Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

Reference to a Deposit of Biological Material

This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference. For complete information see the description.

Background of the Invention

Field of the Invention

The present invention relates to isolated polypeptides having isoamylase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Description of the Related Art

Isoamylase is a debranching enzyme (E. C. 3.2.1.68) that hydrolyzes 1 ,6-alpha-linkages in amylopectin, glycogen, and beta-limit dextrins. Amylopectin is partially degraded by alpha-amylase which hydrolyzes the 1 ,4-alpha-glucosidic linkages into branched and linear oligosaccharides that results in the formation of alpha-limit dextrins. Unlike pullulanase, isoamylase has high activity towards amylopectin and glycogen and very low activity towards pullulan. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by debranching enzyme. The remaining linear oligosaccharides can be rapidly depolymerized to D-glucose by glucoamylase.

US patent No. 4,335,208 discloses a process of saccharifying starch hydrolysate by an enzyme mixture of glucoamylase and an acidophilic isoamylase derived from Pseudomonas amyloderamosa.

EP 1 ,002,062 concerns an isoamylase from Sulfolobus acidocaldarius and the use thereof in a starch conversion process.

WO 2005/121305 (Novozymes) concerns a process for production of beer having a low content of carbohydrates which comprises; a) preparing a mash in the presence of enzyme activities, b) filtering the mash to obtain a wort, and, c) fermenting said wort to obtain a beer, wherein the enzyme activities comprise; an alpha-amylase, a glucoamylase and an isoamylase.

It is an object of the present invention to provide polypeptides having isoamylase activity and polynucleotides encoding the polypeptides. The invention also provides uses of said isoamylase for syrup preparation and other related applications.

Summary of the Invention

The present invention relates to an isolated polypeptide having isoamylase activity, selected from the group consisting of:

(a1 ) a polypeptide comprising an amino acid sequence having at least 93%, preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 2;

(a2) a polypeptide comprising an amino acid sequence having at least 94%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 4;

(a3) a polypeptide comprising an amino acid sequence having at least 93%, preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 6;

(a4) a polypeptide comprising an amino acid sequence having at least 90%, preferably at least 91 %, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or

(iii) a full-length complementary strand of (i) or (ii);

(c1 ) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 1

(c2) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 3;

(c3) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 5

(c4) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 84%, preferably at least 86%, more preferably at least 87%, more preferably at least

88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least

99% identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and

(d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NOs: 2, 4, 6 and/or 8.

The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of the invention. More specifically the invention in this aspect relates to an isolated polynucleotide encoding a polypeptide having isoamylase activity, obtained by: (a) hybridizing a population of DNA under at least high stringency conditions, preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7, or (iii) a full-length complementary strand of (i) or (ii);

(b) isolating the hybridized polynucleotide, which encodes a polypeptide having isoamylase activity.

The present invention also relates to nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the polynucleotides, and methods of producing a polypeptide having isoamylase activity.

The present invention also relates to methods of producing beer and syrups.

The present invention also relates to plants comprising an isolated polynucleotide encoding such a polypeptide having isoamylase activity.

The present invention also relates to methods of producing such a polypeptide having isoamylase activity, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding such a polypeptide having isoamylase activity under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

Definitions

Isoamylase activity: The term "isoamylase activity" is defined herein as an enzyme having glycogen alpha-1 ,6-glucanohydrolase activity (EC number 3.2.1.68) activity that catalyzes the hydrolysis of (1 , 6)-alpha-D-glucosidic branch linkages in glycogen, amylopectin and their beta-limit dextrins. For purposes of the present invention, isoamylase activity is determined according to the procedure as described in the "Materials & Methods"-section below under the heading "Determination of lsoamylase Activity Units (IAU)".

The polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the isoamylase activity of the mature polypeptide of SEQ ID NOS:

2, 4, 6, or 8, respectively.

Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1 % pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE. The term "pure" is in this context used in relation to the polypeptide of the present invention and it is to be understood as an indication of how much other polypeptide material the polypeptide of the present invention is associated with.

Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide having isoamylase activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

In a preferred aspect, the mature polypeptide is amino acids 1 to 750 of SEQ ID NOS: 2, 4,

6 and 8, respectively, based on the SignalP v 3.0 program that predicts amino acids -26 to -1 of SEQ ID NOS: 2, 4, 6 and 8, respectively, are signal peptides.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" is defined herein as a nucleotide sequence that encodes a mature polypeptide having isoamylase activity. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 79 to 2328 of SEQ ID NOS: 1 , 3, 5 and 7, respectively, based on the SignalP v 3.0 program that predicts nucleotides 1 to 78 of SEQ ID NOS: 1 , 3, 5, and 7, respectively, encode signal peptides.

Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,

J. MoI. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package

(EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, Trends in

Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 10O)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Homologous sequence: The term "homologous sequence" is defined herein as a predicted protein that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W.R., 1999, in Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with the Dyella japonica isoamylases (Accession Nos. DSM 22712, DSM 22713, DSM

22714, and DSM 22715), respectively.

Polypeptide fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide of SEQ ID NOS: 2, 4, 6 or 8, respectively; or a homologous sequence thereof; wherein the fragment has isoamylase activity. The term "fragment thereof" used in relation to a polypeptide is in the context of the present invention to be understood as having the same meaning as "polypeptide fragment".

Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5' and/or 3' end of the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 or 7, respectively; or a homologous sequence thereof; wherein the subsequence encodes a polypeptide fragment having isoamylase activity.

Allelic variant: The term "allelic variant" denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term "isolated polynucleotide" as used herein refers to a polynucleotide that is isolated from a source. In a preferred aspect, the polynucleotide is at least 1 % pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis. The term "pure" is in this context used in relation to the polynucleotide of the present invention and it is to be understood as an indication of how much other polynucleotide material the polynucleotide of the present invention is associated with.

Substantially pure polynucleotide: The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, or synthetic origin, or any combinations thereof.

Coding sequence: When used herein the term "coding sequence" means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence.

Nucleic acid construct: The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

Control sequences: The term "control sequences" is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.

Operably linked: The term "operably linked" denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

Expression: The term "expression" includes any step involved in the production of the polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" is defined herein as a linear or circular

DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.

Host cell: The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.

Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NOS: 2, 4, 6 or 8, respectively; or a homologous sequence thereof; as well as genetic manipulation of the DNA encoding such a polypeptide. The modification can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains.

Artificial variant: When used herein, the term "artificial variant" means a polypeptide having isoamylase activity produced by an organism expressing a modified polynucleotide sequence of the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 or 7; or a homologous sequence thereof. The modified nucleotide sequence is obtained through human intervention by modification of the polynucleotide sequence disclosed in SEQ ID NOS: 1 , 3, 5 and/or 7; or a homologous sequence thereof.

Detailed Description of the Invention

Polypeptides Having Isoamylase Activity

In a first aspect, the present invention relates to an isolated polypeptide having isoamylase activity, selected from the group consisting of:

(a1 ) a polypeptide comprising an amino acid sequence having at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 2;

(a2) a polypeptide comprising an amino acid sequence having at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 4;

(a3) a polypeptide comprising an amino acid sequence having at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 6;

(a4) a polypeptide comprising an amino acid sequence having at least 90%, preferably at least 91 %, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99% identity to the mature polypeptide of SEQ ID NO: 8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or (iii) a full-length complementary strand of (i) or (N);

(c1 ) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 1

(c2) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least

92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity (hereinafter "homologous polypeptides") to the mature polypeptide coding sequence of SEQ ID NO: 3;

(c3) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 5

(c4) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 84%, preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and

(d) a variant comprising a substitution, deletion, and/or insertion of one or more

(several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8.

In a preferred aspect, the homologous polypeptides have an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8.

In one embodiment the polypeptide of the present invention comprises an amino acid sequence, wherein said amino acid sequence consists of the amino acid sequence of SEQ ID

NOS: 2, 4, 6, 8 or an amino acid sequence that differs by 1-70 amino acids, such as 1-65 amino acids, or 1-60 amino acids, or 1-55 amino acids, or 1-50 amino acids, or 1-45 amino acids, or 1-40 amino acids, or 1-35 amino acids, or 1-30 amino acids, or 1-25 amino acids, or 1-20 amino acids, or 1-15 amino acids, or 1-10 amino acids, or 1-9 amino acids, or 1-8 amino acids, or 1-7 amino acids, or 1-6 amino acids, or 1-5 amino acids, or 1-4 amino acids, or 1-3 amino acids, or 1-2 amino acids or 1 amino acid from the amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8, wherein the term "differ" means that the given number of amino acids have been substituted, deleted and/or inserted when compared to the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8.

In another embodiment the polypepide of the present invention comprises an amino acid sequence, wherein said amino acid sequence consists of the mature polypeptide of SEQ ID NOS: 2, 4, 6, 8 and/or an amino acid sequence that differs by 1-70 amino acids, such as 1-65 amino acids, or 1-60 amino acids, or 1-55 amino acids, or 1-50 amino acids, or 1-45 amino acids, or 1-40 amino acids, or 1-35 amino acids, or 1-30 amino acids, or 1-25 amino acids, or 1-20 amino acids, or 1-15 amino acids, or 1-10 amino acids, or 1-9 amino acids, or 1-8 amino acids, or 1-7 amino acids, or 1-6 amino acids, or 1-5 amino acids, or 1-4 amino acids, or 1-3 amino acids, or 1-2 amino acids or 1 amino acid from the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8, wherein the term "differ" means that the given number of amino acids have been substituted, deleted and/or inserted when compared to the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8.

In another embodiment the polypepide of the present invention comprises an amino acid sequence, wherein said amino acid sequence consists of amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6, 8 and/or an amino acid sequence that differs by 1-70 amino acids, such as 1-65 amino acids, or 1-60 amino acids, or 1-55 amino acids, or 1-50 amino acids, or 1-45 amino acids, or 1-40 amino acids, or 1-35 amino acids, or 1-30 amino acids, or 1-25 amino acids, or 1-20 amino acids, or 1-15 amino acids, or 1-10 amino acids, or 1-9 amino acids, or 1-8 amino acids, or 1-7 amino acids, or 1- 6 amino acids, or 1-5 amino acids, or 1-4 amino acids, or 1-3 amino acids, or 1-2 amino acids or 1 amino acid from amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6 and/or 8, wherein the term "differ" means that the given number of amino acids have been substituted, deleted and/or inserted when compared to the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8.

A polypeptide of the present invention preferably comprises the amino acid sequence of

SEQ ID NOS: 2, 4, 6 and/or 8 or variants thereof; or fragments thereof having isoamylase activity. The variants may in particular be an allelic variant, an artificial variant or a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 2, 4, 6, and/or 8. In another preferred aspect, the polypeptide comprises amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6 and/or 8 respectively, or a variant thereof; or fragments thereof having isoamylase activity. The variants may in particular be an allelic variant, an artificial variant or a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide comprises amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8; or variants thereof; or fragments thereof having isoamylase activity. The variants may in particular be an allelic variant, an artificial variant or a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide consists of amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6 and/or 8; or a variant thereof; or a fragment thereof having isoamylase activity. The variant may in particular be an allelic variant, an artificial variant or a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, the polypeptide consists of amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6 and/or 8.

In a second aspect, the present invention relates to an isolated polynucleotide encoding polypeptides having isoamylase activity, obtained by: (a) hybridizing a population of DNA under at least high stringency conditions, preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7, or (iii) a full-length complementary strand of (i) or (ii);

(b) isolating the hybridized polynucleotide, which encodes a polypeptide having isoamylase activity.

Therefore, the invention relates to isolated polypeptides having isoamylase activity that are encoded by polynucleotides that hybridize under preferably high stringency conditions, preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , 3, 5, and/or 7, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , 3, 5 and/or 7, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). A subsequence of the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7, respectively, contain at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment having isoamylase activity. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7.

The nucleotide sequence of SEQ ID NOS: 1 , 3, 5 and/or 7; or a subsequence thereof; as well as the amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8; or fragments thereof; may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having isoamylase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length.

For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes may be used, e.g., nucleic acid probes that are preferably at least 600 nucleotides, more preferably at least 700 nucleotides, even more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³² P, ³ H, ³⁵ S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having isoamylase activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NOS: 1 , 3, 5 and/or 7; or subsequences thereof; the carrier material is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7; the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5, and/or 7; its full-length complementary strands; or a subsequence thereof; under high to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of

SEQ ID NOS: 1 , 3, 5 and/or 7. In another preferred aspect, the nucleic acid probe is nucleotides 79 to 2328 of SEQ ID NOS: 1 , 3, 5 and/or 7, respectively. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NOS: 1 , 3, 5 and/or 7. In another preferred aspect, the nucleic acid probe is one of the polynucleotide sequences contained in and obtainable from deposited strains Dyella japonica DSM 22712, Dyella japonica DSM 22713, Dyella japonica DSM 22714 and/or Dyella japonica DSM 22715, wherein the polynucleotide sequence thereof encodes polypeptides having isoamylase activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in and obtainable from deposited strains Dyella japonica DSM 22712, Dyella japonica DSM 22713, Dyella japonica DSM 22714 and/or Dyella japonica DSM 22715. For long probes of at least 100 nucleotides in length, high and very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micro g/ml sheared and denatured salmon sperm DNA, and 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS preferably at 65°C (high stringency), and most preferably at 70 ⁰ C (very high stringency).

For short probes that are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5°C to about 10 ⁰ C below the calculated T _m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCI, 0.09 M Tris-HCI pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.

For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 10 ⁰ C below the calculated T _m .

In a third aspect, the present invention relates to isolated polypeptides having isoamylase activity encoded by polynucleotides comprising or consisting of

(c1 ) a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ;

(c2) a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 3;

(c3) a nucleotide sequence having at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 5;

(c4) a nucleotide sequence having at least 84%, preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least

90%, more preferably at least 92%, even more preferably at least 95%, most preferably at least 97%, and even most preferably at least 99% identity to the mature polypeptide coding sequence of

SEQ ID NO: 7. In a fourth aspect, the present invention relates to artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8; or homologous sequences thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/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 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

The amino acid substitutions in the wild-type polypeptide include both substitutions with the 20 standard amino acids but also non-standard amino acids (such as 4-hydroxyproline, 6-Λ/-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine). A limited number of non- conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., isoamylase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et a/., 1992, Science 255: 306-312; Smith et a/., 1992, J. MoI. Biol. 224: 899-904; Wlodaver et a/., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et a/., 1991 , Biochem. 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et a/., 1986, Gene 46: 145; Ner et a/., 1988, DNA l: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et a/., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1. Sources of Polypeptides Having lsoamylase Activity

A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly. Examples of sources from which a polypeptide of the present invention may be obtained include but are not limited to any of the following described below.

A polypeptide having isoamylase activity of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,

Clostridium, Geobacillus, or Oceanobacillus polypeptide having isoamylase activity, or a Gram negative bacterial polypeptide such as a Dyella, Fulvimonas, Frateuria and Rhodanobacter E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, llyobacter, Neisseria, or Ureaplasma polypeptide having isoamylase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having isoamylase activity.

In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus egu/ subsp. Zooepidemicus polypeptide having isoamylase activity.

In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having isoamylase activity.

In another preferred aspect, the polypeptide is a Pseudomonas amyloderamosa polypeptide, e.g., the one descriped in (Harada et al., 1968; Sugimoto et al., 1974) having isoamylase activity.

In another preferred aspect, the polypeptide is a Frateuria aurantia polypeptide having isoamylase activity.

In another preferred aspect, the polypeptide is a Dyella japonica and Dyella koreensis polypeptide having isoamylase activity.

In a more preferred aspect, the polypeptide is a Dyella japonica polypeptide having isoamylase activity. In a most preferred aspect, the polypeptide is a Dyella japonica DSM 22712 DSM22713, DSM22714 and/or DSM22715 polypeptide having isoamylase activity, e.g., the polypeptides comprising the mature polypeptides of SEQ ID NOS: 2, 4, 6 or 8, respectively.

It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et a/., 1989, supra).

Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide having isoamylase activity from the fusion protein. Examples of cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et a/., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; Svetina et a/., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et a/., 1997, Appl. Environ. Microbiol. 63: 3488- 3493; Ward et a/., 1995, Biotechnology 13: 498-503; and Contreras et a/., 1991 , Biotechnology 9: 378-381 ), an Ne-(GIu or Asp)-Gly-Arg site, which is cleaved by a Factor Xa protease after the arginine residue (Eaton et a/., 1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after the lysine (Collins-Racie et a/., 1995, Biotechnology 13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter et a/., 1989, Proteins: Structure, Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); a Glu-Asn-Leu- Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease after the GIn (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the GIn (Stevens, 2003, supra). Polynucleotides

The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides having isoamylase activity of the present invention.

The polypeptide of the present invention may in particular be encoded by a polynucleotide comprising or consisting of any of the nucleotide sequences of SEQ ID NOS: 1 , 3, 5 and/or 7; or a subsequence thereof encoding a fragment having isoamylase activity.

In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NOS: 1 , 3, 5 and/or 7, respectively. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in the deposited strain: Dyella japonica NN060811 (DSM 22712); Dyella japonica NN060812 (DSM 22713); Dyella japonica NN060813 (DSM 22714); Dyella japonica NN060814 (DSM 22715).

In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7.

The present invention also encompasses nucleotide sequences that encode polypeptides comprising or consisting of the amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8, respectively, or the mature polypeptide thereof, which differ from SEQ ID NOS: 1 , 3, 5 and/or 7 or the mature polypeptide coding sequences thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NOS: 1 , 3, 5 and/or 7 that encode fragments of SEQ ID NOS: 2, 4, 6 and/or 8, respectively, that have isoamylase activity.

The present invention also relates to mutant polynucleotides comprising or consisting of at least one mutation in the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8.

The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., lnnis et a/., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Dyella, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7, respectively.

The present invention also relates to isolated polynucleotides encoding polypeptides having isoamylase activity, obtained by:

(a) hybridizing a population of DNA under at least high stringency conditions, preferably at least very high stringency conditions, with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7, or (iii) a full-length complementary strand of (i) or (N);

(b) isolating the hybridized polynucleotide, which encodes a polypeptide having isoamylase activity.

The isolated polynucleotide of the invention may be the mature polypeptide coding sequence in nucleotides 79-2328 of SEQ ID NOS: 1 , 3, 5 and/or 7.

Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et a/., 1991 , Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide 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 (see, e.g., Cunningham and Wells, 1989, supra). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for isoamylase 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 the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et a/., 1992, supra; Smith et a/., 1992, supra; Wlodaver et a/., 1992, supra).

In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The present invention also relates to a nucleic acid construct comprising an isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of the present invention, operably linked to one or more (several) control sequences that direct the production of the polypeptide in an expression host.

An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene

(sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta- lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences

USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National

Academy of Sciences USA 80: 21-25). Combinations of promoters may also be used, such as the triple promoter system described in WO 99/43835 or example 1 of the present application, which consists of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including stabilizing sequence. Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for

Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae galactokinase (GAL1 ), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1 ), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et a/., 1992, Yeast 8: 423- 488.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

Examples of terminators for bacterial host cells include but are not limited to those obtained from the genes of Bacillus licheniformis BPN' and Bacillus licheniformis amyl.

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et a/., 1992, supra.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha- glucosidase.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta- lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), Bacillus clausii alcaline protease (aprH) and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et a/., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of amino acids -26 to -1 of SEQ ID NOS: 2, 4, 6 and/or 8, respectively. In another preferred aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 78 of SEQ ID NOS: 1 , 3, 6 and/or 8, respectively.

The control sequence may also be a propeptide coding sequence that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, xy/ and trp operator systems.

In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence. Expression Vectors

The present invention also related to a recombinant expression vector comprising a nucleic acid construct of the present invention.

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vectors of the present invention preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the da/ genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and

URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine- 5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβi permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et ai, 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883. More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook ef a/., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The present invention also relates to a recombinant host cell comprising a nucleic acid construct of the present invention.

The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gram negative bacterium. Gram positive bacteria include, but not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram negative bacteria include, but not limited to, E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, llyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens,

Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In a more preferred aspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. In another more preferred aspect, the bacterial host cell is a Bacillus clausii cell. In another more preferred aspect, the bacterial host cell is a Bacillus licheniformis cell. In another more preferred aspect, the bacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell. Streptococcus cells useful in the practice of the present invention include, but are not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcus equisimilis cell. In another preferred aspect, the bacterial host cell is a Streptococcus pyogenes cell. In another preferred aspect, the bacterial host cell is a Streptococcus uberis cell. In another preferred aspect, the bacterial host cell is a Streptococcus equi subsp. Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomyces cells useful in the practice of the present invention include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

In a preferred aspect, the bacterial host cell is a Streptomyces achromogenes cell. In another preferred aspect, the bacterial host cell is a Streptomyces avermitilis cell. In another preferred aspect, the bacterial host cell is a Streptomyces coelicolor cell. In another preferred aspect, the bacterial host cell is a Streptomyces griseus cell. In another preferred aspect, the bacterial host cell is a Streptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 1 11-115), by using competent cells (see, e.g., Young and Spizizen, 1961 , Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56: 209-221 ), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). The introduction of DNA into an E coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. MoI. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171 : 3583-3585), or by transduction (see, e.g., Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios. 68: 189-2070, by electroporation (see, e.g., Buckley et a/., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell. In a preferred aspect, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et a/., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et a/., 1995, supra, page 171 ) and all mitosporic fungi (Hawksworth et a/., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida, Hansenula,

Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis,

Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,

Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et a/., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

In an even more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus,

Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trie hothec bides, or Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al, 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; lto et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is of the genus Dyella. In a more preferred aspect, the cell is Dyella japonica. In a most preferred aspect, the cell is Dyella japonica DSM 22712, Dyella japonica DSM 22713, Dyella japonica DSM 22714 and/or Dyella japonica DSM 22715, respectively.

The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell, as described herein, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to a method of producing the polypeptide of the present invention, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one mutation in the mature polypeptide coding sequence of SEQ ID NOS: 1 , 3, 5 and/or 7, wherein the mutant nucleotide sequence encodes a polypeptide that comprises or consists of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8, respectively, and (b) recovering the polypeptide.

The present invention also relates to a method of producing a polypeptide of of the present invention, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

The polypeptides of the present invention may in a particular embodiment be purified by starch affinity chromatography, e.g. by using amylase-agarose as the affinity material. Methods of performing this include but are not limited to the method described below in example 2.

Plants

The present invention also relates to plants, e.g., a transgenic plant, plant part, or plant cell, comprising an isolated polynucleotide encoding a polypeptide having isoamylase activity of the present invention so as to express and produce the polypeptide in recoverable quantities. The polypeptide may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

The present invention also relates to a transgenic plant, plant part or plant cell transformed with a polynucleotide encoding a polypeptide of the present invention.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part.

Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seeds coats.

Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more (several) expression constructs encoding a polypeptide of the present invention into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed. For instance, the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et a/., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1 , and the rice actin 1 promoter may be used (Franck et a/., 1980, Ce// 21 : 285-294, Christensen et a/., 1992, Plant Mo. Biol. 18: 675-689; Zhang et a/., 1991 , Plant Cell 3: 1155-1165). organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et a/., 1994, Plant MoI. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et a/., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et a/., 1998, Journal of Plant Physiology 152: 708-711 ), a promoter from a seed oil body protein (Chen et a/., 1998, Plant and Cell Physiology 39: 935-941 ), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et a/., 1993, Plant Physiology 102: 991- 1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et a/., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et a/., 1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higher expression of a polypeptide of the present invention in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For instance, Xu et a/., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including /Agroόactemvm-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser ef a/., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et a/., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-med\aled gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275- 281 ; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et a/., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et a/., 1993, Plant Molecular Biology 21 : 415- 428.

Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well-known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptide of the present invention comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide having isoamylase activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. Compositions

The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in such a polypeptide. The term "enriched" indicates that the isoamylase activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1.

The composition may comprise an isoamylase of the present invention as the major enzymatic component, e.g., a mono-component composition. In an embodiment the composition may further comprise one or more glucoamylases, in particular derived from a strain of the genus Aspergillus, Trichoderma, Talaromyces or Trametes, including Aspergillus niger, Trichoderma reesei, Talaromyces emersonii, and Trametes cingulata. In another embodiment the composition further comprising one or more additional enzymes selected from the group of proteases, alpha- amylases, beta-amylases, maltogenic amylases, alpha-glucosidases, pullulanases, hexosyltransferase and branching enzymes.

The additional enzyme(s) may be produced, for example, by a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum; Humicola, preferably Humicola insolens or Humicola lanuginosa; or Trichoderma, preferably Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.

The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using the polypeptides having isoamylase activity, or compositions thereof.

In one embodiment an isoamylase of the invention may be used in beverage, such as beer, mashing process. In an embodiment an isoamylase of the invention may be used in the production of food ingredients from starch. Isoamylase are typically used in combination with other starch-hydrolysing enzymes, such as alpha-amylase, beta-amylase and glucoamylase. Depending on the specific application, isoamylase is used at levels between 50-5000 IAU per gram starch.

In the production of glucose syrup from starch, starch is liquefied using alpha-amylase, such as bacterial alpha-amylase, e.g., one or more Bacillus alpha-amylase. Subsequently, glucoamylase may be added to convert the starch hydrolysate to glucose syrup. The addition of isoamylase of the invention may then result in syrup with higher glucose content while reducing the amount of added glucoamylase.

Thus the present invention also relates to the use of an isoamylase of the present invention or a composition of the present invention for producing a glucose syrup.

The present invention also relates to the use of an isoamylase of the present invention or a composition of the present invention for a process for producing high fructose syrup process, in particular for producing HFCS.

Specific examples of the methods contemplated for high DX glucose syrup (e.g. 97-98 DX) production using mixtures of glucoamylase and isoamylase are described in US patent 4,335,208- A, in particular examples 1 , 3, 4, 5, 6 and 7, which is hereby incorporated by reference.

The present invention also relates to the use of an isoamylase of the present invention or a composition of the present invention in a process for producing maltose or maltitol.

Isoamylase of the invention may also be used in the production of maltose and maltitol.

In such embodiment the isoamylase is added to liquefied starch after being subjected to alpha- amylase. At the same time, beta-amylase may be added. The hydrolysis may be carried out at temperature around 50-60 ⁰ C, e.g at 50-55 ⁰ C, and a pH of around pH 4-6, e.g about 5.0.) The resulting high maltose syrup may subsequently be purified and concentrated and subjected to crystallization to obtain crystalline maltose. Maltose may then be converted to maltitol by the catalytic hydrogenation. Maltitol is used as a sugar substitute in the production of non-cariogenic hard candies, chewing gum, and other confectionary.

Isoamylase of the invention may also be used together with cyclodextrin glucanotransferase (CGTase), malto-oligosyl trehalose synthase, and malto-oligosyl trehalose trehalohydrolase in the production of a disaccharide trehalose from liquefied starch. The reaction product is trehalose syrup, which is subsequently purified and concentrated. Trehalose is used in food (for example, in bakery goods, beverages, confectionery, and breakfast cereals) as a texturizer, stabilizer, humectant, and sweetener.

Isoamylase of the invention may also be used in conjunction with CGTase to enhance the production of cyclodextrins from starch. For example, a 90% yield of beta-cyclodextrin from amylopectin was obtained by applying a mixture of isoamylase, CGTase, and cyclodecanone at around pH 6 and 25 ⁰ C. Cyclodecanone is a complexant that forms an insoluble inclusion complex with a cyclodextrin molecule. Cyclodextrins are used as encapsulating agents for food additives, flavours, and vitamins.

Generally isoamylase activity is preferably used in amounts of 1 to 1.000.000.000 IAU/kg DS, more preferably 10 to 100.000.000 IAU/kg DS, even more preferably 100 to 10.000.000 IAU/kg DS, and most preferably 50.000 to 5.000.000 IAU/kg DS or 0.001 mg to 100000 mg EP/kg DS, preferably in the amount of 0.01 mg to 10000 mg EP/kg DS, more preferably in the amount of 0.1 mg to 1000 mg EP/kg DS, most preferably in the amount of 1 mg to 100 mg EP/kg DS.

The invention also relates to a method of producing glycogen using an isoamylase, branching enzyme and amylomaltase (EC 2.4.1.25), e.g., as described by Kajuura et al (2008), Biocatalysis and Biotransformation, 26(1-2): 133-140.

Materials & Methods Materials

Alpha-Amylase LS: Blend of 2 parts hybrid alpha-amylase comprising 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), with the following substitution: G48A+T49I+G107A+H156Y+A181T+N190F+I201 F+A209V+Q264S and 1 part Bacillus stearothermophilus alpha-amylase with the following mutations: 1181 ^* + G182 ^* + N193F.

Glucoamylase AN: Glucoamylase from Aspergillus niger. Glucoamylase T: Glucoamylase derived from Talaromyces emersonii disclosed in SEQ ID NO: 7 in WO 99/28448 and available from Novozymes A/S, Denmark.

Deposit of Biological Material

The following biological material has been deposited under the terms of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inffenstr. 7 B, D-38124 Braunschweig, Germany, and given the following accession number:

Deposit Accession Number Date of Deposit

Dyella japonica NN060811 DSM 22712 June 24, 2009

Dye//ayapoπ/ca NN060812 DSM 22713 June 24, 2009

Dye//ayapoπ/ca NN060813 DSM 22714 June 24, 2009 Dye//ayapoπ/ca NN060814 DSM 22715 June 24, 2009

The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Origin of deposited donor strains

Deposit Country of Origin Year

Dyella japonica NN060811 Denmark 2007

Dyella japonica NN060812 China 2007

DDyyee/l/laa j/aappoonn/iccaa NNNN006600881133 CChhiinnaa 2007

Dyella japonica NN060814 China 2007

Methods:

Determination of lsoamylase Activity Units (IAU)

The debranching of starch is measured as an increase in iodine binding and thereby increases in blue color due to the fact that linear glucans binds iodine more strongly than branched glucans (amylopectin).

1 unit (U) of isoamylase activity (IAU) is the amount of enzyme which causes an increase in absorbency of 0.01 per minute at 610 nm at 40 ⁰ C, pH 4.5.

Substrate: 1 % waxy corn starch (amylopectin) in 75 mM Na-acetate pH 4.5, 2 mM CaCI ₂ . The substrate should be boiled for 10-15 minutes to solubilise the starch.

Iodine/stop reagent: 10 mM I ₂ /KI in MQ H ₂ O

Sulphuric acid solution: 20 mM H ₂ SO ₄ in MQ H ₂ O

Assay procedure:

Mix

1. 600 microL substrate

2. 100 microL enzyme solution (include a control which should be 100 microL MQ H ₂ O)

3. Incubate at 40 ⁰ C, 30 minutes 4. Withdraw 50 microL after 30 minutes into an eppendorf tube which already contains 50 microL of the iodine solution.

5. Subsequently add 1.5 mL of the sulphuric acid solution and transfer 200 microL to a MTP plate.

6. After 5 minutes read the absorbance at 610 nm

Activity calculation:

U/mL = OD610 (sample) - OD610 (control) / 0.01 / 30 minutes / 50 microL ^* 1000 microL

Glucoamylase activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes

1 micromole maltose per minute under the standard conditions 37°C, pH 4.3, substrate: maltose

23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming

NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

A folder (E B-S M-0131.02/01 ) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference. The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Example 1

Cloning and Expression of Four lsoamylases from Dyella iaponica in Bacillus subtilis.

A linear integration vector-system was used for the expression cloning of four isoamylase genes. The linear integration construct was a PCR fusion product made by fusion of each gene between two Bacillus subtilis homologous chromosomal regions along with a strong promoter and a chloramphenicol resistance marker. The fusion was made by SOE PCR (Horton, R. M., Hunt, H. D., Ho, S. N. , Pullen, J. K. and Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes, gene splicing by overlap extension Gene 77: 61-68). The SOE PCR method is also described in WO 2003/095658. Each gene was expressed under the control of a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including stabilizing sequence. The gene coding for Chloramphenicol acetyl-transferase was used as marker. (Described in e.g. Diderichsen.B.; Poulsen.G.B.; Joergensen.S.T.; A useful cloning vector for Bacillus subtilis. Plasmid 30:312 (1993)). The final gene construct was integrated on the Bacillus chromosome by homologous recombination into the pectate lyase locus.

Chromosomal DNA of the four different Dyella japonica strains was isolated by QIAmp Tissue Kit (Qiagen, Hilden, Germany). For each gene construct three fragments were PCR amplified: the gene fragment from genomic DNA from the 4 Dyella japonica species (all primers used are listed in the Table 1 below), the upstream flanking fragment was amplified with the primers #01-iMB1362 and iMB1362Uni2 and the downstream flanking fragment was amplified with the primers #20-iMB1362 and oth435 from genomic DNA of the strain iMB1362 (described in WO 2003/095658).

The gene fragment was amplified using a proofreading polymerase (Phusion™ High-Fidelity DNA Polymerase, (New England Biolabs, Inc.)). The two flanking DNA fragments were amplified with "Expand High Fidelity PCR System" (Roche-Applied-Science). The PCR reactions were made according to standard procedures (following the manufacturer's recommendations). The PCR conditions were as follows: 94°C for 2 minutes followed by 10 cycles of (94°C for 15 seconds, 50 ⁰ C for 45 seconds, 68°C for 4 minutes) followed by 20 cycles of (94°C for 15 seconds, 50 ⁰ C for 45 seconds, 68°C for 4 minutes (+20 seconds, extension per cycle)) and ending with one cycle at 68°C for 10 minutes. The 3 resulting fragments were mixed in equal molar ratios and a new PCR reaction were run under the following conditions: initial 2 minutes at 94°C, followed by 10 cycles of (94°C for 15 seconds, 50 ⁰ C for 45 seconds, 68°C for 5 minutes), 10 cycles of (94°C for 15 seconds, 50 ⁰ C for 45 seconds, 68°C for 8 minutes), 15 cycles of (94°C for 15 seconds, 50 ⁰ C for 45 seconds, 68°C for 8 minutes in addition 20 seconds extra per cycle). After the 1 ^st cycle the two end primers #01-iMB1362: and #20-iMB1362 were added (2OpMoI of each). Two microL of the PCR product was transformed into Bacillus subtilis and transformants was selected on LB-plates containing chloramphenicol (6 microg/mL medium). A clone containing the construct without mutations leading to amino acid changes was selected for fermentation in liquid media. Table 1. Primers used

Amplification of SPECIFIC PRIMER SPECIFIC PRIMER REVERSE

FORWARD

Dyella japonica 5' 5'

(NN060814) CTTGCTGCCTCATTCTGCAG GGGCCAAGGCCGGTTTTTT gen 1 CCGCGGCCATCAACAGCATG ATGTTTTACTTCGAAATCAA

ACCTTG 3' (SEQ ID NO: 9) CAACAACAGCG 3' (SEQ I D

NO: 10)

Dyella japonica 5' 5'

(NN060812) gen 2 CTTGCTGCCTCATTCTGCAG GGGCCAAGGCCGGTTTTTT

CCGCGACACCGGCCCAGGC ATGTTTTACTTGGAAATCAG GGCCATCAAC 3' (SEQ ID NO: CAGCAGCAACGACTGGC 3' 11 ) (SEQ ID NO: 12)

Dyella japonica 5' 5'

(NN060813) gen 3 CTTGCTGCCTCATTCTGCAG GGGCCAAGGCCGGTTTTTT

CCGCGGCCATCAACAGCATG ATGTTTTACTTGGAGATCAG AGTCTTG 3' (SEQ ID NO. 13) CAGCAACAGC 3' (SEQ ID

NO: 14)

Fermentation

The clones was streaked on an LB-agar plates with 6 micro g/mL chloramphenicol from -80 ⁰ C stock, and grown overnight at 37°C. The colonies were transferred to 100 ml. Cal-18 media in a 500 ml. shaking flask. The culture was shaken at 26°C at 170 rpm for 2 or 3 days. The cells were spun down and the enzyme purified from the supernatant by methods described in Example 2.

Example 2

Purification of Dyella isoamylase by starch affinity chromatography

The crude ferment was filtered through a double filter paper sandwich layer consisting of the following grades of Whatman filter paper, D, A, B, C and F with F at the bottom. This crude filtration was then followed by vacuum filtration with a vacuum cup filter with a 0.2 micro m pore size cut off.

The volume of filtrate was recorded and CaCI ₂ was added under slow stirring to give a final concentration of 1 mM. To 900 ml. of filtrate add 100 ml. of 1 M Sodium phosphate buffer pH 6.5 to give a final concentration of 0.1 M. It was checked that the pH was 6.5 and adjusted if necessary.

The buffer addition may cause some precipitation, in this event the vacuum filtration step was repeated using a vacuum cup filter unit with a 0.2 micro m cut off.

Meanwhile a column was prepared (12 x 2.6 cm) with Amylose-agarose affinity column material (purchased from New England Biolabs) equilibrated with 0.1 M phosphate buffer pH 6.5. Load 200 ml. of buffered culture filtrate mixture on to the column (5ml_s/min) and collect the flow through. The column was washed with 0.1 M phosphate buffer until the UV absorbance was stable

(the wash was collected). The isoamylase was eluted from the column by washing the column with

0.1 M phosphate buffer + 20% Maltose. The peak was collected when the absorbance started to climb over the base line level until the base line became stable again (ca. 50-100 ml. total volume). The column was reconditioned with 0.1 M phosphate until a stable baseline was achieved and the load, wash, elute cycle was repeated until all the isoamylase supernatant was used up. After the first run a 4-20% Tris-glycine SDS page gel was run to check for the presence of the isoamylase in the load, column flow through, column wash and in the eluted fractions.

20 microL of sample was mixed with 20 microL of sample buffer and heated to 95 ⁰ C for 10 minutes. 20 microL was loaded to each lane and the gel was run at 35 mA for one hour. Protein bands were realized using simply Blue safe stain from Invitrogen. The Dyella isoamylase appeared as a single band at 80KD.

Once purity had been achieved the isoamylase fractions were pooled and were then concentrated using Amicon Ultra ultracentrifuge units fitted with a 30 kD cut off membrane (spun at 3,000 rpm x g for 30 minutes, the filtrate was discarded and the load/ concentrate cycle was continued until all the pooled fraction was used). Once concentrated, the concentrate was washed several times with 0.1 M phosphate buffer pH 6.5 to remove the 20% maltose. The concentrated isoamylase was removed with a pipette and saved. The inner membrane was washed several times with 0.1 M phosphate buffer and each wash was pooled with the concentrate (this is important as unspecific binding will result in significant sample loss). The final volume was recorded.

The absorbance was measured at 280 nm using the phosphate buffer to zero the spectrophotometer. If the absorbance was more than 2.0 the sample was diluted in 0.1 M phosphate buffer so that the absorbance reading failed within 0.5-1.5.

The protein concentration was calculated thus: A280 value/molar absorbance (calculated from the amino acid sequence) x dilution factor. The value obtained was in mg/ml.

The sample was formulated by adding glycerol to a final concentration of 50%. Example 3

A spray-dried DE 1 1 maltodextrin, produced from common corn starch liquefied with Alpha-Amylase LS, was dissolved in deionized water and the pH and solids level adjusted to approximately 4.3 and 30% respectively. Aliquots of the substrate containing 15 g dry solids were transferred to 50 mL blue-cap flasks fitted with magnetic stirrers which were then placed in a water bath at 58°C. Different amounts of Glucoamylase AN and Dyella japonica isoamylase (NN060812) were then added according to the scheme below and the pH's adjusted to 4.3.

Samples were taken at set time intervals and the reaction stopped by heating the sample to boiling. After dilution and sterile filtration the samples were analyzed for D-glucose by HPLC.

These data show that it is possible to obtain a higher D-glucose yield when combining Dyella isoamylase with Aspergillus niger glucoamylase. At the same time, the glucoamylase dosage can be significantly reduced.

Example 4

A DE 11 maltodextrin substrate was prepared as described in Example 2. Different amounts of Glucoamylase AN, Glucoamylase T and Dyella japonica isoamylase (NN060812) were added according to the scheme below. The saccharification reaction was carried out at 58°C, pH 4.3. Samples were taken at set time intervals and the reaction stopped by heating the sample to boiling. After dilution and sterile filtration the samples were analyzed for D-glucose by HPLC.

These data show that it is possible to match the performance of Glucoamylase AN (Aspergillus niger glucoamylase) by using a dosage of 0.24 AGU /g DS Glucoamylase T (Talaromyces emersonii glucoamylase) and 100-200 IAU/g DS Dyella japonica isoamylase. The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

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