The present invention relates to novel transglutaminase preparations derivable from the class Oomycetes , a novel transglutaminase derived from Phytophthora cactorum , CBS 618.94 or IFO 30474, a DNA construct encoding the trans¬ glutaminase enzyme, a method of producing the novel transglutaminase and the novel transglutaminase prepara¬ tion, a method for producing a gel or protein gelation composition, and the use thereof.

BACKGROUND OF THE INVENTION

Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the e-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transglu¬ taminases form intramolecular or intermolecular e- (y- Glu)-Lys crosslinks.

This peptide crosslinking activity is useful for a variety of industrial purposes, including gelling of pro¬ teins, reduction of antigenicity of proteins, improvement of baking quality of flour, producing paste type food materia from protein, fat and water, preparation of cheese from milk concentrate, binding of chopped meat product, improvement of taste and texture of food proteins, producing jelly, gel cosmetics etc.

A wide array of transglutaminases have been isolated and characterized from animals and plants. The animal derived TGases are Ca ²⁺ -dependent and often multi-subunit enzy¬ mes. The most widely used mammalian transglutaminase, FXIIIa, is product inhibited, difficult to obtain in high

amounts and thus expensive, and therefore not useful for all applications.

A few microbial TGases have been described, including the Ca ⁺ -independent TGases from Streptovertxcxllxa disclosed in US 5,156,956 and related species disclosed in US 5,252,469.

The yields of the microbial transglutaminases in shake- flasks and fermentors are far below those seen for other industrial enzymes. Thus, better production methods, in¬ cluding new high-yielding producers are needed. Previously, this goal has been pursued by applying con¬ ventional recombinant DNA techniques for cloning and ex- pression in E . coli , S . cerevisiae and S . lividans

(Washizu et al.; Tahekana et al.; Takagi et al.) but wit¬ hout success.

Klein et al. found and partially characterized a trans- glutaminase from the slime mold Physarum polycephalum which is a homodimer having a total molecular weight of 77 kDa. JP 6078783 Kokai relates to the use of this transglutaminase for protein gelation. However, it is well-known that slime molds are unsuited for large-scale industrial fermentation. Further, Physarum is not a fun¬ gus; it belongs to the Myxomycetes (Entrez NIH data base, current version January 1996) . Taxono ically, the only common feature of Oomycetes, Myxomycetes and Eumycota (fungi) is that they all are mitochondrial eukaryotes.

The object of the invention is to provide a novel trans¬ glutaminase, a novel transglutaminase preparation, a met¬ hod for producing the transglutaminase or transglutamina¬ se preparation in a better yield and higher purity than hitherto possible which transglutaminase can be used eit¬ her alone or in combination with other enzymes for indu¬ strial purposes.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that organisms belonging to the class Oomycetes produce transglutaminase and that high-level expression is achievable by growing these coe- nocytium forming organisms.

In particular, isolates belonging to the class Oomycetes have been shown to express transglutaminases in unprece- dented high amounts, including isolates belonging to the order Peronosporales , family Pythiaceae , and the genera Pythium and Phytophthora .

Accordingly, the present invention relates to transgluta- minase preparations producible by cultivation of a trans¬ glutaminase producing strain of the class Oomycetes and to novel transglutaminases derived from transglutaminase producing strains of the class Oomycetes . Preferably, the novel transglutaminase and the transglutaminase prepara- tion of the invention are derived from or producible by transglutaminase producing strains belonging to the class Oomycetes.

Further, the present invention relates to a parent trans- glutaminase derived from or producible by a species se¬ lected from Phytophthora cactorum , CBS 618.94 or IFO 30474, Phytophthora cryptogea , CBS 651.94, Pythium perii- lum (oτ P. periplocum) , CBS 620.94, Pythium irregulare, CBS 701.95, Pythium sp. , CBS 702.95, Pythium intermedium, CBS 703.95, Pythium sp. , CBS 704.95, Pythium ultimum, CBS 705.95 or a functional analogue thereof.

The present invention also relates to a method for the production of a transglutaminase preparation according to the invention by cultivating, in a suitable medium, a strain belonging to the class Oomycetes , preferably be¬ longing to an order selected from Peronosporales , Sapro- legniales, Leptomitales and Lagenidiales, more preferably belonging to a family selected from Pythiaceae, Perono-

sporaceae, Saprolegniaceae , Leptomitacea , Rhiphidiaceae and Lagenidiacea , especially belonging to a genus selec¬ ted from Pythium and Phytophthora .

Further, the present inventors have now surprisingly suc¬ ceeded in isolating and characterizing a DNA sequence from a strain of the oomycetes Phytophthora cactorum ex¬ hibiting transglutaminase activity, thereby making it possible to prepare a recombinant transglutaminase.

Accordingly, in yet another aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting transglutaminase activity, which DNA sequence comprises

a) the DNA sequence shown in SEQ ID No. 1, and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256 or

b) an analogue of the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, which

i) is homologous with the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, or

ii) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, or

iii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, or

iv) encodes a polypeptide which is immunologically

reactive with an antibody raised against the purified transglutaminase encoded by the DNA sequence shown in SEQ ID No 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256.

It is believed that the DNA sequence shown in SEQ ID No.

1 is identical to the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256.

The strain Escherichia coli was deposited under the depo¬ sition number DSM 10256 on 18 September 1995 at the DSM - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maascheroder Weg lb, D-38125 Braunschweig, Germany, according to the Budapest Treaty.

In another aspect, the invention relates to a method of crosslinking proteins comprising contacting a proteinace- ous substrate with a transglutaminase or transglutaminase preparation of the present invention.

In yet another aspect, the invention relates to use of the transglutaminase or transglutaminase preparation of the present invention in flour, baked products, meat pro¬ ducts, fish products, cosmetics, cheese, milk products, gelled food products and leather finishing.

DETAILED DESCRIPTION OF THE INVENTION

In the present specification and claims, the term "trans¬ glutaminase" is intended to be understood as an enzyme capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor.

In the present context the term "derivable" or "derived from" is intended not only to indicate a transglutaminase produced by a strain of the organism in question, but also a transglutaminase encoded by a DNA sequence isola-

ted from such strain and produced in a host organism tra¬ nsformed with said DNA sequence. Furthermore, the term is intended to indicate a transglutaminase which is encoded by a DNA sequence of synthetic an /or cDNA origin and which has the identifying characteristics of the trans¬ glutaminase in question.

The transglutaminase may be a component occurring in an enzyme system produced by a given microorganism, such an enzyme system mostly comprising several different enzyme components. In the present specification and claims, such an enzyme system comprising at least one transglutaminase component is denoted "transglutaminase preparation".

Alternatively, the transglutaminase may be a single com¬ ponent, i.e. a component essentially free of other enzyme components usually occurring in an enzyme system produced by a given microorganism, the single component being a recombinant component, i.e. produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host. The host is preferably a heterologous host, but the host may under certain conditions also be the homologous host. A recombinant transglutaminase may be cloned and expressed according to standard techniques conventional to the skilled person.

According to the present invention, the native or unmodi¬ fied transglutaminase is of microbial origin, more speci- fically obtainable from a strain belonging to the class Oomycetes .

The class Oomycetes comprises the orders Peronosporales , Saprolegnialeε , Leptomitales and Lagenidialeε .

The order Peronosporales comprises the families Pythiace¬ ae, Peronosporaceae, Peronophytophthoraceae and AlJugina- ceae.

The order Saprolegniales comprises the families Saproleg- niaceae, Ectrogellaceae, Thraustochytriaceae, Haliph- thoraceae and Leptolegniellacea .

The order Leptomitales comprises the families Leptomita- ceae and Rhiphidiaceae .

The order Lagenidiales comprises the families Lagenidia- ceae, Olpidiaceae and Sirolpidiaceae .

It is contemplated that all orders and all families taxo- nomically belonging to the class Oomycetes comprise transglutaminase producing strains. In this respect it should be noted that the families Peronophytophthoraceae, Albuginaceae , Ectrogellaceae, Thraustochytriaceae, Ha- liphthoraceae, Leptolegniellaceae , Olpidiaceae and Sirol¬ pidiaceae are small and often highly specialised. Thus, the families Pythiaceae, Peronosporaceae, Saprolegniacea- e, Leptomitaceae , Rhiphidiaceae and Lagenidiaceae should be considered as being representative of the Oomycetes .

In a preferred embodiment, the transglutaminase prepara¬ tion of the present invention is producible by a trans¬ glutaminase producing strain which taxonomically belongs to the family Pythiaceae , preferably to the genus Pythium or the genus Phytophthora , more preferably to a subdivi¬ sion of the genus Pythium Pringsheim (Waterhouse) or a subdivision of the genus Phytophthora deBary (Newhook, Waterhouse and Stamps) . In the following, examples of members of all subdivisions (I-III) of genus Pythium , and all subdivisions (I-VI) of genus Phytophthora are given. Examples of transglutaminase producing species of the genus Pythium are I) P. irregulare, CBS 701.95; 11A,) P. dissotocum;

IIA ₂ ) P. periilum tor P. periplocum) ; P. torulosum; P. aphanidermatum; preferably P. periilum (or P. periplo¬ cum) , CBS 620.94; IIB) P.ultijmun, CBS 705.95;

III) P . intermedium, CBS 703.95.

Examples of transglutaminase producing species of the genus Phytophthora are

I) P . cactorum; preferably P. cactorum , CBS 618.94 and IFO 30474.

II) P . palmivora ;

III) P .porri ;

IV) P. infestans ;

V) P. egasperma ; VI) P. cryptogea; and P. cinnajnotni; preferably P. crypto¬ gea , CBS 651.94.

In another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Peronosporaceae , preferably to the genus Plasmopara , more preferably to the species Plasmo- para halstedii .

In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Saprolegniaceae , preferably to a genus selected from the genera Achlya, Saprolegnia and Aphanomyceε .

In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Leptomitaceae, preferably to a gen¬ us selected from the genera Apodachlya and Leptomitus .

In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Rhiphidiaceae, preferably to a gen¬ us selected from the genera Aqualinderella and Rhiphi- dium .

In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Lagenidiaceae, preferably to a gen- us selected from the genera Lagenidium and Olpidiopsis .

In a preferred aspect of the invention, it is contempla¬ ted that novel transglutaminases are obtainable by or derivable from species selected from the group of genera consisting of Pythium and Phytophthora , more preferably from the species Pyt iutn periilum t/or P. periplocum) , Pythium irregulare, Pythium sp . , Pythium ultimum, Pythium intermedium, Phytophthora cactorum and Phytophthora cryp¬ togea , especially from the species Pythium periilum (Or P. periplocum) deposited at Centraalbureau voor Schimmel¬ cultures, Oosterstraat 1, NL-3742 SK Baarn, The Nether¬ lands on December 20, 1994 under the deposition number CBS 620.94; Phytophthora cactorum deposited at Centraal¬ bureau voor Schimmelcultures under the deposition number CBS 618.94 on December 20, 1994 (and redeposited on 19 October, 1995) and previously at the Institute for Fer¬ mentation, Osaka, under the deposition number IFO 30474; Phytophthora cryptogea deposited at Centraalbureau voor Schimmelcultures on December 27, 1994 under the deposi- tion number CBS 651.94; Pythium irregulare deposited at Centraalbureau voor Schimmelcultures on 19 October, 1995 under the deposition number CBS 701.95; Pythium sp. depo¬ sited at Centraalbureau voor Schimmelcultures on 19 Oc¬ tober, 1995 under the deposition number CBS 702.95; Py- thium intermedium deposited at Centraalbureau voor Schim¬ melcultures on 19 October, 1995 under the deposition num¬ ber CBS 703.95; Pythium sp. deposited at Centraalbureau voor Schimmelcultures on 19 October, 1995 under the depo¬ sition number CBS 704.95; Pythium ultimum deposited at Centraalbureau voor Schimmelcultures on 19 October, 1995 under the deposition number CBS 705.95; all depositions made under the Budapest Treaty.

The transglutaminase component may be derived either from

the homologous or a heterologous host. Preferably, the component is homologous. However, a heterologous compo¬ nent which is immunologically reactive with an antibody raised against a highly purified transglutaminase and which is derived from a specific microorganism is also preferred.

Advantageously, a parent transglutaminase derivable from a strain of the genera Pythium and Phytophthora may be used.

In a preferred embodiment, the parent transglutaminase is selected from the group consisting of a Phytophthora cac¬ torum, CBS 618.94/IFO 30474, transglutaminase; a Pythium periilum (or P. periplocum) , CBS 620.94, transglutamina¬ se; a Pythium irregulare, CBS 701.95, transglutaminase; a Pythium sp. , CBS 702.95, transglutaminase; a Pythium in¬ termedium, CBS 703.95, transglutaminase; a Pythium sp . , CBS 704.95, transglutaminase; a Pythium ultimum, CBS 705.95, transglutaminase and a Phytophthora cryptogea ,

CBS 651.94, transglutaminase; or is a functional analogue of any of said parent transglutaminases which

(i) comprises an amino acid sequence being at least 40%, preferably at least 60%, especially more than 74%, homo¬ logous with the amino acid sequence of the parent trans¬ glutaminase,

(ii) reacts with an antibody raised against the parent transglutaminase, and/or

(iii) is encoded by a DNA sequence which hybridizes with the same probe as a DNA sequence encoding the parent transglutaminase.

Property i) of the analogue is intended to indicate the degree of identity between the analogue and the parent transglutaminase indicating a derivation of the first sequence from the second. In particular, a polypeptide is

considered to be homologous to the parent transglutamina¬ se if a comparison of the respective amino acid sequences reveals an identity of greater than about 40%, such as above 45%, 50%, 55%, 60%, 65%, 70%, 74%, 80%, 85%, 90% or even 95%. Sequence comparisons can be performed via known algorithms, such as the one described by Lipman and Pear¬ son (1985).

The additional properties ii) and iii) of the analogue of the parent transglutaminase may be determined as follows:

Property ii) , i.e. the immunological cross reactivity, may be assayed using an antibody raised against or reac¬ tive with at least one epitope of the parent transgluta- minase. The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunologi¬ cal cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et al. , 1989.

The probe used in the characterization of the analogue in accordance with property iii) defined above, may suitably be prepared on the basis of the full or partial nucleoti¬ de or amino acid sequence of the parent transglutaminase. The hybridization may be carried out under any suitable conditions allowing the DNA sequences to hybridize. For instance, such conditions are hybridization under spec- ified conditions, e.g. involving presoaking in 5xSSC and prehybridizing for lh at -45 ^β C in a solution of 5xSSC, 5xDenhardt ^, s solution, 0.5% SDS, and 100 μg/ml of dena¬ tured sonicated salmon sperm DNA, followed by hybridiza¬ tion in the same solution supplemented with ³² P-dCTP-la- belled probe for 12h at -45°C, or other methods described by e.g. Sambrook et al., 1989.

In the present context, the "analogue" of the DNA sequen¬ ce shown in SEQ ID No. l is intended to indicate any DNA

sequence encoding an enzyme exhibiting transglutaminase activity, which has any or all of the properties i)-iv) of claim 27. The analogous DNA sequence

a) may be isolated from another or related (e.g. the sa¬ me) organism producing the enzyme with transglutaminase activity on the basis of the DNA sequence shown in SEQ ID No. 1, e.g. using the procedures described herein, and thus, e.g. be an allelic or species variant of the DNA sequence comprising the DNA sequences shown herein,

b) may be constructed on the basis of the DNA sequence shown in SEQ ID No. 1, e.g. by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the transglutaminase encoded by the DNA sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. However, in the latter case amino acid changes are preferably of a minor nature, that is conservative amino acid substitu¬ tions that do not significantly affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids; small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 re¬ sidues, or a small extension that facilitates purifica¬ tion, such as a poly-histidine tract, an antigenic epito¬ pe or a binding domain. See in general Ford et al., Pro- tein Expression and Purification 2 : 95-107, 1991.

Examples of conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histidine) , acidic amino acids (such as glutamic acid and aspartic acid) , polar amino acids (such as glutamine and asparagine) , hydrophobic amino acids (such as leucine, isoleucine, valine) , aromatic amino acids (such as pheny¬ lalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methioni¬ ne) .

It will be apparent to persons skilled in the art that such substitutions can be made outside the regions criti¬ cal to the function of the molecule and still result in an active polypeptide. Amino acids essential to the ac- tivity of the polypeptide encoded by the DNA construct of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Sci- ence 244. 1081-1085, 1989) . In the latter technique mu¬ tations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biolo¬ gical (i.e. transglutaminase) activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of crystal structure as deter¬ mined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255; 306-312, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992.

The ho ology referred to in i) above or of claim 27 is determined as the degree of identity between the two se- quences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needle an, S.B. and Wunsch, CD., Journal of Molecular Biology, 4Jϊ: 443- 453, 1970). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the DNA sequence exhibits a degree of identity preferably of at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more pre¬ ferably at least 74%, even more preferably at least 80%, especially at least 90%, with the coding region of the DNA sequence shown in SEQ ID No.1.

The hybridization referred to in ii) above or of claim 27 is intended to indicate that the analogous DNA sequence hybridizes to the same probe as the DNA sequence encoding the transglutaminase enzyme under certain specified con- ditions which are described in detail in the Materials and Methods section hereinafter. Normally, the analogous DNA sequence is highly homologous to the DNA sequence such as at least 70% homologous to the DNA sequence shown in SEQ ID No. 1 encoding an transglutaminase of the in- vention, such as at least 75%, at least 80%, at least

85%, at least 90% or even at least 95% homologous to said DNA sequence.

The homology referred to in iii) above or of claim 27 is determined as the degree of identity between the two se¬ quences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S.B. and Wunsch, CD., Journal of Molecular Biology, 4$: 443- 453, 1970). Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1, the polypeptide encoded by a homologous DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 75%, most preferably at least 80%, especially at least 90%, with the enzyme encoded by a DNA construct comprising the DNA sequence shown in SEQ ID No.l.

In connection with property iv) above or of claim 27 it is intended to indicate a transglutaminase encoded by a DNA sequence isolated from strain CBS 618.94 and produced in a host organism transformed with said DNA sequence or produced by the strain CBS 618.94. The immunological re- activity may be determined by the method described in the Materials and Methods section below.

In further aspects the invention relates to an expression vector harbouring a DNA construct of the invention, a

cell comprising the DNA construct or expression vector and a method of producing an enzyme exhibiting transglu¬ taminase activity which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

In a still further aspect the invention relates to an enzyme exhibiting transglutaminase activity, which enzyme

a) is encoded by a DNA construct of the invention b) produced by the method of the invention, and/or c) is immunologically reactive with an antibody raised against a purified transglutaminase encoded by the DNA sequence shown in SEQ ID No.l.

The transglutaminase mentioned in c) above may be encoded by the DNA sequence isolated from the strain Phytophthora cactorum, CBS 618.94, and produced in a host organism transformed with said DNA sequence or produced by the strain CBS 618.94.

The DNA sequence of the invention encoding an enzyme ex¬ hibiting transglutaminase activity may be isolated by a general method involving - cloning, in suitable vectors, a DNA library from Phy¬ tophthora cactorum,

- transforming suitable yeast host cells with said vec¬ tors,

- culturing the host cells under suitable conditions to express any enzyme of interest encoded by a clone in the DNA library,

- screening for positive clones by determining any transglutaminase activity of the enzyme produced by such clones, and - isolating the enzyme encoding DNA from such clones.

The general method is further disclosed in WO 94/14953 the contents of which are hereby incorporated by referen¬ ce. A more detailed description of the screening method

is given in Example 5 below.

The DNA sequence coding for the enzyme may for instance be isolated by screening a cDNA library of Phytophthora cactorum , and selecting for clones expressing transgluta¬ minase activity, or from Escherichia coli , DSM 10256. The appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Example 5.

It is expected that a DNA sequence coding for a homolo¬ gous enzyme, i.e. an analogous DNA sequence, is obtai¬ nable from other microorganisms. For instance, the DNA sequence may be derived by similarly screening a cDNA library of another fungus, such as a strain of Pythium .

Alternatively, the DNA coding for a transglutaminase of the invention may, in accordance with well-known proce¬ dures, conveniently be isolated from DNA from a suitable source, such as any of the above mentioned organisms, by use of synthetic oligonucleotide probes prepared on the basis of a DNA sequence disclosed herein. For instance, a suitable oligonucleotide probe may be prepared on the basis of the nucleotide sequence shown in SEQ ID No. 1 or any suitable subsequence thereof.

The DNA sequence may subsequently be inserted into a re¬ combinant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA proce- dures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal repli- cation, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integra¬ ted into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the transgluta¬ minase should be operably connected to a suitable promo¬ ter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the transglutaminase, the promoter and the terminator, respectively, and to insert them into sui- table vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., 1989).

The host cell which is transformed with the DNA sequence encoding the enzyme of the invention is preferably a eu- karyotic cell, in particular a fungal cell such as a ye¬ ast or filamentous fungal cell. In particular, the cell may belong to a species of Aspergillus or Trichoderma , most preferably Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the pro¬ toplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238 023 (of Novo Nordisk A/S) , the contents of which are hereby incorporated by reference. The host cell may also be a yeast cell, e.g. a strain of Saccharomyces , in particular Saccharomyces cerevisia , Saccharomyces kluyveri or Saccharomyces uvarum. a strain of Schizosaccaromyces sp., such as Schi- zosaccharomvces pombe. a strain of Hansenula sp. Pichia sp., Yarrowia sp. such as Yarrowia lipolvtica. or Kluv- veromyces sp. such as Kluvveromyces lactis.

In a still further aspect, the present invention relates to a method of producing an enzyme according to the in- vention, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under condi¬ tions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed transglutaminase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, fol¬ lowed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Cloning and expression of a transglutaminase enzyme from Phytophthora cactorum

MATERIALS AND METHODS

Deposited organism: Escherichia coli DSM 10256 containing the plasmid comprising the full length DNA sequence, coding for the transglutaminase of the invention, in the shuttle vector pYES 2.0.

Yeast strain: The Saccharomyces cerevisiae strain used was W3124 (MATα; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prcl::HIS3; prbl: : LEU2; cir+) .

Plasmids:

The Aspergillus expression vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023) . The construction of pHD414 is further described in WO 93/11249.

pYES 2.0 (Invitrogen)

Isolation of the DNA sequence shown in SEQ ID Mo. 1:

The full length DNA sequence, comprising the cDNA sequence shown in SEQ ID No. 1 coding for the transglutaminase of the invention, can be obtained from the deposited organism Escherichia coli DSM 10256 by

extraction of plasmid DNA by methods known in the art (Sambrook et al.).

Extraction of total RNA was performed with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion, and isolation of poly(A) ⁺ RNA was carried out by oligo(dT)-cellulose affinity chromatography using the procedures described in WO 94/14953.

cDNA synthesis: Double-stranded cDNA was synthesized from 5 μg poly(A) ⁺ RNA by the RNase H method (Gubler and Hoffman, Sambrook et al.) using the hair-pin modification developed by F. S. Hagen (pers. comm.). The poly(A) ⁺ RNA (5 μg in 5 μl of DEPC-treated water) was heated at 70°C for 8 min. in a pre-siliconized, RNase-free Eppendorph tube, quenched on ice and combined in a final volume of 50 μl with reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KC1, 3 mM MgCl ₂ , 10 mM DTT, Bethesda Research Laboratories) containing 1 mM of dATP, dGTP and dTTP and 0.5 mM 5-methyl-dCTP (Pharmacia), 40 units human placental ribonuclease inhibitor (RNasin, Promega) , 1.45 μg of oligo(dT) ₁₈ -Not I primer (Pharmacia) and 1000 units Superscript II RNase H reverse transcriptase (Bethesda Research Laboratories) . First-strand cDNA was synthesized by incubating the reaction mixture at 45°C for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a MicroSpin S-400 HR (Pharmacia) spin column according to the manufacturer's instructions.

After the gelfiltration, the hybrids were diluted in 250 μl second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KC1, 4.6 mM MgCl ₂ , 10 mM (NH ₄ ) ₂ S0 ₄ , 0.16 mM 0NAD+) containing 200 μM of each dNTP, 60 units E. coli DNA polymerase I (Pharmacia), 5.25 units RNase H (Promega) and 15 units E. coli DNA ligase (Boehringer Mannheim) . Second strand cDNA synthesis was performed by incubating the reaction tube at 16°C for 2 hours and additional 15 min. at 25°C The reaction was stopped by addition of EDTA to a final concentration of 20 mM followed by phenol

and chloroform extractions.

Mung bean nuclease treatment: The double-stranded cDNA was precipitated at -20°C for 12 hours by addition of 2 vols 96% EtOH, 0.2 vol 10 M NH ₄ Ac, recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 μl Mung bean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnS0 ₄ , 0.35 mM DTT, 2% glycerol) containing 25 units Mung bean nuclease (Pharmacia) . The single-stranded hair-pin DNA was clipped by incubating the reaction at 30°C for 30 min., followed by addition of 70 μl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction and precipitation with 2 vols of 96% EtOH and 0.1 vol 3 M NaAc, pH 5.2 on ice for 30 min.

Blunt-ending with T4 DNA polymerase: The double-stranded cDNAs were recovered by centrifugation and blunt-ended in 30 μl T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM of each dNTP and 5 units T4 DNA polymerase (New England Biolabs) by incubating the reaction mixture at 16°C for 1 hour. The reaction was stopped by addition of EDTA to a final concentration of 20 mM, followed by phenol and chloroform extractions, and precipitation for 12 hours at -20°C by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.

Adaptor ligation, Not I digestion and size selection:

After the fill-in reaction the cDNAs were recovered by centrifugation, washed in 70% EtOH and dried. The cDNA pellet was resuspended in 25 μl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP) containing 2.5 μg non-palindromic BstXI adaptors (Invitrogen) and 30 units T4 ligase (Promega) and incubated at 16°C for 12 hours. The reaction was stopped by heating at 65°C for 20 min. and then cooling on ice for 5 min. The adapted cDNA was digested with Not I restriction enzyme by addition of 20 μl water, 5 μl lOx Not I restriction enzyme buffer (New England Biolabs) and

50 units Not I (New England Biolabs) , followed by incubation for 2.5 hours at 37°C The reaction was stopped by heating at 65°C for 10 min. The cDNAs were size-fractionated by gel electrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarose gel (FMC) in lx TBE to separate unligated adaptors and small cDNAs. The cDNA was size-selected with a cut-off at 0.7 kb and rescued from the gel by use of β-Agarase (New England Biolabs) according to the manufacturer's instructions and precipitated for 12 hours at -20°C by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.

Construction of libraries: The directional, size-selected cDNA was recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 μl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. The cDNAs were desalted by gelfiltration through a MicroSpin S-300 HR (Pharmacia) spin column according to the manufacturer's instructions. Three test ligations were carried out in 10 μl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP) containing 5 μl double-stranded cDNA (reaction tubes #1 and #2) , 15 units T4 ligase (Promega) and 30 ng (tube #1) , 40 ng (tube #2) and 40 ng (tube #3, the vector background control) of BstXI-NotI cleaved pYES 2.0 vector. The ligation reactions were performed by incubation at 16°C for 12 hours, heating at 70°C for 20 min. and addition of 10 μl water to each tube. 1 μl of each ligation mixture was electroperated into 40 μl electrocompetent E. coli DH10B cells (Bethesda research Laboratories) as described (Sambrook et al.). Using the optimal conditions a library was established in E. coli consisting of pools containing 15.000-30.000 colony forming units. Each pool of transformed E. coli was spread on LB+ampicillin agar plates giving 15.000-30.000 colonies/plate after incubation at 37°C for 24 hours. 20 ml LB+ampicillin was added to the plate and the cells were suspended herein. The cell suspension was shaked in a 50 ml tube for 1 hour at 37°C Plasmid DNA was isolated from the cells according to the manufacturer's instructions using QIAGEN

plasmid kit and stored at -20°C.

1 μl aliquots of purified plasmid DNA (100 ng/μl) from individual pools were transformed into S. cerevisiae W3124 by electroporation (Becker and Guarante) and the transformants were plated on SC agar containing 2% glucose and incubated at 30°C.

Identification of positive colonies: After 3-5 days of growth, the agar plates were replica plated onto a set of SC-variant agar plates. These plates were incubated for 6-8 days at 30°C

Round (diameter 8.2 cm) Immobilon PVDF Transfer Membranes for protein blotting (Millipore) were wetted for 1-3 seconds in 96% EtOH and rinsed in water for 1 min. The membranes were incubated for 2 hours in 2% N,N- dimethylcasein, 150 mM NaCl, 0.1 M Trisbuffer pH 7.5 and washed twice (1 min.) in 150 mM NaCl, 0.1 M Trisbuffer pH 7.5.

A casein saturated membrane was placed on each SC-variant agar plate with yeast colonies. The plate was incubated at 30°C over night with 1 ml 0.5 mM 5-(biotinamido)- pentylamine (Pierce), 0.1 M Trisbuffer pH 7.5, 50 mM CaCl ₂ . After 3 washes (15 min.) in 0.1 M Na ₃ P0 ₄ /H ₃ P0 ₄ buffer pH 6.5 the membrane was incubated for 1 hour at room temperature with 10 ml 0.17 μg/ml peroxidase-labeled Streptavidin (Kirkegaard & Perry Laboratories Inc.). After further 3 washes (15 min.) in 0.1 M Na ₃ P0 ₄ /H ₃ P0 ₄ buffer pH 6.5 the membrane was incubated at room temperature with 1 ml 2 mM ABTS (Sigma), 1 mM H ₂ 0 ₂ , 0.1 M Na ₃ P0 ₄ /H ₃ P0 ₄ buffer pH 6.5 until transglutaminase positive colonies were identified by a green or lilac zone.

Cells from enzyme-positive colonies were spread for single colony isolation on agar, and an enzyme-producing single colony was selected for each of the transglutaminase-producing colonies identified.

Characterization of positive clones: The positive clones were obtained as single colonies, the cDNA inserts were amplified directly from the yeast colony using biotinylated polylinker primers, purified by magnetic beads (Dynabead M-280, Dynal) system and characterized individually by sequencing the 5'-end of each cDNA clone using the chain-termination method (Sanger et al.) and the Sequenase system (United States Biochemical) .

Isolation of a cDNA gene for expression in Aspergillus:

A transglutaminase-producing yeast colony was inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube was shaken for 2 days at 30°C The cells were harvested by centrifugation for 10 min. at 3000 rpm.

DNA was isolated according to WO 94/14953 and dissolved in 50 μl water. The DNA was transformed into E. coli by standard procedures. Plasmid DNA was isolated from E . coli using standard procedures, and analyzed by restriction enzyme analysis. The cDNA insert was excised using appropriate restriction enzymes and ligated into an Aspergillus expression vector.

Transformation of Aspergillus oryzae or Aspergillus niger

Protoplasts may be prepared as described in WO 95/02043, p. 16, line 21 - page 17, line 12.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriate DNA in 10 μl of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH = 7.5, 10 mM CaCl ₂ ) . Protoplasts are mixed with p3SR2 (an A. nidulεms amdS gene carrying plasmid) . The mixture is left at room temperature for 25 minutes. 0.2 ml Of 60% PEG 4000 (BDH 29576) , 10 mM CaCl ₂ and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed. The mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol.

After one more sedimentation the protoplasts are spread on minimal plates (Cove) containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37°C spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant.

Test of A. oryzae transformants

Each of the transformants were inoculated in 10 ml YPM and propagated. After 2-5 days of incubation at 37°C, 10 ml supernatant was removed. The transglutaminase activity was identified by the 5-(biotinamido)-pentylamine plate assay described above and the Putrescine assay described in Example 1 below.

Hybridization conditions (to be used in evaluating property ii) of the DNA construct of the invention) : Suitable conditions for determining hybridization between a DNA or RNA or an oligonucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (standard saline citrate) for 10 min. and prehybridizing of the filter in a solution of 5 x SSC (Sambrook et al., 1989), 5 x Denhardt's solution (Sambrook et al., 1989), 0.5 % SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al., 1989) , followed by hybridization in the same solution containing a random-primed (Feinberg and Vogelstein, 1983) ³² P-dCTP labelled (specific activity > 1 x 10 ⁹ cpm/μg) probe for 12 h at -45°C The filter is then washed two times for 30 minutes in 2 x SSC, 0.5% SDS at a temperature preferably not higher than 45°C, more preferably not higher than 50°C, even more preferably not higher than 55°C, even more preferably not higher than 60°C, most preferably not higher than 65°C, especially not higher than 70°C, more preferably not higher than 75°C

A suitable DNA or RNA or an oligonucleotide probe to be used in the hybridization may be prepared on the basis of the DNA sequence shown in SEQ ID No. 1, or on basis of the deduced amino acid sequence shown in SEQ ID No.2.

Immunological cross-reactivity: Antibodies to be used in determining immunological cross-reactivity may be prepared by use of a purified transglutaminase. More specifically, antiserum against the transglutaminase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al.. Chapter 23, or A. Johnstone and R. Thorpe. Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ((NH ₄ ) ₂ S0 ₄ ) , followed by dialysis and ion exchange chromatography, e.g. on DEAE-Sephadex. Immunochemical characterization of proteins may be done either by Outcherlony double- diffusion analysis (0. Ouchterlony) , by crossed immunoe- lectrophoresis (N. Axelsen et al.. Chapters 3 and 4) , or by rocket immunoelectrophoresis (N. Axelsen et al.. Chapter 2) .

Media

YPD: 10 g yeast extract, 20 g peptone, H ₂ 0 to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.

YPM: 10 g yeast extract, 20 g peptone, H ₂ 0 to 900 ml. Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.

10 x Basal salt: 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH, H ₂ 0 ad 1000 ml, sterile filtered.

SC-URA: 100 ml 10 x Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml 1% tryptophan, H ₂ 0 ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20% galactose added.

SC-agar: SC-URA, 20 g/1 agar added.

SC-variant agar: 20 g agar, 20 ml 10 x Basal salt, H ₂ θ ad 900 ml, autoclaved, 10 ml 1% tryptophan, 3.6 ml 5% threonine and 100 ml 20% galactose added.

Compositions of the invention

Although the useful transglutaminase preparation or the recombinant transglutaminase may be added as such it is preferred that it is formulated into a suitable composi¬ tion. The transglutaminase to be used industrially may be in any form suited for the use in question, e.g. in the form of a dry powder or granulate, in particular a non-dusting granulate, a liquid, in particular a stabili- zed liquid, or a protected enzyme. Granulates may be pro¬ duced, e.g. as disclosed in US 4,106,991 and US 4,661,- 452, and may optionally be coated by methods known in the art. Liquid enzyme preparations may, for instance, be stabilized by adding nutritionally acceptable stabilizers such as a sugar, a sugar alcohol or another polyol, lac¬ tic acid or another organic acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The enzyme prepara¬ tion of the invention may also comprise a preservative.

Normally, for inclusion in flour, baking or baked products, meat products, cheese and other milk products, fish products, cosmestics, various gelled food, it may be advantageous that the enzyme preparation is in the form of a dry product, e.g. a non-dusting granulate, whereas for inclusion together with a liquid it is advantageously in a liquid form.

The recombinant transglutaminase and the transglutaminase preparations of the present invention may also be used in baking for improving the development, elasticity and/or stability of dough and/or the volume, crumb structure and/or anti-staling properties of the baked product. Although the transglutaminase may be used for the

preparation of dough or baked products prepared from any type of flour or meal (e.g. based on rye, barley, oat or maize) the present transglutaminases have been found to be particularly useful in the preparation of dough or baked products made from wheat or comprising substantial amounts of wheat. The baked products produced with a tranglutaminase of the invention includes bread, rolls, baguettes and the like. For baking purposes the transglutaminase of the invention may be used as the only or major enzymatic activity, or may be used in combination with other enzymes such as a lipase, an amylase, an oxidase (e.g. glucose oxidaase, peroxidase) , a laccase and/or a protease.

Preferably, the transglutaminase of the invention, especially the recombinant transglutaminase, is used in flour, dough, baked products, meat products, cheese and other milk products, fish products, cosmetics, and vari¬ ous gelled food products in an amount of between 0.01 and 100 mg per kg, more preferably of between 0.1 and 50 mg per kg, most preferably between 0.5 and 30 mg per kg, especially between 1 and 10 mg per kg.

Further, it is contemplated that the recombinant transglutaminase and the transglutaminase preparations of the present invention also can exhibit glutaminase activity, i.e. are capable of glutamine-specific deamidation. Accordingly, a protein substrate essentially free of lysine or at least with a very low content of lysine may be subjected to deamidation by applying the transglutaminase of the invention, such as protein being e.g. gluten or a gluten hydrolysate. In another aspect of the invention, the transglutaminases of the invention can be useful for treatment of food products containing gluten, e.g. for improvement of the palability or other properties of bread and other baked food products, or for reducing the allergenicity of food products containing gluten or gluten hydrolysates.

The invention is further illustrated in the following non-limiting examples.

EXAMPLE 1 Identification of transglutaminase secreting strains be¬ longing to Oomycetes

The oomycetes were inoculated into shake flasks by cut¬ ting out 4-8 small pieces of mycelium (5 mm x 5 mm) from PDA plates (39 g/1 potato dextrose agar) . The shake flasks contain either SFM-4 (4 g/£ meat extract, 4 g/£ yeast extract, 40 g/£ glucose, 8 g/£ tryptone, 0.001 g/£ FeS0 ₄ -7H ₂ 0, 2 tablets/ EBIOS, pH 7.0), ^BP (potato meal 25g/l, barley meal 12.5 g/1, BAN 800 MG 0.013 g/1, Na- casein 2.5 g/1, soy meal 5 g/1, Na ₂ HP04 2.25 g/1, pluro- nic 0.025 ml/1) or FG-4 (soy meal 30 g/1, maltodextrine 15 g/1, bacto peptone 5 g/1, pluronic 0.2 g/1) medium. The cultures were cultured at 26°C for 5-7 days with sha¬ king. The resulting culture broths were centrifuged 10 minutes at 2300 g to give cell-free culture broths (transglutaminase preparations) .

Transglutaminases have been identified in cell-free cul¬ ture broths of several Oomycetes using the assay descri- bed in detail below. It was not possible to detect these transglutaminase activities using the hydroxamate assay (Folk & Cole) as described by others in screening for microbial transglutaminases (EP 0 481504 Al) .

The assay used is a slightly modified version of the ori¬ ginal procedure (Curtis & Lorand) . The transglutaminase activity is measured as incorporation of [1,4- ¹⁴ C]putre- scine into α-casein. The detection limit of the C14- putrescine incorporation assay was found to be 1/20 of the detection limit of the hydroxamate assay.

To 20 μ£ of cell-free culture broth is added 5 μ£ [1,4- ¹⁴ C]putrescine (1.85 MBq/m£ in 2% aqueous ethanol; speci¬ fic activity 4.22 GBq/mmol) and 20 μ£ α-casein (2% in 50

mM Tris-HCl, 100 mM NaCl, pH 7.5). Incubation takes place for 2 h at room temperature following which 30 μ£ of the assay mixture is spotted onto a small round Whatman 3MM filter. The filter is immediately put into a basket sub- merged in cold 10% trichloroacetic acid and washed for 20 min to remove excess radioactivity. After this first wash the filters are washed three times with cold 5% tri¬ chloroacetic acid, one time with cold ethanol:acetone (50:50, v:v) and one time with cold acetone. Each of these washes takes place for 5 min. In all washing steps the amount of washing liquid should be at least 5 m£/fil¬ ter. The washed filters are counted directly in scintil¬ lation vials.

Table 1 shows examples of species belonging to Oomycetes that secrete transglutaminases into the growth medium upon cultivation and the determined enzyme activities are shown in terms of units of transglutaminase activity.

30 Table 1

No. Genus species Units/ Medium ml

CBS 701 .95 Pythium irregulare 0.35 SFM-4

CBS 702.95 Pythium 12 2.5 ₂ BPX

CBS 620.94 Pythium periilum / periplocum 2.5 SFM-4

CBS 703.95 Pythium intermedium 0.83 SFM-4

CBS 704.95 Pythium sp. 1 .5 % BPX

Pythium torulosum 0.72 ¹ / ₂ BPX

CBS 705.95 Pythium ultimum 0.38 SFM-4

Pythium aphanidermatum 0.37 SFM-4

CBS 618.94 Phytophthora cactorum 28.3 SFM-4

Phytophthora palmivora 5.6 SFM-4

Phytophthora cinnamomi 4.9 SFM-4

CBS 651 .94 Phytophthora cryptogea 10.0 FG-4

Units: An enzyme activity which incorporates 1 nmol [ ¹⁴ C] putrescine per hour is defined as 1 U.

EXAMPLE 2

Casein polymerisation

The ability of the transglutaminase present in Phytophthora cactorum culture broth to polymerize α- casein was investigated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) .

To 20 μl of Phytophthora cactorum culture broth was added 20 μl 1.5% α-casein in 0.2 M Tris-HCl, pH 7.5. The mixture was incubated for 2 h at room temperature.

Control samples where the culture broth or the α-casein were substituted with water were incubated in parallel.

SDS-PAGE of 10 μl of each of the three samples clearly showed that only the Phytophthora cactorum culture broth converted the α-casein to high molecular weight polymers.

EXAMPLE 3

Activity dependence in the presence of cysteine or Ca ^t ¬ ions at different temperatures

The effect of reducing agents such as cysteine and Ca ²⁺ - ions on the transglutaminase activity at different temperatures was investigated using a modification of the putrescine assay described in example 1.

The transglutaminase preparations were concentrated approximately 10 times using a Macrosep™ concentrator from Filtron. Following the samples were diluted 10 times in either: a) 50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.5; b) 50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 1 mM cysteine, pH 7.5; c) 50 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl ₂ , pH 7.5; or d) 50 mM Tris-HCl, 100 mM NaCl, 1 mM cysteine, 5 mM CaCl ₂ , pH 7.5.

For activity determination incubation took place for l hour at room temperature, 40°C and 55°C, respectively.

The tables below show the activity dependencies of the different parameters. The enzyme activities are given in relative activities. The activity obtained in buffer + EDTA at room temperature is set to 100. The activity of transglutaminase is dependent on calcium and in most cases the activity measured in the culture broth is further increased by the presence of cysteine.

Strain: Phytophthora cactorum, CBS 618.94

50 mM Tris-HCl, 100 mM NaCl, pH 7.5

Temperature 2mM 2mM EDTA +5mM + 1 mM Cys EDTA +lmM Cys Ca ²⁺ + 5 mM Ca ²⁺

Room temp. 100 125 986 991

40°C 68 85 1954 2350

55°C 70 58 1073 829

Strain: Phytophthora cryptogea, CBS 651.94

50 mM Tris-HCl, 100 mM NaCl, pH 7.5

Temperature 2mM 2mM EDTA + 5 mM + 1 mM Cys EDTA +lmM Cys Ca ²⁺ + 5 mM Ca ²⁺

Room temp. 100 115 1267 2527

40°C 69 69 4372 7423

55°C 78 143 3865 5518

Strain: Pythium sp., CBS 702.95

50 mM Tris-HCl, 100 mM NaCl, pH 7.5

Temperature 2mM 2mM EDTA + 5 mM ⁰ + 1 mM Cys EDTA +lmM Cys Ca ²⁺ + 5 mM Ca ²⁺

Room temp. 100 57 487 991

40°C 0 0 3216 5773

55°C 100 96 4191 5896

Strain: Pythium irregulare, CBS 701.95

50 mM Tris-HCl, 100 mM NaCl, pH 7.5

Temperature 2mM 2mM EDTA + 5 mM + 1 mM Cys EDTA +lmM Cys Ca ²⁺ + 5 mM Ca ²⁺

Room temp. 100 110 87 86

40°C 167 168 462 450

55 ^β C 50 43 130 114

Strain: Pythium ultimum, CBS 705.95

50 mM Tris-HCl, 100 mM NaCl, pH 7.5

Temperature 2mM 2mM EDTA + 5 mM + 1 mM Cys EDTA +lmM Cys Ca ²⁺ + 5 mM Ca ²⁺

Room temp. 100 93 107 141

40°C 142 164 416 483

55°C 15 22 89 88

Strain: Pythium intermedium, CBS 703.95

50 mM Tris-HCl, 100 mM NaCl, pH 7.5

Temperature 2mM 2mM EDTA + 5 mM + 1 mM Cys EDTA +lmM Cys Ca ²⁺ + 5 mM Ca ²⁺

Room temp. 100 138 459 2438

40°C 129 142 3872 6117

55°C 181 180 733 1716

Example 4

pH Dependency of Oomycetes transglutaminases

The pH dependency of the transglutaminase activity

present in the transglutaminase preparation of Pythium irregulare (CBS 701.95), Pythium sp. (CBS 702.95), Pythium periilum (or P. periplocum) (CBS 620.94), Pythium intermedium (CBS 703.95), Pythium sp. (CBS 704.95), Pyt- hium ultimum (CBS 705.95), Phytophthora cactorum (CBS

618.94/IFO 30474) and Phytophthora cryptogea (CBS 651.94) was investigated using a modification of the putrescine assay described in example 1.

A 4% α-casein solution was made in 50 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl ₂ 1 mM cysteine, pH 7.5 and diluted 1:1 in a modified 200 mM Britton-Robinson buffer (0.1M CH ₃ COOH, 0.2 M H ₃ B0 ₃ ) at the pH values mentioned below.

For pH dependency determination incubation takes place at room temperature for 1 hour at pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0, respectively.

The table below shows the pH dependencies of the Oomycetes transglutaminases. The stated enzyme activities are relative activities.

PH

Strains 6.0 6.5 7.0 7.5 8.0 8.5 9.0

Pythium 20 24 36 46 62 100 46 irregulare, CBS 701.95

Pythium 9 16 27 31 48 93 100 sp. ,

CBS 702.95

Pythium 63 90 99 100 95 54 25 interme¬ dium, CBS 703.95

Pythium 23 33 41 72 95 100 78 sp. ,

CBS 704.95

Pythium 28 62 68 68 100 93 69 ultimum, CBS 705.95

Phytoph¬ 28 38 46 59 74 100 92 thora cactorum, CBS 618.94

Phytoph¬ 63 78 86 100 99 93 56 thora cryptogea, CBS 651.94

EXAMPLE 5

Cloning and expression of a transglutaminase from Phytophthora cactorum, CBS 618.94 and IFO 30474

mRNA was isolated from Phytophthora cactorum , CBS 618.94

and IFO 30474, grown in SFM-4 fermentation medium with agitation to ensure sufficient aeration. Mycelia were harvested after 3-5 days' growth, immediately frozen in liquid nitrogen and stored at -80°C. A library from P. cactorum, CBS 618.94 or IFO 30474 consisting of approx. 9x10 ^s individual clones was constructed in E. coli as described with a vector background of 1%. Plasmid DNA from some of the pools was transformed into yeast, and 50-100 plates containing 250-400 yeast colonies were obtained from each pool.

Transglutaminase-positive colonies were identified and isolated on agar plates with the 5-(biotinamido)- pentylamine assay. cDNA inserts were amplified directly from the yeast colonies and characterized as described in the Materials and Methods section above. The DNA sequence of the cDNA encoding the transglutaminase is shown in SEQ ID No. 1 and the corresponding amino acid sequence is shown in SEQ ID No. 2.

The cDNA is obtainable from the plasmid in DSM 10256.

Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E . coli as described above. In order to express the transglutaminase in

Aspergillus , the DNA was digested with Hindlll/Xbal, size fractionated on gel, and a fragment corresponding to the transglutaminase gene was purified. The gene was subsequently ligated to Hindlll/Xbal digested pHD414 resulting in the plasmid pA2TG3.

After amplification of the DNA in E. coli the plasmid was transformed into Aspergillus oryzae as described above.

Test of A. oryzae transformants

Each of the transformants were tested for enzyme activity as described above. Some of the transformants had transglutaminase activity which was significantly larger than the Aspergillus oryzae background. This demonstrates

efficient expression of the transglutaminase in Aspergillus oryzae .

Fed batch fermentation Fermentations were carried out as fed-batch processes with maltose sirup as carbon source and ammonia as nitrogen source. The batch phase was carried out at pH 6.5 and the pH was increased to 7.5 during the fed-batch phase. The temperature was maintained at 34°C during the entire process.

EXAMPLE 6

Production of the transglutaminase from Phytophthora cac- torum, CBS 918.94/IFO 60474

Phytophthora cactorum , CBS 618.94/IFO 30474, was inoculated into 8 I SFM-4 medium and cultured with sha¬ king at 26 °C for 7 days. The resulting culture broth was filtered through Miracloth to give 5 1 of culture filtrate. The transglutaminase activity in the culture filtrate was 22 units/ml.

EXAMPLE 7

Purification and characterisation of native and recombinant Phytophthora cactorum transglutaminase.

Transglutaminase activity measured with putrescine assay: The putrescine assay was in principle performed according to Lorand et al.

The reaction mixture contained: 2 μmoles of CaCl ₂ , 1 μmoles of cysteine, 75 nmoles of [ ¹⁴ C]-putrescine (4.03 GBq/mmol; Amersham), 0.7 mg of α-casein, and 0.6 μg of transglutaminase made up to 1 ml with 0.1 M Tris-HCl, pH 7.9. The incubations were performed at ambient temperature. Aliquots of 30 μl were withdrawn after 60 min of incubation and spotted onto Whatman 3 MM filters

(D = 2 cm) . The filters were immediately put into a ba¬ sket submerged in ice-cold 10% TCA and washed for 20 min. Following the first wash the filters were washed three times with ice-cold 5% TCA and two times with ice-cold acetone. In each washing step there should be at least 5 ml of washing solution per filter. The filters were dried, put into counting vials containing 8 ml of scin¬ tillation fluid (Optiphase, Wallac) and the radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer. Each determination was performed in tripli¬ cate.

Partially purification of native P. cactorum transglutaminase.

The culture broth was germ filtrated and concentrated 5 times by ultrafiltration using a Filtron Minisette membrane with 10 kDa cut off. After dialysis against 20 mM Tris-HCl, pH 8.0 the sample was passed through a Q- Sepharose column equilibrated with 20 mM Tris-HCl, pH 8.0. The transglutaminase was eluted from the column using a linear gradient from 0 to 0.5 M sodium chloride. Fractions with transglutaminase activity (putrescine assay) were pooled and concentrated in an Amicon cell equipped with a 10 kDa Diaflo membrane. This preparation of native transglutaminase was only partially pure.

Purification, specific activity and N-terminal sequencing of recombinant P. cactorum transglutaminase.

The Aspergillus oryzae culture broth was germ filtrated and concentrated 5 times by ultrafiltration using a Filtron Minisette membrane with 10 kDa cut off. After dialysis against 50 mM sodium borate, pH 8.0 the sample was passed through a Q-Sepharose column equilibrated with 50 mM sodium borate, pH 8.0. The transglutaminase was eluted from the column using a linear gradient from 0 to 0.5 M sodium chloride. Fractions that gelate casein were pooled and concentrated in an Amicon cell equipped with a

10 kDa Diaflo membrane.

In Aspergillus oryzae the recombinant transglutaminase is produced as two forms and from SDS-PAGE the molecular weights are judged to be 57 kDa and 43 kDa, respectively. The ratio between the two forms is dependent on the fermentation time. Early in the fermentation the 57 kDa form dominates but this form is during the fermentation processed to the low molecular weight form. Both forms of the transglutaminase are catalytic active. The specific activity of the recombinant transglutaminase was determined in the putrescine assay and found to be 3,000 U/mg.

N-terminal amino acid sequencing of the two forms of the transglutaminase revealed that the 57 kDa form has a blocked N-terminal and that the 43 kDa form starts at Leul68, cf. SEQ ID No.2.

The influence of calcium and cysteine on the activity of recombinant P. cactorum transglutaminase.

The effect of calcium and cysteine (used as a reducing agent) was investigated in the putrescine assay. The results presented below are given as relative activities. The activity obtained in buffer at 25°C is set to 100.

The activity of the transglutaminase is dependent on calcium and the activity is not further increased by the presence of cysteine as reducing agent.

Temp. Buffer 2 mM 1 mM 2 mM 1 mM Cys (°C) EDTA Cys Ca ²⁺ 2 mM Ca ²⁺

25 100 15 180 270 280

30 105 10 210 430 490

40 30 10 75 750 780

55 10 5 75 350 350

The influence of calcium and cysteine on the gelation of casein by P. cactorum transglutaminase.

The influence of calcium and cysteine on the gelation of casein was investigated as described below.

The gelation mixture contained 80 mg Hammarsten casein, 2 μmoles of calcium, 1 μmole of cysteine, and approximately 0.03 mg transglutaminase made up to 1 ml with 0.2 M Tris- HCl, pH 7.5. Following incubation overnight at 37 °C the samples were temperated to ambient temperature and the gelation was judged by visual inspection.

Both native and recombinant transglutaminase are able to gelate casein. Contrary to the native enzyme it is not essential for the recombinant enzyme that cysteine is present as a reducing agent.

Buffer 1 mM 2 mM 1 mM Cys+ Cys Ca ²⁺ 2 mM Ca ²⁺

•

Recombinant +" + P. cactorum

Native +

P. cactorum"

- designates no visible gelation. ^** + designates the formation of a stable gel * partially purified

Temperature profile of P. cactorum transglutaminase.

The temperature profile was determined using the putrescine assay with 0.1 M sodium borate/acetate buffer, pH 7.9 instead of 0.1 M Tris-HCl, pH 7.9.

As can be seen from the table the temperature optimum for both the native and the recombinant transglutaminase is 45 °C.

Temp. Native P. cactorum" Recomb. P. cactorum (°C) Relative activity Relative activity

(%) (%)

25 20 30

30 40 50

35 60 60

40 85 75

45 100 100

50 75 85

55 15 25

partially purified.

pH profile of recombinant P. cactorum transglutaminase.

The pH profile was determined using the putrescine assay with 0.1 M sodium borate/acetate buffer.

pH optimum of the recombinant Phytophthora cactorum transglutaminase is found to be at pH 8.5.

PH Recombinant P. cactorum

Relative activity

(%)

6.5 ^* 10

7.0 15

7.5 35

8.0 45

8.5 100

9.0 85

9.5 80

visible precipitate in the substrate

EXAMPLE 8

Crosslinking of Na-caseinate in solution measured by the viscosity increase as function of time

A 9% protein solution was prepared from Na-caseinate (Miprodan 30, MD Foods, Denmark, 87.8% protein). Calcium chloride was dissolved in the solution to a concentration of 5 mM and pH was adjusted to 7.0, using NaOH. The solution was heated to 40°C.

A Haake Viscosimeter, VT 501 (Haake Mess-Technik GmbH, Germany) was prepared for viscosity measurements at 40°C by sensor system MV1 at speed range H, speed 3.

To the protein solution was added recombinant Phytophthora cactorum transglutaminase, cf. example 7, purified to electrophoretic purity, at a dosage of 0.08% (weight of enzyme/weight of protein) . The solution was immediately transferred to the viscosimeter for measure¬ ment. The viscosity of a control solution without enzyme

addition was subsequently measured.

Results: Viscosity (mPa*s) as funtion of time:

Time Caseinate solution Control (minutes) + enzyme

2.66 17.5 21

7.33 25.4 22.8

12 36.9 27.2

24 88.6 35

36 186.9 41.1

48 355.3 47.3

60 800.2 50.7

The casein solution with enzyme solidified subsequently to a gel within a few minutes, while the viscosity of the control remained constant at 53 mPa*s for 120 minutes.

EXAMPLE 9 Transglutaminase for Gluten Strengthening

The strengthening effect of a given dough conditioner on wheat flour dough or gluten dough may be measured by dynamic rheological measurements. These measurements are able to show the strength of a dough, under oscillation. Both wheat fluor dough and gluten dough are viscoelastic materials. In oscillatory measurements, the viscoelastic properties of a wheat dough and a gluten dough can be divided into two components, the dynamic shear storage modulus G' and the dynamic shear loss modulus G". The ratio of the loss and the storage moduli is numerically equal to the tangent of the viscoelastic phase angle δ . An increase in the storage modulus G' and a decrease in

the phase angle δ indicate a stronger and more elastic dough.

The dynamic shear storage modulus G' and the viscoelastic phase angle δ were measured in the gluten from 3 doughs, which were treated with the recombinant transglutaminase described in example 6 in two dosages, i.e. 4 mg and 10 mg, respectively. The transglutaminase was added to the flour before dough mixing. The gluten was washed out of the flour dough containing the conditioner after the flour dough had been incubated at 32°C for 1% hours. The results of the tests are shown in the table below where the measured values of G' and ό" resulting from the inclusion of 4 mg and 10 mg enzyme per kg of flour, respectively, are presented as index values relative to the control dough (index 100) with no transglutaminase inclusion.

Dosage of G' Index δ Index transglutaminase

4 mg 135 117

10 mg 167 131

From the results it is surprisingly seen that the storage modulus, G', is significantly higher when transglutaminase is present in the dough compared to control without the enzyme. This indicates that the gluten, and thereby also the dough, is significantly strengthened by the action of the enzyme.

Further, it is shown that the viscoelastic phase angle, ό", is lowered relative to the control when tranglutaminase is present in the dough, indicating that a more elastic rheological property of the gluten and thereby the dough is achieved by the action of the enzyme.

REFERENCES CITED IN THE SPECIFICATION

Washizu et al., Bioscience, Biotechnology and

Biochemistry Vol. 58, 1994, pages 82-87.

Tahekana et al., ibid . Vol. 58, 1994, pages 88-92.

Takagi et al., EP-0 481 504 Al.

Klein et al., J. Bacteriol. Vol. 174, 1992, pages 2599- 2605.

Lipman and Pearson, Science 227, 1435 (1985).

Hudson, L. , and Hay, F. , Practical Immunology, Third edi¬ tion (1989) , Blackwell Scientific Publications.

Sambrook et al..Molecular Cloninσ: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, 1989.

Folk, J. E. & Cole, P. W. (1966) J . Biol . Chem . 241 , 5518-5525.

Curtis, C. G. & Lorand, L. (1976) Methods in Enzymology 45 , 177-191.

Lorand, L. , Campbell-Wilkes, L.K., and Cooperstein, L. (1972) Anal . Biochem . , 50, 623-631.

Newhook, F.J., Waterhouse, G.M., and Stamps, D.J. , 1978: Tabular key to the species of Phytophthora De Bary, Mycological Papers No. 143, CAB, Surrey, England.

Waterhouse, G.M. , 1967: Key to Pythium Pringsheim, Mycological Papers No. 109, CAB, Surrey, England.

Ford et al., Protein Expression and Purification 2_: 95- 107, 1991.

Cunningham and Wells, Science 24 . 1081-1085, 1989.

de Vos et al., Science 255: 306-312, 1992.

Smith et al., J. Mol. Biol. 224: 899-904, 1992.

Wlodaver et al., FEBS Lett. 309: 59-64, 1992.

O. Ouchterlony in: Handbook of Experimental Immunology (D.M. Weir, Ed.), Blackwell Scientific Publications, 1967, pp. 655-706.

N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis. Blackwell Scientific Publications, 1973.

A. Johnstone and R. Thorpe, Immunochemistry in Practice. Blackwell Scientific Publications, 1982, pp. 27-31.

Cove, Biochem. Biophys. Acta 113 (1966) 51-56.

WO 95/02043.

WO 94/14953.

Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467.

Becker and Guarante (1991) Methods Enzymol. 194:182-187.

Gubler and Hoffman (1983) Gene 25:263-269.

Feinberg, A.P., and Vogelstein, B. (1983) ANal. Biochem. 132, 6-13.

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

( i ) APPLICANT :

(A) NAME: Novo Nordisk A/S

(B) STREET: Novo Alle

(C) CITY: DK-2880 Bagsvaerd

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP): DK-2880 (6) TELEPHONE: +45 44 44 88 88 (H) TELEFAX: +45 44 49 32 56 (I) TELEX: 37304

(ii) TITLE OF INVENTION: TITLE

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1901 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Phytophthora cactorum

(B) STRAIN: CBS 618.94

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:46..1765

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CGACATCGAG AAGATTACAA ACTCATTGTT GCAGGTTTCA CAACC ATG GTC TAC 54

Met Val Tyr

1

TCA CCC AGC TCC TAC CTC ATC TCC GCC GCC GTG GCT GCG GTG GCC TTC 102 Ser Pro Ser Ser Tyr Leu lie Ser Ala Ala Val Ala Ala Val Ala Phe 5 10 15

CAG ATT CAG CAA GCG ACT GCC GGA TCG CTG TAC TAC GGG GCG TTC TCC 150 Gin lie Gin Gin Ala Thr Ala Gly Ser Leu Tyr Tyr Gly Ala Phe Ser 20 25 30 35

GTG TCC GAC ACG GAT GGC AAA ATC AGC AAC GAC TCT CCT CTT GTC GGT 198 Val Ser Asp Thr Asp Gly Lys lie Ser Asn Asp Ser Pro Leu Val Gly 40 45 50

ACC GAA ATT TCC GAC CAG GAC TGC GCC ATC GAG GTG GAG GTC GAC CCG 246 Thr Glu lie Ser Asp Gin Asp Cys Ala lie Glu Val Glu Val Asp Pro 55 60 65

ACG CTG CCG GAC ATC ACG ACC ATC TCG ACG GTG CCG GTG ACC TAC CCT 294 Thr Leu Pro Asp lie Thr Thr He Ser Thr Val Pro Val Thr Tyr Pro 70 75 80

GAC CTG CTG GCC AAC TTG ACG ACG GCT CCG TCG GAG CCG GTG TTC TCA 342 Asp Leu Leu Ala Asn Leu Thr Thr Ala Pro Ser Glu Pro Val Phe Ser 85 90 95

AAG GTG GGC ACG GTG ATC ATG TCG GAG GAG ACC CCC GCC ACC GAC GCC 390 Lys Val Gly Thr Val He Met Ser Glu Glu Thr Pro Ala Thr Asp Ala 100 105 110 115

GAC CAG GAC GCG TAC ATC GAC TCG ACG CTT CCG TGG ATT GGC ACT GGT 438 Asp Gin Asp Ala Tyr He Asp Ser Thr Leu Pro Trp He Gly Thr Gly 120 125 130

ACG CCG ACC AAG ACG GGT GTG GAG AAG ACC GCC AAG GAC TGC GCT ACT 486 Thr Pro Thr Lys Thr Gly Val Glu Lys Thr Ala Lys Asp Cys Ala Thr 135 140 145

GGG TGG GAG GAG ACC GCG GCC GGC GAT AAG CTC CAG GAG AAG CTC GAA 534 Gly Trp Glu Glu Thr Ala Ala Gly Asp Lys Leu Gin Glu Lys Leu Glu 150 155 160

AAG AAG CGT CGC CTG GAG GAA AAC ACG AAC AGG GAT ATC GCT AGG CTC 582 Lys Lys Arg Arg Leu Glu Glu Asn Thr Asn Arg Asp He Ala Arg Leu 165 170 175

GAG GCC TAC TTT GGC ACC AAG ATG GAG ATG ACC CTG AAG GAC CTG CCT 630 Glu Ala Tyr Phe Gly Thr Lys Met Glu Met Thr Leu Lys Asp Leu Pro 180 185 190 195

ACC CAG GGT GTC CAC ACA CCG TCG CCG TGG GCT GGA CCG TAC TGG CCG 678 Thr Gin Gly Val His Thr Pro Ser Pro Trp Ala Gly Pro Tyr Trp Pro 200 205 210

ACT TAC CAG GAC AGT ATC AAC GTT GTC TGG AGC GAG GGA GAA GCC AGC 726 Thr Tyr Gin Asp Ser He Asn Val Val Trp Ser Glu Gly Glu Ala Ser 215 220 225

CCC GCT GAG AAG TAC GCC AAG GCT TTC GGT CTG GAC GTG ACG GAC TTC 774 Pro Ala Glu Lys Tyr Ala Lys Ala Phe Gly Leu Asp Val Thr Asp Phe 230 235 240

ATG GAC AAG GTG TCG AAG GAC AAT GGT GTG GAC TCT CAG AGC AAA CGC 822 Met Asp Lys Val Ser Lys Asp Asn Gly Val Asp Ser Gin Ser Lys Arg 245 250 255

AGA CAG TGC CAG ACT GAC GAG GGA TGC GAG TCC CTT AAC AAC GCC AGC 870 Arg Gin Cys Gin Thr Asp Glu Gly Cys Glu Ser Leu Asn Asn Ala Ser 260 265 270 275

AAG TGC GCC ATT CGT GCC GGC AAG ACC TCG GGC TAC TGC ATC CCG ACG 918 Lys Cys Ala He Arg Ala Gly Lys Thr Ser Gly Tyr Cys He Pro Thr 280 285 290

TGG TTC GGG ATC TGC CAC GCT TGG GCC CCG GCT GCC ATT CTC GAG GCA 966 Trp Phe Gly He Cys His Ala Trp Ala Pro Ala Ala He Leu Glu Ala 295 300 305

GAG CCG ACC TGC CCG GTG ACG CAC AAC GGC GTG ACG TTC CAG CCG ATT 1014 Glu Pro Thr Cys Pro Val Thr His Asn Gly Val Thr Phe Gin Pro He 310 315 320

GAC ATC AAG GGG CTG ATC TCG GAC GTC TAC GAT GGC GCA GGT GTG GCA 1062 Asp He Lys Gly Leu He Ser Asp Val Tyr Asp Gly Ala Gly Val Ala 325 330 335

ACG GTT TTC ACG GGT GCC CGG TAC AAC GGC GGT GAC GAT GCT GCC GAT 1110 Thr Val Phe Thr Gly Ala Arg Tyr Asn Gly Gly Asp Asp Ala Ala Asp 340 345 350 355

GAG TAT GGC CGT CAC ACG AAT GCC GCC TAC CGC GAC CTG AAC CCT GCC 1158 Glu Tyr Gly Arg His Thr Asn Ala Ala Tyr Arg Asp Leu Asn Pro Ala 360 365 370

TAC TTC CAC ATT GCG TCT GCC AAT ATC CTG GGC AAG CTA AAC GCT ACA 1206 Tyr Phe His He Ala Ser Ala Asn He Leu Gly Lys Leu Asn Ala Thr 375 380 385

TTT GTT GCT GAC GTC GAC GCC GCC GCA GAA GTG TGG AAC CAG CCC GTG 1254 Phe Val Ala Asp Val Asp Ala Ala Ala Glu Val Trp Asn Gin Pro Val 390 395 400

CGC GGT TTC AAG GTG TTC GAG CAG ACC GCC ATG TCG CTC GAG GAG GCC 1302 Arg Gly Phe Lys Val Phe Glu Gin Thr Ala Met Ser Leu Glu Glu Ala 405 410 415

GCT CAG ACC TTC TAC GGC CTT GAG GAG TAC CCG TGG AAT GCC GCC GCC 1350 Ala Gin Thr Phe Tyr Gly Leu Glu Glu Tyr Pro Trp Asn Ala Ala Ala 420 425 430 435

AAG AGC ATT GTG TAC GTC AAG TCG CGT CTC TCG TGG ATC TTC GAG ACG 1398 Lys Ser He Val Tyr Val Lys Ser Arg Leu Ser Trp He Phe Glu Thr 440 445 450

TAC ACC GAC GGT GGC CTG GTG GCC TCG GGT GAG ATC AAC CGA TAC ACA 1446 Tyr Thr Asp Gly Gly Leu Val Ala Ser Gly Glu He Asn Arg Tyr Thr 455 460 465

ACG GGC AAG TAC TAC TAC TAC CTT CTG GAG CTG GAC GAT GCT GGT GAG 1494 Thr Gly Lys Tyr Tyr Tyr Tyr Leu Leu Glu Leu Asp Asp Ala Gly Glu 470 475 480

ATC ATT GGC GGT GAG TGG GTT TAC GAT TCG GAC AGC GAC CAC CCT GAC 1542 He He Gly Gly Glu Trp Val Tyr Asp Ser Asp Ser Asp His Pro Asp 485 490 495

TTC CTG TGG GTG CCC AAG GCG AAG CCT GCT GCG GAC ACG GTG ACC AGC 1590 Phe Leu Trp Val Pro Lys Ala Lys Pro Ala Ala Asp Thr Val Thr Ser 500 505 510 515

ATT GGC CTG AGC TAC GCG GAC GTG AGC ATG CTT CTG GAG AAA TCC GTC 1638 He Gly Leu Ser Tyr Ala Asp Val Ser Met Leu Leu Glu Lys Ser Val 520 525 530

GCT TGC TCC GAC TCC ACT TCG GCT GCC GGC TCC GTG TCG TCC GGA TCG 1686 Ala Cys Ser Asp Ser Thr Ser Ala Ala Gly Ser Val Ser Ser Gly Ser 535 540 545

GTG GGT GAG TCC ACG GAG GCG CCT ACG GAA GTG CCC ACG ACG TCG ACG 1734 Val Gly Glu Ser Thr Glu Ala Pro Thr Glu Val Pro Thr Thr Ser Thr 550 555 560

AGT GCT CCC ACT TCT GGC AGT GGC GCG CTG T AAGTATGTCG CAGCTCGTCT 1785 Ser Ala Pro Thr Ser Gly Ser Gly Ala Leu 565 570

TCCGTATTTG TCGTTGCACA TGAATGTGTA GCTTCGTTTA GATTGCCAGC 1835

TATTCACAAG AAATGGTTCA AAACATGCAC TAAGTTCAAG TTGTAAAAAA 1885

AAAAAAAAAA AAAAAA 1901

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 573 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Val Tyr Ser Pro Ser Ser Tyr Leu He Ser Ala Ala Val Ala Ala 1 5 10 15

Val Ala Phe Gin He Gin Gin Ala Thr Ala Gly Ser Leu Tyr Tyr Gly 20 25 30

Ala Phe Ser Val Ser Asp Thr Asp Gly Lys He Ser Asn Asp Ser Pro 35 40 45

Leu Val Gly Thr Glu He Ser Asp Gin Asp Cys Ala He Glu Val Glu 50 55 60

Val Asp Pro Thr Leu Pro Asp He Thr Thr He Ser Thr Val Pro Val 65 70 75 80

Thr Tyr Pro Asp Leu Leu Ala Asn Leu Thr Thr Ala Pro Ser Glu Pro 85 90 95

Val Phe Ser Lys Val Gly Thr Val He Met Ser Glu Glu Thr Pro Ala 100 105 110

Thr Asp Ala Asp Gin Asp Ala Tyr He Asp Ser Thr Leu Pro Trp He 115 120 125

Gly Thr Gly Thr Pro Thr Lys Thr Gly Val Glu Lys Thr Ala Lys Asp 130 135 140

Cys Ala Thr Gly Trp Glu Glu Thr Ala Ala Gly Asp Lys Leu Gin Glu 145 150 155 160

Lys Leu Glu Lys Lys Arg Arg Leu Glu Glu Asn Thr Asn Arg Asp He 165 170 175

Ala Arg Leu Glu Ala Tyr Phe Gly Thr Lys Met Glu Met Thr Leu Lys 180 185 190

Asp Leu Pro Thr Gin Gly Val His Thr Pro Ser Pro Trp Ala Gly Pro 195 200 205

Tyr Trp Pro Thr Tyr Gin Asp Ser He Asn Val Val Trp Ser Glu Gly 210 215 220

Glu Ala Ser Pro Ala Glu Lys Tyr Ala Lys Ala Phe Gly Leu Asp Val 225 230 235 240

Thr Asp Phe Met Asp Lys Val Ser Lys Asp Asn Gly Val Asp Ser Gin 245 250 255

Ser Lys Arg Arg Gin Cys Gin Thr Asp Glu Gly Cys Glu Ser Leu Asn 260 265 270

Asn Ala Ser Lys Cys Ala He Arg Ala Gly Lys Thr Ser Gly Tyr Cys 275 280 285

He Pro Thr Trp Phe Gly He Cys His Ala Trp Ala Pro Ala Ala He 290 295 300

Leu Glu Ala Glu Pro Thr Cys Pro Val Thr His Asn Gly Val Thr Phe 305 310 315 320

Gin Pro He Asp He Lys Gly Leu He Ser Asp Val Tyr Asp Gly Ala 325 330 335

Gly Val Ala Thr Val Phe Thr Gly Ala Arg Tyr Asn Gly Gly Asp Asp 340 345 350

Ala Ala Asp Glu Tyr Gly Arg His Thr Asn Ala Ala Tyr Arg Asp Leu 355 360 365

Asn Pro Ala Tyr Phe His He Ala Ser Ala Asn He Leu Gly Lys Leu 370 375 380

Asn Ala Thr Phe Val Ala Asp Val Asp Ala Ala Ala Glu Val Trp Asn 385 390 395 400

Gin Pro Val Arg Gly Phe Lys Val Phe Glu Gin Thr Ala Met Ser Leu 405 410 415

Glu Glu Ala Ala Gin Thr Phe Tyr Gly Leu Glu Glu Tyr Pro Trp Asn 420 425 430

Ala Ala Ala Lys Ser He Val Tyr Val Lys Ser Arg Leu Ser Trp He 435 440 445

Phe Glu Thr Tyr Thr Asp Gly Gly Leu Val Ala Ser Gly Glu He Asn 450 455 460

Arg Tyr Thr Thr Gly Lys Tyr Tyr Tyr Tyr Leu Leu Glu Leu Asp Asp 465 470 475 480

Ala Gly Glu He He Gly Gly Glu Trp Val Tyr Asp Ser Asp Ser Asp 485 490 495

His Pro Asp Phe Leu Trp Val Pro Lys Ala Lys Pro Ala Ala Asp Thr 500 505 510

Val Thr Ser He Gly Leu Ser Tyr Ala Asp Val Ser Met Leu Leu Glu 515 520 525

Lys Ser Val Ala Cys Ser Asp Ser Thr Ser Ala Ala Gly Ser Val Ser 530 535 540

Ser Gly Ser Val Gly Glu Ser Thr Glu Ala Pro Thr Glu Val Pro Thr 545 550 555 560

Thr Ser Thr Ser Ala Pro Thr Ser Gly Ser Gly Ala Leu 565 570

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18 September 1995 D SM 10256

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CENTRAALBUREAU VOOR SCHIMMELCULTURES

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Date of deposit Accession Number 19 October 1995 C B S 704 . 95

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CENTRAALBUREAU VOOR SCHIMMELCULTURES

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_C ENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Baarn, the Netherlands

Date of deposit Accession Number

27 December 1994 CBS 651 . 94

C. ADDITIONAL INDICATIONS (leave blank if not applicable) Tbis information is continued on an additional sheet

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the Intemational Bureau later (specify the general nature of the indications eg., 'Accession Number of Deposit")

For International Bureau use only

I [ This sheet was received by the Intemational Bureau on:

Authorized officer

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on page 3 , line 2 7 - 28

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet P]

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Baarn, the Netherlands

Date of deposit Accession Number

20 December 1994 CBS 620 . 94

C. ADDITIONAL INDICATIONS (leave blank if not applicable) Tbis information is continued on an additional sheet Q

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE fif the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed belowwill be submitted to the Intemational Bureau later (specify the general nature of 'the indications e.g., "Accession Number of Deposit")

For intemational Bureau use only

[ I This sheet was received by the Intemational Bureau on:

Authorized officer

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description 3 .. 26 on page . ^lιne

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Baarn, the Netherlands

Date of deposit Accession Number 20 December 1994 CBS 618 . 94

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet [ ^"

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the Intemational Bureau later (specify the general nature of the indications eg., 'Accession Number of Deposit")

For International Bureau use only

I I Tbis sheet was received by the Intemational Bureau on:

Authorized officer