milk and/or may enhance flavour.

In animal feed, for example, the enzyme according to the present invention advantageously may result in one or more the following: enhanced feed utilisation/conversion efficiency within the animal, improved body weight gain of the animal, improved digestibility of the feed, improved nitrogen uptake by the animal, e.g. from the feed, improved metabolisability of dry matter of the feed and improved palatability of feed.

USES

The enzyme according to the present invention has many applications.

In particular, the variant polypeptides according to the present invention may be useful in the preparation of a foodstuff.

For example, the variant polypeptides according to the present invention may be particularly useful in the treatment of egg or egg-based products.

Treatment of egg or egg-based products with a fungal lipolytic enzyme according to the present invention can improve the stability, thermal stability under heat treatment such as pasteurisation and result in substantial thickening. Egg-based products may include, but are not limited to cakes, mayonnaise, salad dressings, sauces, ice creams and the like.

The fungal lipolytic enzymes according to the present invention are particularly useful in the preparation of baked products, such as those prepared from a dough, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies.

The fungal lipolytic enzymes according to the present invention may also be used in bread- improving additive, e.g. dough compositions, dough additive, dough conditioners, pre-mixes and similar preparations conventionally added to the flour and/or the dough during processes for making bread or other baked products to provide improved properties to the bread or other baked products.

Thus, the present invention further relates to a bread-improving composition and/or a dough- improving composition comprising a variant polypeptide according to the present invention; and also to a dough or baked product comprising such a bread-improving and/or dough- improving composition.

The bread-improving composition and/or dough-improving composition may comprise, in addition to a fungal lipolytic enzyme according to the present invention, other substances, which substances are conventionally used in baking to improve the properties of dough and/or baked products.

The bread-improving composition and/or dough-improving composition may comprise one or more conventional baking agents, such as one or more of the following constituents: a milk powder, gluten, an emulsifier, granulated fat, an oxidant, an amino acid, a sugar, a salt, flour or starch.

Examples of suitable emulsifiers are: monoglycerides, diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, sugar esters, sodium stearoyl lactylate (SSL) and lecithins.

The bread and/or dough improving composition may further comprise another enzyme, such as one or more other suitable food grade enzymes, including starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phospholipases and hexose oxidase, proteases and acyltransferases (such as those described in WO04/064987 for instance).

The term "improved properties" as used herein means any property which may be improved by the action of the variant polypeptide of the present invention. In particular, the use of a variant polypeptide according to the present invention results in one or more of the following characteristics: increased volume of the baked product; improved crumb structure of the baked product; anti-staling properties in the baked product; increased strength, increased stability, reduced stickiness and/or improved machinability of the dough.

The improved properties are evaluated by comparison with a dough and/or a baked product prepared without addition of the variant polypeptide according to the present invention or by comparison with a dough and/or baked product prepared with the addition of a wild-type enzyme (e.g. KLMl ; comprising the amino acid sequence shown herein as amino acids 33- 296 (suitably 31-304 or 31-305) of SEQ ID No. 2).

The term "baked product" as used herein includes a product prepared from a dough. Examples of baked products (whether of white, light or dark type) which may be advantageously produced by the present invention include one or more of the following: bread (including white, whole-meal and rye bread), typically in the form of loaves or rolls or toast, French baguette-type bread, pitta bread, tortillas, tacos, cakes, pancakes, biscuits, crisp bread, pasta, noodles and the like.

The dough in accordance with the present invention may be a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways such as by adding sodium bicarbonate or the like, or by adding a suitable yeast culture such as a culture of Saccharomyces cerevisiae (baker's yeast).

The present invention further relates to the use of fungal lipolytic enzymes in accordance with the present invention to produce a pasta dough, preferably prepared from durum flour or a flour of comparable quality. The variant polypeptides according to the present invention are suitable for use in the enzymatic degumming of vegetable or edible oils. In processing of vegetable or edible oil the edible or vegetable oil is treated with a fungal lipolytic enzyme according to the present invention so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid). Preferably, the fatty acyl groups are hydrolysed from the polar lipids. The degumming process typically results in the reduction of the content of the polar lipids, particularly of phospholipids, in an edible oil due to hydrolysis of a major part (i.e. more than 50%) of the polar lipid, e.g. glycolipid and/or phospholipid. Typically, the aqueous phase containing the hydrolysed polar lipid (e.g. phospholipid and/or glycolipid) is separated from the oil. Suitably, the edible or vegetable oil may initially (pre-treatment with the enzyme according to the present invention) have a phosphorus content of 50-250 ppm.

Furthermore, the present invention is directed to the use of the variant polypeptides according to the present invention for treatment of cheese products.

The variant polypeptides according to the present invention are also particularly suitable for use in the preparation of an animal feed.

As the skilled person is aware, the term "degumming" as used herein means the refining of oil by converting phosphatides (such as lecithin, phospholipids and occluded oil) into hydratable phosphatides. Oil which has been degummed is more fluid and thus has better handling properties than oil which has not been degummed.

The following table is merely for general guidance and provides an overview of the dosage level for a variant polypeptide according to the present invention which may be needed in different applications. The table further provides guidance in respect of the dosage level for a lipolytic enzyme according to the present invention when used in combination with an emulsifier for example. Of course, as would be apparent to the person of ordinary skill in the art optimisation of enzyme dosage, reaction temperature and reaction time may be readily determined, using routine experimentation, for any given application.

GLYCOSYLATION

It has surprisingly been found that by introducing even one glycosylation site into a lipolytic enzyme (particularly those that do not comprise any glycosylation sites naturally), e.g. such as the lipolytic enzymes taught herein (e.g. the Fusarium heterosporum enzyme (sometimes designated herein as KLMl) and/or the Fusarium oxysporum lipolytic enzyme (sometimes designated herein as Lipopan F™)) the results on the level of expression and/or the functionality and/or the activity of the enzyme is much improved compared with the wild-type enzymes - particularly when the host cell is Trichoderma spp., such as T. reesei. Therefore the present invention provides modifying a lipolytic enzyme (particularly one comprising amino acids 40-290 (preferably 35-300, more preferably 31 -305) of the amino acid SEQ ID No. 2) or the nucleotide sequence encoding a lipolytic enzyme (such as the nucleotide sequence shown herein as SEQ ID No. 1) to substitute or insert amino acids in the sequence to produce variant polypeptides comprising an added glycosylation site. Notably, the wild-type enzyme shown as SEQ ID No. 2 (preprosequence for KLMl) or comprising amino acids 33-296 (suitably 31-304 or 31 -305) of SEQ ID No. 2 for the mature sequence does not naturally comprise any glycosylation sites. Suitably, the lipolytic enzyme is modified to substitute or insert one or more amino acids selected from Asn, Ser, Thr for the purpose of introducing at least one glycosylation site (suitably more than one glycosylation site).

Notably the wild-type enzyme from Fusarium heterosporum CBS 782.83 (designated herein as KLMl and having the preprosequence shown as SEQ ID No. 2 with the mature sequence comprise amino acids 33-296 (such as 31-304 or 31-305) of SEQ ID No. 2) has no glycosylation sites. It was surprising for the inventors that even a modest addition, i.e. the addition of one glycosylation site, could bring about such a significant improvement with regard to expression, functionality and/or activity.

The term "glycosylation site" as used herein means a sequence Asn-Xxx-Ser or Asn-Xxx-Thr wherein Xxx is any amino acid residue except proline.

When we refer to glycosylation site herein we may mean potential glycosylation site. In other words we provide the appropriate consensus sequence, i.e. Asn-Xxx-Ser or Asn-Xxx-Thr wherein Xxx is any amino acid residue except proline in the variant enzyme and when such a protein carrying the consensus sequence is secreted by the fungal host there is a high probability that the γ-amide of the asparagine will be glycosylated (although tertiary structure of the protein may modulate the efficiency of glycosylation).

Glycosylation is the enzymatic process that links saccharides to produce glycans, either free or attached to proteins and lipids. This enzymatic process produces one of four fundamental components of all cells (along with nucleic acids, proteins, and lipids) and also provides a co- translational and post-translational modification mechanism that modulates the structure and function of membrane and secreted proteins. The majority of proteins synthesized in the rough ER undergo glycosylation. It is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation. Glycosylation is also present in the cytoplasm and nucleus as the O-GlcNAc modification. Six classes of glycans are produced: TV-linked glycans attached to the amide nitrogen of asparagine side chains, O-linked glycans attached to the hydroxy oxygen of serine and threonine side chains; glycosaminoglycans attached to the hydroxy oxygen of serine; glycolipids in which the glycans are attached to ceramide, hyaluronan which is unattached to either protein or lipid, and GPI anchors which link proteins to lipids through glycan linkages.

In the present invention when we refer to glycosylation we are only referring to TV-linked glycosylation. In other words the present invention is not intended to relate to O-linked glycosylation.

For TV-linked oligosaccharides, a 14-sugar precursor is first added to the asparagine in the polypeptide chain of the target protein. The structure of this precursor is common to most eukaryotes, and contains 3 glucose, 9 mannose, and 2 TV-acetylglucosamine molecules. A complex set of reactions attaches this branched chain to a carrier molecule called dolichol, and then it is transferred to the appropriate point on the polypeptide chain as it is translocated into the ER lumen.

There are three major types of TV-linked saccharides: high-mannose oligosaccharides, complex oligosaccharides and hybrid oligosaccharides.

High-mannose is, in essence, just two TV-acetylglucosamines with many mannose residues, often almost as many as are seen in the precursor oligosaccharides before it is attached to the protein.

Complex oligosaccharides are so named because they can contain almost any number of the other types of saccharides, including more than the original two TV-acetylglucosamines. Proteins can be glycosylated by both types of oligos on different portions of the protein. Whether an oligosaccharide is high-mannose or complex is thought to depend on its accessibility to saccharide-modifying proteins in the Golgi. If the saccharide is relatively inaccessible, it will most likely stay in its original high-mannose form. If it is accessible, then it is likely that many of the mannose residues will be cleaved off and the saccharide will be further modified by the addition of other types of group as discussed above.

The oligosaccharide chain is attached by oligosaccharyltransferase to asparagine occurring in the tripeptide consensus sequence Asn-Xxx-Ser or Asn-Xxx-Thr where X could be any amino acid except Pro. This sequence is also known as a glycosylation sequon. After attachment, once the protein is correctly folded, the three glucose residues are removed from the chain and the protein is available for export from the ER. The glycoprotein thus formed is then transported to the Golgi where removal of further mannose residues may take place.

In the present invention when we discuss modifying a lipolytic (backbone) enzyme, e.g. a wild-type enzyme such as KLMl or Lipopan F or the nucleotide sequence encoding same to substitute or insert one or more amino acids such that the variant polypeptide formed comprises at least one glycosylation site (or at least one additional glycosylation site compared with the backbone enzyme) - we mean substituting the amino acids in the backbone enzyme or inserting one or more amino acids into the backbone such that one or more consensus sequences Asn-Xxx-Ser or Asn-Xxx-Thr where X could be any amino acid except Pro are introduced.

In one embodiment, suitably the modification may be the substitution or introduction of a single amino acid in the backbone sequence. For example the backbone sequence may comprise the following: Yyy-Xxx-Ser or Yyy-Xxx-Thr or Asn-Xxx-Zzz (where Xxx is not Pro, Yyy is not Asn and Zzz is not Ser or Thr) - and may simply require the substitution of amino acid Yyy with Asn and the substitution of Zzz with either Ser or Thr or the insertion of Asn after Yyy or the insertion of Ser or Thr before Zzz.

Alternatively, two or three of the amino acids in the backbone may be changed to produce a single glycosylation consensus sequence. For example, the backbone may be modified to substitute or insert three amino acids with Asn-Xxx-Ser or Asn-Xxx-Thr where X could be any amino acid except Pro.

Suitably more than one glycosylation site (potential glycosylation site) may be introduced into the back bone sequence such that the variant polypeptide comprises more than one glycosylation site (or more than additional glycosylation sites compared with the backbone sequence). Obviously the overall number of glycosylation sites (or potential glycosylation sites) in the variant polypeptide will depend on the number of glycosylation sites in the backbone enzyme and then number of glycosylation sites added. For the avoidance of doubt the wild-type KLMl enzyme (shown herein as SEQ ID No. 2 does not comprise any glycosylation sites and therefore the number of glycosylation sites in the variant polypeptide will be determined by the number of added glycosylation sites.

STABILISATION OF THE C-TERMINAL Suitably, the variant polypeptides according to the present invention may include C-terminal processing, preferably to render the polypeptide more stable. Suitably the C-terminal processing may comprise removal of the C-terminal KEX2 sites. Without wishing to be bound by theory it is thought that removal of the KEX2 site causes cessation or a decrease in the rate of proteolytic processing and improved stability of the enzyme without compromising its activity. One KEX2 site may be found at position 306 (when aligned with SEQ ID No. 2). Another KEX2 site may be found at positions 311-312 (when aligned with SEQ ID No. 2).

Suitably the C-terminal of the polypeptide commences at amino acid position 306 onwards.

EXTERNAL LOOPS DISTAL TO THE ACTIVE SITE

In the present application it is taught that amino acid modifications (e.g. substitutions and/or insertions) are made to surface amino acids of the polypeptide which are located in external loops distal to the active site of the polypeptide in order to either introduce hydrophilic amino acids or to introduce one or more glycosylation sites.

When selecting a site for modification preferably the site is a) a surface location, b) a non- conservative amino acid, and c) at a location not in the immediate vicinity of the active site (catalytic tria) or the active site lid. By the term "external loops" it is meant that a portion of the amino acid sequence which in the tertiary structure of the protein is exposed on the outer surface of the protein in a loop. The external loops are not involved in forming the catalytic triad or the lid region of the enzyme.

The term "active site" as used herein is synonymous with the term catalytic triad. For the avoidance of doubt the catalytic triad in the lipolytic enzymes taught herein are at positions The catalytic triad of the KLMl enzyme is formed from S 174, D228 and H287.

Preferably the external loops are more than about 15 A, preferably more than about 16 A, such as more than about 17 A, more than about 18 A, more than about 19 A, preferably more than about 2θA from the α-carbon of Serl74. For the avoidance of doubt both the catalytic triad and the lid region of the enzyme are less than 15A from the α-carbon of Serl74.

In one embodiment the external loops distal to the active site of the polypeptide correspond with the one or more of the following amino acid regions (with the amino acid positions Corresponding with the numbering shown in SEQ ID No. 2 - i.e. obtained by aligning the lipolytic enzyme with SEQ ID No. 2 shown herein): 54-66, 75-79, 99-103, 127-135, 162-167, 188-195 and 213-221.

By the term "distal to the active site" it is meant remote to or at a distance from the active site of the protein. Preferably the external loops are at least about 15 A, preferably more than about 16 A, such as more than about 17A, more than about 18A ₅ more than about 19A, preferably more than about 2θA from the α-carbon of Ser 174.

HYDROPHILIC AMINO ACIDS

Depending on the polarity of the side chain, amino acids vary in their hvdrophilic or hydrophobic character. Selecting amino acids which are more hydrophilic than other amino acids should be routine a person of ordinary skill in the art. In any event, guidance is provided in the below table: Side chain Side chain charge Hj

Amino Acid 3-Letter 1 -Letter polarity (pH 7) index

Alanine Ala A nonpolar neutral 1.8

Arginine Arg R polar positive -4.5

Asparagine Asn N polar neutral -3.5

Aspartic acid Asp D polar negative -3.5

Cysteine Cys C nonpolar neutral 2.5

Glutamic acid GIu E polar negative -3.5

Glutamine GIn Q polar neutral -3.5

Glycine GIy G nonpolar neutral -0.4

Histidine His H polar positive -3.2

Isoleucine lie I nonpolar neutral 4.5

Leucine Leu L nonpolar neutral 3.8

Lysine Lys K polar positive -3.9

Methionine Met M nonpolar neutral 1.9

Phenylalanine Phe F nonpolar neutral 2.8

Proline Pro P nonpolar neutral -1.6

Serine Ser S polar neutral -0.8

Threonine Thr T polar neutral -0.7

Tryptophan Trp W nonpolar neutral -0.9

Tyrosine Tyr Y polar neutral -1.3

Valine VaI V nonpolar

LIPOLYTIC ENZYME

Preferably, the lipolytic enzyme or variant lipolytic enzyme according to the present invention has hydrolytic activity towards an ester bond in a polar lipid. Preferably, the lipolytic enzyme or variant lipolytic enzyme according to the present invention hydrolyses polar lipids (e.g. glycolipids and/or phospholipids). In other words the variant lipolytic enzyme according to the present invention preferably has phospholipase activity (e.g. phospholipase A2 (E.C. 3.1.1.4) activity and/or phospholipase Al (E.G. 3.1.1.32) activity) and/or galactolipase or glycolipase (E.C. 3.1.1.26) activity. The variant lipolytic enzyme according to the present invention may additionally hydrolyse triglycerides. In other words the variant lipolytic enzyme according to the present invention may additional have triglyceride lipase activity (E.C. 3.1.1.3).

The term "glycolipase activity" as used herein encompasses "galactolipase activity". The terms glycolipids and galactolipids may be used interchangeably herein and include hydrolysis of DGDG and MGDG, which are hydrolysed to DGMG or MGMG, respectively.

The term "polar lipids" as used herein means phospholipids and/or glycolipids. Preferably, the term "polar lipids" as used herein means both phospholipids and glycolipids.

Suitably the variant polypeptide according to the present invention may have phospholipase activity (e.g. phospholipase A2 (E.C. 3.1.1.4) activity and/or phospholipase Al (E.C. 3.1.1.32) activity) and/or galactolipase or glycolipase (E.C. 3.1.1.26) activity.

The glycolipase activity, phospholipase activity and triacylglyceride lipase activity of an enzyme can be determined using the assays presented hereinabove.

In some embodiments the lipolytic enzyme prior to modification in accordance with the present invention does not comprise a glycosylation site.

In one embodiment the lipolytic enzyme prior to modification in the accordance with the present invention (i.e. the fungal lipolytic enzyme) is one which belongs to the family 23 of alpha/beta hydrolases, more specifically to the subfamily 23.01 (as classified by the lipase engineering database from the University of Stuttgart - see http://www.led.uni-stuttgai1.de/ ). This database integrates information on the sequence and structure of lipases and related proteins sharing the same a/b hydrolase fold. ISOLATED

In one aspect, preferably the sequence is in an isolated form. The term "isolated" means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.

In one embodiment the polypeptides and/or nucleotides sequences of the present invention are isolated.

PURIFIED In one aspect, preferably the sequence is in a purified form. The term "purified" means that the sequence is in a relatively pure state - e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.

In one embodiment the polypeptides and/or nucleotides sequences of the present invention are purified.

NUCLEOTIDE SEQUENCE

The scope of the present invention encompasses nucleotide sequences encoding enzymes having the specific properties as defined herein.

The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti- sense strand.

The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for the present invention.

In a preferred embodiment, the nucleotide sequence when relating to and when encompassed by the per se scope of the present invention does not include the native nucleotide sequence according to the present invention when in its natural environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence", hi this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism. Preferably, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.

PREPARATION OF THE NUCLEOTIDE SEQUENCE

Typically, the nucleotide sequence encompassed by scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH βt al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al, (1980) Nuc Acids Res Symp Ser 225-232).

A nucleotide sequence encoding an enzyme which has the specific properties as defined herein may be identified and/or isolated and/or purified from any cell or organism producing said enzyme. Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme or a part of the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme- encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used. Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for the enzyme (e.g. maltose for a glucosidase (maltase) producing enzyme), thereby allowing clones expressing the enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S. L. et al, (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al, (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.

The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K e^ al., (Science (1988) 239, pp 487-491).

Due to degeneracy in the genetic code, nucleotide sequences may be readily produced in which the triplet codon usage, for some or all of the amino acids encoded by the original nucleotide sequence, has been changed thereby producing a nucleotide sequence with low homology to the original nucleotide sequence but which encodes the same, or a variant, amino acid sequence as encoded by the original nucleotide sequence. For example, for most amino acids the degeneracy of the genetic code is at the third position in the triplet codon (wobble position) (for reference see Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN 0-7167-1920-7) therefore, a nucleotide sequence in which all triplet codons have been "wobbled" in the third position would be about 66% identical to the original nucleotide sequence. However, the amended nucleotide sequence would encode for the same, or a variant, primary amino acid sequence as the original nucleotide sequence. Therefore, the present invention further relates to any nucleotide sequence that has alternative triplet codon usage for at least one amino acid encoding triplet codon, but which encodes the same, or a variant, polypeptide sequence as the polypeptide sequence encoded by the original nucleotide sequence.

Furthermore, specific organisms typically have a bias as to which triplet codons are used to encode amino acids. Preferred codon usage tables are widely available, and can be used to prepare codon optimised genes. Such codon optimisation techniques are routinely used to optimise expression of transgenes in a heterologous host.

AMINO ACID SEQUENCES

The scope of the present invention also encompasses amino acid sequences of enzymes having the specific properties as defined herein.

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

The enzyme encompassed in the present invention may be used in conjunction with other enzymes. Thus the present invention also covers a combination of enzymes wherein the combination comprises the enzyme of the present invention and another enzyme, which may be another enzyme according to the present invention.

Preferably the amino acid sequence when relating to and when encompassed by the per se scope of the present invention is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence. SEQUENCE IDENTITY OR SEQUENCE HOMOLOGY

Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.

In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95, 98% or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95, 98% or 99% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTl Advance™ 1 1 (Invitrogen Corp.). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), and FASTA (Altschul et al 1990 J. MoI. Biol. 403-410). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the Vector NTI Advance™ 1 1 program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; and FEMS Microbiol Lett 1999 177(1): 187-8.). Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI Advance™ 11 package.

Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI Advance™ 11 (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

Should Gap Penalties be used when determining sequence identity, then preferably the default parameters for the programme are used for pairwise alignment. For example, the following parameters are the current default parameters for pairwise alignment for BLAST 2:

In one embodiment, preferably the sequence identity for the nucleotide sequences and/or amino acid sequences may be determined using BLAST2 (blastn) with the scoring parameters set as defined above.

For the purposes of the present invention, the degree of identity is based on the number of sequence elements which are the same. The degree of identity in accordance with the present invention for amino acid sequences may be suitably determined by means of computer programs known in the art such as Vector NTI Advance™ 1 1 (Invitrogen Corp.). For pairwise alignment the scoring parameters used are preferably BLOSUM62 with Gap existence penalty of 1 1 and Gap extension penalty of 1.

Suitably, the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.

Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienyl alanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences.

The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PiIeUp program is widely used. The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides .

Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

HYBRIDISATION

The present invention also encompasses the use of sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.

The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the subject sequences discussed herein, or any derivative, fragment or derivative thereof.

The present invention also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences discussed herein.

Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.

Maximum stringency typically occurs at about Tm-5°C (5 ⁰ C below the Tm of the probe); high stringency at about 5°C to 10 ⁰ C below Tm; intermediate stringency at about 10 ⁰ C to 2O ⁰ C below Tm; and low stringency at about 20 ⁰ C to 25 ⁰ C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.

Preferably, the present invention encompasses the use of sequences that are complementary to sequences that are capable of hybridising under high stringency conditions or intermediate stringency conditions to nucleotide sequences encoding polypeptides having the specific properties as defined herein.

More preferably, the present invention encompasses the use of sequences that are complementary to sequences that are capable of hybridising under high stringency conditions (e.g. 65 ⁰ C and O.lxSSC {lxSSC = 0.15 M NaCl ₅ 0.015 M Na-citrate pH 7.0}) to nucleotide sequences encoding polypeptides having the specific properties as defined herein.

The present invention also relates to the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).

The present invention also relates to the use of nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).

Also included within the scope of the present invention are the use of polynucleotide sequences that are capable of hybridising to the nucleotide sequences discussed herein under conditions of intermediate to maximal stringency.

In a preferred aspect, the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g. 5O ⁰ C and 0.2xSSC).

In a more preferred aspect, the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringency conditions (e.g. 65 ⁰ C and O.lxSSC).

BIOLOGICALLY ACTIVE Preferably, the variant sequences etc. are at least as biologically active as the sequences presented herein. As used herein "biologically active" refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.

RECOMBmANT

In one aspect the sequence for use in the present invention is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.

SYNTHETIC

In one aspect the sequence for use in the present invention is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.

EXPRESSION OF ENZYMES

The nucleotide sequence for use in the present invention may be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in enzyme form, in and/or from a compatible host cell.

Expression may be controlled using control sequences e.g. regulatory sequences.

The enzyme produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences may be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane. EXPRESSION VECTOR

The term "expression vector" means a construct capable of in vivo or in vitro expression.

Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.

The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.

The vectors for use in the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention.

The choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or more selectable marker genes such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Alternatively, the selection may be accomplished by co-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell.

Thus, in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.

The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBl 10, pE194, pAMBl and pIJ702. In one embodiment the pTrex 3 expression vector may be used as described in the Examples.

REGULATORY SEQUENCES

In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.

The teπn "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions.

Preferably, the nucleotide sequence according to the present invention is operably linked to at least a promoter.

Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.

CONSTRUCTS

The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the ShI -intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.

The construct may even contain or express a marker, which allows for the selection of the genetic construct.

For some applications, preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.

HOST CELLS

The term "host cell" - in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein.

Thus, a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells. Preferably, the host cells are not human cells.

Examples of suitable bacterial host organisms are gram positive or gram negative bacterial species.

Depending on the nature of the nucleotide sequence encoding the enzyme of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.

The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.

The host cell may be a protease deficient or protease minus strain.

The genotype of the host cell may be modified to improve expression.

Examples of host cell modifications include protease deficiency, supplementation of rare tRNA's, and modification of the reductive potential in the cytoplasm to enhance disulphide bond formation.

For example, the host cell E. coli may overexpress rare tRNA's to improve expression of heterologous proteins as exemplified/described in Kane (Curr Opin Biotechnol (1995), 6, 494-500 "Effects of rare codon clusters on high-level expression of heterologous proteins in E, coli"). The host cell may be deficient in a number of reducing enzymes thus favouring formation of stable disulphide bonds as exemplified/described in Bessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 " Efficient folding of proteins with multiple disulphide bonds in the Escherichia coli cytoplasm").

In a preferred embodiment the host cell is a fungal host cell, preferably a filamentous fungi host cell, preferably from the genus Trichoderma. In a preferred embodiment preferably the host cell is Trichoderma reesei.

It has surprisingly been found that the expression of the variant polypeptides of the present invention can be substantially increased by using T. reesei as the host cell.

Before the present invention lipolytic enzymes from Fusarium heterosporum (i.e. the wt KLMl) had been produced by expression in the yeast Hansenula polymorpha. When considering alternative expression systems with the aim to scale the production for mass enzyme production expression in T. reesei was considered. However, the wt KLMl enzyme is inefficiently produced in T. reesei.

Surprisingly, however, it has been found that variant polypeptides according to the present invention have significantly improved expression levels in T. reesei - 12 to 25 times better than the wild type KLM 1 enzyme. This is a significant improvement which can lead to significant cost reductions in production on a commercial scale of the enzyme.

In one embodiment the present invention provides a method for expressing variant lipolytic enzymes in Trichoderma reesei comprising transforming T. reesei with a nucleotide sequence encoding a polypeptide having hydro] ytic activity towards an ester bond in a polar lipid (such as a nucleotide sequence according to the present invention) and culturing the T. reesei to obtain expression of the nucleotide sequence and harvesting the polypeptide.

ORGANISM

The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism.

Suitable organisms may include a prokaryote, fungus, yeast or a plant.

The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.

The term "transgenic organism" does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment. Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof.

For example the transgenic organism may also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter.

TRANSFORMATION OF HOST CELLS/ORGANISM

As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis.

Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.

Filamentous fungi cells may be transformed using various methods known in the art - such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known. The use of Aspergillus as a host microorganism is described in EP 0 238 023. In one embodiment preferably Trichoderma reesei is the host organism.

Another host organism can be a plant. A review of the general techniques used for transforming plants may be found in articles by Potrykus (Annu Rev Plant Physiol Plant MoI Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17- 27). Further teachings on plant transformation may be found in EP-A-0449375.

General teachings on the transformation of fungi, yeasts and plants are presented in following sections. TRANSFORMED FUNGUS

A host organism may be a fungus - such as a filamentous fungus. Examples of suitable such hosts include any member belonging to the genera Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like. In one embodiment preferably Trichoderma is the host organism, preferably T. reesei.

Teachings on transforming filamentous fungi are reviewed in US-A-5741665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N. crassa is found, for example in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143.

Further teachings on transforming filamentous fungi are reviewed in US-A-5674707.

Gene expression in filamentous fungi has been reviewed in Punt et al. (2002) Trends Biotechnol 2002 May;20(5):200-6, Archer & Peberdy Crit Rev Biotechnol (1997) 17(4):273- 306.

TRANSFORMED YEAST

In another embodiment, the transgenic organism can be a yeast.

A review of the principles of heterologous gene expression in yeast are provided in, for example, Methods MoI Biol (1995), 49:341 -54, and Curr Opin Biotechnol (1997) Oct;8(5):554-60

In this regard, yeast — such as the species Saccharomyces cerevisiae or Pichia pastoris or Hansenula polymorpha (see FEMS Microbiol Rev (2000 24(l):45-66), may be used as a vehicle for heterologous gene expression.

A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, VoI 5, Anthony H Rose and J Stuart Harrison, Eds., 2nd edition, Academic Press Ltd.). For the transformation of yeast, several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al., (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells may be selected using various selective markers - such as auxotrophic markers dominant antibiotic resistance markers.

TRANSFORMED PLANTS/PLANT CELLS

A host organism suitable for the present invention may be a plant. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant MoI Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/ April 1994 17-27).

CULTURING AND PRODUCTION

Host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded enzyme and which facilitate recovery of the enzyme from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the enzyme.

The protein produced by a recombinant cell may be displayed on the surface of the cell.

The enzyme may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.

SECRETION

Often, it is desirable for the enzyme to be secreted from the expression host into the culture medium from where the enzyme may be more easily recovered. According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention. Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenuld) or the α-amylase gene (Bacillus).

By way of example, the secretion of heterologous proteins in E. coli is reviewed in Methods Enzymol (1990) 182:132-43.

DETECTION A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.

A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures.

Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A- 3,817,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345; US-A-4,277,437; US-A- 4,275,149 and US-A-4,366,241.

Also, recombinant immunoglobulins may be produced as shown in US-A-4,816,567.

FUSION PROTEINS The amino acid sequence for use according to the present invention may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (β-galactosidase). It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences.

Preferably, the fusion protein will not hinder the activity of the protein sequence.

Gene fusion expression systems in E. coli have been reviewed in Curr Opin Biotechnol (1995) 6(5):501-6.

In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.

LARGE SCALE APPLICATION

In one preferred embodiment of the present invention, the amino acid sequence is used for large scale applications.

Preferably the amino acid sequence is produced in a quantity of from Ig per litre to about 25g/litre, preferably from above 2.5g/litres to about 18 g/litre, preferably above 8 g per litre of the total cell culture volume after cultivation of the host organism.

FOOD/FOODSTUFF

The composition of the present invention may be used as - or in the preparation of - a food or foodstuff. Here, the term "food" or "foodstuff is used in a broad sense - and covers food for humans as well as food for animals (i.e. a feed). In a preferred aspect, the food is for human consumption.

The food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

FOOD INGREDIENT

The composition of the present invention may be used as a food ingredient. As used herein the term "food ingredient" includes a formulation, which is or can be added to functional foods or foodstuffs and includes formulations which can be used at low levels in a wide variety of products that require, for example, acidifying or emulsifying.

The food ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

FOOD PRODUCTS The composition of the present invention can be used in the preparation of food products such as one or more of: confectionery products, dairy products, poultry products, fish products and bakery products.

The present invention also provides a method of preparing a food or a food ingredient, the method comprising admixing a lipolytic enzyme according to the present invention with another food ingredient.

EXAMPLES

The present invention will now be described, by way of example only, in which reference may be made to the following figures:

Figure 1 shows SEQ ID No. 1 a synthetic DNA fragment encoding a lipolytic enzyme from

Fusarium heterosporum CBS 782.83 (wild type);

Figure 2 shows a protein preprosequence SEQ ID No. 2 of a lypolytic enzyme from Fusarium heterosporum CBS 782.83 (wild type) - the preprosquence undergoes translational modification such that the mature form of the enzyme comprises amino acids 31-305 of SEQ ID No. 2, and preferably consists of amino acids 31-305 of SEQ ID No. 2. However in some host organisms the protein may be N-terminally processes such that a number of additional amino acids are added onto the N- or C-terminus. Thus the mature form of the enzyme may an enzyme comprising at least amino acids 31-305 of the SEQ ID No. 2. The mature form of the enzyme may be referred to herein as KLM 1. This enzyme is considered to be the wild- type enzyme; Figure 3 shows SEQ ID No. 3 the DNA sequence of expression vector pTrex3 which is shown in graphical from in Figure 23;

Figure 4 shows SEQ ID No. 4 which is the DNA sequence for the polypeptide variant designated "mut 3"; Figure 5 shows SEQ ID No. 5 which is the protein preprosequence for the polypeptide variant designated "mut 3";

Figure 6 shows SEQ ID No. 6 which is the DNA sequence for the polypeptide variant designated "mut 4";

Figure 7 shows SEQ ID No. 7 which is the protein preprosequence for the polypeptide variant designated "mut 4";

Figure 8 shows SEQ ID No. 8 which is the DNA sequence for the polypeptide variant designated "mut 5";

Figure 9 shows SEQ ID No. 9 which is the protein preprosequence for the polypeptide variant designated "mut 5"; Figure 10 shows SEQ ID No. 10 which is the DNA sequence for the polypeptide variant designated "mut 345";

Figure 1 1 shows SEQ ID No. 1 1 which is the protein preprosequence for the polypeptide variant designated "mut 345";

Figure 12 shows SEQ ID No. 12 which is the DNA sequence for the polypeptide variant designated "mut 3459";

Figure 13 shows SEQ ID No. 13 which is the protein preprosequence for the polypeptide variant designated "mut 3459";

Figure 14 shows SEQ ID No. 14 which is the DNA sequence for the polypeptide variant designated "mut 9"; Figure 15 shows SEQ ID No. 15 which is the protein preprosequence for the polypeptide variant designated "mut 9";

Figure 16 shows SEQ ID No. 16 which is the DNA sequence for the polypeptide variant designated "mut 10";

Figure 17 shows SEQ ID No. 17 which is the protein preprosequence for the polypeptide variant designated "mut 10";

Figure 18 shows SEQ ID No. 18 which is the DNA sequence for the polypeptide variant designated "mut 11"; Figure 19 shows SEQ ID No. 19 which is the protein preprosequence for the polypeptide variant designated "mut 1 1";

Figure 20 shows SEQ ID No. 20 which is the DNA sequence for the polypeptide variant designated "mut 12"; Figure 21 shows SEQ ID No. 21 which is the protein preprosequence for the polypeptide variant designated "mut 12";

Figure 22 shows an alignment of the acid sequences of the polypeptide variants

(preprosequences) and wild type enzymes (shown herein as a) SEQ ID No. 2 - designated as the preprosequence for KLM lwt, b) the Fusarium oxysporum lipase as taught in EP 0 867 167 - shown herein as SEQ ID No. 22; this enzyme is sometimes referred to herein as

Lipopan F™ or "F.ox EP"; and a further amino acid sequence for a Fusarium oxysporum lipase as taught in US 7,465,570 - shown herein as SEQ ID No. 23; this enzyme is sometimes referred to herein as Lipopan F™ or "F.ox US".

Figure 23 depicts the structure of the expression vector pTrex3 in graphical form wherein the synthetic DNA fragment encoding the lipase from Fusarium heterosporum CBS 782.83

(DNA sequence SEQ ID No: 1 has been digested with Sacll and Ascl and cloned between

Saclϊ and Ascl restriction sites;

Figure 24 shows the expression of wild type lipolytic enzyme and four single-site glycosylation mutants in microliter plates; Figure 25 shows the expression of wild type KLM 1 lipolytic enzyme, C-terminal processing site mutant and two single-site glycosylation mutants in fed batch fermentation (184 h);

Figure 26 shows expression levels of multiply glycosylated and/or modified in the C-terminal processing area mutants of lipolytic enzyme from CBS 782.83 measured in DGGR assay

(Example 1). Activities are expressed relative to the activity of wt lipolytic enzyme; Figure 27 shows expression levels of multiply glycosylated and/or modified in the C-terminal processing area mutants of lipolytic enzyme from CBS 782.83. KLMl : wild type lipolytic enzyme. 5, 345, 3459, 9, 10, 1 1 and 12: mutants MUT 5, MUT 345, MUT 3459, MUT 9,

MUT 10, MUT 11, MUT 12 expressed in quad-deleted T. reesei strain. 1 lε and 12ε: MUTl 1 and MUT 12 expressed in Endo-T deleted strain of T, reesei; Figure 28 show the results of the first baking trial - showing bread volume (ml/g) as a function of lipolytic enzyme (KLMl, Mut4, Mut5 and Mut9) and dose (TIPU/kg flour);

Figure 29 shows the results from the second baking trial, bread volume (ml/g) as a function of lipolytic enzyme (KLMl, Mut4, Mut5 and Mut9) and dose (TIPU/kg flour); Figure 30 shows the amino acid sequence for Fusarium oxysporum lipase as taught in EP 0 867 167 - shown herein as SEQ ID No. 22; this enzyme is sometimes referred to herein as Lipopan F™ or "F.ox EP".

Figure 31 shows the results of baking trials for Mut 9, Mut345, Mut3459 and Mut 1 1, the graph depicting relative bread volume (%) of bread baked with different variants in different doses (mg/kg flour);

Figure 32 shows a stereoview comparing the homology model of residues 33-296 of the KLMl lipolytic enzyme (dark lines) with the structure of the Thermomyces lipase (pdb entry 1 DT3) in light lines. The two structure share a high conservation of secondary structure with the catalytic traid of the homology model found at the location relative to common features of secondary structure found in the Thermomyces lipase;

Figure 33 shows a stereoview showing the relative location of the substitutions at positions 63, 78 and 190 (shown in space filing representation) relative to the catalytic triad shown in the stick representation. It can be seen that these position are found in Loops that are distal to the catalytic triad;

Figure 34 shows a stereoview showing the location of distal loops in the KLMl lipolytic enzyme based on the homology model. These loops incorporate the position of substitutions at positions 63, 78 and 190 shown in space filling representation and are distal to the catalytic triad shown as stick figures. These loops comprise residues 54-66, 75-79, 99-103, 127-135, 162-167, 188-195 and 213-221;

Figure 35 shows an amino acid sequence for a Fusarium oxysporum lipase as taught in US 7,465,570 as SEQ ID No. 1 - shown herein as SEQ ID No. 23; this enzyme is sometimes referred to herein as Lipopan F™ or "F.ox US"; Figure 36 shows a nucleotide sequence for the Fusarium oxysporum lipase as taught in EP 0 867 167 - shown herein as SEQ ID No. 24; this enzyme is sometimes referred to herein as Lipopan F™ or "F.ox EP";

Figure 37 shows SEQ ID No. 25 which is the protein preprosequence for the polypeptide variant designated "mut 1 "; and Figure 38 shows SEQ ID No. 26 which is the DNA sequence for the polypeptide variant designated "mut 1". Example 1 : Lipase assay using l,2-0-dilauryl-rac-glycero-3-glutaric-resorufϊn ester (DGGR assay).

A substrate solution was prepared by mixing of 4 parts of buffer (50 mM HEPES pH 8, 0.4 mg/ml MgCl ₂ , 1.2 mg/ml CaCl ₂ , 2% Gum Arabic) and 1 part of substrate (664 μM 1,2-0- dilauryl-rac-glycero-S-glutaric-resorufm ester (DGGR, Fluka) in dimethylsulfoxide). A suitably diluted aliquot of lipase was added to 200 μl of the substrate solution in a well of a microtiter plate. The hydrolysis of DGGR results in a change of absorption at 572 run that was followed in real time using a microtiter plate reader.

Example 2: Expression of the wild-type lipolytic enzyme from Fusarium heterosporum CBS 782.83 (KLMl).

A synthetic DNA fragment encoding the lipolytic enzyme from Fusarium heterosporum CBS 782.83 (DNA sequence - SEQ ID No: 1 ; prepro protein sequence SEQ ID No: 2) has been digested with Sacll and Ascl and cloned between Saclϊ and Ascl restriction sites of the expression vector pTrex3. pTrex3 comprises the following functional regions:

1. The T. reesei cbhl promoter and part of the coding region. This DNA sequence begins at a naturally occurring Xba\ site approximately 1500 bp upstream of the coding region and ends at the naturally occurring Sfil site within the cbhl gene coding sequence corresponding to the signal peptide of CBHI.

2. An engineered Ascl site followed by the T. reesei cbhl transcription terminator region (approx. 0.36 kb)

3. A 2.75 kb fragment of Aspergillus nidulans genomic DNA including the promoter, coding region and terminator of the amdS (acetamidase) gene. A natural Xbal site occurs near the 3'- end of this fragment

4. About 3.2 kb fragment of bacterial DNA comprising the colEl origin of replication and ampicillin resistance gene.

Figure 23 depicts the structure of pTrex3 in graphical form. The DNA sequence of pTrex3 is listed as SEQ ID No: 3 (see Figure 3). The vector resulting from cloning the lipolytic enzyme gene in pTrex3 (pTrex3(KLMl)) has been digested with Xbal and Sspl and a 4.5 kb Xbal-Xbal DNA fragment comprising the lipolytic expression cassette and amdS marker has been purified by agarose gel electrophoresis. The purified fragment was used to transform the spores of a quad-deleted strain of T. reesei (Acbhl, Δcbh2, Aegll , Δeg/2, described in WO05/001036) by electroporation (A. N. Miasnikov, S. Kim. Transformation of T. reesei spores by electroporation. Poster No 598. Abstracts of 25 ^th Fungal Genetics Conference at Asilomar. March 17-22, 2009, p 266). The transformants were selected on a medium containing acetaniide as a sole source of nitrogen (acetamide 0,6 g/1; cesium chloride 1,68 g/1; glucose 20 g/1; potassium dihydrogen phosphate 15 g/1; magnesium sulfate heptahydrate 0,6 g/1; calcium chloride dihydrate 0,6 g/1; iron (II) sulfate 5mg/l; zinc sulfate 1,4 mg/1; cobalt (II) chloride lmg/1; manganese (II) sulfate 1,6 mg/1; agar 20 g/1; pH 4,25). Transformed colonies appeared in about 1 week. Individual transformants were transferred onto fresh acetamide selective plates and allowed to grow for 2-4 days. Isolates showing stable growth on selective medium were used to inoculate 0.2 ml of glucose-sophorose medium (1% sophorose, 0.6% glucose, 0.6% glycine, 3.3% PIPPS buffer, 0.47 % (NH ₄ ) ₂ SO ₄ , 0.5% KH ₂ PO ₄ , 0.3% citric acid, 0.1% MgSO ₄ , 500 mg/1 FeSO ₄ , 40 mg/1 ZnSO ₄ , 8 mg/1 CuSO ₄ , 3.5 mg/1 MnSO ₄ , 2 mg/1 boric acid) in the wells of a microtiter plate equipped with a microfilter at the bottom (Millipore Multiscreen -GV™). The plates were incubated for 4-6 days at 25- 28 ⁰ C in an atmosphere of pure oxygen. The culture media were separated by filtration and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS PAGE) or the DGGR assay. A number of transformants produced a new protein band on the SDS gels. The estimated molecular weight of this band (about 28 kDa) corresponded to the expected molecular weight of the N- and C-terminals (Nagao et al. J. Biochem. 124, 1124-1129 (1998)) processed KLMl lipase (28.6 kDa). Culture media of transformant clones that produced the 28 kDa band also contained substantial lipase activity (measured in DGGR assay). Essentially no lipase activity was detectable in the culture medium of the untransformed T. reesei strain used as the recipient in transformation. Example 3 : Expression of mutant forms of lipolytic enzyme from Fusarium heterosporum CBS 782.83

The mutant forms of lipolytic enzyme gene were constructed using standard PCR-based techniques. The DNA and protein sequences of the mutant genes and the prepro-forms of lipolytic enzymes are listed as indicated by Table 1. AU mutants carry R306S mutation

(Mutant 0). Mutants 1 , 3, 4 and 5 have a single N-linked glycosylation site consensus sequence introduced at different locations (the wild-type lipolytic enzyme (KLMl) has no N- linked glycosylation sites). Mutant 9 carries a deletion of two amino acid residues (K _{3 I} iR _3I2 ). All other mutants contain multiple glycosylation sites.

Table 1 Fusarium heterosporum CBS 782.83 lipolytic enzyme mutants: nucleotide and prepro protein sequences and expression vectors

* Confirmed by DNA sequencing.

The modifications in each of the mutant or variant lipolytic enzymes compared with the wild- type enzyme (KLMl; SEQ ID No. 2) is shown below in Table 2. All numbering is according to the sequence of wt prepro-KLMl (shown herein as SEQ ID No. 2) Table 2:

All of the mutant forms of the lipolytic enzyme gene have been cloned in pTrex3 in the same way as the wild type lipolytic enzyme gene (using Sacll and Ascl restrictions sites). Transformation of T. reesei, selection and cultivation of transformants were done as described in Example 2. At least 50 stable transformants expressing each of the mutants were analyzed. One transformant of each type producing the highest level of lipase was selected.

Example 4: Construction of a disruption cassette for the Endo T gene of T. reesei Endo T gene was identified in the genomic sequence of T. reesei (http://genome.i gi- psf.org/Trire2/Trire2.home.html) using the information of the patent application WO

2006/050584. Its 5' flanking region (1.9 Kb) was amplified by PCR using primers SK915

(S'-CTGATATCCTGGCATGGTGAATCTCCGTG-S') and SK916 (5'-

CATGGCGCGCCGAGGCAGATAGGCGGACGAAG-3'). The 3' flanking region (1.7 Kb) was amplified by PCR using primers SK917 (5'-

CATGGCGCGCCGTGTAAGTGCGTGGCTGCAG-3') and SK918 (5'-

CTGAT ATCGATCGAGTCGAACTGTCGCTTC-3'). Pfull Ultra (Stratagene) was used as the polymerase in all PCR reactions. The products of the PCR reaction were purified with the

QIAquick PCR purification kit (Qiagen) by following the protocol listed in the manual. Both amplified DNA fragments were digested with restriction endonuclease Ascl, followed by purification of digested DNA using QIAquick kit. The two DNA fragments were mixed and used as a template for a fusion PCR reaction with primers SK915 and SK918. The product of this reaction, a 3.6 kb DNA fragment, was cloned into pCR-Blunt Il TOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The structure of the resulting plasmid (pCR-BluntII-TOPO(5'-3' flank)) was confirmed by restriction analysis. A mutant form of the T. reesei acetolactate synthase (ALS) gene conferring resistance to chlorimuron ethyl (WO 2008/039370) has been amplified using PCR primers SK949 (5'- GTTTCGCATGGCGCGCCTGAGACAATGG-3') and SK946 (5'-

C AC AGGCGCGCCGATCGCCATCCCGTCGCGTC-S') and pTrex-Glucoamylase vector (WO 2008/039370, Example 2) as the template. The product of the PCR reaction was purified with QIAquick kit, digested with Ascl, purified again and ligated with pCR-Blunfll- TOPO(5'-3' flank) digested with the same enzyme and purified similarly. The orientation of the insert in the resulting plasmid pCR-BluntII-TOPO(5' flank- ALS marker-3' flank) was established by restriction analysis. An additional fragment of T. reesei chromosomal sequence (referred to as "3 '-repeat") was amplified using the same techniques and primers MC40 (5'-CTATGACATGCCCTGAGGCGATGCTGGCCAGGTACGAGCTG-S') and MC41 (5'-CAGCCTCGCGGTCACAGTGAGAGGAACGGGGTGAAGTCGTATAAG-S'). This sequence is located on T. reesei chromosome further downstream of the 3 '-flank area that is contained within pCR-BluntII-TOPO(5'-3' flank). The 0.46 kb product of this PCR (3 '-repeat) was cloned upstream of the ALS gene in the pCR-BluntII-TOPO(5'flank-ALS marker-3 'flank) using In-Fusion Dry-Down PCR Cloning Kit (Clontech). pCR-Bluntll- TOPO(5 'flank- ALS marker-3 ' flank) was digested with Pasl and BsiEiI for insertion of the 3 ' repeat. The resulting construct pCR-BluntII-TOPO(5' flank- ALS marker-3' repeat-3' flank) was used as the template for a PCR with primers SK 1008 (CTAGCG ATCGCGTGTGC ACA TAGGTGAGTTCTCC) and SK 1009: (CTAGCGATCGCGCAGACTGGCATGCCTCAAT CAC). The 7.5 kb DNA product was cloned into pCR-Bluntlϊ-TOPO vector using the corresponding kit from Invitrogen. The resulting plasmid was digested with AsiSl and a 7.5 kb DNA fragment (the Endo-T deletion cassette) was purified by preparative agarose gel electrophoresis.

Example 5: Disruption of the Endo-T gene in T. reesei and transformation of the resulting mutant with lipolytic enzyme expression constructs

A quad deleted strain of T. reesei (AcM 1, Δcbh2, Δegll, Aegl2) is described in WO05/001036 This strain was transformed with the deletion cassette (of Example 4) using transformation method described by Penttila et al. (Penttila M. et α/.1987. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61: 155-164). The transformants were selected on a Modified Vogel's medium containing 200 ppm chlorimuron ethyl (WO 2008/039370). Transformants were cultured in liquid medium and culture supernatants were analyzed by SDS gel electrophoresis. Two clones (#1 1 and #74) displaying an upward shift in mobility of most of the protein bands on the gel were identified. Chromosomal DNA was isolated from these two strains as well as the parent quad deleted strain of T. reesei. PCR analyses were performed on these DNA preparations using primer pairs MC 42 plus MC 48 (5'-CTCGCCATCTGACAACCTACAAATC-S ' and 5'- CT AGTACCCTGAGTTGTCTCGCCTCC-S') and MC 45 plus MC 50 (5'- CCTCT ACCAT AACAGGATCCATCTG-3' and 5'-CGTGAGCTGATGAAGGAGAGAAC AAAGG-3'). Products of the expected size (2.9 and 2.3 kb) were obtained with DNA isolated from clone # 74. This clone was subjected to two successive rounds of purification (by isolation of progeny of a single spore). DNA was isolated from the purified transformant #74. PCR analyses were repeated confirming successful deletion of the Endo-T gene. The resulting mutant strain of T. reesei was transformed with pTrex3(MUT10), pTrex3(MUTll) and pTrex3(MUT12). Screening and spore-purification of the transformants were done as described in Example 3.

Example 6: Production of the mutant forms of lipolytic enzyme from Fusarium heterosporum CBS 782.83 carrying single engineered glycosylation site

The best selected transformants expressing wild-type lipolytic enzyme as well as mutants 3, 4 and 5 (see Examples 2 and 3) were cultivated for 4 days in micro titer plates as described above (in Example 2). Production of two of these mutants as well as the wild-type lipolytic enzyme and MUT 0 (carrying only R306S mutation) was also tested in a fermentor using the standard fed-batch process (WO 2004/035070). All three mutants containing a single engineered glycosylation site were expressed at higher level than wild-type lipolytic enzyme, especially under conditions of prolonged (184 hours) fed batch cultivation in fermentor (see Figure 24 and Figure 25 and Table 3). Table S:

(* Relative activity values (wild type=l) measured in DGGR assay (Example 1)

Example 6: Production of the mutant forms of lipolytic enzyme from Fusarium heterosporum CBS 782.83 carrying multiple glycosylation sites and modification of the C-terminal proteolytic processing site

Genes encoding mutants MUT345, MUT3459, MUT9, MUTlO, MUTI l and MUT12 (Table 1) were expressed in quad deleted strain of T. reesei as described in Example 2. Genes encoding mutants MUT 1 1 and MUT 12 were additionally expressed in Endo-T deleted strain of T. reesei (see Example 5). At least 50 stable transformants were screened for lipolytic enzyme production as described in Example 2. Typically, each set of transformants would contain 5-6 clones expressing the lipolytic enzyme at similar level, highest for the given mutant. One such transformant was selected for each type of mutant. Selected best transformants were all cultivated in MTP for 7 days in one experiment and analysed by SDS PAGE and activity assays (see Example 1). The results (Figure 25 and Table 4) indicate that all tested mutants are expressed at higher level than wild-type lipolytic enzyme. Multiply glycosylated mutants as a group do not show a substantial improvement in expression level compared to MUT 5 that carries only a single engineered N-linked glycosylation site. A notable exception is MUT 12 that produces about twice as much recombinant protein as MUT 5 or other mutants in this series. Table 4:

Relative activity values (wild type=l) measured in DGGR assay (Example 1)

Example 7: Application of mutant forms of lipolytic enzyme from Fusaήum heterosporum CBS 782.83

To evaluate the functionality of the variants of the lipolytic enzyme, these were evaluated in pilot baking applications as described below.

MATERIAL AND METHODS

Enzymes

The following enzymes were used for the baking trials (Table 5)

Table 5. Enzyme samples used in application trials and their activity (TIPU/ml). KLMl is identical to the mature form of wild-type lipolytic enzyme from Fusarium heterosporum CBS 782.93 expressed in T. reesei with the amino acid comprising amino acids 31-305 of the sequence shown herein as SEQ ID No. 2, Mut4, Mut5 and Mut9 are the variants described in Example 3 expressed in T. reesei and thus post-translationally modified into the mature form of the enzyme. The expression product was used in the following trials.

Enzymes assays (TIPU)

Phospholipase activity determination of the enzymes used was performed using the following assay:

1 TIPU (Titration Phospholipase Unit) is defined as the amount of enzyme, which liberates lμmol free fatty acid per minute at the assay conditions.

Phospholipase Al and A2 catalyse the conversion of lecithin to lyso-lecithin with release of the free fatty acid from position 1 and 2, respectively. Phospholipase activity can be determined by continuous titration of the fatty acids liberated from lecithin during enzymation, since the consumption of alkali equals the amount of fatty acid liberated.

Substrate:

4% lecithin, 4% Triton-X 100, and 6 mM CaC12: 12 g lecithin powder (Avanti Polar Lipids

#44160) and 12 g Triton-X 100 (Merck 108643) was dispersed in approx. 200 ml demineralised water during magnetic stirring. 3.0 ml 0.6 M CaC12 (p.a. Merck 1.02382) was added. The volume was adjusted to 300 mL with demineralised water and the emulsion was homogenised using an Ultra Thurax. The substrate was prepared freshly every day.

Assay procedure:

An enzyme solution was prepared to give a slope on the titration curve between 0.06 and 0.18 ml/min with an addition of 300 μL enzyme.

A control sample of known activity is included.

The samples were dissolved in demineralised water and stirred for 15 min. at 300 rpm.

25.00 ml substrate was thermostatted to 37.0 ⁰ C for 10-15 minutes before pH was adjusted to

7.0 with 0.05 M NaOH. 300 μL enzyme solution was added to the substrate and the continuous titration with 0.05 M NaOH was carried out using a pH-Stat titrator (Phm 290,

Mettler Toledo). Two activity determinations are made on each scaling.

After 8 minutes the titration is stopped and the slope of the titration curve is calculated between 5 and 7 minutes. The detection limit is 3 TIPU/ml enzyme solution.

Calculations: The phospholipase activity (TIPU/g enzyme) was calculated in the following way:

Where: α is the slope of the titration curve between 5 and 7 minutes of reaction time (ml/min). N is the normality of the NaOH used (mol/l). Vl is the volume in which the enzyme is dissolved (ml), m is the amount of enzyme added to Vl (g). V2 is the volume of enzyme solution added to the substrate (ml).

Baking protocol: Recipe:

Equipment:

Mixer: Diosna

Heating cabinet Moulding: Glimek rounder

Proofing cabinet

Oven: / MIWE

Procedure:

1. Mix all dry ingredients in the bowl for 1 min. - add water 2. Mixing program: 2 min. slow - 5.5 min. fast

3. Dough temperature must be approx. 26 ⁰ C

4. Scale 135O g - mould

5. Rest in heating cabinet for 10 min. at 3O ⁰ C

6. Mould on "Glimek rounder" — settings according to table on machine 7. Proof for 45 min. at 34°, 85% RH

8. Bake

9. After baking cool the rolls for 25 min. before scaling and measuring of the volume

5 Baking trials:

Two baking trials were conducted using the wildtype- and the variant lipases. Trial setup are listed in Table 6 and Table 7.

Table 6. First baking trial set including 12 doughs dosed with different lipolytic enzymes 10 (KLM 1 (wt), Mut4, Mut5 or Mut9) at different doses (TIPU/kg flour).

Baking Variant ID TIPU/kg flour

1 KLM1 225

2 KLM 1 450

3 KLM1 900

4 Control 0

5 MUT4 225

6 MUT4 450

7 MUT4 900

8 MUT5 225

9 MUT5 450

10 Control 0

11 MUT5 900

12 MUT9 225

Table 7. Second baking trial set including 12 doughs dosed different lipolytic enzymes (KLMl (wt), Mut4, Mut5 or Mut9) at different doses (TIPU/kg flour).

Baking Mutant ID TIPU/kg flour

1 KLM 1 225

2 KLM 1 450

3 MUT4 56

4 MUT4 112

5 MUT4 225

6 MUT5 56

7 MUT5 112

8 MUT5 225

9 Control 0

10 MUT9 56

11 MUT9 112 J ₅ 12 MUT9 225 Analysis of lipid modification:

Dough were collected after proofing (see baking recipe) and immediately frozen. Hereafter the frozen dough samples were lyophilised and milled in a coffee mill. Lipids were extracted from the dough samples and analysed by the following protocol:

Lipid extraction

6.0 mL water saturated Butanol:Ethanol (85:15 (v/v)) was added to the 1 g sample and then mixed for 15 sec. on a vortex before being placed on a rotormixer at 35 rpm for 5 min. Afterwards the sample was placed in a water bath at 97°C for 10 minutes, followed by mixing on a rotormixer at 35 rpm for one hour. The sample was then centrifuged at 1370g for ten minutes. The supernatant being the organic phase containing the extracted lipid was then transferred to new glass tube.

For HPTLC 1.5 mL of the extracted lipid was evaporated at 7O ⁰ C under nitrogen cover and then redispersed in 400 μL hexane:isopropanol (3:2 (v/v)). 3 μL redispersed extracted lipid was applied to the TLC plate, see below.

HPTLC Procedure:

HPTLC plates (20 x 10 cm, Merck no. 1.05641) were activated by drying (160 ⁰ C, 20-30 minutes) and standard and samples were applied using an Automatic HPTLC Applicator (ATS4, CAMAG). Plate elution was performed using an Automatic Developing Chamber (ADC2, CAMAG) (7 cm). After elution, plates were dried (160 ⁰ C, 10 minutes), cooled, and immersed (10 seconds) in developing fluid (6 % cupric acetate in 16 % H ₃ PO ₄ ). After drying (16O ⁰ C, 6 minutes) plates were evaluated visually using a TLC scanner (TLC Scanner 3, CAMAG).

RESULTS:

Results from the first baking trial is represented in table 8 and Figure 28.

Table 8. First baking trial. Bread volume (ml/g) as a function of lipolytic enzymes (KLMl, Mut4, Mut5 and Mut9) and dose (TIPU/kg flour) Bread volume = f(Enzyme x dose) TIPU/kg flour KLM1 IMUT4 MUT5 MUT9

0 6,6 6,6 6,6 6,6

225 6,56 7,9 7,84 7,55

450 7,66 7,54 7,79 na.

900 7,57 6,82 7,77 na.

Figure 28 show the results of the first baking trial - showing bread volume (ml/g) as a function of lipolytic enzyme (KLMl, Mut4, Mut5 and Mut9) and dose (TIPU/kg flour).

As can be see from Table 8 and Figure 28, all three lipolytic variants increased the bread volume at significantly lower enzyme dose than the wildtype (KLMl). Based on the results obtained in the first baking trial, a second baking trial was performed, dosing the lipolytic variant lower than the wildtype lipase (KLMl). Results from this experiment are represented in Table 9 and Figure 29.

Table 9. Second baking trial: Bread volume (ml/g) as a function of lipases (KLMl , Mut4, Mut5 and Mut9) and dose (TIPU/kg flour)

Bread volume = f(Enzyme x dose) TIPU/kg flour KLMI MUT4 MUT5 MUT9

0 7,05 7,05 7,05 7,05

56 na 7,94 6,95 7,06

112 na 8,05 7,57 8,28

225 7,32 8,32 7,94 8,53

450 8,46 na na na

Figure 29 shows the results from the second baking trial, bread volume (ml/g) as a function of lipolytic enzyme (KLMl, Mut4, Mut5 and Mut9) and dose (TIPU/kg flour)

As can be seen from the second baking trial, the lipolytic enzyme variants facilitate the same bread volume as the wildtype lipolytic enzyme with much less activity dosed, indicating that their performance in breadmaking is superior to the wildtype lipolytic enzyme.

The above baking performance of the variant lipolytic enzyme and the wildtype (KLMl) correlated nicely to lipid modification demonstrated by the lipid analysis. AU three variant lipolytic enzymes facilitated a significantly higher modification of the galactolipid fraction (di-galactosyl-di-glyceride (DGDG)) generating the resulting lyso component (di-galactosyl- mono-glyceride (DGMG)).

In addition to the above trials, a baking trial was also conducted using the same methods and materials for Mut 9, Mut345, Mut3459 and Mut 1 1 and Figure 31 shows the results of this trial, namely the relative bread volume (%) of bread baked with different variants in different doses (mg/kg flour).

As can be seen from these data the baking performance of the variant lipolytic enzymes facilitate the same or better bread volume as the wild type lipolytic enzyme with much less activity dosed, indicating that their performance in bread making is superior to the wildtype lipolytic enzyme (KLMl).

Example 8: Homology model of KLMl lipolytic enzyme

A 3-D model showing the 3-D structure of the KLMl lipolytic enzyme was prepared in order to identify sites for modification.

The amino acid sequence for the lipolytic enzyme (shown herein as SEQ. ID No. 2 (KLMl) was compared to all known enzyme structures in the Protein Data Bank (www.rcsb.org) and the known structure having the highest sequence homology was found to be the Thermomyces lanuginosa lipase entry 1DT3. The amino acid sequence of the KLMl lipolytic enzyme shares only 40% sequence identity with the Thermomyces lipase over the 269 residues present in the protein data bank structure.

Using the Homology modelling features of the Computer program suite MOE© provided by Chemical Computing Group of Montreal, Quebec Canada, a model of the residues 33-296 of the KLMl lipolytic enzyme was generated, using program defaults in the MOE program suite. The resulting model is compared with the basis structure of the Thermomyces lipase in Figure 32. Overall there is very good agreement of the overall fold and the catalytic triad residues of the KLMl lipolytic enzyme, S 174, D228 and H287 superpose the Thermomyces lipase triad.

The catalytic triad for KLMl is S 174, D228 and H287. Figure 32 shows a stereoview comparing the homology model of residues 33-296 of the KLMl lipolytic enzyme (dark lines) with the structure of the Thermomyces lipase (pdb entry 1DT3) in light lines. The two structure share a high conservation of secondary structure with the catalytic traid of the homology model found at the location relative to common features of secondary structure found in the Thermomyces lipase.

The positions of the three substitutions (namely at K63, G78 and A 190) were located in the homology model.

K63N, G78N and A 190N introduce glycosylation sites in loops that are distal to the catalytic triad. The locations of these sites relative to the catalytic triad are shown in Figure 33. The substitution at position 63 is in a loop formed by a disulfide bond between C54 and C66.

Position 78 is found in an adjacent loop between an expected helix ending at position 75 and a β strand of the central mixed β sheet beginning with residue 79. Position 190 is found in another loop extending from the residue 188 and the end of a helix and residue 195 which is the start of another β strand of the central mixed β sheet.

Figure 33 shows a stereoview showing the relative location of the substitutions at positions 63, 78 and 190 (shown in space filing representation) relative to the catalytic triad shown in the stick representation. It can be seen that these position are found in Loops that are distal to the catalytic triad.

In addition to the loops having substitutions at positions 63, 78 and 190 several others loops can be identified in the model that share a similar juxtaposition relative the active site of the enzyme. Three of these loops are found between loops 75-79 and 188-195, these are formed by residues 99-103 occurring between two β strands of the central sheet, between Cl 29-Cl 35 forming another disulfide linked loop and 162-167 which occurs between the end of a helix and the beginning of another strand of the central helix. There is one other such loop again between the helix and a strand of the central helix residues 213-221. The location of these loops relative to the catalytic triad are shown in Figure 34. Figure 34 shows a stereoview showing the location of distal loops in the KLMl lipolytic enzyme based on the homology model. These loops incorporate the position of substitutions at positions 63, 78 and 190 shown in space filling representation and are distal to the catalytic triad shown as stick figures. These loops comprise residues 54-66, 75-79, 99-103, 127-135, 162-167, 188-195 and 213-221.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.