阅读说明：本技术 热稳定的葡糖淀粉酶及其使用方法 (Thermostable glucoamylases and methods of use thereof ) 是由 J·F·克拉米尔 葛晶 M·A·B·科尔克曼 I·尼古拉耶夫 J·K·舍蒂 唐忠美 W· 于 2017-11-09 设计创作，主要内容包括：公开了具有葡糖淀粉酶活性的多肽、包含此类多肽的组合物、以及应用此类多肽和组合物的方法。(Polypeptides having glucoamylase activity, compositions comprising such polypeptides, and methods of using such polypeptides and compositions are disclosed.)

1. A polypeptide having glucoamylase activity, selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity to the polypeptide of SEQ ID No. 3;

(b) a polypeptide encoded by a polynucleotide that hybridizes under at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency conditions, and even most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of seq id no

(i) The mature polypeptide coding sequence of SEQ ID NO. 1,

(ii) 1, or a genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO, or

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

(c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity to the mature polypeptide coding sequence of SEQ ID No. 1;

(d) a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., several) amino acids of the polypeptide of SEQ ID NO. 3; and

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

2. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2 operably linked to one or more control sequences that control the production of the polypeptide in an expression host.

4. A recombinant host cell comprising the polynucleotide of claim 2.

5. The host cell of claim 4, which is an ethanologenic microorganism.

6. The host cell of claim 4 or 5, which further expresses and secretes one or more additional enzymes selected from the group consisting of proteases, hemicellulases, cellulases, peroxidases, lipolytic enzymes, metallolipolytic enzymes, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, alpha-amylases, pullulanases, phytases, tannases, pentosanases, maleases (malanases), beta-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, transferases, or combinations thereof.

7. A method for saccharifying starch-containing material, the method comprising the steps of: i) contacting the starch-containing material with an alpha-amylase; and ii) contacting the starch-containing material with a glucoamylase at a temperature of at least 70 ℃; wherein the process produces at least 70% free glucose from the starch-containing material (substrate).

8. The method of claim 7, wherein step (ii) is carried out at a temperature of at least 75 ℃, preferably at least 80 ℃, for between 12 and 96 hours, preferably for between 18 and 60 hours.

9. The process of claim 7 or 8, wherein the glucoamylase maintains at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% relative activity at a temperature of at least 70 ℃, and/or at a pH between 2.0 and 7.0, preferably between pH 4.0 and pH 6.0, more preferably between pH 4.5 and pH 5.5.

10. The method of any one of claims 7-9, wherein the method comprises performing step (i) and step (ii) sequentially or simultaneously.

11. The method of any one of claims 7-10, wherein the method further comprises a pre-saccharification prior to saccharification step ii).

12. The method of any one of claims 7-11, wherein the glucoamylase is the polypeptide of claim 1.

13. The method of any one of claims 7-12, wherein step (i) is carried out at or below the gelatinization temperature of the starch-containing material.

14. The method of any one of claims 7-13, wherein no additional debranching enzyme is present in step (i) and/or step (ii).

15. The method of claim 14, wherein the debranching enzyme is a pullulanase.

16. A saccharide produced by the method of any one of claims 7-15.

17. A method of producing a fermentation product from the sugar of claim 16, wherein the sugar is fermented by a fermenting organism.

18. The method of claim 17, wherein the fermentation process is carried out sequentially or simultaneously with step (ii).

19. The method of claim 17 or 18, wherein the fermentation product comprises ethanol.

20. The method of claim 17 or 18, wherein the fermentation product comprises a non-ethanol metabolite.

21. The method of claim 20, wherein the metabolite is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, organic acids, glucono delta-lactone, sodium erythorbate, omega 3 fatty acids, butanol, isobutanol, amino acids, lysine, tyrosine, threonine, glycinol, itaconic acid, 1, 3-propanediol, vitamins, enzymes, hormones, isoprene, or other biochemicals or biomaterials.

22. A method of using the polypeptide of claim 1 in brewing.

23. A method of using the polypeptide of claim 1 to produce beer or malt-based beverages.

Technical Field

The present disclosure relates to polypeptides having glucoamylase activity and compositions comprising such polypeptides. The disclosure further relates to polynucleotides encoding such polypeptides, vectors and host cells comprising genes encoding such polypeptides, which are also capable of producing such polypeptides. The disclosure also relates to methods of saccharifying starch-containing material using or applying the polypeptides or compositions, and saccharides produced by the methods. Further, the present disclosure relates to methods of producing fermentation products and fermentation products produced by the methods.

Background

Glucoamylase (1, 4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing end of starch or related oligo-and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts.

The primary application of glucoamylases is to saccharify partially processed starch/dextrin into glucose, which is an essential substrate for many fermentation processes. The glucose can then be converted directly or indirectly into a fermentation product using a fermenting organism. Examples of commercial fermentation products include alcohols (e.g., ethanol, methanol, butanol, 1, 3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid, 2, 5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gas (e.g. H)₂And CO₂) (ii) a And more complex compounds including, for example, antibiotics (e.g., penicillin and tetracycline); an enzyme; vitamins (e.g. riboflavin, B)₁₂Beta-carotene); hormones, and other compounds that are difficult to produce synthetically. Fermentation processes are also commonly used in the consuming alcohol (e.g., beer and wine), dairy (e.g., for producing yogurt and cheese), leather, beverage, and tobacco industries.

The end product may also be a syrup. The end product may be, for example, glucose, but may also be converted, for example, by glucose isomerase to fructose or a mixture of almost equal amounts of glucose and fructose. This mixture, or a mixture further enriched in fructose, is the most commercially used High Fructose Corn Syrup (HFCS) worldwide.

Although various microorganisms have been reported to produce glucoamylases (because they secrete large amounts of enzyme extracellularly), glucoamylases for commercial purposes have traditionally been produced using filamentous fungi. However, commercially used fungal glucoamylases have certain limiting factors, such as moderate thermal stability, acidic pH requirements, and slow catalytic activity, which increases process costs. Therefore, there is a need to find new glucoamylases to improve the temperature optima, and thus the catalytic efficiency to shorten the saccharification time or to obtain higher final product yields.

It is an object of the present disclosure to provide certain polypeptides having glucoamylase activity, polynucleotides encoding the polypeptides, nucleic acid constructs useful for producing such polypeptides, compositions comprising the same, and methods of applying such polypeptides to various industrial applications.

Disclosure of Invention

The polypeptides, compositions, and methods of saccharifying starch-containing material using or applying the polypeptides or compositions of the invention. Aspects and embodiments of the polypeptides, compositions and methods are described in the following independently numbered paragraphs.

1. In one aspect, a polypeptide having glucoamylase activity, selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity to the polypeptide of SEQ ID No. 3;

(b) a polypeptide encoded by a polynucleotide that hybridizes under at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency conditions, and even most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1,

(ii) 1, or a genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO, or

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

(c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, even most preferably at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity to the mature polypeptide coding sequence of SEQ ID No. 1;

(d) variants comprising substitution, deletion, and/or insertion of one or more (e.g., several) amino acids of the mature polypeptide of SEQ ID No. 2; and

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

2. In another aspect, a polynucleotide comprising a nucleotide sequence encoding the polypeptide of paragraph 1.

3. In some embodiments of the polynucleotide of paragraph 2, the polynucleotide is operably linked to one or more control sequences that control the production of the polypeptide in an expression host.

4. In another aspect, a recombinant host cell comprising the polynucleotide of paragraph 2.

5. In some embodiments of the host cell of paragraph 4, the host cell is an ethanologenic microorganism.

6. In some embodiments of the host cell of paragraphs 4 or 5, the host cell further expresses and secretes one or more additional enzymes selected from the group consisting of a protease, a hemicellulase, a cellulase, a peroxidase, a lipolytic enzyme, a metallolipolytic enzyme, a xylanase, a lipase, a phospholipase, an esterase, a perhydrolase, a cutinase, a pectinase, a pectate lyase, a mannanase, a keratinase, a reductase, an oxidase, a phenoloxidase, a lipoxygenase, a ligninase, an alpha-amylase, a pullulanase, a phytase, a tannase, a pentosanase, a malease, a beta-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, a transferase, or a combination thereof.

7. In another aspect, a method for saccharifying a starch-containing material, the method comprising the steps of: i) contacting the starch-containing material with an alpha-amylase; and ii) contacting the starch-containing material with a glucoamylase at a temperature of at least 70 ℃; wherein the process produces at least 70% free glucose from the starch-containing material (substrate).

8. In some embodiments of the method according to paragraph 7, wherein step (ii) is carried out at a temperature of at least 75 ℃, preferably at least 80 ℃ for between 12 and 96 hours, preferably for between 18 and 60 hours.

9. In some embodiments of the method of paragraph 7 or 8, wherein the glucoamylase retains at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% relative activity at a temperature of at least 70 ℃, and/or at a pH between 2.0 and 7.0, preferably between pH 4.0 and pH 6.0, more preferably between pH 4.5 and pH 5.5.

10. In some embodiments of the method of any of paragraphs 7-9, wherein the method comprises performing step (i) and step (ii) sequentially or simultaneously.

11. In some embodiments of the methods of any of paragraphs 7-10, wherein the method further comprises a pre-saccharification prior to saccharification step ii).

12. In some embodiments of the method of any of paragraphs 7-11, wherein the glucoamylase is the polypeptide of claim 1.

13. In some embodiments of the method of any of paragraphs 7-12, wherein step (i) is performed at or below the gelatinization temperature of the starch-containing material.

14. In some embodiments of the method of any one of paragraphs 7-13, wherein no additional debranching enzyme is present in step (i) and/or step (ii).

15. In some embodiments of the method of paragraph 14, wherein the debranching enzyme is a pullulanase.

16. In another aspect, a saccharide produced by the method of any one of paragraphs 7-15.

17. In another aspect, a method of producing a fermentation product from the sugar of paragraph 16, wherein the sugar is fermented by a fermenting organism.

18. In some embodiments of the method of paragraph 17, wherein the fermentation process is performed sequentially or simultaneously with step (ii).

19. In some embodiments of the methods of paragraphs 17 or 18, wherein the fermentation product comprises ethanol.

20. In some embodiments of the methods of paragraphs 17 or 18, wherein the fermentation product comprises a non-ethanol metabolite.

21. In some embodiments of the method of paragraph 20, wherein the metabolite is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, an organic acid, glucono delta-lactone, sodium erythorbate, omega 3 fatty acids, butanol, isobutanol, an amino acid, lysine, tyrosine, threonine, glycinol, itaconic acid, 1, 3-propanediol, vitamins, enzymes, hormones, isoprene, or other biochemicals or biomaterials.

22. In another aspect, a method of using the polypeptide of paragraph 1 in brewing.

Drawings

FIG. 1 is a plasmid map of pJG 580.

FIG. 2 is a graph of the product profile of PruGA1 after 95 hours fermentation.

FIG. 3 is a comparison of DP1 of PruGA1-0.3X, PruGA1-1X, AfuGA-1X, and AnGA-1X at 60 deg.C, 65 deg.C, and 70 deg.C after 72-h incubation.

FIG. 4 is a comparison of PruGA1 and TrGA activity on crude starch at pH 3.5.

FIG. 5 is a comparison of PruGA1 and TrGA activity on crude starch at pH 4.5.

Detailed Description

The present disclosure relates to polypeptides having glucoamylase activity and compositions comprising such polypeptides. The disclosure further relates to polynucleotides encoding such polypeptides, vectors and host cells comprising genes encoding such polypeptides, which are also capable of producing such polypeptides. The disclosure also relates to methods of saccharifying starch-containing material using or applying the polypeptides or compositions, and saccharides produced by the methods. Further, the present disclosure relates to methods of producing fermentation products and fermentation products produced by the methods.

Before describing the compositions and methods in detail, the following terms and abbreviations are defined.

Unless defined otherwise, all technical and scientific terms used have their ordinary meaning in the relevant scientific field. Singleton, et al, Dictionary of Microbiology and Molecular Biology [ Dictionary of Microbiology and Molecular Biology ], 2 nd edition, John Wiley and Sons [ John Willi-father publishing Co., Ltd ], New York (1994), and Hale and Markham, Harper Collins Dictionary of Biology [ Harburkholds Biodictionary ], Harper Perennial [ Huper permanent Press ], New York State (1991) provide common meanings for many of the terms describing the present invention.

Definition of

The term "glucoamylase (1, 4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) activity" is defined herein as an enzymatic activity that catalyzes the release of D-glucose from the non-reducing end of starch or related oligo-and polysaccharide molecules.

The polypeptide of the invention has a glucoamylase activity of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the mature polypeptide of SEQ ID No. 2.

The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein", and "peptide", and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". The amino acid sequence is represented in the standard amino-terminal-to-carboxyl-terminal orientation (i.e., N → C) using the conventional single-letter or three-letter code for amino acid residues.

The term "mature polypeptide" is defined herein as a polypeptide that is in its final form after translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one aspect, the mature polypeptide is amino acids 22 to 614 of SEQ ID NO:2 based on the SignalP (Nielsen et al 1997 Protein Engineering 10:1-6) program which predicts that amino acids 1 to 21 of SEQ ID NO:2 are signal peptides.

The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid may be single-stranded or double-stranded, and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the invention encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5 '-to-3' orientation.

The term "coding sequence" means a nucleotide sequence that directly specifies the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which typically begins with an ATG start codon or alternative start codon (e.g., GTG and TTG) and ends with a stop codon (e.g., TAA, TAG, and TGA). The coding sequence may be a DNA, cDNA, synthetic or recombinant nucleotide sequence.

The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription of a mature spliced mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The early initial RNA transcript is a precursor of mRNA that is processed through a series of steps before it appears as mature spliced mRNA. These steps include the removal of intron sequences by a process known as splicing. Thus, cDNA derived from mRNA lacks any intron sequences.

The term "hybridization" refers to the process by which a strand of nucleic acid forms a duplex (i.e., a base pair) with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65 ℃ and 0.1XSSC (where 1XSSC ═ 0.15M NaCl, 0.015M trisodium citrate, pH 7.0). The hybridized double-stranded nucleic acid is characterized by a melting temperature (T)_m) Wherein half of the hybridized nucleic acids are not paired with complementary strands. Mismatched nucleotide in duplex decreases T_m。

"synthetic" molecules are produced by in vitro chemical or enzymatic synthesis and not by organisms.

A "host strain" or "host cell" is an organism into which has been introduced an expression vector, phage, virus or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an amylase). Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeasts) capable of expressing a polypeptide of interest and/or fermenting a sugar. The term "host cell" includes protoplasts produced from a cell.

The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

The term "vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

By "expression vector" is meant a DNA construct comprising a DNA sequence encoding a polypeptide of interest, operably linked to suitable control sequences capable of effecting the expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences which control termination of transcription and translation.

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

The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in the intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is controlled by the regulatory sequence.

A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks a signal sequence that is cleaved off during secretion.

"biologically active" refers to a sequence having a specified biological activity (e.g., enzymatic activity).

The term "specific activity" refers to the number of moles of substrate that can be converted to a product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is usually expressed as units (U)/mg protein.

By "percent sequence identity" is meant that a particular sequence has at least a certain percentage of amino acid residues that are identical to amino acid residues in a designated reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al (1994) Nucleic acids SRes [ Nucleic acids research ]22: 4673-one 4680. The default parameters for the CLUSTAL W algorithm are:

the term "homologous sequence" is defined herein as a predicted protein having an E-value (or expected score) of less than 0.001 in the tfasty search with the Penicillium lansselli (Penicillium russelii) glucoamylase of SEQ ID NO:3 (Pearson, W.R.,1999, in Bioinformatics methods and Protocols, S.Misener and S.A.Krawetz, eds., 185-219 page).

The term "polypeptide fragment" is defined herein as a polypeptide having one or more (e.g., several) amino acids deleted from the amino and/or carboxy terminus of SEQ ID NO:3, or a homologous sequence thereof, wherein the fragment has glucoamylase activity.

With respect to polypeptides, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polypeptide that does not comprise human substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polynucleotide that does not contain human nucleoside changes. However, it is noted that a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to a naturally occurring polynucleotide and encompasses any polynucleotide encoding a wild-type, parent, or reference polypeptide.

The terms "thermostable" and "thermostability" with respect to an enzyme refer to the ability of the enzyme to retain activity after exposure to elevated temperatures. Thermostability of an enzyme (e.g.an amylase) by its half-life (t) given in minutes, hours or days_1/2) During which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring, for example, the residual alpha-amylase activity after exposure (i.e., challenge) to elevated temperatures.

"pH range" in reference to an enzyme refers to the range of pH values at which the enzyme exhibits catalytic activity.

The terms "pH stable" and "pH stability" in reference to an enzyme relate to the ability of the enzyme to retain activity for a predetermined period of time (e.g., 15min, 30min, 1h) at a wide range of pH values.

The term "pre-saccharification" is defined herein as the process prior to complete saccharification or Simultaneous Saccharification and Fermentation (SSF). The pre-saccharification is usually carried out at a temperature of between 30 ℃ and 65 ℃ and about 60 ℃ for 40 to 90 minutes.

The phrase "Simultaneous Saccharification and Fermentation (SSF)" refers to a biochemical production process in which a microorganism, e.g., an ethanologenic microorganism, and at least one enzyme, e.g., an amylase, are present in the same process step. SSF involves simultaneous hydrolysis of starch substrates (granular, liquefied, or solubilized) to sugars (including glucose) and fermentation of the sugars to alcohols or other biochemicals or biomaterials in the same reaction vessel.

A "slurry" is an aqueous mixture comprising insoluble starch particles in water.

The term "total sugar content" refers to the total soluble sugar content present in a starch composition including monosaccharides, oligosaccharides, and polysaccharides.

The term "dry solids" (ds) refers to dry solids dissolved in water, dry solids dispersed in water, or a combination of both. Thus dry solids include granular starch and its hydrolysates, including glucose.

"dry solids" content refers to the percentage of dissolved and dispersed dry solids by weight percent relative to the water in which the dry solids are dispersed and/or dissolved. The initial dry solids content of the starch is the weight of granular starch converted to water content divided by the weight of granular starch plus the weight of water. The subsequent dry solids content may be determined from the initial content adjusted for any added or lost water and chemical gain. The subsequent dissolved dry solids content can be measured by refractive index as shown below. 8

The term "high DS" refers to an aqueous starch slurry having a dry solids content greater than 38% (wt/wt).

"dry starch" refers to the dry starch content of a substrate, such as a starch slurry, and can be determined by subtracting the contribution of any non-starch components, such as protein, fiber, and water, from the substrate mass. For example, if the granular starch slurry has a water content of 20% (wt/wt) and a protein content of 1% (wt/wt), then 100kg of granular starch has a dry starch content of 79 kg. Dry substance starch can be used to determine the number of enzyme units to be used.

"Degree of Polymerization (DP)" refers to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP4+ (> DP3) represents polymers with a degree of polymerization greater than 3.

The term "contacting" refers to placing reference components (including, but not limited to, an enzyme, a substrate, and a fermenting organism) in sufficiently close proximity to affect the intended result, e.g., the enzyme acts on the substrate or the fermenting organism ferments the substrate. One skilled in the art will recognize that mixing the solutions may cause "contact". "ethanologenic microorganism" refers to a microorganism having the ability to convert sugars or other sugars into ethanol.

The term "biochemical" refers to a metabolite of a microorganism, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acids, butanol, isobutanol, amino acids, lysine, itaconic acid, other organic acids, 1, 3-propanediol, vitamins, or isoprene, or other biological material.

The term "pullulanase" also refers to debranching enzymes (e.c.3.2.1.41, amylopectin 6-glucanohydrolase) that are capable of hydrolyzing alpha-1-6-glucosidic bonds in amylopectin molecules.

The term "about" refers to ± 15% of a reference value.

The term "comprising" and its cognates are used in their inclusive sense; i.e., equivalent to the term "comprising" and its corresponding cognate terms.

EC enzyme Committee

CAZy carbohydrate active enzyme

w/v weight/volume

w/w weight/weight

v/v volume/volume

wt%

DEG C

g or gm gram

Microgram of μ g

mg of

kg kilogram

μ L and μ L microliter

mL and mL mL

mm

Micron diameter of

mol mole of

mmol millimole

M mol

mM millimole

Micromolar at μ M

nm nanometer

U unit

parts per million ppm

hr and h hours

EtOH ethanol

ds dry solids

Polypeptides having glucoamylase activity

In a first aspect, the present invention relates to a polypeptide comprising an amino acid sequence having preferably at least 90%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as at least 96%, 97%, 98%, 99% or 100% identity to the polypeptide of SEQ ID No. 3, said polypeptide having glucoamylase activity.

In some embodiments, the polypeptide of the invention is a homologous polypeptide comprising an amino acid sequence that differs from the polypeptide of SEQ ID No. 3 by ten amino acids, preferably by nine amino acids, preferably by eight amino acids, preferably by seven amino acids, preferably by six amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid.

In some embodiments, the polypeptide of the invention is a variant of the polypeptide of SEQ ID NO. 3, or a fragment thereof having glucoamylase activity.

In some embodiments, the polypeptides of the invention are thermostable and maintain glucoamylase activity at elevated temperatures. The polypeptides of the invention exhibit thermal stability at pH values ranging from about 2.5 to about 8.0 (e.g., about 3.0 to about 7.5, about 3.0 to about 7.0, about 3.0 to about 6.5, etc.). For example, at a pH of about 3.0 to about 7.0 (e.g., about 3.5 to about 6.5, etc.), a polypeptide of the invention retains a majority of glucoamylase activity at an elevated temperature (e.g., a temperature of at least 50 ℃, at least 55 ℃, at least 60 ℃, at least 65 ℃, at least 70 ℃, at least 75 ℃, at least 80 ℃, at least 85 ℃, at least 90 ℃ or more) for an extended period of time (e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 5 hours, or even longer). For example, a polypeptide of the invention retains at least about 35% (e.g., a percentage of at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or more) of glucoamylase activity when incubated at an increased temperature at a pH of from about 3.5 to about 6.5 for at least 1 hour, 3 hours, 5 hours, or even longer.

In some embodiments, a polypeptide of the invention has a maximum activity at a pH of about 5, 90% or more of the maximum activity at a pH of about 3.5 to a pH of about 6.0, and 70% or more of the maximum activity at a pH of about 2.8 to a pH of about 7.0, at a temperature of 50 ℃, as measured by the assay described herein. Exemplary pH ranges for the enzyme are pH 2.5-7.0, 3.0-7.0, 3.5-7.0, 2.5-6.0, 3.0-6.0, 3.5-6.0.

In some embodiments, a polypeptide of the invention has a maximum activity at a temperature of about 75 ℃ and greater than 70% of the maximum activity at a temperature of about 63 ℃ to about 79 ℃ at a pH of 5.0 as measured by the assay described herein. Exemplary temperature ranges for using the enzyme are 50 ℃ to 82 ℃, 50 ℃ to 80 ℃, 55 ℃ to 82 ℃, 55 ℃ to 80 ℃ and 60 ℃ to 80 ℃. In a second aspect, the present invention relates to a polypeptide having glucoamylase activity encoded by a polynucleotide that hybridizes under preferably very low stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) a genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) the full-length complementary strand of (i) or (ii) (J.Sambrook, E.F.Fritsch and T.Maniatis,1989, molecular cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, N.Y.).

The nucleotide sequence of SEQ ID NO. 1 can be used; or fragments thereof, to identify and clone DNA encoding a polypeptide having glucoamylase activity against strains from different genera or species according to methods well known in the art. In particular, such probes can be used to hybridize to genomic or cDNA of a genus or species of interest following standard southern blotting procedures in order to identify and isolate the corresponding gene therein. Such probes can be much shorter than the complete sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. However, it is preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probes may be at least 200 in lengthNucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides. Even longer probes may be used, for example nucleic acid probes of preferably at least 600 nucleotides, more preferably at least 800 nucleotides, even more preferably at least 1000 nucleotides, even more preferably at least 1500 nucleotides, or most preferably at least 1800 nucleotides in length. Both DNA and RNA probes may be used. The probes are typically labeled (e.g., with)³²P、³H、³⁵S, biotin, or avidin) to detect the corresponding gene. The invention also encompasses such probes.

Thus, genomic DNA or cDNA libraries prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having glucoamylase activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or isolated DNA may be transferred and immobilized on nitrocellulose (nitrocellulose) or other suitable carrier material. For the identification of clones or DNAs which are homologous to SEQ ID NO:1 or subsequences thereof, use of vector materials is preferred in southern blotting.

In a third aspect, the present invention relates to a polypeptide having glucoamylase activity encoded by a polynucleotide, the polynucleotide comprises a nucleotide sequence identical to SEQ ID NO:1 has preferably at least 60%, more preferably at least 63%, more preferably at least 65%, more preferably at least 68%, more preferably at least 70%, more preferably at least 72%, more preferably at least 75%, at least 77%, more preferably at least 79%, more preferably at least 81%, more preferably at least 83%, more preferably at least 85%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity, encoding a polypeptide having glucoamylase activity.

In a fourth aspect, the glucoamylase of the invention comprises conservative substitutions of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO 3. Exemplary conservative amino acid substitutions are listed in table 1. Some conservative mutations may be generated by genetic manipulation, while others are generated by introducing synthetic amino acids into the polypeptide by other means.

TABLE 1 conservative amino acid substitutions

In some embodiments, the glucoamylase of the invention comprises a deletion, substitution, insertion or addition of one or several amino acid residues relative to the amino acid sequence of SEQ ID No. 3 or a homologous sequence thereof. In some embodiments, the glucoamylases of the invention are derived from the amino acid sequence of SEQ ID NO 3 by conservative substitution of one or several amino acid residues. In some embodiments, the glucoamylase of the invention is derived from the amino acid sequence of SEQ ID NO. 3 by deletion, substitution, insertion, or addition of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO. 3. In all cases, the expression "one or several amino acid residues" means 10 or fewer, i.e. 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 amino acid residues.

Alternatively, the amino acid change has one property: altering the physicochemical properties of the polypeptide. For example, amino acid changes can improve the thermostability, change substrate specificity, change the pH optimum, etc. of a polypeptide.

Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known mutagenesis, recombination and/or shuffling methods, followed by a related screening procedure, such as that described by Reidhaar-Olson and Sauer,1988, Science [ Science ]241: 53-57; bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochem. [ biochemistry ]30: 10832-; 10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al, 1986, Gene [ Gene ]46: 145; Ner et al, 1988, DNA 7: 127).

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

The amino acid substitutions, deletions and/or insertions in the mature polypeptide of SEQ ID No. 2 may be up to 10, preferably up to 9, more preferably up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5, more preferably up to 4, even more preferably up to 3, most preferably up to 2, and even most preferably up to 1.

A glucoamylase may be a "chimeric" or "hybrid" polypeptide in that it includes at least a portion from a first glucoamylase, and at least a portion from a second amylase, glucoamylase, beta-amylase, alpha-glucosidase, or other starch degrading enzyme, or even other glycosyl hydrolases, such as, but not limited to, cellulases, hemicellulases, and the like (including chimeric amylases recently "rediscovered" as domain exchange amylases). The glucoamylase of the invention may further comprise a heterologous signal sequence, i.e. an epitope allowing tracking or purification etc.

Production of glucoamylase

The glucoamylase of the invention may be produced in a host cell, e.g., by secretion or intracellular expression. After secretion of the glucoamylase into the cell culture medium, a cultured cell material (e.g., whole cell broth) comprising the glucoamylase can be obtained. Optionally, the glucoamylase may be isolated from the host cell, or even from the cell culture broth, depending on the desired purity of the final glucoamylase. The gene encoding glucoamylase may be cloned and expressed according to methods well known in the art. Suitable host cells include bacteria, fungi (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae, Trichoderma reesei or myceliophthora Thermophila. Other host cells include bacterial cells such as Bacillus subtilis or Bacillus licheniformis (B.licheniformis), and Streptomyces (Streptomyces).

In addition, the host may express one or more coenzymes, proteins, peptides. These may be beneficial for liquefaction, saccharification, fermentation, SSF, and downstream processing. Furthermore, in addition to enzymes used to digest various feedstocks, host cells may also produce ethanol and other biochemicals or biomaterials. Such host cells can be used in fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need for enzyme addition.

Composition comprising a metal oxide and a metal oxide

The invention also relates to compositions comprising the polypeptides of the invention. In some embodiments, polypeptides comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity to the amino acid sequence of SEQ ID No. 1 can also be used in the enzyme compositions. Preferably, these compositions are formulated to provide desirable characteristics such as light color, low odor, and acceptable storage stability.

These compositions may comprise the polypeptide of the invention as the main enzymatic component, e.g. a one-component composition. Alternatively, the composition may comprise multiple enzymatic activities such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, hyperammoniase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-amylase, isoamylase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, pullulanase, ribonuclease, transglutaminase, xylanase, or combinations thereof, which enzymes may be added in effective amounts well known to those skilled in the art.

Polypeptide compositions can be prepared according to methods known in the art, and can be in the form of liquid or dry compositions. For example, a composition comprising a glucoamylase of the invention may be an aqueous or non-aqueous formulation, granule, powder, gel, slurry, paste, or the like, which may further comprise any one or more of the additional enzymes listed herein, as well as buffers, salts, preservatives, water, co-solvents, surfactants, and the like. Such compositions may function in combination with endogenous enzymes or other ingredients already present in the slurry, water bath, washing machine, food or beverage product, etc. (e.g., endogenous plant (including algae) enzymes, residual enzymes from previous processing steps, etc.). The polypeptides included in the composition may be stabilized according to methods known in the art.

The composition can be a cell that expresses the polypeptide, including a cell that is capable of producing a product from fermentation. Such cells can be provided in cream or dry form together with suitable stabilizers. Such cells may further express additional polypeptides, such as those mentioned above.

Examples of preferred uses of the polypeptides or compositions of the invention are given below. The dosage of the polypeptide composition of the present invention and other conditions for using the composition can be determined based on methods known in the art.

The above compositions are suitable for use in liquefaction, saccharification, and/or fermentation processes, preferably starch conversion, particularly for producing syrups and fermentation products (e.g., ethanol).

Use of

The invention is also directed to the use of a polypeptide or composition of the invention in a liquefaction process, a saccharification process, and/or a fermentation process. The polypeptide or composition may be used in a single process, for example in a liquefaction process, a saccharification process, or a fermentation process. The polypeptide or composition may also be used in a combination of processes, for example in a liquefaction and saccharification process, in a liquefaction and fermentation process, or in a saccharification and fermentation process, preferably in connection with starch conversion.

1. Liquefaction

As used herein, the term "liquefaction" or liquify "means a process of converting gelatinized starch into a lower viscosity liquid containing shorter chain soluble dextrins, optionally with the addition of a liquefaction inducing enzyme and/or saccharifying enzyme. In some embodiments, the prepared starch substrate is slurried with water. The starch slurry may contain starch in a weight percent dry solids of about 10% -55%, about 20% -45%, about 30% -40%, or about 30% -35%. For example, alpha-amylase (EC 3.2.1.1) may be added to the slurry with a metering pump. Alpha-amylases commonly used for this application are thermostable bacterial alpha-amylases, e.g., Geobacillus stearothermophilus (Geobacillus stearothermophilus) alpha-amylase, Cytophaga (Cytophaga) alpha-amylase, and the like, e.g., Spezyme(DuPont), Spezyme AA (DuPont),Fred (DuPont), Clearflow AA (DuPont), Spezyme Alpha PF (DuPont), and Spezyme Powerliq (DuPont) may all be used herein.

The starch plus alpha-amylase slurry may be pumped continuously through a jet cooker (which heats its steam to 80-110 ℃, depending on the source of the starch-containing feedstock). Under these conditions, gelatinization occurs rapidly and the enzyme activity, in combination with significant shear forces, begins to hydrolyze the starch substrate. The residence time in the jet cooker is short. The partially gelatinized starch can then be passed into a series of holding tubes maintained at 105-110 ℃ and held for 5-8min to complete the gelatinization process ("primary liquefaction"). Hydrolysis to the desired DE is accomplished in a holding tank at 85-95 ℃ or higher for about 1 to 2 hours ("secondary liquefaction"). The slurry was then allowed to cool to room temperature. This cooling step may be from 30 minutes to 180 minutes, for example from 90 minutes to 120 minutes. Liquefied starch is typically in the form of a slurry having a dry solids content (w/w) of about 10% to 50%; about 10% to 45%; about 15% -40%; about 20% to about 40%; about 25% -40%; or about 25% to 35%.

In a conventional enzymatic liquefaction process, a thermostable alpha-amylase is added and degrades long-chain starch into shorter units (maltodextrins) that are branched and linear, but no glucoamylase is added. The glucoamylase of the invention is highly thermostable, so it is advantageous to add the glucoamylase during liquefaction.

2. Saccharification

The liquefied starch may be saccharified into a syrup rich in low DP (e.g., DP1+ DP2) sugars using an alpha-amylase and a glucoamylase (optionally in the presence of another enzyme or enzymes). The exact composition of the saccharified product depends on the combination of enzymes used and the type of starch processed. Advantageously, the syrup obtainable using the provided glucoamylase may contain more than 30% by weight of DP2 of the total oligosaccharides in the saccharified starch, e.g. 45-65% or 55-65%. The weight percentage of (DP1+ DP2) in the saccharified starch may be more than about 70%, for example 75-85% or 80-85%.

Liquefaction is usually carried out as a continuous process, whereas saccharification is usually carried out as a batch process. The saccharification conditions depend on the nature of the liquefact and the type of enzyme available. In some cases, the saccharification process may involve a temperature of about 60 ℃ to 65 ℃ and a pH of about 4.0 to 4.5 (e.g., pH 4.3). Saccharification can be carried out at a temperature of, for example, about 40 ℃, about 50 ℃, or about 55 ℃ to about 60 ℃ or about 65 ℃, necessitating cooling of the liquefied mass. The pH can be adjusted as desired. Saccharification is typically carried out in a stirred tank, which may take several hours to fill or empty. The enzyme is usually added to the dry solid at a fixed ratio (when the tank is filled) or in a single dose (at the beginning of the filling phase). The saccharification reaction to prepare the syrup is usually carried out for about 24 to 72 hours, for example 24 to 48 hours. However, pre-saccharification is usually carried out only at temperatures between 30 ℃ and 65 ℃ (usually about 60 ℃) for usually 40-90 minutes, followed by complete saccharification in Simultaneous Saccharification and Fermentation (SSF). The glucoamylases of the invention are highly thermostable, so that the presaccharification and/or saccharification of the invention can be carried out at higher than conventional presaccharification and/or saccharification temperatures. In one embodiment, the process of the invention comprises pre-saccharifying starch-containing material prior to a Simultaneous Saccharification and Fermentation (SSF) process. The presaccharification can be carried out at elevated temperatures (e.g., 50 ℃ to 85 ℃, preferably 60 ℃ to 75 ℃) prior to moving into the SSF. Preferably, saccharification is optimally conducted at a higher temperature range of about 30 ℃ to about 75 ℃ (e.g., 45 ℃ to 75 ℃ or 50 ℃ to 75 ℃). By performing the saccharification process at a higher temperature, the process may be performed in a shorter period of time, or alternatively may be performed using a lower enzyme dosage. Furthermore, when the liquefaction and/or saccharification process is carried out at higher temperatures, the risk of microbial contamination is reduced.

In a preferred aspect of the invention, the liquefaction and/or saccharification comprises a liquefaction and saccharification process performed sequentially or simultaneously.

3. Fermentation of

Soluble starch hydrolysates (especially glucose-rich syrups) can be fermented by contacting the starch hydrolysate with a fermenting organism, usually at a temperature of about 32 ℃ (e.g. from 30 ℃ to 35 ℃). "fermenting organism" refers to any organism (including bacterial and fungal organisms) suitable for use in a fermentation process and capable of producing a desired fermentation product. Particularly suitable fermenting organisms are capable of fermenting (i.e., converting) a sugar (such as glucose or maltose) directly or indirectly into the desired fermentation product. Examples of fermenting organisms include yeasts expressing alcohol dehydrogenase and pyruvate decarboxylase, such as Saccharomyces cerevisiae, and bacteria, such as Zymomonas mobilis. The ethanologenic microorganisms may express xylose reductase and xylitol dehydrogenase, both of which convert xylose to xylulose. For example, improved ethanologenic microbial strains that can withstand higher temperatures are known in the art and can be used. See Liu et al, (2011) ShengWu Gong Cheng Xue Bao [ bioengineered reports ]27: 1049-56. Commercially available yeasts include, for example, Red Star (TM)/Lesfre (Lesafre) ethanol Red (available from Red Star/Lesfre (Red Star/Lesafre) Inc., USA), FALI (available from Fleischmann's Yeast, a division of Burnswick Food Co., Ltd. (Burns Philp Food Inc.), SUPERSTART (available from Altech), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIL IOL (available from DSM Specialties). The temperature and pH of the fermentation will depend on the fermenting organism. Microorganisms that produce other metabolites such as citric acid and lactic acid by fermentation are also known in the art. See, e.g., papagiani (2007) biotechnol. adv. [ biotechnological progress ]25: 244-63; john et al (2009) Biotechnol. adv. [ biotechnological progress ]27: 145-52.

The saccharification and fermentation process may be performed as an SSF process. The SSF process can be performed with fungal cells that continuously express and secrete glucoamylase throughout the SSF. The fungal cell expressing a glucoamylase may also be a fermenting microorganism, such as an ethanologenic microorganism. Thus, ethanol production can be performed using fungal cells expressing sufficient glucoamylase to require less or no exogenous addition of enzyme. The fungal host cell may be from an appropriately engineered fungal strain. In addition to glucoamylases, fungal host cells expressing and secreting other enzymes can be used. Such cells may express amylases and/or pullulanases, phytases, alpha-glucosidases, isoamylases, beta-amylase cellulases, xylanases, other hemicellulases, proteases, beta-glucosidases, pectinases, esterases, oxidoreductases, transferases, or other enzymes. Ethanol can be recovered after fermentation.

4. Hydrolysis of crude starch

The present invention provides the use of a glucoamylase of the invention for producing glucose and the like from raw or granular starch. Generally, the glucoamylases of the invention are useful, alone or in the presence of alpha-amylase, in Raw Starch Hydrolysis (RSH) or Granular Starch Hydrolysis (GSH) processes for the production of desired sugars and fermentation products. The granular starch is solubilized by enzymatic hydrolysis below the gelatinization temperature. Such "low temperature" systems (also referred to as "no cook" or "cold cook") are reported to be capable of handling higher concentrations of dry solids (e.g., up to 45%) than conventional systems.

The "raw starch hydrolysis" process (RSH) differs from conventional starch treatment processes by the sequential or simultaneous saccharification and fermentation of granular starch at or below the gelatinization temperature of the starch substrate, typically in the presence of at least a glucoamylase and/or an amylase. Starch heated in water begins to gelatinize between 50 ℃ and 75 ℃, the exact temperature of gelatinization depending on the particular starch. For example, the gelatinization temperature may vary depending on the plant species, the particular variety of the plant species, and the growth conditions. In the context of the present invention, the gelatinization temperature of the Starch is given as the temperature at which the loss of birefringence of the Starch granules is 5% using the method described in Gorinstein.S. and Lii.C., Starch/Starke, Vol.44 (12), pp.461-466 (1992).

The glucoamylases of the invention may also be used in combination with enzymes that hydrolyze only alpha- (1,6) -glucosidic bonds in molecules containing at least four glucose residues. Preferably, the glucoamylase of the invention is used in combination with a pullulanase or an isoamylase. The use of isoamylases and pullulanases for Starch debranching, the molecular nature of the enzymes, and the potential use of the enzymes with glucoamylase are described in G.M.A. van Beynum et al, Starch Conversion Technology, Marcel Dekker, Massel Dekker, New York, 1985, 101-142.

In another aspect, the invention relates to the use of a glucoamylase of the invention, including converting starch into, for example, syrup beverages and/or fermentation products (including ethanol).

5. Fermentation product

The term "fermentation product" means a product produced by a process that includes a fermentation process using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (e.g., arabitol, butanol, ethanol, glycerol, methanol, ethylene glycol, 1, 3-propanediol [ propylene glycol ]]Butylene glycol, glycerin, sorbitol, and xylitol); organic acids (e.g. acetic acid)Acids, acetonic acid, adipic acid, ascorbic acid, citric acid, 2, 5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); olefins (e.g., pentene, hexene, heptene, and octene); gases (e.g. methane, hydrogen (H)₂) Carbon dioxide (CO)₂) And carbon monoxide (CO)); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g. riboflavin, B)₁₂Beta-carotene), and hormones. In a preferred aspect, the fermentation product is ethanol, e.g., fuel ethanol; drinking alcohol, i.e. drinkable neutral alcohol; or products used in the industrial ethanol or consumer alcohol industries (e.g. beer and liquor), dairy industries (e.g. fermented dairy products), leather industry and tobacco industry. Preferred types of beer include ale (ale), stout, porter, lagoon (lager), bitter, malt (malt liquour), high alcohol, low calorie or light beer. The fermentation process preferably used includes an alcohol fermentation process well known in the art. Preferred fermentation processes are anaerobic fermentation processes, which are well known in the art.

6. Brewing

The glucoamylases of the invention are highly thermostable, so that they can be used for starch hydrolysis at high temperatures to prepare fermented malt beverages. For example, the glucoamylase of the invention may be added to hot mash using elevated temperatures to increase reaction rates and increase the yield of fermentable sugars prior to the addition of yeast. In combination with amylase and optionally pullulanase and/or isoamylase, glucoamylase helps to convert starch into dextrins and fermentable sugars, reducing residual non-fermentable carbohydrates in the final beer. The glucoamylase of the invention is added in an effective amount that can be readily determined by one skilled in the art.

Processes for making beer are well known in the art. See, for example, Wolfgang Kunze (2004) "Technology Brewing and malt [ technical Brewing and Malting ]" Research and Teaching Institute of Brewing and Teaching, Berlin [ Institute of Brewing and Teaching of Berlin ] (VLB), 3 rd edition. Briefly, the process involves: (a) preparing a mash, (b) filtering the mash to prepare a wort, and (c) fermenting the wort to obtain a fermented beverage (e.g. beer).

A brewing composition comprising a glucoamylase in combination with an amylase and optionally a pullulanase and/or isoamylase may be added to the mash of step (a) above, i.e. during the preparation of the mash. Alternatively or in addition, the brewing composition may be added to the mash of step (b) above, i.e. during filtration of the mash. Alternatively or in addition, the brewing composition may be added to the wort of step (c) above, i.e. during fermentation of the wort.

All references cited herein are hereby incorporated by reference in their entirety for all purposes. To further illustrate the compositions and methods and advantages thereof, the following specific examples are given with the understanding that they are illustrative and not limiting.

Examples of the invention

Example 1

Sequence of penicillium lanssei glucoamylase (PruGA1)

The strain of penicillium lanssei that is useful for industrial applications is selected as a potential source of various enzymes. The entire genome of the strain of penicillium lanssei was sequenced and the nucleotide sequence of a putative glucoamylase (designated "PruGA 1") was determined by sequence identity. The gene for encoding PruGA1 is shown in SEQ ID NO: 1:

the amino acid sequence of the PruGA1 precursor protein is shown in SEQ ID NO: 2:

the amino acid sequence of the mature form of PruGA1, as demonstrated by N-terminal Edman (Edman) degradation, is shown in SEQ ID NO: 3:

example 2

Expression and purification of Penicillium lansselli Glucoamylase (PruGA1)

The nucleotide sequence of the synthesized PruGA1 gene from the penicillium lanssense is shown as SEQ ID NO: 4:

the DNA sequence of PruGA1 was optimized for expression of PruGA1 in trichoderma reesei and inserted into the pTrex3gM expression vector (described in U.S. published application 2011/0136197a 1) to yield pJG580 (fig. 1).

Plasmid pJG580 was transformed into a strain of Trichoderma reesei (described in WO 05/001036) using protoplast transformation (Te' o et al, J.Microbiol. methods [ J.Microbiol. methods ]51: 393-992002). The transformants were selected and fermented by the method described in WO 2016/138315. Supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis and enzyme activity assay.

A seed culture of the above transformed cells was then grown in a 2.8L fermentor in defined medium. The fermentation broth was sampled for SDS-PAGE analysis at fermentation times of 42, 65 and 95 hours to measure dry cell weight, residual glucose and extracellular protein concentration. FIG. 2 shows the product profile of PruGA1 fermentation at 95 hours. After 95 hours of fermentation, followed by centrifugation, filtration and concentration, 500mL of concentrated sample was obtained. The protein concentration was determined to be 10.7g/L by the BCA method.

200mL of clarified culture broth was loaded onto a 20-mL beta-cyclodextrin coupled Sepharose 6 column (pre-equilibrated with 20mM sodium acetate (pH 5.0), 150mM NaCl) and then washed with 3 column volumes of the same buffer. Elution was performed using 5 column volumes of 10mM α -cyclodextrin in 20mM sodium acetate (pH 5.0) containing 150mM NaCl. Fractions were collected and assayed for glucoamylase activity and subjected to SDS-PAGE. Fractions containing the target protein were pooled and concentrated in an Amicon Ultra-15 unit with 10K MWCO using 20mM sodium acetate (pH 5.0) containing 150mM NaCl. The purified samples were more than 99% pure and stored in 40% glycerol at-80 ℃ until use.

Example 3

Specific activity of PruGA1 on soluble starch

Glucoamylase specific activity was determined based on the release of glucose from soluble starch by glucoamylase using a coupled glucose oxidase/peroxidase (GOX/HRP) method (anal. biochem.105(1980), 389-397).

The substrate solution was prepared by mixing 9mL of soluble starch (1% aqueous solution, w/w) and 1mL of 0.5M sodium acetate buffer pH 5.0 in a 15-mL conical tube. A solution of conjugated enzyme with ABTS (GOX/HRP) was prepared in 50mM sodium acetate buffer (pH 5.0) with final concentrations of 2.74mg/mL ABTS, 0.1U/mL HRP, and 1U/mL GOX.

Serial dilutions of glucoamylase samples and glucose standards were prepared in purified water. Each glucoamylase sample (10. mu.L) was transferred to a new microtiter plate (Corning 3641) containing 90. mu.L of substrate solution preincubated at 600rpm at 50 ℃ for 5 min. The reaction was carried out at 50 ℃ for 10min (shaking in a thermal mixer (Eppendorf) (600rpm)), 10. mu.L of the reaction mixture and 10. mu.L of serial dilutions of the glucose standard were transferred rapidly to a new microtiter plate (Corning 3641), respectively, and then 100. mu.L of the ABTS/GOX/HRP solution was added. The absorbance at 405nm was immediately measured using a SoftMax Pro microplate reader (Molecular Device) at 11 second intervals for 5 min. The output is the reaction rate (Vo) for each enzyme concentration. Linear regression was used to determine the slope of the curve for Vo versus enzyme dosage. Specific activity of glucoamylase was calculated based on glucose standard curve using equation 1:

specific activity (unit/mg) slope (enzyme)/slope (standard) × 1000 (1),

where 1 unit is 1 μmol glucose/min.

Using the above method, the specific activity of PruGA1 was determined and compared to a benchmark AnGA (glucoamylase from aspergillus niger). The results are shown in table 2. PruGA1 has a specific activity of 197U/mg for soluble starch, about 2-fold higher than that of AnGA, another glucoamylase.

TABLE 2 specific activity of purified PruGA1 versus soluble starch compared to AnGA

Example 4

Amylopectin hydrolyzing activity of glucoamylase PruGA1

The activity of glucoamylase on amylopectin was determined using the same protocol as described above for determining the specific activity of glucoamylase PruGA1 on soluble starch (except that the enzyme was dosed at 10 ppm). Table 3 summarizes the amylopectin hydrolysis activities of PruGA1 and benchmark AnGA. PruGA1 was approximately 6-fold more active on amylopectin than AnGA.

TABLE 3 amylopectin hydrolysis activity of PruGA1 compared to AnGA.

Example 5

Effect of pH and temperature on PruGA1 glucoamylase Activity

The effect of pH (2.0 to 10.0) on PruGA1 activity was monitored using soluble starch (1% aqueous solution, w/w) as substrate. The buffered working solution consisted of a combination of glycine/sodium acetate/HEPES (250mM) with a pH varying between 2.0 and 10.0. The substrate solution was prepared by mixing soluble starch (1% aqueous solution, w/w) with 250mM buffer solution in a ratio of 9: 1. An enzyme working solution was prepared in water at a certain dose (showing a signal in the linear range according to the dose response curve). All incubations were performed at 50 ℃ for 10min according to the same protocol as the glucoamylase PruGA1 specific activity on soluble starch described above. The enzyme activity at each pH was reported as relative activity compared to the enzyme activity at the optimal pH. The pH profile of PruGA1 is shown in table 4.PruGA1 was found to have an optimal pH at about 5.0 and to maintain over 70% of the maximum activity between pH 2.8 and 7.0.

TABLE 4 pH profile of PruGA1

The effect of temperature (from 40 ℃ to 84 ℃) on PruGA1 activity was monitored using soluble starch (1% aqueous solution, w/w) as substrate. The substrate solution was prepared by mixing 9mL of soluble starch (1% aqueous solution, w/w) and 1mL of 0.5M buffer (pH 5.0 sodium acetate) in a 15mL conical tube. An enzyme working solution was prepared in water at a certain dose (showing a signal in the linear range according to the dose response curve). Incubation was carried out for 10min at a temperature of 40 ℃ to 84 ℃ respectively, according to the same protocol as described above for the specific activity of glucoamylase PruGA1 on soluble starch. The activity at each temperature was reported as relative activity compared to the enzyme activity at the optimal temperature. The temperature profile of PruGA1 is shown in table 5.PruGA1 exhibited optimal activity at 75 ℃ and maintained greater than 70% of maximum activity between 63 ℃ and 79 ℃.

TABLE 5 temperature-Activity curves of PruGA1

Temperature (. degree.C.)	Relative Activity (%)
		40	24
44.7	34
		49.4	42
55	54
		59.7	63
65	77
		69.2	90
74.6	100
		80	58
85	19

Example 6

Saccharification performance of PruGA1 at pH 4.5 at various temperatures

The activities of PruGA1, AnGA and AfuGA (described in WO 2014092960) were evaluated under saccharification conditions at different incubation temperatures at pH 4.5. Evaluation of DP1 was measured by analyzing sugar compositions with equal enzyme doses. Alpha-amylase pretreated corn starch liquefact (34.9% ds, prepared at pH 3.8) was used asStarting substrate. Incubation of glucoamylase (dosed at 0.121mg/gds as 1 × dose) and corn starch liquefact (34% ds) was performed at 60 deg.C, 65 deg.C and 70 deg.C, respectively, at pH 4.5. Samples were collected at 16, 24, 40, 64 and 72h, respectively. All incubations were quenched by heating at 100 ℃ for 15 min. Transfer sample supernatant and at 5mM H₂SO₄Diluted 400-fold and run at 85 ℃ for HPLC analysis using an Agilent 1200 series system with Fast Fruit column (100 mm. times.7.8 mm). A10. mu.L sample was loaded onto the column and separated with an equal gradient of purified water as the mobile phase at a flow rate of 1.0 mL/min. The oligosaccharide product was detected using a refractive index detector. The glycogenic activity (glucogenic activity) of the samples is summarized in table 6. DP 1% (FIG. 3) after 72-h incubation was chosen as an example, and PruGA1 at 0.3 × dose (40 μ g/gds) was superior to AfuGA-1 × dose (121 μ g/gds) and AnGA-1 × dose (121 μ g/gds) at all three temperatures tested. PruGA1 maintained its excellent performance in DP1 production even when the incubation temperature was raised to 70 ℃. The glycogenic activity of the samples is summarized in table 6.

TABLE 6 DP1 production of PruGA1, AfuGA, and AnGA (1X dose set at 121. mu.g/gds) incubated with corn starch liquefact at pH 4.5 at different temperatures.

Example 7

PruGA1 saccharification evaluation at pH5.5 and 70 ℃

The glycogenic activity of PruGA1 was evaluated at elevated temperatures (aimed at shortening the saccharification time). Corn starch liquefact (32% ds, pH 3.9) was obtained from corn starch liquefact from alpha-amylase pretreatment. Incubations with different doses of PruGA1 and corn starch liquefact (32% ds) were performed at pH5.5, 70 ℃. Samples were collected at 18, 26, 42, 50, 66 and 72 h. All incubations were quenched by heating at 100 ℃ for 15 min. Transfer the supernatant of the sample and at 5mM H₂SO₄Middle thinRelease 400-fold, using the same conditions as shown in example 6 for HPLC analysis. The glycogenic activity of the samples is summarized in table 7. The results show that PruGA1 (administered at 30. mu.g/gds) was achieved after two days incubation at pH5.5 and 70 ℃>95% DP1 production.

TABLE 7 corn starch liquefact as substrate under saccharification conditions at pH5.5 and 70 ℃,

sugar production of PruGA1(20 to 50. mu.g/gds) at different doses

Example 8

Crude starch Activity of PruGA1

The activity of PruGA1 on crude starch was measured and compared to the activity of trichoderma reesei glucoamylase (TrGA) for Granular Starch Hydrolase (GSHE) fermentation and direct starch hydrolysis to glucose/maltose process (DSTG/DSTM). In this assay, alpha-amylase and glucoamylase were mixed at a ratio of 1: 6.6. Aspergillus kawachii (Aspergillus kawachii) amylase (AkAA, described in WO 2013169645) was used. Sugar profiling was performed using Fast front HPLC columns (Waters) and the enzymatic crude starch hydrolysis capacity was determined using glucose (final product).

mu.L of corn starch substrate (1% in 50mM acetate buffer pH 3.5/pH 4.5) was dispensed into 0.5mL microtiter plates using wide mouth tips. Add 10. mu.L of amylase and 10. mu.L of glucoamylase per well to set the final dosage of AkAA and glucoamylase to 1.5ppm and 10ppm, respectively. The samples were incubated in an iEMS incubator set at 32 ℃ and 900rpm for 6, 20 and 28 h. The reaction was quenched by adding 50 μ L of 0.5M NaOH and suspending the starch plug by placing the plate on a shaker for 2 min. After that, the plate was sealed and centrifuged at 2500rpm for 3 min. For HPLC analysis, 0.01N H was used₂SO₄The supernatant was diluted 10-fold. Using Agile equipped with refractive index detectorThe nt 1200 series HPLC analysis 10. mu.L of sample. The column used was a Phenomenex Rezex-RFQFast Fruit column (Cat. No. 00D-0223-K0) with a Phenomenex Rezex ROA organic acid guard column (Cat. No. 03B-0138-K0). The mobile phase was 0.01N H₂SO₄And a flow rate of 1.0mL/min at 85 ℃. The results are shown in fig. 4 and 5.PruGA1 showed comparable activity to the benchmark glucoamylase TrGA at both pH 3.5 and pH 4.5 for crude starch.

Example 9

Evaluation of PruGA1 in Low pH fermentations

The glycogenic activity of PruGA1 under low pH fermentation conditions was evaluated. PruGA1 and TrGA were tested for performance at the same protein concentration (0.25 mg/gds). An amylase treated corn starch liquefact (34.9% ds, pH 3.8) was used as substrate. The pH of the corn starch liquefact (32% ds, amylase pre-treatment) was adjusted to pH 3.0 and 10g was transferred to a 50mL glass bottle. Incubations were performed at 32 ℃ and 55 ℃. Samples were collected at 17, 24, 41, 48, 63, 72 h. All incubations were quenched by heating at 100 ℃ for 15 min. Transfer the supernatant of the sample and at 5mM H₂SO₄Diluted 400-fold and used for HPLC analysis using the same conditions as shown in example 7.

The values reported in table 7 reflect the peak area percentage per DPn as a small fraction of the total DP1, DP2, DP3 and DP3 +. The data in table 8 show that PruGA1 exhibits higher glycogenic activity than TrGA when dosed at equal protein concentrations and incubated at 32 ℃, pH 3. When the incubation temperature was raised to 55 ℃, PruGA1 hydrolyzed the high DP sugars very efficiently, leaving only 5% DP3+ after 17h of incubation, while TrGA remained 18%.

To further evaluate the performance of PruGA1 at low pH, another test was performed on starch liquefacts at even lower pH (pH 2.0). The screening procedure was the same as at pH 3.0 (except that the enzyme was fed at 0.2 mg/gds) and samples were collected at 4, 21, 29, 45, 53, 70 h. As shown in table 9, PruGA1 released 77.4% DP1 and TrGA released 54% after 70h at pH2 and 32 ℃. PruGA1 released 26.2% of DP1 at 55 deg.C, while the TrGA response was only 3.2%.

TABLE 8 analysis of sugar composition of PruGA1 and TrGA hydrolyzed starch liquefacts at pH 3, 32 deg.C or 55 deg.C

TABLE 9 analysis of sugar composition of PruGA1 and TrGA hydrolyzed starch liquefacts at pH2, 32 deg.C or 55 deg.C

Example 10

High temperature leach saccharification with glucoamylase on wort substrates

The glucogenic activity of PruGA1 during the high temperature leach saccharification process was evaluated in comparison to other glucoamylase benchmarks for brewing application wort substrates.

Using 55% Beltson malt (Belsner malt; Danish Fogger Mulberry (Fuglding) Inc., batch 13.01.2016) and 45% corn grist (northern European Brandt Nordic; NordgetreideGmBH L ü bec, Germany, batch 02.05.2016), a mashing operation was performed using a water to grist ratio of 4.0:1, the Belson malt was ground on a Buhler Miag malt grinder (0.5mm set up), crude corn (1.35g), malt (ground Belson malt, 1.65g) was mixed in a Wheaton cup (covered Wheaton glass container), tap water was pre-incubated with 12.0g, pH was adjusted to pH 5.4 with 2.5M sulfuric acid, the resulting substrate (15% ds, pH 5.4) was then used for the starch performance evaluation, Prussn 1(10 uGA1) was added to the other glucoamylase variants (Trueger GA) in a microtiter plate (Trueger glucoamylase) (Trueger Langeri Mig GA 52. GA) was evaluated in a PCR with the other variants (Truese glucoamylase variants) (Trueger Miag)Generation D44R and D539R, 10 μ L of a 2mg/mL stock solution) and aspergillus niger glucoamylase (AnGA, 10 μ L of a 1mg/mL stock solution). All incubations were performed at 64 ℃ for 4h, or for even higher saccharification temperatures, incubations were performed at 70 ℃ for 2 h; then 15min at 79 ℃. After quenching the reaction at 95 ℃ for 10min, the reaction mixture was centrifuged at 3700rpm for 10 min. Transfer supernatant samples and at 5mM H₂SO₄Diluted 20-fold for HPLC analysis. HPLC separation was carried out using a Fast front column (100 mm. times.7.8 mm) at 85 ℃ using an Agilent 1200 series HPLC system. The sample (10. mu.L) was loaded onto an HPLC column and separated with an equal gradient of purified water as the mobile phase at a flow rate of 1.0 mL/min. The oligosaccharide product was detected using a refractive index detector. The glycogenic activity of the samples is summarized in table 10. The 100ppm PruGA1 sample exhibited comparable performance to TrGA variant a (TrGA va) glucoamylase at 200 ppm. The PruGA1 enzyme also showed superior performance to baseline when the incubation temperature was increased to 70 ℃ and the incubation time was shortened to 2 h.

TABLE 10 analysis of saccharide composition of glucoamylase incubated with wort substrate at pH 5.4

Example 11

High temperature leach saccharification using glucoamylase for malt and corn

The glucoamylase pruga1 was tested in a mashing operation with 55% pilsner malt (pilsner malt; denmark fugersan, batch 13.01.2016) and 45% corn grist (northern european brigtag; nordgeriede GmBH L ü bec, germany, batch 02.05.2016) using a water to grist ratio of 4.0: 1. pilsner malt was ground using a braler (Buhler mig) malt grinder (0.5mm set-up).

Crude corn (1.35g) and malt (milled pilson malt, 1.65g) were mixed in Wheaton cups (Wheaton glass containers with lids), pre-incubated with 12.0g of tap water, and the pH adjusted to pH 5.4 with 2.5M sulfuric acid. Glucoamylase was added on a ppm active protein basis (1.0 ml total) and water was added as an enzyme-free control. Wheaton cups were placed in Drybath (Thermo Scientific Stem station) from Seimer science, magnetic stirring and applying the following saccharification procedure; heating the sample to 64 ℃ for 30 minutes, holding at 64 ℃ for 15 minutes; heating to 79 ℃ by increasing the temperature at 1 ℃/min for 15 min; held at 79 ℃ for 15 minutes; heating to 90 ℃ for 11 minutes by increasing the temperature at 1 ℃/minute, holding at 90 ℃ for 15 minutes; cooling to 79 ℃ for 15 minutes, and finally heated to 79 ℃ for 15 minutes and washed out (massed off.) 10ml of sample was transferred to a ferkin tube and boiled at 100 ℃ for 20 minutes to ensure complete inactivation of the enzyme, at 10 ℃, the cereals were separated from the wort by centrifugation at 4500rpm for 20 minutes in Heraeus Multifuge X3R the supernatant was collected for HPLC sugar analysis using standard methods the results are shown in table 11.

TABLE 11 determination of the relative distribution of sugars (DP1 to DP5+) in the wort by HPLC

It is clear that PruGA1 promotes high yield of DP1 in a dose-dependent manner. A DP1 of up to 83.44% was produced at a dose of 750ppm enzyme.

Example 12

Saccharification of 100% corn by extraction with glucoamylase at elevated temperature

The purpose of this experiment was to evaluate the glucogenic activity of PruGA1 during high temperature saccharification using corn and malt compared to industry standards the saccharification operation was performed with 100% corn flour (Nordgereide GmBH L ü bec, Germany, batch: 02.05.2016) using a water to flour ratio of 4.0: 1.

Crude corn (3.0g) was added to Wheaton cups (Wheaton glass containers with lids), pre-incubated with 12.0g of tap water and the pH adjusted to pH 5.4 with 2.5M sulfuric acid. Glucoamylase was added on a ppm active protein basis (1.0 ml total), or water was added as an enzyme-free control. A fixed concentration of 5.0ppm of alpha-amylase (from DuPont) was applied to all samples5T) and 0.21ppm of beta-glucanase (from Dupont)750) To promote liquefaction and filterability. Wheaton cups were placed in Drybath (Seimer science workbench), magnetically stirred and three different mashing procedures were applied. According to curve 1, the sample is heated to 64 ℃; held at 64 ℃ for 80 minutes; heating to 80 ℃ by raising the temperature at 1.6 ℃/min for 10 minutes; kept at 80 ℃ for 30 minutes and then washed out. According to curve 2, the sample is heated to 70 ℃; held at 70 ℃ for 80 minutes; heating to 80 ℃ by raising the temperature at 1.0 ℃/minute for 10 minutes; kept at 80 ℃ for 30 minutes and then washed out; according to curve 3, the sample is heated to 75 ℃; held at 75 ℃ for 80 minutes; heating to 80 ℃ by raising the temperature at 0.5 ℃/minute for 10 minutes; kept at 80 ℃ for 30 minutes and then washed out. 10ml samples were transferred to a Forken tube and boiled at 100 ℃ for 20 minutes to ensure complete inactivation of the enzyme. The cereals were separated from the wort by centrifugation at 4500rpm in Heraeus Multifuge X3R for 20 minutes at 10 ℃. The supernatant was collected for HPLC sugar analysis. The glycogenic activity of the samples is summarized in table 12.

TABLE 12 analysis of glycocomposition of glucoamylase after saccharification by extraction using 100% corn using various temperatures at pH 5.4.

PruGA1 showed enhanced performance (final concentration: 18ppm) at 70 ℃ and 75 ℃ saccharification curves compared to TrGA (wild-type from Trichoderma reesei glucoamylase) (final concentration: 18 ppm).