WO2000075296A1 - Novel glucoamylase - Google Patents

Abstract

The present invention relates to isolated nucleic acid sequences encoding polypeptides having glucoamylase activity derived from a strain of Thermoascus crustaceus. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides, and the use of the glucoamylase of the invention.

Description

NOVEL GLUCOAMYLASE

Field of the Invention

The present invention relates to isolated nucleic acid sequences encoding polypeptides having glucoamylase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.

Background of the Invention Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme, which catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts, with those from Aspergillus being commercially most important.

Commercially, the glucoamylase enzyme is used to convert com starch which is already partially hydrolyzed by an alpha-amylase to glucose. The glucose is further converted by glucose isomerase to a mixture composed almost equally of glucose and fructose. This mixture, or the mixture further enriched with fructose, is the commonly used high fructose corn syrup commercialized throughout the world. This syrup is the world's largest tonnage product produced by an enzymatic process. The three enzymes involved in the conversion of starch to fructose are among the most important industrial enzymes produced.

One of the main problems exist with regard to the commercial use of glucoamylase in the production of high fructose corn syrup is the relatively low thermal stability of glucoamylase. Glucoamylase is not as thermally stable as alpha-amylase or glucose isomerase and it is most active and stable at lower pH's than either alpha-amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH.

Summary of the Invention

The object of the present invention is to provide a novel glucoamylase backbone with a higher thermostablility and/or higher specific activity and/or decreased glucose reversion tendency (i.e., decreased tendency to produce isomaltose from glucose) in comparison to the A. niger Gl glucoamylase. The glucoamylase of the invention is suitable for use in, e.g., the saccharification step in starch conversion processes.

The inventors of the present invention have cloned a parent glucoamylase from a strain of Thermoascus crustaceus with higher thermostability and/or higher specific activity in comparison to the, e.g., the commercial available Aspergillus niger Gl glucoamylase (Boel et al. (1984), EMBO J. 3 (5), 1097-1102) or the truncated A. niger G2 glucoamylase shown in

SEQ ID NO: 4.

The present invention relates to isolated nucleic acid sequences encoding polypeptides having glucoamylase activity, selected from the group consisting of:

(a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity with the mature part of SEQ ID NO:2;

(b) a nucleic acid sequence having at least 50% homology with nucleotides for nucleic acid sequence encoding mature polypeptide nucleotides of SEQ ID NO: 1 ;

(c) a nucleic acid sequence which hybridizes under very low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:l, (ii) the cDNA sequence of SEQ ID NO:l, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);

(d) an allelic variant of (a), (b), or (c);

(e) a subsequence of (a), (b), (c), or (d), wherein the subsequence encodes a polypeptide fragment which has glucoamylase activity; and (f) a polypeptide having higher thermostability than the A. niger Gl glucoamylase shown in SEQ ID NO: 4.

The protein sequence shown in SEQ ID NO: 2 is deduced from the DNA sequence shown in SEQ ID NO: 1 cloned from Thermoascus crustaceus. When expressing the DNA sequence shown in SEQ ID NO: 1 in, e.g., an Aspergillus host cell, such as A. niger host cell, as described below, the protein sequence shown in SEQ ID NO: 2 will be expressed. Introns and the like in the DNA sequence will be cut out (see Fig. 1).

The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides. The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides. Brief Description of the Drawing

Fig. 1 shows the nucleotide sequence of the glucoamylase gene, and the deduced amino acid sequence of the glucoamylase precursor from Thermoascus crustaceus. The intron sequences are underlined and shown in lower case letters.

Detailed Description of the Invention Polypeptides Having Glucoamylase Activity

Glucoamylase activity (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is for the purposes of the present invention determined using the AGU assay described below in the "Materials & Methods" section.

Homology of Enzyme

In a first embodiment, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature part of SEQ ID NO:2 of at least about 50%, preferably at least about 60%, more preferably at least about 70%>, even more preferably at least about 80%, even more preferably at least about 90%o, even more preferably at least about 93%, more preferably at least about 95%, even more preferably at least 97%, and most preferably 99% which have glucoamylase activity (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the mature amino acids of SEQ ID NO:2.

The amino acid sequence homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art. Thus, GAP provided in GCG version 8 (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453) may be used for a pairwise alignment of the sequences and calculation of the degree of identity or degree of homology using the default settings.

Homology To Other Glucoamylase A homology search showed low homology for the sequence of the invention determined as descπbed above to known sequences

Preferably, the polypeptides of the present invention compπse the amino acid sequence of SEQ ID NO 2 or an allelic vaπant thereof, or a fragment thereof that has glucoamylase activity SEQ ID NO 2 show the glucoamylase of the invention

A fragment of SEQ ID NO 2 is a polypeptide having one or more ammo acids deleted from the ammo and/or carboxyl terminus of this amino acid sequence

An allelic vanant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus Allelic vaπation aπses 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 vaπant of a polypeptide is a polypeptide encoded by an allelic vaπant of a gene

The ammo acid sequences of the homologous polypeptides may differ from the amino acid sequence of SEQ ID NO 2 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues Preferably, amino acid changes are of a minor nature, that is conservative ammo acid substitutions 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 methionme residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates punfication by changing net charge or another function, such as a poly-histidine tract, an antigemc epitope or a binding domain

Examples of conservative substitutions are within the group of basic ammo acids (arginine, lysme and histidine), acidic amino acids (glutamic acid and aspartic acid), polar ammo acids (glutamine and asparagine), hydrophobic ammo acids (leucine, isoleucine and vahne), aromatic amino acids (phenylalanme, tryptophan and tyrosine), and small ammo acids

(glycine, alanine, seπne, threonme and methiomne) Amino acid substitutions which do not generally alter the specific activity are known in the art and are descπbed, for example, by H Neurath and R L Hill, 1979, In, The Proteins, Academic Press, New York The most commonly occurπng exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these m reverse In a second embodiment, the present invention relates to isolated polypeptides having glucoamylase activity which are encoded by nucleic acid sequences which hybridize under low stringency conditions, more preferably medium stringency conditions, even more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (i) the nucleic acid sequence of SEQ ID NO: l, (ii) the cDNA sequence of SEQ ID NO: l , (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratoiy Manual, 2d edition, Cold Spring Harbor, New York). The subsequence of SEQ ID NO: l may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment, which has glucoamylase activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have glucoamylase activity.

The nucleic acid sequence of SEQ ID NO:l or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO:2 or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having glucoamylase 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 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³³S, biotin, or avidin). Such probes are encompassed by the present invention. Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA, which hybridizes with the probes described above and which encodes a polypeptide having glucoamylase activity. Genomic or other DNA from such other organisms 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 which is homologous with SEQ ID NO: l or subsequences thereof, the carrier matenal is used in a Southern blot For purposes of the present invention, hybπdization indicates that the nucleic acid sequence hybndizes to a nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO 1, its complementary strand, or a subsequence thereof, under very low to very high stπngency conditions Molecules to which the nucleic acid probe hybndizes under these conditions are detected using X-ray film

In another prefened embodiment, the nucleic acid probe is the nucleotides of SEQ ID NO 1 which encodes a mature polypeptide having glucoamylase activity shown in SEQ ID NO 2

For long probes of at least 100 nucleotides in length, very low to very high stπngency conditions are defined as prehybndization and hybndization at 42°C in 5X SSPE, 0 3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stπngencies, 35% formamide for medium and medium-high stnngencies, or 50% formamide for high and very high stnngencies, following standard Southern blotting procedures For long probes of at least 100 nucleotides in length, the earner matenal is finally washed three times each for 15 minutes using 2 x SSC, 0 2% SDS preferably at least at 45°C (very low stnngency), more preferably at least at 50°C (low stringency), more preferably at least at 55°C (medium stnngency), more preferably at least at 60°C (medium-high stnngency), even more preferably at least at 65°C (high stnngency), and most preferably at least at 70°C (very high stnngency)

For short probes which are about 15 nucleotides to about 70 nucleotides in length, stnngency conditions are defined as prehybndization, hybndization, and washing post- hybndization at 5°C to 10°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 NaCl, 0 09 M Tns-HCl pH 7 6, 6 mM EDTA, 0 5% NP-40, IX 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 short probes, which are about 15 nucleotides to about 70 nucleotides in length, the earner matenal 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°C below the calculated T_m

The present invention also relates to isolated nucleic acid sequences produced by (a) hybridizing a DNA under very low, low, medium, medium-high, high, or very high stringency conditions with the sequence of SEQ ID NO: l. or its complementary strand, or a subsequence thereof; and (b) isolating the nucleic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence, which encodes a polypeptide fragment, which 5 has glucoamylase activity.

The polypeptides of the present invention have at least 20%, preferably at least 40%>, more preferably at least 60%, even more preferably at least 80%), even more preferably at least 90%, and most preferably at least 100%o of the glucoamylase activity of the mature polypeptide of SEQ ID NO: 2. 0 A polypeptide of the present invention may be obtained from organism or 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 the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted. 5 A polypeptide of the present invention may be a bacterial or fungal polypeptide, in particular from the genus Thermoascus, especially a strain of the species Thermoascus crustaceus .

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, o 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 5 (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 o well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).

As defined herein, an "isolated" polypeptide is a polypeptide which is essentially free

5 of other non-glucoamylase polypeptides, e.g., at least about 20% pure, preferably at least about 40%) pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by

SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present invention also include 0 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 nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the 5 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.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequences, which encode a o polypeptide of the present invention. In a preferred embodiment, the nucleic acid sequence is set forth in SEQ ID NO: l. In another prefened embodiment, the nucleic acid sequence is the mature polypeptide-coding region of SEQ ID NO: 1. The present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO:2 which differ from SEQ ID NO: 1 by virtue of the degeneracy of the genetic code. 5 The present invention also relates to subsequences of SEQ ID NO: l which encode fragments of SEQ ID NO: 2 that have glucoamylase activity.

Subsequences of SEQ ID NO: 1 are nucleic acid sequences encompassed by SEQ ID NO:l except that one or more nucleotides from the 5' and/or 3' end have been deleted.

The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide o are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences 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., Innis et al, 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 nucleic acid sequence- based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain of the genus Thermoascus, especially a strain of the species Thermoascus crustaceus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence. The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Homology of DNA sequences encoding the enzyme

The present invention also relates to nucleic acid sequences which have a degree of homology to the polypeptide coding sequence of SEQ ID NO: l, especially the mature part, of at least about 50%, more preferably about 60%, even more preferably about 70%, even more preferably about 80%, even more preferably about 90%o, even more preferably about 93%, even more preferably about 95%, even more preferably about 97%, and most preferably about 99%) homology, which encodes an active polypeptide. The DNA sequence homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (descnbed above) Thus, Gap GCGv8 may be used with the following default parameters GAP creation penalty of 5 0 and GAP extension penalty of 0 3, default scoring matnx GAP uses the method of Needleman/Wunsch/Sellers to make alignments

Modification of a nucleic acid 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 , variants that differ in specific activity, thermostability, pH optimum, or the like The variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO 1, e g , a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give nse to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different ammo acid sequence For a general descnption of nucleotide substitution, see, e g , Ford et al , 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 cntical to the function of the molecule and still result in an active polypeptide Ammo acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence 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-scannmg mutagenesis (see, e g , Cunningham and Wells, 1989, Science 244 1081-1085) In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for glucoamylase 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 labelling (see, e g , de Vos et al , 1992, Science 255

306-312, Smith et al , 1992, Journal of Molecular Biology 224 899-904, Wlodaver et al , 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated nucleic acid sequences encoding a polypeptide of the present invention, which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, even more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with the nucleic acid sequence of SEQ ID NO: l or its complementary strand; or allelic variants and subsequences thereof (Sambrook et al, 1989, supra), as defined herein. The present invention also relates to isolated nucleic acid sequences produced by (a) hybridizing a DNA under very low, low, medium, medium-high, high, or very high stringency conditions with the sequence of SEQ ID NO: l, or their complementary strands, or a subsequence thereof; and (b) isolating the nucleic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence, which encodes a polypeptide fragment, which has glucoamylase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences, which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post- transcriptional modification, translation, post-translational modification, and secretion.

"Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" is defined herein as a portion of a nucleic acid sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence 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 nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. 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 nucleic acid sequence encoding a polypeptide. The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence, which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which 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 coehcolor agarase gene (dagA), Bacillus subti s levansucrase gene (sacB), Bacillus hcheniformis alpha-amylase gene (amvL), Bacillus stearothermophilus maltogemc amylase gene (amvM), Bacillus amvlohquefaciens alpha- amylase gene (amyQ), Bacillus hcheniformis penicilhnase gene (penP), Bacillus subtihs xvlA 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) Further promoters are descnbed in "Useful proteins from recombinant bactena" in Scientific American, 1980, 242: 74-94, and in Sambrook et al , 1989, supra. Examples of suitable promoters for directing the transcnption of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oiyzae TA-KA 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 orvzae alkaline protease, Aspergillus oryzae tnose phosphate isomerase, Aspergillus mdulans acetamidase, Fusarium oxγsporum trypsin-hke protease (WO 96/00787), as well as the NA2-tpι promoter (a hybnd of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus orvzae tnose phosphate isomerase), and mutant, truncated, and hybnd promoters thereof In a yeast host, useful promoters are obtained from the genes for Saccharomvces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomvces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells are descnbed by Romanos et al, 1992, Yeast 8. 423-488 The control sequence may also be a suitable transcnption terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator, which is functional in the host cell of choice, may be used in the present invention. Preferred terminators for filamentous fungal host cells are obtained from the genes for

Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus mdulans anthranilate synthase, Aspergillus mgei alpha-glucosidase, and Fusarium oxvsporum trypsm- hke protease

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

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA, which is important for translation by the host cell The leader sequence is operably linked to the 5' terminus of the nucleic acid 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 orvzae TAKA amylase and Aspergillus mdulans tnose phosphate isomerase

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

The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcnbed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA

Any polyadenylation sequence, which is functional in the host cell of choice may be used m the present invention

Preferred polyadenylation sequences for filamentous fungal host cells are obtamed from the genes for Aspergillus orvzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus mdulans anthranilate synthase, Fusarium oxvsporum trypsin-hke protease, and

Aspergillus niger alpha-glucosidase

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

The control sequence may also be a signal peptide-codmg region that codes for an ammo acid sequence linked to the ammo terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention. Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837maltogenιc amylase, Bacillus stear other mophύus alpha-amylase, Bacillus hcheniformis subtihsm, Bacillus hcheniformis beta-lactamase, Bacillus stear othermoplulus neutral proteases (nprT, nprS, nprM), and Bacillus subtihs prsA. Further signal peptides are descπbed by Simonen and Palva, 1993, Microbiological Reviews 57 109-137.

Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus orvzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase Useful signal peptides for yeast host cells are obtained from the genes for

Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae mvertase. Other useful signal peptide coding regions are descnbed by Romanos et al , 1992, supra

The control sequence may also be a propeptide coding region 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 region may be obtained from the genes for Bacillus subtihs alkaline protease (aprE), Bacillus subtihs neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Mycehophthora thermophύa laccase (WO 95/33836).

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

It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which 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, 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 the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those, which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.

The present invention also relates to nucleic acid constructs for altering the expression of a nucleic acid sequence of the present invention that is endogenous to a cell. The constructs may contain the minimal number of components necessary for altering expression of the endogenous gene. In one embodiment, the nucleic acid constructs preferably contain (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, and (d) a splice-donor site. Upon introduction of the nucleic acid construct into a cell, the construct inserts by homologous recombination into the cellular genome at the endogenous gene site. The targeting sequence directs the integration of elements (a)-(d) into the endogenous gene such that elements (b)-(d) are operably linked to the endogenous gene. In another embodiment, the nucleic acid constructs contain (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a)-(f) such that elements (b)-(f) are operably linked to the endogenous gene. However, the constructs may contain additional components such as a selectable marker.

In both embodiments, the introduction of these components results in production of a new transcription unit in which expression of the endogenous gene is altered. In essence, the new transcription unit is a fusion product of the sequences introduced by the targeting constructs and the endogenous gene. In one embodiment in which the endogenous gene is altered, the gene is activated. In this embodiment, homologous recombination is used to replace, disrupt, or disable the regulatory region normally associated with the endogenous gene of a parent cell through the insertion of a regulatory sequence which causes the gene to be expressed at higher levels than evident in the coπesponding parent cell. The activated gene can be further amplified by the inclusion of an amplifiable selectable marker gene in the construct using methods well known in the art (see, for example, U.S. Patent No. 5,641,670). In another embodiment in which the endogenous gene is altered, expression of the gene is reduced.

The targeting sequence can be within the endogenous gene, immediately adjacent to the gene, within an upstream gene, or upstream of and at a distance from the endogenous gene. One or more targeting sequences can be used. For example, a circular plasmid or DNA fragment preferably employs a single targeting sequence, while a linear plasmid or DNA fragment preferably employs two targeting sequences.

The regulatory sequence of the construct can be comprised of one or more promoters, enhancers, scaffold-attachment regions or matrix attachment sites, negative regulatory elements, transcription binding sites, or combinations of these sequences. The constructs further contain one or more exons of the endogenous gene. An exon is defined as a DNA sequence, which is copied into RNA and is present in a mature mRNA molecule such that the exon sequence is in-frame with the coding region of the endogenous gene. The exons can, optionally, contain DNA, which encodes one or more amino acids and/or partially encodes an amino acid. Alternatively, the exon contains DNA which corresponds to a 5' non-encoding region. Where the exogenous exon or exons encode one or more amino acids and/or a portion of an amino acid, the nucleic acid construct is designed such that, upon transcription and splicing, the reading frame is in-frame with the coding region of the endogenous gene so that the appropriate reading frame of the portion of the mRNA derived from the second exon is unchanged. The splice-donor site of the constructs directs the splicing of one exon to another exon.

Typically, the first exon lies 5' of the second exon. and the splice-donor site overlapping and flanking the first exon on its 3' side recognizes a splice-acceptor site flanking the second exon on the 5' side of the second exon. A splice-acceptor site, like a splice-donor site, is a sequence, which directs the splicing of one exon to another exon. Acting in conjunction with a splice-donor site, the splicing apparatus uses a splice-acceptor site to effect the removal of an intron.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid 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) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid 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 which 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 which, 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 which 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 selectable markers, which permit easy selection of transformed 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 bactenal selectable markers are the dal genes from Bacillus subtihs or Bacillus hcheniformis, or markers, which confer antibiotic resistance such as ampicillm, kanamycm, chloramphenicol or tetracychne resistance Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3 A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothncin acetyltransferase), hygB (hygromycm phosphotransferase), niaD (nitrate reductase), pyrG (orotιdιne-5 '-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof Prefened for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus mdulans or Aspergillus orvzae and the bar gene of Streptomγces hygroscopicus The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell

For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise locatιon(s) in the chromosome(s) To increase the likelihood of integration at a precise location, the mtegrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination The mtegrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell Furthermore, the mtegrational elements may be non-encodmg or encoding nucleic acid 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 ongm of replication enabling the vector to replicate autonomously in the host cell in question Examples of bacteπal ongins of replication are the oπgins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E coli, and pUBHO, pE194, pTA1060, and pAMBl permitting replication in Bacillus Examples of ongins of replication for use in a yeast host cell are the 2 micron ongin of replication, ARSl, ARS4, the combination of ARSl and CEN3, and the combination of ARS4 and CEN6 The ongin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e g , Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75 1433)

More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product An increase m the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amphfiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropπate selectable agent

The procedures used to gate 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 et al , 1989, supra)

Host Cells

The present invention also relates to recombinant host cells, compnsing a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides A vector comprising a nucleic acid sequence 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 descnbed 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 duπng replication The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source The host cell may be a unicellular microorganism, e g , a prokaryote, or a non- unicellular microorganism, e g , a eukaryote

Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e g , Bacillus alkalophilus, Bacillus amylohquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausu, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus hcheniformis, Bacillus megaterium, Bacillus stear othermophύus, Bacillus subtihs, and Bacillus thuringiensis, or a Streptomvces cell, e g , Streptomyces hvidans or Streptomyces murinus, or gram negative bactena such as E coli and Pseudomonas sp In a prefeπed embodiment, the bactenal host cell is a Bacillus lentus, Bacillus hcheniformis, Bacillus stear othet mophύus or Bacillus subtihs cell In another preferred embodiment, the Bacillus cell is an alkalophihc Bacillus

The introduction of a vector into a bactenal host cell may, for instance, be effected by protoplast transformation (see, e g , Chang and Cohen, 1979, Molecular General Genetics

168 1 11-115), using competent cells (see, e g , Young and Spizizm, 1961, Journal of Bacteriology 81 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular

Bιolog\' 56 209-221), electroporation (see, e g , Shigekawa and Dower, 1988, Biotechmques 6 742-751), or conjugation (see, e g , Koehler and Thome, 1987, Journal of Bacter iology

169 5771-5278)

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

In a more preferred embodiment, the fungal host cell is a yeast cell "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes) Since the classification of yeast may change in the future, for the purposes of this mvention, yeast shall be defined as descnbed 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 embodiment, the yeast host cell is a Candida, Hansenula, Kluvveromyces, Pichia, Saccharomvces, Schizosaccharomyces, or Yarrowia cell

In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasu, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomvces oviformis cell. In another most prefeπed embodiment, the yeast host cell is a Kluyveromvces lactis cell.

In another most prefeπed embodiment, the yeast host cell is a Yarrowia hpolytica cell

In another more preferred embodiment, 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 al , 1995, supra). The filamentous fungi are characteπzed by a mycehal wall composed of chitm, cellulose, glucan, chitosan, mannan, and other complex polysacchandes Vegetative growth is by hyphal elongation and carbon catabohsm is obhgately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabohsm may be fermentative

In an even more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicil um, Thielavia, Tolypocladium, or Trichoderma

In a most prefeπed embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus , Aspergillus japomcus, Aspergillus mdulans, Aspergillus niger or Aspergillus oryzae cell. In another most prefeπed embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerea s, 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 trichothecioides, or Fusarium venenatum cell In an even most prefeπed embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov ) cell. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal host cell is a Mucor miehei cell In another most prefeπed embodiment, the filamentous fungal host cell is a Myceliophthora thermophύa cell In another most prefeπed embodiment, the filamentous fungal host cell is a Neurospora crassa cell In another most preferred embodiment, the filamentous fungal host cell is a Penicillium purpurogenum cell In another most prefeπed embodiment, the filamentous fungal host cell is a Thielavia terrestris cell In another most preferred embodiment, the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningu, 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 host cells are descπbed 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 descnbed by Malardier et al , 1989, Gene 78 147-156 and WO 96/00787 Yeast may be transformed using the procedures descnbed 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, Ito et al , 1983, Journal of Bacteriology 153 163, and Hmnen et al , 1978, Proceedings of the National Academy of Sciences USA 75 1920

Methods of Production

The present invention also relates to methods for producing a polypeptide compnsmg (a) cultivating a host cell under conditions suitable for production of the polypeptide, and (b) recovering the polypeptide

The present invention also relates to methods for producing a polypeptide of the present invention compnsmg (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell compnses a mutant nucleic acid sequence having at least one mutation m the mature polypeptide coding region of SEQ ID NO 1, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of the ammo acids of SEQ ID NO 2, and (b) recovenng the polypeptide

The present invention further relates to methods for producing a polypeptide compnsmg (a) cultivating a homologously recombinant cell, having incorporated therein a new transcnption unit compnsmg a regulatory sequence, an exon, and/or a splice donor site operably linked to a second exon of a nucleic acid sequence of the present invention which is endogenous to a cell, under conditions suitable for production of the polypeptide encoded by the endogenous nucleic acid sequence, and (b) recoveπng the polypeptide The methods are based on the use of gene activation technology, for example, as descnbed in U S Patent No 5,641,670 In the production methods of the present invention, the cells are cultivated in a nutπent medium suitable for production of the polypeptide using methods known in the art For example, the cell may be cultivated by shake flask cultivation, 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 nutnent 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 Ameπcan Type Culture Collection) If the polypeptide is secreted into the nutπent medium, the polypeptide can be recovered directly from the medium If the polypeptide is not secreted, 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 descπbed herein

The resulting polypeptide may be recovered by methods known in the art For example, the polypeptide may be recovered from the nutnent medium by conventional procedures including, but not limited to, centnfugation, filtration, extraction, spray-drymg, evaporation, or precipitation The polypeptides may be puπfied by a vanety of procedures known m the art including, but not limited to, chromatography (e g , ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e g , preparative isoelectnc 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) Plants

The present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleic acid sequence encoding a polypeptide having glucoamylase 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 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 (com).

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. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. 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. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

Conveniently, the expression construct is a nucleic acid construct which comprises a nucleic acid sequence encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleic acid 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, descnbed by Tague et al , 1988, Plant Physiology 86 506 For constitutive expression, the 35S-CaMV promoter may be used (Franck et al ,

1980, Cell 21 285-294) 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 menstems (Ito et al , 1994, Plant Mol Biol 24 863-878), a seed specific promoter such as the glutehn, prolamm, globulin, or albumin promoter from πce (Wu et al , 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 al , 1998, Journal of Plant Physiology 152 708-71 1), a promoter from a seed oil body protein (Chen et al , 1998, Plant and Cell Physiology 39 935-941, the storage protein napA promoter from Biassica napus, or any other seed specific promoter known in the art, e g , as descnbed in WO 91/14772 Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al , 1993, Plant Physiology 102 991-1000, the chlorella vims adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26 85-93, or the aldP gene promoter from nee (Kagaya et al , 1995, Molecular and General Genetics 248 668-674), or a wound inducible promoter such as the potato pιn2 promoter (Xu et al , 1993, Plant Molecular Biology 22 573- 588

A promoter enhancer element may also be used to achieve higher expression of the enzyme in the plant For instance, the promoter enhancer element may be an mtron, which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention For instance, Xu et al , 1993, supra disclose the use of the first mtron of the nee actm 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 Agrobacterium-mediated transformation, vims-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et αl, 1990, Science 244: 1293; Potrykus, 1990,

Bio/Technology 8: 535; Shimamoto ef αl., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, 0 Plant Molecular Biology 19: 15-38). However it can also be used for transforming monocots, although other transformation methods are generally preferred 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 5 Opinion Biotechnology 5: 158-162; Vasil et al, 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al, 1993, Plant Molecular Biology 21 : 415-428.

Following transformation, the transformants having incorporated therein the expression constmct are selected and regenerated into whole plants according to methods o well-known in the art.

The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a polypeptide having [enzyme] activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering 5 the polypeptide.

Uses

The present invention is also directed to methods of using the polypeptides having glucoamylase activity. o The polypeptides of the present invention may be used in starch conversion processes, especially in the production of high fructose com syrup. The glucoamylase of the invention may also be used in detergents, including laundry detergents, dish wash detergents, and hard surface cleaning compositions.

The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Starch conversion

The present invention provides a method of using the novel glucoamylase backbone of the invention for producing glucose and the like from starch. Generally, the method includes the steps of partially hydrolyzing precursor starch in the presence of α-amylase and then further hydrolyzing the release of D-glucose from the non-reducing ends of the starch or related oligo- and polysaccharide molecules in the presence of glucoamylase by cleaving a-(l- 4) and alpha-(l-6) glucosidic bonds.

The partial hydrolysis of the precursor starch utilizing alpha-amylase provides an initial breakdown of the starch molecules by hydrolyzing internal alpha-(l-4)-linkages. In commercial applications, the initial hydrolysis using α-amylase is mn at a temperature of approximately 105°C. A very high starch concentration is processed, usually 30% to 40% solids. The initial hydrolysis is usually carried out for five minutes at this elevated temperature. The partially hydrolyzed starch can then be transfened to a second tank and incubated for approximately one hour at a temperature of 85° to 90°C to derive a dextrose equivalent (D.E.) of 10 to 15.

The step of further hydrolyzing the release of D-glucose from the non-reducing ends of the starch or related oligo- and polysaccharides molecules in the presence of glucoamylase is normally carried out in a separate tank at a reduced temperature between 30° and 60°C. Preferably the temperature of the substrate liquid is dropped to between 55° and 60°C. The pH of the solution is dropped from 6 to 6.5 to a range between 3 and 5.5. Preferably, the pH of the solution is 4 to 4.5. The glucoamylase is added to the solution and the reaction is carried out for 24-72 hours, preferably 36-48 hours.

By using a thermostable glucoamylase of the invention saccharification processes may be carried out at a higher temperature than traditional batch saccharification processes. According to the invention saccharification may be carried out at temperatures in the range from above 60-80°C, preferably 63-75°C. This applies both for traditional batch processes (descπbed above) and for continuous sacchaπfication processes

Actually, continuous sacchanfication processes including one or more membrane separation steps, 1 e , filtration steps, must be earned out at temperatures of above 60°C to be able to maintain a reasonably high flux over the membrane or to minimize microbial

5 contamination Therefore, the thermostable glucoamylase of the invention provides the possibility of carrying out large-scale continuous saccharification processes at a fair pnce and or at a lower enzyme protein dosage within and penod of time acceptable for industrial saccharification processes According to the invention the sacchanfication time may even be shortened 0 As the thermal stability of the glucoamylase of the invention is improved a minor amount of glucoamylase need to be added to replace the glucoamylase being inactivated dunng the sacchanfication process More glucoamylase is maintained active dunng sacchanfication process according to the present invention Furthermore, the πsk of microbial contamination is also reduced when carrying the sacchanfication process at temperature above 5 63°C

The glucose yield from a typical sacchanfication tnal with glucoamylase and pullulanase is 95 5-96 5%> The remaining carbohydrates typically consist of 1% maltose, 1 5-

2% isomaltose and 1-1 5% higher ohgosachandes The disacchandes are produced since the glucoamylase at high concentrations of glucose and high dry-solid levels has a tendency to o form reversion products

A glucoamylase with an increased specific activity towards sacchandes present in the solution after liquefaction and sacchandes formed dunng sacchanfication would be an advantage as a reduced enzyme protein dosage or a shorter process time then could be used

In general, the glucoamylase has a preference for substrates consisted of longer 5 sacchandes compared to short chain sacchandes and the specific activity towards e g maltoheptaose is therefore approximately 6 times higher than towards maltose An increased specific activity towards short chain sacchandes such as maltose (without reducing the activity towards ohgosacchaπdes) would therefore also permit using a lower enzyme dosage and/or shorter process time o Furthermore, a higher glucose yield can also be obtained with a glucoamylase with an increased alpha- 1,4 hydrolytic activity (if the alpha- 1,6 activity unchanged or even decreased), since a reduced amount of enzyme protein is being used, and alpha- 1 ,6 reversion product formation therefore is decreased (less isomaltose).

The specific activity may be measured using the method described in the Materials and Methods section at 37°C or 60°C. An example of saccharification process wherein the glucoamylase of the invention may be used include the processes described in JP 3-224493; JP 1-191693 ;JP 62-272987; and EP 452,238.

The glucoamylase of the invention may be used in the present inventive process in combination with an enzyme that hydrolyzes only alpha-(l-6)-glucosidic bonds in molecules with at least four glucosyl residues. Preferentially, the glucoamylase of the invention can be used in combination with pullulanase or isoamylase. The use of isoamylase and pullulanase for debranching, the molecular properties of the enzymes, and the potential use of the enzymes with glucoamylase is set forth in G.M.A. van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, 101-142. In a further aspect the invention relates to the use of a glucoamylase of the invention in a starch conversion process.

Further, the glucoamylase of the invention may be used in a continuous starch conversion process including a continuous saccharification step.

The glucoamylase of the invention may also be used in immobilised form. This is suitable and often used for producing specialty symps, such as maltose symps, and further for the raffinate stream of oligosaccharides in connection with the production of fructose symps.

The glucoamylase of the invention may also be used in a process for producing ethanol for fuel or beverage (drinking ethanol) or may be used in a fermentation process for producing organic compounds, such as citric acid, ascorbic acid, lysine, glutamic acid.

DETERGENT COMPOSITIONS

The glucoamylase of the invention may also be used in detergents, including laundry detergent compositions, dish wash compositions and/or hard surface cleaning compositions.

The AMG of the invention may be added to and thus become a component of a detergent composition.

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations. In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase. In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274. Prefeπed commercially available protease enzymes include Alcalase®, Savinase®,

Primase®, Duralase®, Esperase®, and Kannase® (Novo Nordisk A/S), Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect OxP®, FN2®, and FN3® (Genencor International Inc.). Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from

Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (jp 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include LipolaseTM and Lipolase UltraTM (Novo Nordisk A/S).

Amylases: Suitable amylases (a and/or b) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, a- amylases obtained from Bacillus, e.g. a special strain of B. Hcheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novo Nordisk A S), RapidaseTM and PurastarTM (from Genencor International Inc.). Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299. Commercially available cellulases include Celluzyme®, and Carezyme® (Novo Nordisk

A/S), Clazinase®, and Puradax HAO (Genencor International Inc.), and KAC-500(B)0 (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GuardzymeO (Novo Nordisk A/S). The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Prefeπed detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60%) by weight.

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefmsulfonate, alkyl sulfate

(fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.

When included therein the detergent will usually contain from about 0.2%o to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides").

The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst). The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers. The detergent may contain a bleaching system, which may comprise a H2O2 source such as perborate or percarbonate, which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount coπesponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202, which is hereby incorporated as reference.

MATERIALS & METHODS Methods:

Determination of AGU activity

One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the following standard conditions:

Substrate maltose

Temperature 25°C pH 4.3 (acetate buffer)

Reaction time. . . . 30 minutes A detailed description of the analytical method (AF22) is available on request. Determining the specific activity

750 microL substrate is incubated 5 minutes at selected temperature, such as 37°C or 60°C. 50 microL enzyme diluted in sodium acetate is added.

Ahquots of 100 microL are removed after 0, 3, 6, 9 and 12 minutes and transfened to 100 microL 0 4M Sodium hydroxide to stop the reaction. A blank is included. 20 microL is transfeπed to a Micro titre plates and 200 microL GOD-Peπd solution is added Absorbance is measured at 650 nm after 30 minutes incubation at room temperature Glucose is used as standard and the specific activity is calculated as kcat (sec.-l)

Transformation of Aspergillus orvzae (general procedure)

100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics, Cold Spπng Harbor Laboratory) are inoculated with spores of A oryzae and incubated with shaking for about 24 hours. The mycelium is harvested by filtration through miracloth and washed with 200 ml of 0 6 M MgSO4. The mycelium is suspended in 15 ml of 1.2 M MgSO4, 10 mM NaH₂PO₄, pH 5.8. The suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozym™ 234 is added After 5 min., 1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubation with gentle agitation continued for 1.5-2.5 hours at 37C until a large number of protoplasts is visible in a sample inspected under the microscope

The suspension is filtered through miracloth, the filtrate transferred to a stenle tube and overlayed with 5 ml of 0.6 M sorbitol, 100 mM Tns-HCl, pH 7 0. Centnfugation is performed for 15 min. at 1000 g and the protoplasts are collected from the top of the MgSO4 cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tns-HCl, pH 7 5, 10 mM CaC12) are added to the protoplast suspension and the mixture is centnfugated for 5 mm. at 1000 g. The protoplast pellet is resuspended in 3 ml of STC and repelleted. This is repeated Finally, the protoplasts are resuspended in 0.2-1 ml of STC

100 micro 1 of protoplast suspension are mixed with 5-25 micro g of p3SR2 (an A. mdulans amdS gene carrying plasmid descnbed in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. 8, 1430-1439, Aug. 1983) in 10 microl of STC. The mixture is left at room temperature for 25 mm. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaC12 and 10 mM Tns-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution are added and carefully mixed. The mixture is left at room temperature for 25 min., spun at 2.500 g for 15 min. and the pellet is resuspended in 2 ml of 1.2M sorbitol. After one more sedimentation the protoplasts are spread on minimal plates (Cove, (1966), Biochem. Biophys. Acta 113, 51- 56) containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37C spores are picked, suspended in sterile water and spread for single colonies. This procedure is repeated and spores of a single colony after the second re-isolation are stored as a defined transformant.

Fed batch fermentation

Fed batch fermentation is performed in a medium comprising maltodextrin as a carbon source, urea as a nitrogen source and yeast extract. The fed batch fermentation is performed by inoculating a shake flask culture of A. oryzae host cells in question into a medium comprising 3.5%> of the carbon source and 0.5%> of the nitrogen source. After 24 hours of cultivation at pH 5.0 and 34°C the continuous supply of additional carbon and nitrogen sources are initiated. The carbon source is kept as the limiting factor and it is secured that oxygen is present in excess. The fed batch cultivation is continued for 4 days, after which the enzymes can be recovered by centrifugation, ultrafiltration, clear filtration and germ filtration. Further purification may be done by anionexchange chromatographic methods known in the art.

Purification

The culture broth is filtrated and added ammoniumsulphate (AMS) to a concentration of 1.7 M AMS and pH is adjusted to pH 5. Precipitated material is removed by centrifugation on the solution containing glucoamylase activity is applied on a Toyo Pearl Butyl column previously equilibrated in 1.7 M AMS, 20 mM sodium acetate, pH 5. Unbound material is washed out with the equilibration buffer. Bound proteins are eluted with 10 mM sodium acetate, pH 4.5 using a linear gradient from 1.7 - 0 M AMS over 10 column volumes. Glucoamylase containing fractions are collected ad dialysed against 20 mM sodium acetate, pH 4.5. The enzyme solution is then subjection to anion exchange chromatography using the

Q-sepharose column previously equilibrated in 20 mM sodium acetate, pH 5.5. Unbound material is washed out with the equilibration buffer. Bound proteins are eluted with a linear gradient from 0-0.5 M sodium chloride in the equilibration buffer over 10 column volumes. The glucoamylase-containing fraction is collected and separated on a Superdex 200 gel filtration column. Purity is confirmed by SDS page.

Thermal Stability determination

The thermal stability of the glucoamylase of the invention is tested using the following method: 950 microliter 50 mM sodium acetate buffer (pH 4.3) (NaOAc) is incubated for 5 minutes at 70°C. 50 microliter enzyme in buffer (4 AGU/ml) is added. 2 x 40 microliter samples are taken at 0 and 40 minutes and chilled on ice. The activity (AGU/ml) measured before incubation (0 minutes) is used as reference (100%). The decline in percent is calculated as a function of the incubation time. Maltose is used as the substrate.

Thermal Stability or T_1/2 (half-life) Assay The thermal stability and/or T,,₂ is measured by incubating the enzyme (ca. 0.2

AGU/ml) in question in 30%> glucose, 50 mM Sodium acetate at pH 4.5 at the temperature in question (e.g., 70°C). Samples are withdrawn at set time intervals and chilled on ice and residual enzyme activity measured by the pNPG method (as described below).

The % residual glucoamylase activity is determined at different times. T,,-, is the period of time until which the % relative activity is decreased to 50%o.

Residual Enzyme Activity (pNPG method) pNPG reagent:

0.2 g pNPG (p-nitrophenylglucopyranoside) is dissolved in 0.1 M acetate buffer (pH 4.3) and made up to 100 ml.

Borate solution:

3.8 g Na_^B^ 10 H₂O is dissolved in Milli-Q water and made up to 100 ml.

25 microL samples are added 50 microL substrate and incubated 2 hr at 50°C. The reaction is stopped by adding 150 micoL ml borate solution. The optical density is measured at 405 nm, and the residual activity calculated.

Glucose reversion. AMG-catalyzed condensation of a 35% (w/w) glucose solution is followed at 60?C and pH 4.5 using 50 mM sodium acetate. An enzyme dosage coπesponding to 2 AGU/g DS is added and aliquots (40 micro 1) is taken out at appropriate time intervals (t: 0, 2, 4, 24, and 48 hours), heat inactivated, and the oligosaccharide content determined using HPLC. The glucose reversion is determined as the condensation rate.

EXAMPLES

Example 1 0 Determination of Thermal Stability of wild-type Thermoascus crustaceus glucoamylase A strain of Thermoascus crustaceus was fermented under standard condition. The glucoamylase present in the fermentation broth was desalted using a G-25 column (from Pharmacia) using methods described in the "Materials & Methods" section above. The thermal stability of the desalted wild-type glucoamylase was determined as described in the "Materials & Methods" section using pNPG as substrate.

The residual activity after 40 minutes at 70°C, pH 4.5, incubation was determined to 84%o. Under the same conditions the A. niger glucoamylase Gl had 8%₀ residual activity.

Example 2 0 Determination of Specific Activity

The specific activity of the purified (purified as described in the "Materials & Methods" section) wild-type Thermoascus crustaceus glucoamylase is determined as described in the "Materials & Methods" section. The specific activity of the Thermoascus crustaceus glucoamylase is compared to the specific activity of the niger glucoamylase Gl determined 5 under the same conditions.

Example 3

Determination of Glucose Reversion

The glucose reversion (condensation rate - tendency to make isomaltose from glucose) of the o desalted wild-type Thermoascus crustaceus glucoamylase described in Example 1 is determination as described in the "Materials & Methods" section. The glucose reversion of the Thermoascus crustaceus glucoamylase is compared to the glucose reversion of the A. niger glucoamylase Gl determined under the same conditions.

Example 4

Expression of the cloned Thermoascus crustaceus glucoamylase in A. niger

The Thermoascus crustaceus glucoamylase is cloned, transformed into A. niger, expression and purified using standard methods description and the "Materials & Method" section.

References

Ausubel, F. M. et al. (eds.); Current protocols in Molecular Biology; 1995; John Wiley and

Sons. Sambrook et al.; Molecular Cloning: A Laboratory Manual; 1989,; Cold Spring Harbor Lab.;

Cold Spring Harbor; NY.

Harwood C. R., and Cutting S. M. (eds.); Molecular Biological Methods for Bacillus; 1990;

John Wiley and Sons.

Diderichsen B., Wedsted U., Hedegaard L., Jensen B. R., Sjøholm C; Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis; J. Bacteriol.,

1990, vol. 172, pp. 4315-4321.

Pitcher D. G., Saunders N. A., Owen R. J.; Rapid extraction of bacterial genomic DNA with guanidium thiocyanate; Lett. Appl. Microbiol; 1989; vol. 8; pp. 151-156.

Dretzen G., Bellard M., Sassone-Corsi P., Chambon P.; A reliable method for the recovery of DNA fragments from agarose and acrylamide gels; Anal. Biochem.; 1981 ; vol. 112; pp. 295-

298.

Claims

Claims What is claimed is:

1. An isolated polypeptide having glucoamylase activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 50%o identity with the amino acids of SEQ ID NO:2;

(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: l, (ii) the cDNA sequence of SEQ ID NO: l , (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);

(c) an allelic variant of (a) or (b);

(d) a fragment of (a), (b) or (c) that has glucoamylase activity.

2. The polypeptide of claim 1 , which enzyme has higher thermostability than the A. niger Gl glucoamylase shown in SEQ ID NO: 4.

3. The polypeptide of claim 1, having an amino acid sequence which has at least 50% identity with mature part of amino acids in SEQ ID NO: 2.

4. The polypeptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:2 or a fragment thereof.

5. The polypeptide of claim 1 , which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:l, (ii) the cDNA sequence of SEQ ID NO:l, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii).

6. A polypeptide having the same glucoamylase activity as the polypeptide of any of claims 1-5.

7. An isolated nucleic acid sequence comprising a nucleic acid sequence, which encodes the polypeptide of any of claims 1-5.

8. The isolated nucleic acid sequence of claim 7 produced by (a) hybridizing a DNA under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: l, (ii) the cDNA sequence of SEQ ID NO: l, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); and (b) isolating the nucleic acid sequence.

9. A recombinant expression vector comprising the nucleic acid constmct of claims 7-8.

10. A recombinant host cell comprising the nucleic acid constmct of claim 9.

11. A method for producing the polypeptide of any of claims 1-5 comprising (a) cultivating a strain to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.

12. A method for producing the polypeptide of any of claims 1-5 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding the polypeptide under conditions suitable for production of the polypeptide; and (b) recovering the polypeptide.

13. Use of a glucoamylase of any of claims 1-5 in a starch conversion process.

14. Use of a glucoamylase of any of claims 1-5 in a continuous starch conversion process.

15. Use of a glucoamylase of any of claims 1-5 in a process for producing oligosaccharides.

16. Use of a glucoamylase of any of claims 1-5 in a process for producing specialty symps.

17. Use of a glucoamylase of any one of claims 1-5 in a process for producing ethanol for fuel or drinking ethanol (portable ethanol).

18. Use of a glucoamylase of any one of claims 1-5 in a process for producing a beverage.

19. Use of a glucoamylase of any one of claims 1-5 in a fermentation process for producing organic compounds, such as citric acid, ascorbic acid, lysine, glutamic acid.

20. Use of a glucoamylase of any one of claims 1-5 in detergents.