WO2015021601A1 - Simultanenous liquifaction and malto-saccharification - Google Patents

Simultanenous liquifaction and malto-saccharification Download PDF

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Publication number
WO2015021601A1
WO2015021601A1 PCT/CN2013/081390 CN2013081390W WO2015021601A1 WO 2015021601 A1 WO2015021601 A1 WO 2015021601A1 CN 2013081390 W CN2013081390 W CN 2013081390W WO 2015021601 A1 WO2015021601 A1 WO 2015021601A1
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WIPO (PCT)
Prior art keywords
starch
tthamyl
amylase
variant
maltose
Prior art date
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PCT/CN2013/081390
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French (fr)
Inventor
Collette Michelle BLAKE
Ling Hua
Sung Ho Lee
Jianhua Li
Zhen Qian
Vivek Sharma
Jayarama K. Shetty
Mingmin SONG
Bruce A. Strohm
Zhongmei TANG
Zhengzheng ZOU
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Danisco Us Inc.
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Priority to PCT/CN2013/081390 priority Critical patent/WO2015021601A1/en
Publication of WO2015021601A1 publication Critical patent/WO2015021601A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A23L29/37—Sugar alcohols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2425—Beta-amylase (3.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01002—Beta-amylase (3.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K7/00—Maltose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C2200/00—Special features
    • C12C2200/05—Use of genetically modified microorganisms in the preparation of beer

Definitions

  • Starch is composed of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w).
  • Amylose contains linear chains of a-1 ,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000.
  • MW molecular weight
  • Amylopectin is a branched polymer containing a-1 ,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
  • maltose an alpha 1 -4 linked glucosyl glucose, a di-saccharide that is widely used in many industrial and consumers' applications.
  • maltose is used as a sweetener or in the prepration of maltitol, a low calorie sweetener.
  • the majority sugar in high maltose syrup is maltose. It is less sweet than high fructose corn syrup. It is also used in brewing to increase through-put and known to reduce haze caused by varying quality of wort. Since maltose has a low freezing point, high maltose syrup is useful in frozen desserts.
  • Maltose solution exhibits lower viscosity and less humictant than equal concentrations of glucose solution and finds application in candy formulation to reduce the stickyness.
  • Maltitol is a sugar alcohol produced by the hydrogenation of maltose.
  • maltose is prepared by a two step process via the enzymatic hydrolysis of an aqueous slurry (25-32% ds, pH 5.5- 6.0) of starch substrate subjected to a high temperature jet cooking process (>100 °C) using a thermostable alpha amylase to solubilize and hydrolyze the insoluble starch into soluble dexrins, and then treating with an enzyme called beta amylase at 55 °C, pH 5.5, thereby producing a syrup containing high levels of maltose, ⁇ - Amylase (EC 3.2.1 .2) hydrolyses the alpha-1 -4- glucan bonds in amylosaccharide chains from non-reducing ends and generates the di- saccharide maltose, which is linked by alpha-1 -4 glucosidic linkages between two glucosyl residues, ⁇ amylases are well-characterized in higher plants and microbial sources.
  • a thermostable alpha amylase to solubilize and hydro
  • ⁇ amylase extracted from plant e.g. barley (OPTIMALT® BBA from DuPont Industrial Biosciences), wheat, and soy bean are routinely used on a commercial scale for producing high maltose syrup.
  • High maltose syrup is a food additive used as a sweetener and preservative in many food formulations.
  • thermostable liquefying alpha amylase for example SPEZYME® Fred, CLEARFLOW® from DuPont Industrial Biosciences, Liquozyme® Supra from Novozymes
  • a conventional high temperature jet cooking process >100 °C
  • the liquefact is then further hydrolyzed by incubating soluble dextrins with ⁇ amylase (OPTIMALT® BBA from DuPont Industrial Biosciences) at 55 °C for 24 to 48 hours, resulting in a sugar syrup containing high maltose.
  • ⁇ amylase OPTIMALT® BBA from DuPont Industrial Biosciences
  • a maltose syrup containing greater than 65% maltose content typically requires the addition of a debranching enzyme, for example a pullulanase such as OPTIMAX® L-1000 from DuPont Industrial Biosciences, or Promozyme from Novozymes and Amano Pullulanse) to increase the maltose content from 55% to up to 85%.
  • a debranching enzyme for example a pullulanase such as OPTIMAX® L-1000 from DuPont Industrial Biosciences, or Promozyme from Novozymes and Amano Pullulanse
  • the beta amylases used in the maltose process are primarily produced from plant sources by the extraction of either barley (OPTIMALT® BBA), wheat, and/or soy bean.
  • the plant ⁇ amylases from barley and wheat are not thermostable (less than 58 °C) and exhibit maximum activity at pH between 5.4 to 5.6. The low temperature for incubation or a longer time (24 to 48
  • incubation pH of greater than 5.0 are unfavorable for maltose producers due to high risk of microbial infection.
  • beta amylases employed in most industrial applications are primarily obtained from plant sources by extraction and purification processes, attempts have been made to obtain more active, thermostable and extracellular beta amylases from micro-organisms, primarily from bacillus species.
  • Many bacterial sources have been cited and described in the past, including as Bacillus flexus (Matsunaga, Okada and Yamaguchi, 2009, Matsunaga, A., Okada, M., and Yamaguchi S.
  • beta-amylase provides an opportunity to produce more stable and consistent enzyme strains.
  • none of the past beta amylases have shown any improvement in terms of pH and temperature properties over commercially used beta amylase from plant origins. Therefore, the plant beta amylases continue to be used for maltose production, even though they suffer from unreliability and instablility during storage and transportation.
  • thermophilic beta-amylase Yamada et.al US patent # 5,082,781 , Zeikus et.al, 1987, US Patent # 4,647,538, Nanmori et al.1993; US Patent # 5,188,956). Discovery and expression of organism such as Clostridium thermosulfurogenes have been undertaken and discussed (see for example Zeikus et al.1987 and Yamagata et al.1993) where they have demonstrated the beta-amylase activity at 70 °C in absence of substrate and activity at 80 °C in presence of substrate.
  • PCT Patent application #WO201 1/058105A1 describes a process on the application of an alpha amylase, maltogenic enzyme, and/or a thermostable beta amylase from Clostridium thermosulfurogenes being used during mashing and lautering in brewing processes.
  • the present teachings provide a one-step process, offering a simultaneous liquefaction and malto-saccharification of starch substrates into a maltose rich sugar syrup using a thermostable beta amylase at a temperature range of 80-95 °C. Rest TBC upon finalization of claims.
  • FIG. 1 depicts a plasmid according to some embodiments of the present teachings.
  • FIG. 2 depicts a flowchart according to some embodiments of the present teachings.
  • FIG. 3 depicts the pH profile of the TthAmyl enzyme of the present teachings.
  • FIG. 4 depicts the temperature profile of the TthAmyl enzyme of the present teachings.
  • SEQ ID NO: 1 sets forth the full length amino acid sequence of TthAmyl , with the predicted signal peptide shown in italics.
  • SEQ ID NO: 2 sets forth the mature full length DNA sequence of TthAmyl .
  • SEQ ID NO: 3 sets forth the mature TthAmy amino acid sequence.
  • SEQ ID NO: 4 sets forth the mature AmyE Combi L amino acid sequence.
  • a-Amylases are hydrolases that cleave the a-D-(1 ⁇ 4) O-glycosidic linkages in starch.
  • a- amylases (EC 3.2.1 .1 ; a-D-(1 ⁇ 4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving a-D-(1 ⁇ 4) O-glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (1 -4)-a-linked D- glucose units.
  • maltohexaosidases (EC 3.2.1 .98) can produce malto-oligosaccharides of a specific length or enriched syrups of specific maltooligosaccharides.
  • glucoamylase (EC 3.2.1 .3) (otherwise known as glucan 1 ,4-a-glucosidase; glucoamylase; amyloglucosidase; ⁇ -amylase; lysosomal a- glucosidase; acid maltase; exo-1 ,4-a-glucosidase; glucose amylase; y-1 ,4-glucan glucohydrolase; acid maltase; 1 ,4-a-D-glucan glucohydrolase; or 4-a-D-glucan glucohydrolase) refers to a class of enzymes that catalyze the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides.
  • exo-acting enzymes which release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules.
  • the enzymes also hydrolyze alpha-1 , 6 and alpha -1 , 3 linkages although at much slower rates than alpha-1 , 4 linkages.
  • hydrolysis of starch refers to the cleavage of glucosidic bonds with the addition of water molecules.
  • pullulanase (E.C. 3.2.1 .41 , pullulan 6-glucanohydrolase) refers to a class of enzymes that are capable of hydrolyzing alpha 1 -6 glucosidic linkages in an amylopectin molecule.
  • B-amylase (E.C. 3.2.1 .2) refers to a class of enzymes that hydrolyze the alpha-1 -4-glucan bonds in amylosaccahride chains from non-reducing ends and generate a di-saccharide maltose, linked by alpha -1 -4 glucosidic linkages two glucosy resdidues.
  • Beta amylases are well charcterized in higher plants and microbial sources. For example Beta amylase extracted from plants, e.g. barley
  • B-amylases as used in the present methods include those described as PsPAmy9, disclosed in co-filed patent application "B-amylase and
  • thermoresistant maltose-producing enzyme refers to any B- amylase, alpha amylase, or maltogenic amylase, or the like, that retains 50% activity after treatment at 60 °C for 10 minutes at a pH of 4.5-5.5, and is capable of producing at least 30% maltose in a simultaneous liquifaction and malto-saccharification process according to the present teachings.
  • Enzyme units herein refers to the amount of product formed per time under the specified conditions of the assay.
  • a "glucoamylase activity unit” GAU
  • a "soluble starch unit” SSU
  • SSU soluble starch unit
  • Thermostable Beta Amylase activity Unit was determined using a colorimetric assay to monitor the release of reducing sugars from potato amylopectin. The activity is reported as equivalents of glucose released per minute.
  • Substrate solutions were prepared by mixing 9 ml_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 101 18), 1 ml_ of 0.5 M buffer (pH 5.0 sodium acetate), and 40 ⁇ _ of 0.5 M CaCI 2 into a 15-mL conical tube.
  • Stock solutions of purified beta- amylase samples were made by diluting original samples to 0.4 mg/mL (400 ppm) in water.
  • Total reducing sugars present in each well were measured using a PAHBAH method: 80 ⁇ _ of 0.5 N NaOH was aliquoted into a microtiter plate, followed by the addition of 20 ⁇ _ of PAHBAH reagent (5% w/v 4-hydroxybenzoic acid hydrazide in 0.5 N HCI) and 10 ⁇ _ of each reaction mixture. Plates were incubated at 95°C for 5 min and cooled down at 4°C for 5 sec. Samples (80 ⁇ _) were then transferred to polystyrene microtiter plates (Costar 9017) and absorbance was read at 410 nm.
  • PAHBAH method 80 ⁇ _ of 0.5 N NaOH was aliquoted into a microtiter plate, followed by the addition of 20 ⁇ _ of PAHBAH reagent (5% w/v 4-hydroxybenzoic acid hydrazide in 0.5 N HCI) and 10 ⁇ _ of each reaction mixture. Plates were incubated at 95°C for 5 min and
  • Thermostable beta amylase activity (TBA) unit is defined as one micromole of glucose equivalent released per min at pH 5.0, 50°C .
  • PROMase Activity Units is an activity definition based on a colorimetric method that utilizes a soluble red-pullulan substrate for the
  • APU Acid Stable Pullulanase Unit
  • dry solids content refers to the total solids of a slurry in a dry weight percent basis.
  • slurry refers to an aqueous mixture containing insoluble solids.
  • high ds refers to an aqueous starch slurry containing dry solids greater than 38%.
  • Brix refers to a well known hydrometer scale for measuring the sugar content of a solution at a given temperature. The Brix scale measures the number of grams of sucrose present per 100 grams of aqueous sugar solution (the total solubilized solid content). Brix measurements are frequently made by use of a hydrometer or refracto meter.
  • degree of polymerization refers to the number (n) of anhydro- glucopyranose 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.
  • a DP4+ (>DP3) denotes polymers with a degree of polymerization of greater than 3, and contains everything that is not retained on the column - protein, lipids, carbs, etc).
  • DE or “dextrose equivalent,” is defined as the
  • reducing sugar i.e., D-glucose
  • concentration of total reducing sugars It is an industry standard for the concentration of total reducing sugars, and is expressed as % D-glucose on a dry weight basis.
  • Unhydrolyzed granular starch has a DE that is essentially 0 and D-glucose has a DE of 100.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and
  • amylopectin with the formula (C 6 H 0 O5) x wherein X can be any number.
  • the term includes plant-based materials such as grains, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, sweet sorghum brans, cassava, millet, potato, sweet potato, and tapioca.
  • starch includes granular starch.
  • granular starch refers to raw, i.e., uncooked starch, e.g., starch that has not been subject to gelatinization.
  • glucose syrup refers to an aqueous composition containing glucose solids.
  • Glucose syrup will have a DE of at least 20. In some embodiments, glucose syrup will not contain more than 21 % water and will not contain less than 25% reducing sugar calculated as dextrose. In one embodiment, glucose syrup will include at least 90% D-glucose and in another embodiment glucose syrup will include at least 95% D-glucose. In some embodiments, the terms glucose and glucose syrup are used interchangeably.
  • maltose syrup refers to an aqueous composition containing maltose solids. The maltose syrup will have maltose content of greater than 25% on a dry solids basis.
  • total sugar content refers to the total sugar content present in a starch composition.
  • RIDS Refractive Index Dry Substance
  • wild-type refers to a naturally-occurring polypeptide that does not include a man-made
  • wild-type refers to a naturally-occurring polynucleotide that does not include a man-made nucleoside change.
  • a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.
  • a "mature" polypeptide means a polypeptide or variant thereof from which a signal sequence is absent.
  • the signal sequence may be cleaved during expression of the polypeptide.
  • variant refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally-occurring or man-made substitutions, insertions, or deletions of an amino acid.
  • variant refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.
  • activity refers to enzymatic activity, which can be measured as described, herein.
  • nucleic acid when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
  • Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector.
  • Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences.
  • isolated refers to a compound, protein (polypeptides), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature.
  • isolated enzyme, or variant thereof includes, but is not limited to, a culture broth containing secreted enzyme expressed in a heterologous host cell.
  • purified refers to material ⁇ e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.
  • thermoostable and “thermostability,” with reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated
  • thermostability of an enzyme is measured by its half-life (ti /2 ) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions.
  • the half-life may be calculated by measuring residual glucoamylase activity following exposure to (i.e., challenge by) an elevated temperature.
  • a "pH range,” with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.
  • pH stable and “pH stability,” with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).
  • amino acid sequence is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an "enzyme.”
  • amino acid sequences exhibit activity, they may be referred to as an "enzyme.”
  • the conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N ⁇ C).
  • nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may contain chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
  • hybridization refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques.
  • Hybridized, duplex nucleic acids are characterized by a melting temperature (T m ), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the T m .
  • a "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.
  • the terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
  • the term “introduced” in the context of inserting a nucleic acid sequence into a cell means “transfection,” “transformation” or “transduction,” as known in the art.
  • a “host strain” or “host cell” is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest has been introduced.
  • Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast, such as T. reesei) capable of expressing the polypeptide of interest and/or fermenting saccharides.
  • the term "host cell” includes protoplasts created from cells.
  • heterologous with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.
  • endogenous with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
  • the term "expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.
  • a "selective marker” or “selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials ⁇ e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.
  • a “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
  • an "expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
  • control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences that control termination of transcription and translation.
  • operably linked means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
  • a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.
  • a “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell.
  • the mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.
  • biologically active refer to a sequence having a specified biological activity, such an enzymatic activity.
  • Percent sequence identity means that a variant has at least a certain percentage of amino acid residues identical to a reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:
  • Gap extension penalty 0.05
  • Toggle end gap separation penalty OFF Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of a mature 617 residue polypeptide would have a percent sequence identity of 99% (612/617 identical residues ⁇ 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having "at least 99% sequence identity" to a mature polypeptide.
  • Fusion polypeptide sequences are connected, i.e., operably linked, via a peptide bond between the two polypeptide sequences.
  • filamentous fungi refers to all filamentous forms of the subdivision
  • SSF saccharification and fermentation
  • ethanologenic microorganism refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.
  • the term "fermented beverage” refers to any beverage produced by a method comprising a fermentation process, such as a microbial fermentation, e.g., a bacterial and/or yeast fermentation.
  • Beer is an example of such a fermented beverage, and the term “beer” is meant to comprise any fermented wort produced by fermentation/brewing of a starch- containing plant material. Often, beer is produced exclusively from malt or adjunct, or any combination of malt and adjunct.
  • beers include: full malted beer, beer brewed under the "Rösgebot,” ale, IPA, lager, bitter, Happoshu (second beer), third beer, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like, but also alternative cereal and malt beverages such as fruit flavored malt beverages, e.g., citrus flavored, such as lemon-, orange-, lime-, or berry-flavored malt beverages, liquor flavored malt beverages, e.g., vodka-, rum-, or tequila-flavored malt liquor, or coffee flavored malt beverages, such as caffeine-flavored malt liquor, and the like.
  • fruit flavored malt beverages e.g., citrus flavored, such as lemon-, orange-, lime-, or berry-flavored malt beverages
  • liquor flavored malt beverages e.g., vodka-, rum-, or tequi
  • malt refers to any malted cereal grain, such as malted barley or wheat.
  • adjunct refers to any starch and/or sugar containing plant material which is not malt, such as barley or wheat malt.
  • adjuncts include common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, cassava and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like.
  • biomass refers to an aqueous slurry of any starch and/or sugar containing plant material, such as grist, e.g., comprising crushed barley malt, crushed barley, and/or other adjunct or a combination thereof, mixed with water later to be separated into wort and spent grains.
  • wort refers to the unfermented liquor run-off following extracting the grist during mashing.
  • Iodine-positive starch or "IPS” refers to (1 ) amylose that is not hydrolyzed after liquefaction and saccharification, or (2) a retrograded starch polymer.
  • IPS a retrograded starch polymer.
  • starch retrogradation refers to changes that occur spontaneously in a starch paste or gel on ageing.
  • the present teachings provide an improvement in the conversion of starch substrate to high maltose sugar syrup.
  • the process comprises contacting an aqueous slurry of starch substrate (25 to 40% ds, pH 4.5-pH 6.5) with a liquefying thermostable alpha amylase and a thermoresistant maltose-producing enzyme; and, conducting the incubation at a temperature zero to 30°C above the starch gelatinization temperature to produce a high maltose syrup.
  • the present teachings provide a recombinant host cell expressing a TthAmyl or variant thereof having at least 80% sequence identity to SEQ ID NO:2.
  • the TthAmy 1 or variant comprises 85%, 90%, 95%,
  • the recombinant host cell ⁇ sTrichoderma reesei.
  • the recombinant host cell is a Bacillus.
  • the recombinant TthAmyl or variant thereof has at least 70% activity at a pH range of 5.1 -7.1 as assayed under standard conditions of 50 °C, wherein the TthAmyl has at least 80%, 85%, 90%, 95%,
  • the recombinant 98%, or 99% identity to SEQ ID NO:3.
  • the recombinant 98%, or 99% identity to SEQ ID NO:3.
  • TthAmyl or variant thereof has an optimum temperature of about 95°C.
  • the recombinant TthAmyl comprises an amino acid sequence with at least 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO:3.
  • the recombinant TthAmyl is made in a T. reesei or Bacillus host cell, such as Bacillus subtilis.
  • the present teachings provide a method for producing a recombinant TthAmy or variant thereof, comprising: (a) providing a T. reesei host cell or Bacillus host cell that expresses a recombinant TthAmyl or variant thereof having at least 80% sequence identity to SEQ ID NO:2; and, (b) culturing said host cell under conditions which permit the production of said recombinant TthAmyl or variant thereof.
  • the host cell further expresses and secretes an alpha-amylase.
  • the host cell further expresses and secretes a pullulanase and/or isoamylase.
  • the composition comprising starch is contacted with said host cell.
  • a method is provided of malto- saccharifying a composition comprising starch to produce a composition comprising maltose, wherein said method comprises: (i) contacting a starch composition with a thermoresistant maltose-producing enzyme, and an alpha amylase; and (ii) malto- saccharifying the starch composition to produce said composition comprising maltose.
  • the composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch.
  • the malto-saccharifying is conducted at a temperature range of about 65 °C to about 90 °C. In some embodiments, the temperature range is 75 °C - 85 °C. In some embodiments, the malto-saccharifying is conducted over a pH range of pH 4.0 - pH 6.0. In some embodiments, the pH range is pH 4.5 - pH 5.5. In some embodiments, the alpha-amylase comprises AmyE. In some embodiments, the alpha-amylase comprises SEQ ID NO:4. In some embodiments, the method further comprises contacting a starch composition with a pullulanase.
  • the method further comprises fermenting the maltose composition to produce an End of Fermentation (EOF) product.
  • the method further comprises adding an additional enzyme, wherein the additional enzyme is a glucoamylase, a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a protease, a pullulanase, an additional alpha amylase, an additional beta amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, an alpha-glucosidase, a beta-glucosidase, a ly
  • thermoresistant maltose-producing enzyme is added at a dosage of 5-100 TBAs/g dss. In some embodiments, the thermoresistant maltose- producing enzyme is added at a dosage of 20-60TBAs/g dss. In some embodiments, the thermoresistant maltose-producing enzyme is added at a dosage of 30-50TBAs/g dss.
  • the pullulanase is added at a dosage of 0.1 -10 kg/MT. In some embodiments, the pullulanase is added at a dosage of 1 -8 kg/MT.
  • the present teachings provide a composition comprising maltose produced by the method of the present teachings, wherein the composition comprises a DP2 level of at least 42%, and wherein at least 90% of the starch is solubilized, wherein the malto-saccarifying occurs for between 2-12 hours.
  • the DP2 level is at least 45%, 50%, 60%, or 70%.
  • the malto-saccarifying occurs from between 4-12 hours, 6-10 hours, or 8-12 hours.
  • the method of the present teachings in some embodiments can be employed to produce a liquefied starch, or a fermented beverage, or a high maltose syrup.
  • the present teachings provide a method of producing a food composition, comprising combining (i) one or more food ingredients, and (ii) an isolated TthAmyl or variant thereof of the present teachings, wherein said pullulanase and said isolated TthAmyl or variant thereof catalyze the hydrolysis of starch
  • the food composition is selected from the group consisting of a food product, a baking composition, a food additive, an animal food product, a feed product, a feed additive, an oil, a meat, and a lard.
  • the one or more food ingredients comprise a baking ingredient or an additive.
  • the one or more food ingredients is selected from the group consisting of flour; an anti-staling amylase; a phospholipase; a phospholipid; a maltogenic alpha-amylase or a variant, homologue, or mutants thereof which has maltogenic alpha-amylase activity; a bakery xylanase (EC 3.2.1 .8); and a lipase.
  • the one or more food ingredients is selected from the group consisting of (i) a maltogenic alpha-amylase from Bacillus stearothermophilus, (ii) a bakery xylanase is from Bacillus, Aspergillus, Thermomyces or Trichoderma, (iii) a glycolipase from Fusarium heterosporum.
  • the starch composition in some embodiments of the present teachings, the starch composition
  • the TthAmyl or variants thereof may be "precursor,” “immature,” or “full- length,” in which case they include a signal sequence, or “mature,” in which case they lack a signal sequence.
  • the variant enzymes may also be truncated at the N- or C- termini, so long as the resulting polypeptides retain maltose generating activity. Cloning and expression of Thermoanaerobacterium thermosulfurigenes beta- amylase (TthAmyl ) was cloned and expressed as follows. TthAmyl (NCBI accession No.
  • P19584.1 is a beta-amylase from the bacterium Thermoanaerobacterium thermosulfurigenes.
  • the protein sequence is depicted in SEQ ID NO:1 .
  • the protein has a signal peptide with a length of 32 amino acids as predicted by SignalP-NN (Emanuelsson et al., Nature Protocols, 2:953-971 , 2007).
  • SignalP-NN SignalP-NN
  • TthAmyl gene was placed in a replicating plasmid pHPLT. As shown in the plasmid map of Figure 1 , the TthAmyl gene was under the control of the thermostable amylase LAT promoter (pLAT). A signal peptide from Bacillus licheniformis strain DSM13 was used to direct protein secretion. The plasmid was amplified using lllustra TempliPhi 100 Amplification Kit (GE Healthcare Life Sciences, NJ). Competent B. subtilis cells were transformed with the amplification product, and the cells were plated on Luria Agar plates supplemented with 10 ppm neomycin. Expression evaluation of TthAmyl showed that the enzyme expresses very well in Bacillus subtilis.
  • pLAT thermostable amylase LAT promoter
  • TthAmyl The amino acid sequence of TthAmyl is set forth as SEQ ID NO:1 .
  • the predicted native signal peptide is shown in italics.
  • the mature TthAmyl gene sequence that was placed in plasmid pHPLT-TthAmyl is set forth as SEQ ID NO:2.
  • SEQ ID NO:3 siapnfkvfvmgplekvtdfnafkdqlitlknngvygittdiwwgyvenagenqfdwsyyktyadtvraaglkwvpimsth acggnvgdtvnipipswvwtkdtqdnmqykdeagnwdneavspwysgltqlynefyssfasnfssykdiitkiyisggp sgelrypsynpshgwtypgrgslqcyskaaitsfqnamkskygtiaavnsawgtsltdfsqispptdgdnfftngykttygn dfltwyqsvltnelaniasvahscfdpvfnvpigakia
  • thermostable beta-amylase TthAmyl To begin the fermentation of TthAmyl , a seed culture ⁇ Bacillus subtilis) was grown in a 250-mL shake flask that contained 50 mL of LBG media consisting of 10g/L soytone, 5 g/L yeast extract, 10 g/L NaCI, 1 1 g/L glucose monohydrate, 1 .67 drop/L Mazu 6000 and 10ug/mL kanamycin. The seed culture was grown at 37°C with shaking at 250 rpm. After 5 hours growth, OD550 was checked.
  • LBG media consisting of 10g/L soytone, 5 g/L yeast extract, 10 g/L NaCI, 1 1 g/L glucose monohydrate, 1 .67 drop/L Mazu 6000 and 10ug/mL kanamycin.
  • the seed culture was grown at 37°C with shaking at 250 rpm. After 5 hours growth, OD550 was checked.
  • the fermentation was initiated at 3.5 kg working volume and controlled at pH 6.9 and temperature 37 °C. DO was controlled above 20% by adjusting aeration and agitation.
  • the feed of sterile 600 g/kg glucose solution was started at 5 EFT hour with feed rate of 0.25 g/min, and feed rate was ramped linearly in 14 hours to 0.71 g/min and maintained at this rate for the rest of the time.
  • the residual glucose would be controlled at 0 g/L.
  • feed rate would be decreased if DO was still below 20% even though agitation was increased and air was up to 2VVM.
  • Fermentation broth was sampled at 18.8 h and 46.3 h to run residual glucose measurement and SDS-PAGE analysis. Fermentation was terminated after 46.3 hours. Following centrifugation, filtration and ultrafiltration, 540 mL of concentrated sample was obtained. BCA assay (protein quantification kit, Shanghai Generay Biotech CO., Ltd) illustrated that total protein in concentrated sample was 22.56 g/L.
  • thermostable beta-amylase TthAmyl Purification of thermostable beta-amylase TthAmyl
  • TthAmyl was purified via beta-cyclodextrin coupled Sepharose 6 affinity chromatography, taking advantage of its carbohydrate binding domain.
  • the 500 ml_ crude broth from the fermentor was concentrated by ultrafiltration and buffer exchanged with 20 mM HEPES pH 7.5.
  • the solution was then loaded onto a 60-mL beta- cyclodextrin coupled Sepharose 6 column which was pre-equilibrated with 20 mM HEPES pH 7.5 (buffer A).
  • the column was applied with a gradient of 0-100% buffer B (buffer A containing 10 mM alpha-cyclodextrin) in 2 column volumes, followed by 4 column volumes of 100% buffer B.
  • the target protein was eluted in the gradient step.
  • the fractions were pooled according to the activity and SDS-PAGE analysis results. After that, Gel Filtration chromatography was performed to get a high purity product.
  • the active fractions from the superdex-75 column were pooled and concentrated using an Amicon Ultra-15 device with 10 K MWCO. The sample showed above 98% purity and was stored in 40% glycerol at -80°C until usage.
  • aqueous slurry containing 38% DS refined starch (Cargill, Minneapolis, NM), containing 10 ppm Ca 2+ , and 100 ppm sulfur dioxide (S0 2 ) was prepared by stirring overnight.
  • the pH of the slurry was adjusted using sodium carbonate solution (20% w/v).
  • the Baume (degrees) of the slurry were approximately 22.3.
  • High temperature jet liquefaction was performed using 0.55 Kg SPEZYME® FRED (DuPont Industrial Biosciences) per Metric Ton of corn starch at pH 5.6.
  • the slurry with the enzyme was sent through a pilot plant jet at 0.5 gpm with six-minute residence time and cooked at about 108 °C -1 10 °C for the primary cook. Secondary liquefaction was performed at 95°C for 120 minutes.
  • the DE and refractive index (Rl) were measured i.e., a DE value of at least 10 in 90-100 min and used in these studies.
  • Aminadase for the purpose of this invention is a naturally occurring alpha-amylase. (EC 3.2.1 .1 ; 1 , 4-a-D-glucan glucanohydrolase) from B. subtilis.) referred to herein also as AmyE Combi L.
  • the AmyE sequence used for the experiments described herein is SEQ ID NO: 4, is set forth below, and is herein also referred to as AmyE Combi L.
  • Amy E sequences can be used according to the present teachings, including the AmyE having the amino acid sequence disclosed in NCBI Accession No.20 ABW75769, NCBI Accession Nos. ABK54355, AAF14358, AAT01440,
  • the alpha-amylase as contemplated herein may be a Bacillus subtilis alpha-amylase (AmyE) having an amino acid sequence of above or an alpha-amylase having at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to it.
  • AmyE Bacillus subtilis alpha-amylase
  • thermostable beta-amylase activity was monitored using the PAHBAH assay protocol as described above for the thermostable beta-amylase specific activity measurement.
  • Buffer working solutions contained the combination of glycine/sodium acetate/HEPES (250 mM), with pH varying from 3.0 to 1 0.0.
  • Substrate solutions were prepared by mixing 896 ⁇ _ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 1 01 1 8), 1 00 ⁇ _ of 250 mM buffer working solution (pH from 3.0 to 1 0.0), and 4 ⁇ _ of 0.5 M CaCI 2 .
  • Enzyme working solutions were prepared in water at a certain dose (showing signal within linear range as per dose response curve). All the incubations were done using the same protocol as described above for beta- amylase activity assay. The absorbance from a control (water-only) was subtracted, and the resulting values were converted to percentages of relative activity, by defining the activity at the optimal pH as 1 00% (Table 1 , and see FIG. 1 ). The optimal pH range can be defined as keeping >70% of activity and was determined as 5.1 -7.1 . Table 1 shows the pH profile of TthAmyl .
  • thermostable beta-amylase activity was monitored using the PAHBAH assay protocol as described above for the thermostable beta-amylase specific activity measurement.
  • Substrate solutions were prepared by mixing 3.6 ml_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 101 18), 0.4 mL of 0.5 M buffer (pH 5.0 sodium acetate), and 1 6 ⁇ _ of 0.5 M CaCI 2 into a 15-mL conical tube. Enzyme working solutions were prepared in water at a certain dose (showing signal within linear range as per dose response curve).
  • SPEZYME® Xtra (DuPont Industrial Biosciences) was added at constant dose of 0.13 Kg per MT ds starch and the TthAmyl was added at 0.02 and 49.9 TBAs/g dss.
  • the starch slurry was maintained at 80 °C for 7.5 hrs and samples were drawn at 1 .25, 3.5 and 7.5 hrs to analyze the percent solubility and saccharide profile.
  • the saccharide distribution for SPEZYME® Xtra and TthAmyl mediated single step liquefaction and malto- saccharifcation process at 80 °C and pH 5.5 are shown in Table 3.
  • TthAmyl maintains a significant amount of maltogenic activity for 7.5 hours at 80 °C and pH 5.5, as evidenced by continuous generation of maltose, as well as continued increase in % soluble solids.
  • An increased amount of TthAmyl results in increased rate of maltose generation and % solubilization.
  • alpha amylases were added as shown in Table 5, with SPEZYME® Xtra (B. stearothermophilus alpha amylase)
  • the starch slurry was maintained at 80 °C for 4 hrs and samples were drawn at 1 , 2, 3 and 4 hrs to analyze the percent solubility and saccharide profile.
  • the saccharide distribution for different dosage of AmyE for single step liquefaction and malto- saccharifcation process is shown in Table 6.
  • AmyE As shown in Table 6, increasing doses of AmyE resulted in increased amount of DP1 in the maltose rich syrup. AmyE also helps to increase in % soluble solids in the single step liquefaction and malto-saccharification process.
  • the starch slurry was maintained at 80 °C for 4 hrs and samples were drawn at 1 , 2, 3 and 4 hrs to analyze the percent solubility and saccharide profile.
  • the slurry with the enzyme(s) added was sent through a pilot plant jet cooker (Hydro-thermal Corporation, Waukesha, Wl) at 0.5 gpm with six-minute residence time and cooked at about 1 08 °C - 1 1 0 °C for the primary cook.
  • Secondary liquefaction was performed at 95°C for achieving -1 0 DEs.
  • the DE and refractive index (Rl) were measured at various time points during the secondary liquefaction.
  • the active alpha amylase in the liquefact was deactivated by reducing the pH to 4.0 and incubating at 95 °C for 30 min. After that the liquefact was cooled down to 60 °C and pH was adjusted to 6.0.
  • the sample B was incubated at 60 °C with addition of barley beta- amylase OPTIMALT® BBA dosed at 0.5 kg/MT ds and pullulanase OPIMAX® L-1000 dosed at 1.0 kg/MT.
  • the sample C was incubated at 60 °C with addition of pullulanase OPIMAX® L-1000 dosed at 1.0 kg/MT.
  • the saccharide distribution for different enzyme treatments in single step liquefaction and malto-saccharification process is shown in Table 9.
  • variant TthAmyl polypeptides are those that retain beta-amylase activity. They may have a specific activity higher or lower than the wild-type TthAmyl polypeptide.
  • TthAmyl variant Additional characteristics include stability, pH range,
  • the variant may be pH stable for 24-60 hours from pH 3 to about pH 8, e.g., pH 3.0 - 7.8; e.g., pH 3.0 - 7.5; pH 3.5 - 7.0; p H 4.0 - 6.7; or pH 5.0.
  • a TthAmyl variant can be expressed at higher levels than the wild-type TthAmyl , while retaining the performance
  • TthAmyl variants also may have altered oxidation stability in comparison to the parent glucoamylase. For example, decreased oxidation stability may be advantageous in compositions for starch liquefaction.
  • the variant TthAmyl can have altered temperature profile compared to the wild-type beta amylase. Such TthAmyl variants are advantageous for use in baking or other processes that require elevated temperatures. Levels of expression and enzyme activity can be assessed using standard assays known to the artisan skilled in this field, including those disclosed below.
  • the TthAmyl or variant thereof can be isolated from a host cell, for example by secretion of the TthAmyl or variant from the host cell.
  • a cultured cell material comprising TthAmyl or variant thereof can be obtained following secretion of the
  • TthAmyl or variant from the host cell.
  • the TthAmyl or variant is optionally purified prior to use.
  • the TthAmyl gene can be cloned and expressed according to methods well known in the art.
  • Suitable host cells include bacterial, plant, or yeast cells, e.g., filamentous fungal cells. Particularly useful host cells include Trichoderma reesei and Bacillus subtilus. Others include Bacillus licheniformis, and yeasts. In some
  • Trichoderma reesei and Bacillus subtilus host cells can express TthAmyl at higher, or at least comparable, levels to natively expressed TthAmyl .
  • the host cell may further express a nucleic acid encoding a homologous or heterologous beta amylase, i.e., a beta amylase that is not the same species as the host cell, or one or more other enzymes.
  • the beta amylase may be a variant amylase.
  • the host may express one or more accessory enzymes, proteins, peptides. These may benefit liquefaction, saccharification, fermentation, SSF, etc. processes.
  • the host cell may produce biochemicals in addition to enzymes used to digest the carbon feedstock(s). Such host cells may be useful for fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need to add enzymes.
  • a DNA construct comprising a nucleic acid encoding a TthAmyl or variant thereof can be constructed to be expressed in a host cell.
  • Representative nucleic acids that encode TthAmyl include SEQ ID NO: 2. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding a TthAmyl or variant thereof can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below. The vector may be any vector that can be transformed into and replicated within a host cell.
  • a vector comprising a nucleic acid encoding a TthAmyl or variant thereof can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector.
  • the vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional TthAmyl or variant thereof.
  • Host cells that serve as expression hosts can include filamentous fungi.
  • the Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, aiwww.fgsc.net (last modified January 17, 2007).
  • a nucleic acid encoding a TthAmyl or a variant thereof can be operably linked to a suitable promoter, which allows transcription in the host cell.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a- amylase, A.
  • a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable
  • promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. Additional discussion of promoters can be found in Liu et al. (2008) "Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl ) promoter optimization," Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158- 65.
  • the coding sequence can be operably linked to a signal sequence.
  • the DNA encoding the signal sequence may be the DNA sequence naturally associated with the TthAmyl gene to be expressed.
  • the DNA may encode the TthAmyl signal sequence of operably linked to a nucleic acid encoding a TthAmyl or a variant thereof.
  • the DNA encodes a signal sequence from a species other than T.
  • thermosulfurigenes A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source.
  • the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.
  • An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a TthAmyl or variant thereof. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYCI 77, pUB1 10, pE194, pAMB1 , and plJ702.
  • the vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • a selectable marker e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD, and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art.
  • Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of a TthAmyl or variant thereof for subsequent purification.
  • Extracellular secretion of the TthAmy or variant thereof into the culture medium can also be used to make a cultured cell material comprising the isolated TthAmyl or variant thereof.
  • the expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes.
  • the expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes.
  • the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the TthAmyl or variant thereof to a host cell organelle such as a peroxisome, or to a particular host cell compartment.
  • Such a targeting sequence includes but is not limited to the sequence serine-lysine-leucine (SKL), which is a known peroxisome target signal.
  • SSL serine-lysine-leucine
  • the nucleic acid sequence of the TthAmyl or variant thereof is operably linked to the control sequences in proper manner with respect to expression.
  • a Trichoderma reesei host cell either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of a TthAmyl or variant thereof.
  • the cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell.
  • Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination.
  • the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein.
  • Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2, egl1, and egl2 genes.
  • Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.
  • Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction;
  • transfection e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion.
  • General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001 ), supra.
  • the expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No.
  • Trichoderma sp. for transformation may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56.
  • the mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts.
  • the protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium.
  • stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like.
  • concentration of these stabilizers varies between 0.8 M and 1 .2 M, e.g., a 1 .2 M solution of sorbitol can be used in the suspension medium.
  • Uptake of DNA into the host Trichoderma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50 mM CaCI 2 is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.
  • TE buffer 10 mM Tris, pH 7.4; 1 mM EDTA
  • MOPS pH 6.0
  • polyethylene glycol polyethylene glycol
  • Trichoderma sp. usually transformation of Trichoderma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 10 5 to 10 7 /mL, particularly 2x10 6 /ml_. A volume of 100 ⁇ of these protoplasts or cells in an permeability treatment, typically at a density of 10 5 to 10 7 /mL, particularly 2x10 6 /ml_. A volume of 100 ⁇ of these protoplasts or cells in an
  • appropriate solution e.g., 1 .2 M sorbitol and 50 mM CaCI 2
  • appropriate solution e.g., 1 .2 M sorbitol and 50 mM CaCI 2
  • PEG poly(ethylene glycol)
  • a high concentration of PEG is added to the uptake solution.
  • From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension.
  • Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725.
  • a method of producing a TthAmyl or variant thereof may comprise cultivating a Trichoderma reesei host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.
  • Trichoderma reesei host cells express TthAmyl at higher, or at least comparable, levels to natively expressed TthAmyl .
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of a TthAmyl or variant thereof. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
  • An enzyme secreted from the host cells can be used in a whole broth preparation.
  • the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of a glucoamylase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the glucoamylase to be expressed or isolated.
  • the term "spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term "spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
  • An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity
  • the polynucleotide encoding TthAmyl or a variant thereof in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • the control sequences may be modified, for example by the addition of further
  • control sequences may in particular comprise promoters.
  • Host cells may be cultured under suitable conditions that allow expression of the TthAmyl or variant thereof.
  • Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose.
  • an inducer substance for example dexamethasone or IPTG or Sophorose.
  • Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNTTM (Promega) rabbit reticulocyte system.
  • An expression host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25 °C to about 78°C (e.g., 30 °C to 45 °C), depending on the needs of the host and production of the desired TthAmyl or variant thereof. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of a TthAmyl or variant thereof.
  • assays can measure the expressed protein, corresponding mRNA, or maltose- producing activity.
  • suitable assays include Northern blotting, reverse transcriptase polymerase chain reaction, and in situ hybridization, using an
  • Suitable assays also include measuring
  • TthAmyl activity in a sample for example, by assays directly measuring reducing sugars such as maltose in the culture media.
  • Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated TthAmyl or variant glucoamylase polypeptide-containing solution.
  • a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.
  • TthAmyl or variant polypeptide-containing solution It is desirable to concentrate a TthAmyl or variant polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions can require increased incubation time in order to collect the purified enzyme precipitate.
  • the enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of purification include but are not limited to rotary vacuum filtration and/or ultrafiltration.
  • the enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated TthAmyl or variant polypeptide-containing solution is at a desired level.
  • Concentration may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent.
  • a precipitation agent such as a metal halide precipitation agent.
  • Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides, and blends of two or more of these metal halides.
  • Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide, and blends of two or more of these metal halides.
  • the metal halide precipitation agent, sodium chloride can also be used as a preservative.
  • the metal halide precipitation agent is used in an amount effective to precipitate the TthAmyl or variant thereof.
  • the selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.
  • At least about 5% w/v (weight/volume) to about 25% w/v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w/v.
  • metal halide precipitation agent Generally, no more than about 25% w/v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w/v.
  • concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific TthAmyl or variant polypeptide and on its concentration in the concentrated enzyme solution.
  • organic compound precipitating agents include: 4- hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4- hydroxybenzoic acid, and blends of two or more of these organic compounds.
  • the addition of said organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.
  • the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyi esters of 4-hydroxybenzoic acid, wherein the alkyi group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds.
  • the organic compound precipitation agents can be, for example, linear or branched alkyi esters of 4-hydroxybenzoic acid, wherein the alkyi group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds.
  • Exemplary organic compounds are linear alkyi esters of 4-hydroxybenzoic acid, wherein the alkyi group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds.
  • Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used.
  • Additional organic compounds also include but are not limited to 4-hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both amylase preservative agents.
  • methyl PARABEN 4-hydroxybenzoic acid methyl ester
  • propyl PARABEN 4-hydroxybenzoic acid propyl ester
  • additive of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature,
  • TthAmyl or variant polypeptide concentration TthAmyl or variant polypeptide concentration, precipitation agent concentration, and time of incubation.
  • the organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent.
  • the selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing.
  • at least about 0.01 % w/v of organic compound precipitation agent is added to the concentrated enzyme solution and usually at least about 0.02% w/v.
  • no more than about 0.3% w/v of organic compound precipitation agent is added to the concentrated enzyme solution and usually no more than about 0.2% w/v.
  • the concentrated polypeptide solution containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be purified.
  • the pH is adjusted at a level near the isoelectric point of the glucoamylase.
  • the pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pi) up to about 2.5 pH units above the isoelectric point.
  • the incubation time necessary to obtain a purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less than about 10 hours and in most cases even about 6 hours.
  • the temperature during incubation is between about 4°C and about 50 °C.
  • the method is carried out at a temperature between about 10°C and about 45 °C ⁇ e.g., between about 20 °C and about 40 °C).
  • the optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used.
  • the overall recovery of purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound.
  • the agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.
  • the purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further purification of the purified enzyme precipitate can be obtained by washing the precipitate with water. For example, the purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.
  • a TthAmyl or variant polypeptide accumulates in the culture broth.
  • the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme purification.
  • the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation- precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column, and eluted to recover the enzyme-active fraction.
  • a conventional procedure such as ion exchange chromatography may be used.
  • Purified enzymes as well as partially purified enzymes, are useful for laundry and cleaning applications. For example, they can be used in laundry detergents and spot removers. They can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).
  • a TthAmyl or variant polypeptide can be partially purified as generally described above by removing cells via flocculation with polymers.
  • the enzyme can be purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment.
  • the enzyme does not need to be purified, and whole broth culture can be lysed and used without further treatment; or the broth can be clarified, and optionally concentrated and/or further processed, and used.
  • the enzyme can then be processed, for example, into granules.
  • a useful starch substrate may be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically, the granular starch may be obtained from corn, cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, sweet sorghum, rice, peas, bean, banana, or potatoes. Corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch;
  • wheat contains about 60-65% starch; and polished rice contains 70-72% starch.
  • starch substrates are corn starch and wheat starch.
  • the starch from a grain may be ground or whole and includes corn solids, such as kernels, bran and/or cobs.
  • the starch may be highly refined raw starch or feedstock from starch refinery processes.
  • Various starches also are commercially available.
  • corn starch is available from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is available from Sigma; sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).
  • the starch substrate can be a crude starch from milled whole grain, which contains non-starch fractions, e.g., germ residues and fibers.
  • Milling may comprise either wet milling or dry milling or grinding.
  • wet milling whole grain is soaked in water or dilute acid to separate the grain into its component parts, e.g., starch, protein, germ, oil, kernel fibers.
  • Wet milling efficiently separates the germ and meal (i.e., starch granules and protein) and is especially suitable for production of syrups.
  • whole kernels are ground into a fine powder and often processed without fractionating the grain into its component parts. In some cases, oils from the kernels are recovered.
  • Dry ground grain generally will comprise significant amounts of non- starch carbohydrate compounds, in addition to starch. Dry grinding of the starch substrate can be used for production of ethanol and other biochemicals.
  • the starch to be processed may be a highly refined starch quality, for example, at least 90%, at least 95%, at least 97%, or at least 99.5% pure.
  • the soluble starch hydrolysate can be fermented by contacting the starch hydrolysate with a fermenting organism typically at a temperature around 32°C, such as from 30 °C to 35 °C.
  • EOF products include
  • metabolites such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, lysine and other amino acids, omega 3 fatty acid, butanol, isoprene, 1 ,3-propanediol and other
  • Ethanologenic microorganisms include yeast, such as Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis, expressing alcohol dehydrogenase and pyruvate decarboxylase.
  • the ethanologenic microorganism can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose.
  • Improved strains of ethanologenic microorganisms which can withstand higher temperatures, for example, are known in the art and can be used. See Liu et al. (201 1 ) Sheng Wu Gong Cheng Xue Bao 27(7): 1049-56.
  • yeast Commercial sources of yeast include ETHANOL RED® (LeSaffre); Thermosacc® (Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); and SUPERSTART® (Alltech).
  • Microorganisms that produce other metabolites, such as citric acid and lactic acid, by fermentation are also known in the art. See, e.g., Papagianni (2007) "Advances in citric acid fermentation by Aspergillus niger. biochemical aspects, membrane transport and modeling," Biotechnol. Adv. 25(3): 244- 63; John et al. (2009) "Direct lactic acid fermentation: focus on simultaneous
  • the saccharification and fermentation processes may be carried out as an SSF process. Fermentation may comprise subsequent purification and recovery of ethanol, for example. During the fermentation, the ethanol content of the broth or "beer" may reach about 8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to produce enriched, e.g., 96% pure, solutions of ethanol. Further, C0 2 generated by fermentation may be collected with a C0 2 scrubber, compressed, and marketed for other uses, e.g., carbonating beverage or dry ice production. Solid waste from the fermentation process may be used as protein-rich products, e.g., livestock feed.
  • an SSF process can be conducted with fungal cells, such as Trichoderma reesei, that express and secrete TthAmyl or its variants continuously throughout SSF.
  • the fungal cells expressing TthAmyl or its variants also can be the fermenting microorganism, e.g., an ethanologenic microorganism. Ethanol production thus can be carried out using a fungal cell that expresses sufficient TthAmyl or its variants so that less or no enzyme has to be added exogenously.
  • the fungal host cell can be from an appropriately engineered fungal strain. Fungal host cells that express and secrete other enzymes, in addition to TthAmyl or its variants, also can be used.
  • Such cells may express a-amylase and/or a pullulanase, phytase, alpha- glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, £>efa-glucosidase, pectinase, esterase, redox enzymes, transferase, a glucoamylase other than TthAmy 1 or other enzyme.
  • a-amylase and/or a pullulanase phytase, alpha- glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, £>efa-glucosidase, pectinase, esterase, redox enzymes, transferase, a glucoamylase other than
  • a variation on this process is a "fed-batch fermentation" system, where the substrate is added in increments as the fermentation progresses.
  • Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium.
  • the actual substrate concentration in fed-batch systems is estimated by the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases, such as C0 2 . Batch and fed-batch fermentations are common and well known in the art.
  • Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing.
  • Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
  • Continuous fermentation permits modulation of cell growth and/or product concentration. For example, a limiting nutrient such as the carbon source or nitrogen source is maintained at a fixed rate and all other parameters are allowed to moderate. Because growth is maintained at a steady state, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation.
  • compositions comprising TthAmyl or Variants Thereof
  • thermoresistant maltose-producing enzyme that is not TthAmyl , phytase, protease, pullulanase, ⁇ -amylase, isoamylase, a-amylase, alpha-glucosidase, cellulase, xylanase, hemicellulase, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, or a combination thereof.
  • thermoresistant maltose-producing enzymes provided herein can be used in a direct starch to maltose process as described in US2013/30384, Method for Making High Maltose Syrup, filed March 12, 2013, claiming priority to US Provisional 61 /61 6990, filed March 28, 2012.

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Abstract

The present teachings provide a one step process for producing high maltose syrup. The process comprises contacting starch substrate with a starch liquefying thermostable alpha amylase (s) and a thermoresistant maltose-producing enzyme at a temperature zero to thirty degree above the starch gelatinization temperature resulting in simultaneously solubilizing and hydrolyzing the starch substrate to a high maltose syrup. In some embodiments, the thermoresistant maltose-producing enzyme is a recombinantly-expressed TthAmy

Description

SIMULTANENOUS LIQUIFACTION AND MALTO-SACCHARIFICATION
FIELD OF THE INVENTION
Methods of making maltose, and recombinant proteins for use thereof. BACKGROUND
Starch is composed of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose contains linear chains of a-1 ,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000. Amylopectin is a branched polymer containing a-1 ,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
Starch can be broken down to make maltose, an alpha 1 -4 linked glucosyl glucose, a di-saccharide that is widely used in many industrial and consumers' applications. For instance, maltose is used as a sweetener or in the prepration of maltitol, a low calorie sweetener. The majority sugar in high maltose syrup is maltose. It is less sweet than high fructose corn syrup. It is also used in brewing to increase through-put and known to reduce haze caused by varying quality of wort. Since maltose has a low freezing point, high maltose syrup is useful in frozen desserts.
Maltose solution exhibits lower viscosity and less humictant than equal concentrations of glucose solution and finds application in candy formulation to reduce the stickyness. Maltitol is a sugar alcohol produced by the hydrogenation of maltose.
Historically, maltose is prepared by a two step process via the enzymatic hydrolysis of an aqueous slurry (25-32% ds, pH 5.5- 6.0) of starch substrate subjected to a high temperature jet cooking process (>100 °C) using a thermostable alpha amylase to solubilize and hydrolyze the insoluble starch into soluble dexrins, and then treating with an enzyme called beta amylase at 55 °C, pH 5.5, thereby producing a syrup containing high levels of maltose, β- Amylase (EC 3.2.1 .2) hydrolyses the alpha-1 -4- glucan bonds in amylosaccharide chains from non-reducing ends and generates the di- saccharide maltose, which is linked by alpha-1 -4 glucosidic linkages between two glucosyl residues, β amylases are well-characterized in higher plants and microbial sources. For example, β amylase extracted from plant, e.g. barley (OPTIMALT® BBA from DuPont Industrial Biosciences), wheat, and soy bean are routinely used on a commercial scale for producing high maltose syrup. High maltose syrup is a food additive used as a sweetener and preservative in many food formulations. Commercial processes for producing high maltose syrup using starch begins by hydrolyzing insoluble granular starch primarily from corn, wheat or cassava by a thermostable liquefying alpha amylase (For example SPEZYME® Fred, CLEARFLOW® from DuPont Industrial Biosciences, Liquozyme® Supra from Novozymes) using a conventional high temperature jet cooking process (>100 °C) to produce a soluble hydrolyzed liquefact. The liquefact is then further hydrolyzed by incubating soluble dextrins with β amylase (OPTIMALT® BBA from DuPont Industrial Biosciences) at 55 °C for 24 to 48 hours, resulting in a sugar syrup containing high maltose. A maltose syrup containing greater than 65% maltose content typically requires the addition of a debranching enzyme, for example a pullulanase such as OPTIMAX® L-1000 from DuPont Industrial Biosciences, or Promozyme from Novozymes and Amano Pullulanse) to increase the maltose content from 55% to up to 85%. The beta amylases used in the maltose process are primarily produced from plant sources by the extraction of either barley (OPTIMALT® BBA), wheat, and/or soy bean. The plant β amylases from barley and wheat are not thermostable (less than 58 °C) and exhibit maximum activity at pH between 5.4 to 5.6. The low temperature for incubation or a longer time (24 to 48 hours), and the
incubation pH of greater than 5.0 are unfavorable for maltose producers due to high risk of microbial infection.
Even though beta amylases employed in most industrial applications are primarily obtained from plant sources by extraction and purification processes, attempts have been made to obtain more active, thermostable and extracellular beta amylases from micro-organisms, primarily from bacillus species. Many bacterial sources have been cited and described in the past, including as Bacillus flexus (Matsunaga, Okada and Yamaguchi, 2009, Matsunaga, A., Okada, M., and Yamaguchi S. 2009), Bacillus polymyxa (Kawazu et al., 1987, Kawazu, T., Nakanishi, Y., Uozumi, N., Sasaki, T., Yamagata, H., and Tsukagoshi, N. 1987. J. Bacteriol. (1 69) 1564-1570) and Clostridium thermosulfurogenes (Kitamoto, 1988, Kitamoto, N., Yamagata, H ; Kato, T ., Tsukagoshi, N., and Udaka, S. 1988. J. Bacteriol. (170)5848-5854).
Microbially-produced beta-amylase provides an opportunity to produce more stable and consistent enzyme strains. However, none of the past beta amylases have shown any improvement in terms of pH and temperature properties over commercially used beta amylase from plant origins. Therefore, the plant beta amylases continue to be used for maltose production, even though they suffer from unreliability and instablility during storage and transportation.
There has been a historical effort on gene coding and expression of thermophilic beta-amylase (Yamada et.al US patent # 5,082,781 , Zeikus et.al, 1987, US Patent # 4,647,538, Nanmori et al.1993; US Patent # 5,188,956). Discovery and expression of organism such as Clostridium thermosulfurogenes have been undertaken and discussed (see for example Zeikus et al.1987 and Yamagata et al.1993) where they have demonstrated the beta-amylase activity at 70 °C in absence of substrate and activity at 80 °C in presence of substrate. PCT Patent application #WO201 1/058105A1 describes a process on the application of an alpha amylase, maltogenic enzyme, and/or a thermostable beta amylase from Clostridium thermosulfurogenes being used during mashing and lautering in brewing processes.
There remains an un-met need for shortening processing time by eliminating one of the steps of the process for making maltose. In particular, opportunites for improvement exist along several avenues, including savings on energy, reducing risks of process contamination, reducing processing time down to 2 to 4 hours vs the customary 24 to 36 hours, elimination of unit operation, reduced jet cooking resulting in energy saving and liquefaction steps, and increasing capacity without additional capital costs. The present teachings address these un-met needs.
SUMMARY
The present teachings provide a one-step process, offering a simultaneous liquefaction and malto-saccharification of starch substrates into a maltose rich sugar syrup using a thermostable beta amylase at a temperature range of 80-95 °C. Rest TBC upon finalization of claims. BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings are incorporated in and constitute a part of this specification and illustrate various methods and compositions disclosed herein. In the drawings: FIG. 1 depicts a plasmid according to some embodiments of the present teachings.
FIG. 2 depicts a flowchart according to some embodiments of the present teachings.
FIG. 3 depicts the pH profile of the TthAmyl enzyme of the present teachings. FIG. 4 depicts the temperature profile of the TthAmyl enzyme of the present teachings.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 sets forth the full length amino acid sequence of TthAmyl , with the predicted signal peptide shown in italics. SEQ ID NO: 2 sets forth the mature full length DNA sequence of TthAmyl .
SEQ ID NO: 3 sets forth the mature TthAmy amino acid sequence.
SEQ ID NO: 4 sets forth the mature AmyE Combi L amino acid sequence.
DETAILED DESCRIPTION
Definitions & Abbreviations In accordance with this detailed description, the following abbreviations and definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All refererences and sequences herein are incorporated by reference in their entirety. The following terms are provided below.
The terms "amylase" or "amylolytic enzyme" refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch. a-Amylases are hydrolases that cleave the a-D-(1→4) O-glycosidic linkages in starch. Generally, a- amylases (EC 3.2.1 .1 ; a-D-(1→4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving a-D-(1→4) O-glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (1 -4)-a-linked D- glucose units. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1 .2; a-D-(1→4)-glucan maltohydrolase) and some product-specific amylases like maltogenic a-amylase (EC 3.2.1 .133) cleave the polysaccharide molecule from the non- reducing end of the substrate, β-amylases, a-glucosidases (EC 3.2.1 .20; a-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1 .3; a-D-(1→4)-glucan glucohydrolase), and product-specific amylases like the maltotetraosidases (EC 3.2.1 .60) and the
maltohexaosidases (EC 3.2.1 .98) can produce malto-oligosaccharides of a specific length or enriched syrups of specific maltooligosaccharides.
As used herein, the term "glucoamylase" (EC 3.2.1 .3) (otherwise known as glucan 1 ,4-a-glucosidase; glucoamylase; amyloglucosidase; γ-amylase; lysosomal a- glucosidase; acid maltase; exo-1 ,4-a-glucosidase; glucose amylase; y-1 ,4-glucan glucohydrolase; acid maltase; 1 ,4-a-D-glucan glucohydrolase; or 4-a-D-glucan glucohydrolase) refers to a class of enzymes that catalyze the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides. These are exo-acting enzymes, which release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules. The enzymes also hydrolyze alpha-1 , 6 and alpha -1 , 3 linkages although at much slower rates than alpha-1 , 4 linkages. The term
"hydrolysis of starch" refers to the cleavage of glucosidic bonds with the addition of water molecules.
The term "pullulanase" (E.C. 3.2.1 .41 , pullulan 6-glucanohydrolase) refers to a class of enzymes that are capable of hydrolyzing alpha 1 -6 glucosidic linkages in an amylopectin molecule. The term "B-amylase" (E.C. 3.2.1 .2) refers to a class of enzymes that hydrolyze the alpha-1 -4-glucan bonds in amylosaccahride chains from non-reducing ends and generate a di-saccharide maltose, linked by alpha -1 -4 glucosidic linkages two glucosy resdidues. Beta amylases are well charcterized in higher plants and microbial sources. For example Beta amylase extracted from plants, e.g. barley
(OPTIMALT® BBA,OPTIMALT DBA from DuPont Industrial Biosciences), wheat
(Novozymes-WBA ), and soy bean (Nagase Biochemicals, Japan) have already been used on a commercial scale for producing high maltose syrup and food applications. In some embodiments, B-amylases as used in the present methods include those described as PsPAmy9, disclosed in co-filed patent application "B-amylase and
Methods of Use."
The term "thermoresistant maltose-producing enzyme" refers to any B- amylase, alpha amylase, or maltogenic amylase, or the like, that retains 50% activity after treatment at 60 °C for 10 minutes at a pH of 4.5-5.5, and is capable of producing at least 30% maltose in a simultaneous liquifaction and malto-saccharification process according to the present teachings.
"Enzyme units" herein refers to the amount of product formed per time under the specified conditions of the assay. For example, a "glucoamylase activity unit" (GAU) is defined as the amount of enzyme that produces 1 g of glucose per hour from soluble starch substrate (4% DS) at 60°C, pH 4.2. A "soluble starch unit" (SSU) is the amount of enzyme that produces 1 mg of glucose per minute from soluble starch substrate (4% DS) at pH 4.5, 50°C. DS refers to "dry solids."
As used herein "Thermostable Beta Amylase activity Unit (TBA Unit)" was determined using a colorimetric assay to monitor the release of reducing sugars from potato amylopectin. The activity is reported as equivalents of glucose released per minute. Substrate solutions were prepared by mixing 9 ml_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 101 18), 1 ml_ of 0.5 M buffer (pH 5.0 sodium acetate), and 40 μΙ_ of 0.5 M CaCI2 into a 15-mL conical tube. Stock solutions of purified beta- amylase samples were made by diluting original samples to 0.4 mg/mL (400 ppm) in water. Serial dilutions of enzyme samples and glucose standard were prepared in water in non-binding microtiter plates (MTP, Corning 3641 ). Then 90 μΙ_ of substrate solution (preincubated at 50°C for 5 min at 600 rpm) and 10 μΙ_ of the enzyme serial dilution were added and mixed in non-binding microtiter plates (MTP, Corning 3641 ). All the incubations were done at 50°C for 10 min at 600 rpm in a thermomixer (Eppendorf). After incubation, 50 μΙ_ of 0.5 N NaOH were added to each well to stop the reaction. Total reducing sugars present in each well were measured using a PAHBAH method: 80 μΙ_ of 0.5 N NaOH was aliquoted into a microtiter plate, followed by the addition of 20 μΙ_ of PAHBAH reagent (5% w/v 4-hydroxybenzoic acid hydrazide in 0.5 N HCI) and 10 μΙ_ of each reaction mixture. Plates were incubated at 95°C for 5 min and cooled down at 4°C for 5 sec. Samples (80 μΙ_) were then transferred to polystyrene microtiter plates (Costar 9017) and absorbance was read at 410 nm. Resulting absorbance values were plotted against enzyme concentration and linear regression was used to determine the slope of the linear region of the plot Accordingly, Thermostable beta amylase activity (TBA) unit is defined as one micromole of glucose equivalent released per min at pH 5.0, 50°C .
As used herein, "Pullulanase Activity Units (ASPU)" is an activity definition based on a colorimetric method that utilizes a soluble red-pullulan substrate for the
determination of pullulanase activity. As the pullulanase enzyme hydrolyze the substrate, soluble fragments of the dyed substrate are released into the reaction solution. Enzyme reaction is terminated by precipitating the substrate with an ethanol solution. The supernatant is evaluated for the color intensity with a spectrophotometer. The degree of color intensity is proportional to the enzyme activity. One Acid Stable Pullulanase Unit (ASPU) is defined as the amount of enzyme which liberates one equivalent reducing potential as glucose per minute from pullulan at pH 4.5 and a temperature of 60°C.
As used herein "dry solids" content refers to the total solids of a slurry in a dry weight percent basis. The term "slurry" refers to an aqueous mixture containing insoluble solids. The term "high ds" refers to an aqueous starch slurry containing dry solids greater than 38%. The term "Brix" refers to a well known hydrometer scale for measuring the sugar content of a solution at a given temperature. The Brix scale measures the number of grams of sucrose present per 100 grams of aqueous sugar solution (the total solubilized solid content). Brix measurements are frequently made by use of a hydrometer or refracto meter.
The term "degree of polymerization" (DP) refers to the number (n) of anhydro- glucopyranose 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. A DP4+ (>DP3) denotes polymers with a degree of polymerization of greater than 3, and contains everything that is not retained on the column - protein, lipids, carbs, etc). The term "DE," or "dextrose equivalent," is defined as the
percentage of reducing sugar, i.e., D-glucose, as a fraction of total carbohydrate in a syrup. It is an industry standard for the concentration of total reducing sugars, and is expressed as % D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE that is essentially 0 and D-glucose has a DE of 100.
As used herein the term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and
amylopectin with the formula (C6H 0O5)x, wherein X can be any number. The term includes plant-based materials such as grains, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, sweet sorghum brans, cassava, millet, potato, sweet potato, and tapioca. The term "starch" includes granular starch. The term "granular starch" refers to raw, i.e., uncooked starch, e.g., starch that has not been subject to gelatinization.
The term "glucose syrup" refers to an aqueous composition containing glucose solids. Glucose syrup will have a DE of at least 20. In some embodiments, glucose syrup will not contain more than 21 % water and will not contain less than 25% reducing sugar calculated as dextrose. In one embodiment, glucose syrup will include at least 90% D-glucose and in another embodiment glucose syrup will include at least 95% D-glucose. In some embodiments, the terms glucose and glucose syrup are used interchangeably. The term "maltose syrup" refers to an aqueous composition containing maltose solids. The maltose syrup will have maltose content of greater than 25% on a dry solids basis.
The term "total sugar content" refers to the total sugar content present in a starch composition.
The term "Refractive Index Dry Substance" (RIDS) is defined as the determination of the refractive index of a starch solution at a known DE at a controlled temperature, then converting the Rl to dry substance using an appropriate relationship, such as the Critical Data Tables of the Corn Refiners Association. The term "contacting" refers to the placing of the respective enzymes in sufficiently close proximity to the respective substrate to enable the enzymes to convert the substrate to the end product. Those skilled in the art will recognize that mixing solutions of the enzyme with the respective substrates can effect contacting.
The terms, "wild-type," "parental," or "reference," with respect to a polypeptide, refers to a naturally-occurring polypeptide that does not include a man-made
substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wild-type," "parental," or "reference," with respect to a polynucleotide, refers to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.
Reference to the wild-type protein is understood to include the mature form of the protein. A "mature" polypeptide means a polypeptide or variant thereof from which a signal sequence is absent. For example, the signal sequence may be cleaved during expression of the polypeptide.
The term "variant," with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally-occurring or man-made substitutions, insertions, or deletions of an amino acid. Similarly, the term "variant," with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.
In the case of the present enzymes, such as a beta amylase, "activity" refers to enzymatic activity, which can be measured as described, herein.
The term "recombinant," when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state.
Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. The terms "recovered," "isolated," and "separated," refer to a compound, protein (polypeptides), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature. An "isolated" enzyme, or variant thereof includes, but is not limited to, a culture broth containing secreted enzyme expressed in a heterologous host cell.
As used herein, the term "purified" refers to material {e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.
The terms "thermostable" and "thermostability," with reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated
temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (ti/2) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual glucoamylase activity following exposure to (i.e., challenge by) an elevated temperature. A "pH range," with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.
As used herein, the terms "pH stable" and "pH stability," with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).
As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an "enzyme." The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N→C).
The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation. As used herein, "hybridization" refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs 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 °C and 0.1 X SSC (where 1 X SSC = 0.15 M NaCI, 0.015 M Na3 citrate, pH 7.0).
Hybridized, duplex nucleic acids are characterized by a melting temperature (Tm), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the Tm.
As used herein, a "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism. As used herein, the terms "transformed," "stably transformed," and "transgenic," used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations. The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection," "transformation" or "transduction," as known in the art.
A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast, such as T. reesei) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.
The term "heterologous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell. The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
As used herein, the term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation. A "selective marker" or "selectable marker" refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials {e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell. A "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
An "expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting 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 suitable ribosome binding sites on the mRNA, enhancers and sequences that control termination of transcription and translation.
The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.
A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process. As used herein, "biologically active" refer to a sequence having a specified biological activity, such an enzymatic activity.
"Percent sequence identity" means that a variant has at least a certain percentage of amino acid residues identical to a reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:
Gap opening penalty: 10.0
Gap extension penalty: 0.05
Protein weight matrix: BLOSUM series
DNA weight matrix: IUB
Delay divergent sequences %: 40
Gap separation distance: 8
DNA transitions weight: 0.50
List hydrophilic residues: GPSNDQEKR
Use negative matrix: OFF
Toggle Residue specific penalties: ON
Toggle hydrophilic penalties: ON
Toggle end gap separation penalty OFF. Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of a mature 617 residue polypeptide would have a percent sequence identity of 99% (612/617 identical residues χ 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having "at least 99% sequence identity" to a mature polypeptide.
"Fused" polypeptide sequences are connected, i.e., operably linked, via a peptide bond between the two polypeptide sequences. The term "filamentous fungi" refers to all filamentous forms of the subdivision
Eumycotina.
The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microbial organism, such as an ethanologenic microorganism, and at least one enzyme, such as glucoamylase or a variant thereof, are present during the same process step. SSF includes the
contemporaneous hydrolysis of starch substrates (granular, liquefied, or solubilized) to saccharides, including glucose, and the fermentation of the saccharides into alcohol or other biochemical or biomaterial in the same reactor vessel.
As used herein "ethanologenic microorganism" refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.
The term "fermented beverage" refers to any beverage produced by a method comprising a fermentation process, such as a microbial fermentation, e.g., a bacterial and/or yeast fermentation.
"Beer" is an example of such a fermented beverage, and the term "beer" is meant to comprise any fermented wort produced by fermentation/brewing of a starch- containing plant material. Often, beer is produced exclusively from malt or adjunct, or any combination of malt and adjunct. Examples of beers include: full malted beer, beer brewed under the "Reinheitsgebot," ale, IPA, lager, bitter, Happoshu (second beer), third beer, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like, but also alternative cereal and malt beverages such as fruit flavored malt beverages, e.g., citrus flavored, such as lemon-, orange-, lime-, or berry-flavored malt beverages, liquor flavored malt beverages, e.g., vodka-, rum-, or tequila-flavored malt liquor, or coffee flavored malt beverages, such as caffeine-flavored malt liquor, and the like.
The term "malt" refers to any malted cereal grain, such as malted barley or wheat.
The term "adjunct" refers to any starch and/or sugar containing plant material which is not malt, such as barley or wheat malt. Examples of adjuncts include common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, cassava and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like.
The term "mash" refers to an aqueous slurry of any starch and/or sugar containing plant material, such as grist, e.g., comprising crushed barley malt, crushed barley, and/or other adjunct or a combination thereof, mixed with water later to be separated into wort and spent grains.
The term "wort" refers to the unfermented liquor run-off following extracting the grist during mashing. "Iodine-positive starch" or "IPS" refers to (1 ) amylose that is not hydrolyzed after liquefaction and saccharification, or (2) a retrograded starch polymer. When saccharified starch or saccharide liquor is tested with iodine, the high DPn amylose or the retrograded starch polymer binds iodine and produces a characteristic blue color. The saccharide liquor is thus termed "iodine-positive saccharide," "blue saccharide," or "blue sac."
The terms "retrograded starch" or "starch retrogradation" refer to changes that occur spontaneously in a starch paste or gel on ageing.
The term "about" refers to ± 5% to the referenced value. Exemplary Embodiments
The present teachings provide an improvement in the conversion of starch substrate to high maltose sugar syrup. The process comprises contacting an aqueous slurry of starch substrate (25 to 40% ds, pH 4.5-pH 6.5) with a liquefying thermostable alpha amylase and a thermoresistant maltose-producing enzyme; and, conducting the incubation at a temperature zero to 30°C above the starch gelatinization temperature to produce a high maltose syrup.
In some embodiments, the present teachings provide a recombinant host cell expressing a TthAmyl or variant thereof having at least 80% sequence identity to SEQ ID NO:2. In some embodiments, the TthAmy 1 or variant comprises 85%, 90%, 95%,
98%, or 99% percent identity to SEQ ID NO:2. In some embodiments, the recombinant host cell \sTrichoderma reesei.
In some embodiments, the recombinant host cell is a Bacillus.
In some embodiments of the present teachings, the recombinant TthAmyl or variant thereof has at least 70% activity at a pH range of 5.1 -7.1 as assayed under standard conditions of 50 °C, wherein the TthAmyl has at least 80%, 85%, 90%, 95%,
98%, or 99% identity to SEQ ID NO:3. In some embodiments, the recombinant
TthAmyl or variant thereof has an optimum temperature of about 95°C. In some embodiments, the recombinant TthAmyl comprises an amino acid sequence with at least 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO:3. In some embodiments, the recombinant TthAmyl is made in a T. reesei or Bacillus host cell, such as Bacillus subtilis.
In some embodiments, the present teachings provide a method for producing a recombinant TthAmy or variant thereof, comprising: (a) providing a T. reesei host cell or Bacillus host cell that expresses a recombinant TthAmyl or variant thereof having at least 80% sequence identity to SEQ ID NO:2; and, (b) culturing said host cell under conditions which permit the production of said recombinant TthAmyl or variant thereof.
In some embodiments, the host cell further expresses and secretes an alpha-amylase.
In some embodiments, the host cell further expresses and secretes a pullulanase and/or isoamylase. In some embodiments, the composition comprising starch is contacted with said host cell. In some embodiments of the present teaching, a method is provided of malto- saccharifying a composition comprising starch to produce a composition comprising maltose, wherein said method comprises: (i) contacting a starch composition with a thermoresistant maltose-producing enzyme, and an alpha amylase; and (ii) malto- saccharifying the starch composition to produce said composition comprising maltose. In some embodiments, the composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch. In some embodiments, the malto-saccharifying is conducted at a temperature range of about 65 °C to about 90 °C. In some embodiments, the temperature range is 75 °C - 85 °C. In some embodiments, the malto-saccharifying is conducted over a pH range of pH 4.0 - pH 6.0. In some embodiments, the pH range is pH 4.5 - pH 5.5. In some embodiments, the alpha-amylase comprises AmyE. In some embodiments, the alpha-amylase comprises SEQ ID NO:4. In some embodiments, the method further comprises contacting a starch composition with a pullulanase. In some embodiments, the method further comprises fermenting the maltose composition to produce an End of Fermentation (EOF) product. In some embodiments, the method further comprises adding an additional enzyme, wherein the additional enzyme is a glucoamylase, a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a protease, a pullulanase, an additional alpha amylase, an additional beta amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, an alpha-glucosidase, a beta-glucosidase, a lyase, a hydrolase, or a combination thereof, to said starch composition.
In some embodiments, thermoresistant maltose-producing enzyme is added at a dosage of 5-100 TBAs/g dss. In some embodiments, the thermoresistant maltose- producing enzyme is added at a dosage of 20-60TBAs/g dss. In some embodiments, the thermoresistant maltose-producing enzyme is added at a dosage of 30-50TBAs/g dss.
In some embodiments, the pullulanase is added at a dosage of 0.1 -10 kg/MT. In some embodiments, the pullulanase is added at a dosage of 1 -8 kg/MT.
In some embodiments, the present teachings provide a composition comprising maltose produced by the method of the present teachings, wherein the composition comprises a DP2 level of at least 42%, and wherein at least 90% of the starch is solubilized, wherein the malto-saccarifying occurs for between 2-12 hours. In some embodiments, the DP2 level is at least 45%, 50%, 60%, or 70%. In some embodiments, the malto-saccarifying occurs from between 4-12 hours, 6-10 hours, or 8-12 hours.
The method of the present teachings in some embodiments can be employed to produce a liquefied starch, or a fermented beverage, or a high maltose syrup.
In some embodiments, the present teachings provide a method of producing a food composition, comprising combining (i) one or more food ingredients, and (ii) an isolated TthAmyl or variant thereof of the present teachings, wherein said pullulanase and said isolated TthAmyl or variant thereof catalyze the hydrolysis of starch
components present in the food ingredients to produce maltose. In some embodiments, the food composition is selected from the group consisting of a food product, a baking composition, a food additive, an animal food product, a feed product, a feed additive, an oil, a meat, and a lard. In some embodiments, the one or more food ingredients comprise a baking ingredient or an additive. In some embodiments, the one or more food ingredients is selected from the group consisting of flour; an anti-staling amylase; a phospholipase; a phospholipid; a maltogenic alpha-amylase or a variant, homologue, or mutants thereof which has maltogenic alpha-amylase activity; a bakery xylanase (EC 3.2.1 .8); and a lipase. In some embodiments, the one or more food ingredients is selected from the group consisting of (i) a maltogenic alpha-amylase from Bacillus stearothermophilus, (ii) a bakery xylanase is from Bacillus, Aspergillus, Thermomyces or Trichoderma, (iii) a glycolipase from Fusarium heterosporum.
In some embodiments of the present teachings, the starch composition
comprises a starch slurry of 25 to 35% ds, 28 to 33%, or 30%-35%.
TthAmyl Beta Amylase Examples
The TthAmyl or variants thereof may be "precursor," "immature," or "full- length," in which case they include a signal sequence, or "mature," in which case they lack a signal sequence. The variant enzymes may also be truncated at the N- or C- termini, so long as the resulting polypeptides retain maltose generating activity. Cloning and expression of Thermoanaerobacterium thermosulfurigenes beta- amylase (TthAmyl ) was cloned and expressed as follows. TthAmyl (NCBI accession No. : P19584.1 ) is a beta-amylase from the bacterium Thermoanaerobacterium thermosulfurigenes. The protein sequence is depicted in SEQ ID NO:1 . At the N- terminus, the protein has a signal peptide with a length of 32 amino acids as predicted by SignalP-NN (Emanuelsson et al., Nature Protocols, 2:953-971 , 2007). The presence of a signal sequence suggests that TthAmyl is a secreted enzyme. A synthetic
TthAmyl gene was placed in a replicating plasmid pHPLT. As shown in the plasmid map of Figure 1 , the TthAmyl gene was under the control of the thermostable amylase LAT promoter (pLAT). A signal peptide from Bacillus licheniformis strain DSM13 was used to direct protein secretion. The plasmid was amplified using lllustra TempliPhi 100 Amplification Kit (GE Healthcare Life Sciences, NJ). Competent B. subtilis cells were transformed with the amplification product, and the cells were plated on Luria Agar plates supplemented with 10 ppm neomycin. Expression evaluation of TthAmyl showed that the enzyme expresses very well in Bacillus subtilis.
The amino acid sequence of TthAmyl is set forth as SEQ ID NO:1 . The predicted native signal peptide is shown in italics.
SEQ ID NO:1 migafkrlgqklfltlltaslifassivtanas\apnfkvfvmgp\eM
dwsyyktyadtvraaglkwvpimsthacggnvgdtvnipipswvwtkdtqdnmqykdeagnwdneavspwysgltq lynefyssfasnfssykdiitkiyisggpsgelrypsynpshgwtypgrgslqcyskaaitsfqnamkskygtiaavnsawg tsltdfsqispptdgdnfftngykttygndfltwyqsvltnelaniasvahscfdpvfnvpigakiagvhwlynsptmphaae ycagyynystlldqfkasnlamtftclemddsnayvspyysapmtlvhyvanlannkgivhngenalaisnnnqayvnc aneltgynfsgftllrlsnivnsdgsvtsemapfvinivtltpngtipvtftinnattyygqnvyivgstsdlgnwnttyargpasc pnyptwtitlnllpgeqiqfkavkidssgnvtweggsnhtytvptsgtgsvtitwqn
The mature TthAmyl gene sequence that was placed in plasmid pHPLT-TthAmyl is set forth as SEQ ID NO:2.
SEQ ID NO:2 agcattgcgccgaactttaaagtgtttgtgatgggcccgctggaaaaagtgacggattttaacgcgtttaaagatcagctgat tacgctgaaaaataatggcgtgtatggcattacgacggatatttggtggggctatgtggaaaatgcgggcgaaaatcagttt gattggagctattataaaacgtatgcggatacggtgcgtgcggcgggcctgaaatgggtgccgattatgagcacgcatgc gtgtggcggcaatgtgggcgatacggtgaatattccgattccgagctgggtgtggacgaaagatacgcaggataacatgc agtataaagatgaagcgggcaattgggataatgaagcggtgagcccgtggtatagcggcctgacgcagctgtataacg aattttatagcagctttgcgagcaattttagcagctataaagatatcatcacgaaaatctatattagcggcggcccgagcgg cgaactgcgctatccgagctataatccgagccacggctggacgtatccgggccgtggcagcctgcaatgctatagcaaa gcggcgattacgagctttcagaatgcgatgaaaagcaaatatggcacgattgcggcggtgaatagcgcgtggggcacg agcctgacggactttagccagattagcccgccgacggatggcgataatttttttacgaacggctataaaacgacgtatggc aacgattttctgacgtggtatcagagcgtgctgacgaatgaactggccaatattgcgagcgtggcgcatagctgctttgatc cggtgtttaatgtgccgattggcgcgaaaattgcgggcgtgcattggctgtataatagcccgacgatgccgcatgcggcgg aatattgcgcgggctattataattatagcacgctgctggatcagtttaaagcgagcaatctggccatgacgtttacgtgcctg gaaatggatgatagcaatgcgtatgtgagcccgtattatagcgcgccgatgaccctggtgcattatgtggcgaatctggcc aataataaaggcattgtgcataatggcgaaaatgcgctggccattagcaataataatcaggcgtatgtgaattgcgcgaat gaactgacgggctataattttagcggctttacgctgctgcgcctgagcaatattgtgaatagcgatggcagcgtgacgagc gaaatggcgccgtttgtgattaatattgtgacgctgacgccgaatggcacgattccggtgacgttcacgattaataacgcga cgacgtattatggccagaacgtgtatattgtgggcagcacgagcgatctgggcaattggaatacgacgtatgcgcgtggc ccggcgagctgtccgaattatccgacgtggacgatcacgctgaatctgctgccgggcgaacaaatccagtttaaagccgt gaaaattgatagcagcggcaatgtgacgtgggaaggcggcagcaatcatacgtatacggtgccgacgagcggcacgg gcagcgttacgattacgtggcagaactaa
The mature TthAmyl protein sequence is set forth as SEQ ID NO:3. SEQ ID NO:3 siapnfkvfvmgplekvtdfnafkdqlitlknngvygittdiwwgyvenagenqfdwsyyktyadtvraaglkwvpimsth acggnvgdtvnipipswvwtkdtqdnmqykdeagnwdneavspwysgltqlynefyssfasnfssykdiitkiyisggp sgelrypsynpshgwtypgrgslqcyskaaitsfqnamkskygtiaavnsawgtsltdfsqispptdgdnfftngykttygn dfltwyqsvltnelaniasvahscfdpvfnvpigakiagvhwlynsptmphaaeycagyynystlldqfkasnlamtftcle mddsnayvspyysapmtlvhyvanlannkgivhngenalaisnnnqayvncaneltgynfsgftllrlsnivnsdgsvts emapfvinivtltpngtipvtftinnattyygqnvyivgstsdlgnwnttyargpascpnyptwtitlnllpgeqiqfkavkidssg nvtweggsnhtytvptsgtgsvtitwqn
Fermentation of thermostable beta-amylase TthAmyl To begin the fermentation of TthAmyl , a seed culture {Bacillus subtilis) was grown in a 250-mL shake flask that contained 50 mL of LBG media consisting of 10g/L soytone, 5 g/L yeast extract, 10 g/L NaCI, 1 1 g/L glucose monohydrate, 1 .67 drop/L Mazu 6000 and 10ug/mL kanamycin. The seed culture was grown at 37°C with shaking at 250 rpm. After 5 hours growth, OD550 was checked. When OD550 was between 0.8-1 .2, 100 mL of seed culture (50 mL in each of 250-mL shake flask) was transferred to a fermenter. Fermentation medium (3.5 kg) was added in a 7-L fermenter (Applikon), containing 2 g/kg soytone, 1 .4 g/kg yeast extract, 8 g/kg KH2P04, 8 g/kg NaH2PO4-H2O, 2.8 g/kg MgSO4-7H2O, 0.1 g/kg CaCI2-2H20, 0.25 FeCI2-4H20, 0.22 g/kg MnS04-4H20, 13.9 ml/kg 4N H2S04, 5.7 g/kg glucose, 2 mL Mazu 6000, and 10 mL/kg Trace metals 100X stock (52.5 g/kg citric acid-H20, 2.18 g/kg ZnS04-7H20, 2.0 g/kg CoCI2-6H20, 2.0 g/kgNa2Mo04-H20, 1 .9 g/kg CuS04-5H20 and 0.5 g/kg H3B03).
Following inoculation with 100 mL of seed culture, the fermentation was initiated at 3.5 kg working volume and controlled at pH 6.9 and temperature 37 °C. DO was controlled above 20% by adjusting aeration and agitation. The feed of sterile 600 g/kg glucose solution was started at 5 EFT hour with feed rate of 0.25 g/min, and feed rate was ramped linearly in 14 hours to 0.71 g/min and maintained at this rate for the rest of the time. During the fed-batch fermentation period, the residual glucose would be controlled at 0 g/L. In addition, feed rate would be decreased if DO was still below 20% even though agitation was increased and air was up to 2VVM. Fermentation broth was sampled at 18.8 h and 46.3 h to run residual glucose measurement and SDS-PAGE analysis. Fermentation was terminated after 46.3 hours. Following centrifugation, filtration and ultrafiltration, 540 mL of concentrated sample was obtained. BCA assay (protein quantification kit, Shanghai Generay Biotech CO., Ltd) illustrated that total protein in concentrated sample was 22.56 g/L.
Purification of thermostable beta-amylase TthAmyl
TthAmyl was purified via beta-cyclodextrin coupled Sepharose 6 affinity chromatography, taking advantage of its carbohydrate binding domain. The 500 ml_ crude broth from the fermentor was concentrated by ultrafiltration and buffer exchanged with 20 mM HEPES pH 7.5. The solution was then loaded onto a 60-mL beta- cyclodextrin coupled Sepharose 6 column which was pre-equilibrated with 20 mM HEPES pH 7.5 (buffer A). The column was applied with a gradient of 0-100% buffer B (buffer A containing 10 mM alpha-cyclodextrin) in 2 column volumes, followed by 4 column volumes of 100% buffer B. The target protein was eluted in the gradient step. The fractions were pooled according to the activity and SDS-PAGE analysis results. After that, Gel Filtration chromatography was performed to get a high purity product. The solution collected in the previous step was concentrated into 10 ml_ and was subjected to the Hiload TM 26/600, Superdex-75 column (1 column volume = 320 ml_) which was pre-equilibrated with 20 mM sodium phosphate containing 0.15 M NaCI (pH 7.0). The active fractions from the superdex-75 column were pooled and concentrated using an Amicon Ultra-15 device with 10 K MWCO. The sample showed above 98% purity and was stored in 40% glycerol at -80°C until usage.
Enzyme Liquefied starch substrate:
An aqueous slurry containing 38% DS refined starch (Cargill, Minneapolis, NM), containing 10 ppm Ca2+, and 100 ppm sulfur dioxide (S02) was prepared by stirring overnight. The pH of the slurry was adjusted using sodium carbonate solution (20% w/v). The Baume (degrees) of the slurry were approximately 22.3. High temperature jet liquefaction was performed using 0.55 Kg SPEZYME® FRED (DuPont Industrial Biosciences) per Metric Ton of corn starch at pH 5.6. The slurry with the enzyme was sent through a pilot plant jet at 0.5 gpm with six-minute residence time and cooked at about 108 °C -1 10 °C for the primary cook. Secondary liquefaction was performed at 95°C for 120 minutes. The DE and refractive index (Rl) were measured i.e., a DE value of at least 10 in 90-100 min and used in these studies.
AMY E:
Purified AMY E was produced as described in US patent US 8,323,945, US patent application 2010-0003366 A1 , US patent application 2009-0305935 A1 , and US patent application 2009-0305360 A1 . "AmyE" for the purpose of this invention is a naturally occurring alpha-amylase. (EC 3.2.1 .1 ; 1 , 4-a-D-glucan glucanohydrolase) from B. subtilis.) referred to herein also as AmyE Combi L. The AmyE sequence used for the experiments described herein is SEQ ID NO: 4, is set forth below, and is herein also referred to as AmyE Combi L.
SEQ ID NO: 4
LTAPSIKSGTILHAWNWSFNTLKHNMKDIHDAGYTAIQTSPINQVKEGNQGDKSMSNW YWLYQPTSYQIGNRYLGTEQEFKEMCAAAKEYGIKVIVDAVMNHTTSDYAAISNEVKSI PNWTHGNTPIKNWSDRWDVTQNSLLGLYDWNTQNTQVQSYIKRFLDRALNDGADGF RFDAAKHIELPDDGSYGSSFWPNITNNSAEFQYGEILQDSVSRDAAYANYMDITASNY GHSIRSALKNRNLGVSNISHYAIDVEADKLVTWVESHDTYANDDEESTWMSDDDIRLG WAVIASRSGSTPLFFSRPEGGGNGVRFPGKSQIGDRGSALFEDQAITAVNRFHNVMA GQPEELSNPNGNNQIFMNQRGSHGVVLANAGSSSVSINTATKLPDGRYDNKAGAGSF QVNDGKLTGTINARSVAVLYPD
Any of a variety of Amy E sequences can be used according to the present teachings, including the AmyE having the amino acid sequence disclosed in NCBI Accession No.20 ABW75769, NCBI Accession Nos. ABK54355, AAF14358, AAT01440,
AAZ30064, AAQ83841 , and BAA31528. The alpha-amylase as contemplated herein may be a Bacillus subtilis alpha-amylase (AmyE) having an amino acid sequence of above or an alpha-amylase having at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to it.
The present invention is described in further detail in the following examples, which are not to be interpreted as a limitation thereof, or in any limiting fashion.
EXAMPLES
Example 1
The effect of pH (from 3.0 to 10.0) on thermostable beta-amylase activity was monitored using the PAHBAH assay protocol as described above for the thermostable beta-amylase specific activity measurement. Buffer working solutions contained the combination of glycine/sodium acetate/HEPES (250 mM), with pH varying from 3.0 to 1 0.0. Substrate solutions were prepared by mixing 896 μΙ_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 1 01 1 8), 1 00 μΙ_ of 250 mM buffer working solution (pH from 3.0 to 1 0.0), and 4 μΙ_ of 0.5 M CaCI2. Enzyme working solutions were prepared in water at a certain dose (showing signal within linear range as per dose response curve). All the incubations were done using the same protocol as described above for beta- amylase activity assay. The absorbance from a control (water-only) was subtracted, and the resulting values were converted to percentages of relative activity, by defining the activity at the optimal pH as 1 00% (Table 1 , and see FIG. 1 ). The optimal pH range can be defined as keeping >70% of activity and was determined as 5.1 -7.1 . Table 1 shows the pH profile of TthAmyl .
Table 1
Figure imgf000025_0001
Example 2
The effect of temperature (from 30 °C to 95°C) on thermostable beta-amylase activity was monitored using the PAHBAH assay protocol as described above for the thermostable beta-amylase specific activity measurement. Substrate solutions were prepared by mixing 3.6 ml_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 101 18), 0.4 mL of 0.5 M buffer (pH 5.0 sodium acetate), and 1 6 μΙ_ of 0.5 M CaCI2 into a 15-mL conical tube. Enzyme working solutions were prepared in water at a certain dose (showing signal within linear range as per dose response curve). Incubations were done at temperatures from 30 to 95°C, respectively, for 10 min at 600 rpm in a thermomixer (Eppendorf). After incubation, samples were quenched and measured using the same protocol as described above for beta-amylase activity assay. The absorbance from a control (water-only) was subtracted, and the resulting values were converted to percentages of relative activity, by defining the activity at the optimal temperature at 100% (Table 2, and see FIG. 2). The optimal temperature range can be defined as keeping >70% of activity and was determined as 73°C-95°C. The temperature profile of TthAmy 1 is shown in Table 2.
Table 2
Figure imgf000026_0001
Example 3
An evaluation of the single step liquefaction and malto-saccharifaction process at pH 5.5, 80 °C was done. This example describes a process where liquefaction and malto-saccharification were carried out in a single step at high temperature of 80 °C and pH 5.5. The experiments were carried out using 30% ds granular tapioca starch, with Dl water making up the 100% of the liquid phase. Water (19.65 g) and starch (10.35 g; at 86.9% ds) were mixed and the pH of the slurry was increased to pH 5.5 using sodium carbonate. The starch slurry was placed in a waterbath and maintained at 80 °C with continuous stirring and enzyme(s) were added. As shown in Table 3, SPEZYME® Xtra (DuPont Industrial Biosciences) was added at constant dose of 0.13 Kg per MT ds starch and the TthAmyl was added at 0.02 and 49.9 TBAs/g dss. The starch slurry was maintained at 80 °C for 7.5 hrs and samples were drawn at 1 .25, 3.5 and 7.5 hrs to analyze the percent solubility and saccharide profile. The saccharide distribution for SPEZYME® Xtra and TthAmyl mediated single step liquefaction and malto- saccharifcation process at 80 °C and pH 5.5 are shown in Table 3.
Table 3
Incubation % Sugar composition
%
Enzymes time,
DP1 DP2 DP3 DP4+ Solubility Hr
0.13 kg/MT dss SPEZYME® 1 .25 0.29 41 .92 10.32 47.47 97.6
Xtra 3.5 0.46 53.40 12.43 33.71 99.1
+ 24.95 TBAs/g dss TthAmyl 7.5 0.59 57.15 12.45 29.81 99.7
0.13 Kg/MT. dss SPEZYME® 1 .25 0.28 50.49 9.06 40.18 97.6
Xtra 3.5 0.46 59.07 9.90 30.57 99.1
+ 49.9 TBAs/g dss TthAmyl 7.5 0.60 61 .18 9.91 28.31 99.7
As shown in Table 3, the TthAmyl maintains a significant amount of maltogenic activity for 7.5 hours at 80 °C and pH 5.5, as evidenced by continuous generation of maltose, as well as continued increase in % soluble solids. An increased amount of TthAmyl results in increased rate of maltose generation and % solubilization.
Example 4
Evaluation of the single step liquefaction and malto-saccharifaction process at pH 5.0, 70°C with pullulanase OPTIMAX L -1000 was performed. The experiments were carried out using 30% ds granular tapioca starch adjusted to pH 5.0 using sodium carbonate. The starch slurry was placed in a water bath maintained at 70 °C with continuous stirring and enzyme(s) were added. As shown in Table 4, SPEZYME® Xtra (DuPont Industrial Biosciences) was added at constant dose of 0.13 Kg per MT per dss, the TthAmyl was added at constant dose of 24.95 TBAs/g dss and OPTIMAX® L-1 000 (DuPont Industrial Biosciences) was added at 1 .0 kg/MT to one of the two reactions. The starch slurry was maintained at 70 °C for 24 hrs and samples were drawn at 4, 6 and 24 hrs to analyze the percent solubility and saccharide profile. The saccharide distribution for SPEZYME® Xtra and TthAmyl mediated single step liquefaction and malto-saccharifcation process with pullulanase OPTIMAX L -1 000 at 70 °C and pH 5.0 are shown in Table 4.
Table 4
Incubation % Sugar composition
%
Enzymes time,
DP1 DP2 DP3 DP4+ Solubility Hr
0.1 3 Kg. per MT. dss 4 0.42 53.90 1 2.65 33.03 83.8
SPEZYME® Xtra 6 0.46 56.35 1 2.82 30.38 85.9
+ 24.95 TBAs/g dss TthAmyl 24 0.76 59.65 1 2.87 26.72 90.4
0.1 3 Kg. per MT. dss 0.45 54.83 1 3.77 30.95 84.7 SPEZYME® Xtra 0.51 58.03 14.23 27.23 86.9 + 24.95 TBAs/g dss TthAmyl
+ 1 .0 kg/MT OPTIMAX® L- 24 0.77 62.28 14.61 22.34 92.7
1 000
As shown in Table 4, addition of pullulanase resulted in increasing amount of maltose in the syrup as well as increased solubilization of soluble solids.
Example 5
Evaluation of different alpha amylases for the the single step liquefaction and malto-saccharifaction process at pH 6.0, 80 °C was performed. The experiments were carried out using 30% ds granular tapioca starch adjusted to pH 6.0 using sodium carbonate. The starch slurry was placed in a water bath maintained at 80 °C with
continuous stirring and enzyme(s) were added. The alpha amylases were added as shown in Table 5, with SPEZYME® Xtra (B. stearothermophilus alpha amylase)
(DuPont Industrial Biosciences) added at 0.1 3 Kg/ MT dss, SPEZYME® Fred (R
licheniformis alpha amylase) added at 1 0 LUs/ gdss and AmyE (B. subtilis alpha
amylase) (SEQ I D NO: 4) added at 3.0 μg/g dss. The TthAmyl was added at 49.9
TBAs/g dss. The starch slurry was maintained at 80 °C for 4 hrs and samples were drawn at 1 , 2, 3 and 4 hrs to analyze the percent solubility and saccharide profile.
Saccharide distribution for different sources of alpha amylases for single step liquefaction and malto-saccharifcation process are shown in Table 5.
Table 5
Incubation % Sugar composition
%
Enzymes time,
DP1 DP2 DP3 DP4+ Solubility Hr
1 0. .41 43. .59 12.42 43. .59 95. .1
0.13 Kg. per MT dss
2 0. .64 49. .65 15.05 34. .66 96. .9
SPEZYME® Xtra
3 0. .81 51 . .65 15.45 32. .09 99. .4
+ 49.9 TBAs/g dss TthAmyl
4 0. .96 53. .25 15.58 30. .21 99. .7
1 0. .27 47. .64 9.23 42. .85 95. .8
10.0 LUs/g dss SPEZYME®
2 0. .36 53. .64 10.43 35. .57 96. .6
Fred + 49.9 TBAs/g dss
3 0. .42 55. .95 10.64 32. .99 99. .7
TthAmyl
4 0. .46 57. .45 10.66 31 . .43 99. .9
1 0. .23 37. .32 12.40 50. .06 97. .3
13.89 AAU/g dss AmyE + 2 0. .52 44. .30 15.92 39. .28 99. .4
49.9 TBAs/g dss TthAmyl 3 0. .62 46. .37 1 6.85 36. .15 99. .7
4 0. .75 47. .72 17.37 34. .17 99. .9
As shown in Table 5, the single step liquefaction and malto-saccharification resulted in maltose rich syrup with different commercial liquefying alpha amylases in 4 hours of reaction time with high % solubilization.
Example 6:
Evaluation of different dosages of AmyE for the the novel single step liquefaction and malto-saccharifaction process at pH 6.0, 80 °C. Alpha amylase AmyE was
evaluated with increasing dosages for the single step liquefaction and malto- saccharification process. The experiments were carried out using 30% ds granular tapioca starch adjusted to pH 6.0 using sodium carbonate. The starch slurry was placed in a waterbath maintained at 80 °C with continuous stirring and enzyme(s) were added. The AmyE was added as shown in Table 6, at 1 3.89 AAU/g dss, 55.56 AAU/g dss, and 1 1 5.75 AAU/g dss. The TthAmyl was added at a constant dose of 49.9 TBAs/g dss. The starch slurry was maintained at 80 °C for 4 hrs and samples were drawn at 1 , 2, 3 and 4 hrs to analyze the percent solubility and saccharide profile. The saccharide distribution for different dosage of AmyE for single step liquefaction and malto- saccharifcation process is shown in Table 6.
Table 6
Incubation % Sugar composition q
Enzymes - time, DP4
DP1 DP2 DP3 Solubility
Hr +
37\3 5O0
1 0.23 1 2.40 97.3
2 6
44.3 39.2
2 0.52 1 5.92 99.4 1 3.89 AAU/g dss AmyE + 0 8
49.9 TBAs/g dss TthAmyl 46.3 36.1
3 0.62 1 6.85 99.7
7 5
47.7 34.1
4 0.75 1 7.37 99.9
2 7
34.7 45.4
1 0.90 1 8.91 99.1
8 1
55.56 AAU/g dss Amy E+ 42.1 35.1
2 1 .79 20.90 99.4 49.9 TBAs/g dss TthAmyl 1 9
44.6 32.0
3 2.27 21 .04 99.7
0 9 45.9 30.6
20.78 99 1 9
36.5 39.9
1 2.20 21 .30 97.1
7 2
42.7 32.0
2 3.74 21 .42 98.4 1 15.75 AAU/g dss AmyE + 9 5
49.9 TBAs/g dss TthAmyl 44.7 30.2
3 4.74 20.32 99.9
0 4
46.3 28.4
4 5.66 19.53 99.9
8 3
As shown in Table 6, increasing doses of AmyE resulted in increased amount of DP1 in the maltose rich syrup. AmyE also helps to increase in % soluble solids in the single step liquefaction and malto-saccharification process.
Example 7
Evaluation of different sources of granular starch in the single step liquefaction and malto-saccharifaction process at pH 6.0, 80 °C. Granular starch from corn and tapioca was treated with SPEZYME® Xtra and TthAmyl . The experiments were carried out using 30% ds granular starch adjusted to pH 6.0 using sodium carbonate. The starch slurry was placed in a waterbath maintained at 80 °C with continuous stirring and enzyme(s) were added. As shown in Table 6, SPEZYME® Xtra (DuPont Industrial Biosciences) was added at 0.13 Kg per MT dss and the TthAmyl was added at 49.9 TBAs/g dss. The starch slurry was maintained at 80 °C for 4 hrs and samples were drawn at 1 , 2, 3 and 4 hrs to analyze the percent solubility and saccharide profile. The saccharide distribution for different sources of starch in single step liquefaction and malto-saccharifcation process. Table 7
Incubatio % Sugar composition
%
Enzymes n time, DP4
DP1 DP2 DP3 Solubility Hr
1 0.41 43.59 1 2.42 43.59 95.1
2 0.64 49.65 1 5.05 34.66 96.9
Tapioca starch
3 0.81 51 .65 1 5.45 32.09 99.4
4 0.96 53.25 1 5.58 30.21 99.7
1 0.26 45.71 9.64 44.39 1 00
2 0.35 52.08 1 1 .41 36.17 1 00
Corn starch
3 0.42 54.74 1 1 .92 32.92 1 00
4 0.46 56.49 1 2.08 30.98 1 00
As shown in Table 7, the single step liquefaction and malto-saccharification process resulted in high maltose using starch from different sources.
Example 8
An evaluation of TthAmyl with starch liquefact prepared with jet liquefaction system and comparison to the single step liquefaction and malto-saccharifaction process at pH 6.0, 80 °C was done. A 38% DS refined starch (Cargill, Minneapolis, MN) slurry containing 1 0 ppm Ca2+' and 1 00 ppm sulfur dioxide (S02) was prepared in a metal bucket with overnight stirring. The pH of the slurry was adjusted to pH 5.8 using sodium carbonate solution. The slurry Baume (degrees) was approximately 22.3. The liquefaction was carried out with SPEZYME® Fred 10 LUs/g ds. The slurry with the enzyme(s) added was sent through a pilot plant jet cooker (Hydro-thermal Corporation, Waukesha, Wl) at 0.5 gpm with six-minute residence time and cooked at about 1 08 °C - 1 1 0 °C for the primary cook. Secondary liquefaction was performed at 95°C for achieving -1 0 DEs. The DE and refractive index (Rl) were measured at various time points during the secondary liquefaction. The active alpha amylase in the liquefact was deactivated by reducing the pH to 4.0 and incubating at 95 °C for 30 min. After that the liquefact was cooled down to 60 °C and pH was adjusted to 6.0. A 30 g aliquot of this liquefact at 34% ds was weighed out in a glass bottle and TthAmyl was added at 49.9 TBAs/g dss. The sample was incubated at 80 °C for 24 hours. Samples were drawn at 1 , 5 and 24 hours to evaluate the saccharide distribution. The saccharide distribution for liquefact prepared with conventional jet liquefaction and high temperature malto- saccharification process is shown in Table 8.
Table 8
Incubation % Sugar composition
Enzymes time,
DP1 DP2 DP3 DP4+
Hr
SPEZYME® Fred liquefact at 1 0.55 44.89 1 1 .48 43.09
10 DE 5 0.6 56.65 1 1 .16 31 .58 + 49.9 TBAs/g dss TthAmyl 24 0.71 58.42 1 1 .1 1 29.77
As shown in Table 8, the liquefact prepared with SPEZYME® Fred using conventional jet liquefaction also resulted in comparable amount of maltose from one step process for maltose production.
Example 9
An evaluation of different enzyme treatments for the novel single step process high in malto-saccharifaction process at pH 6.0, 80 °C was done. Granular starch from tapioca was treated with SPEZYME® Fred and TthAmyl for the single step liquefaction and malto-saccharification process. Followed by the single step liquefaction and malto- saccharification process for 4 hours the maltose rich syrup was divided in three samples, namely, 1 ) A, 2) B and 3) C. The sample A was incubated at 60 °C without any additional enzymes. The sample B was incubated at 60 °C with addition of barley beta- amylase OPTIMALT® BBA dosed at 0.5 kg/MT ds and pullulanase OPIMAX® L-1000 dosed at 1.0 kg/MT. The sample C was incubated at 60 °C with addition of pullulanase OPIMAX® L-1000 dosed at 1.0 kg/MT. The saccharide distribution for different enzyme treatments in single step liquefaction and malto-saccharification process is shown in Table 9.
Table 9
Extra Incubation % Sugar composition Enzymes time,
DP1 DP2 DP3 DP4+ hr
1 0 .34 41.58 11.08 47
2 0 .59 47.86 14.77 36.78
3 0 .78 50.41 15.63 33.17
4 0 .93 51.53 15.91 31.64
Tapioca starch with
1A 5.5 0 .99 51.98 15.98 31.05 SPEZYME® Fred 10 LUs/g
1B 5.5 1 .03 53.43 16.08 29.46 dss and various enzyme
1C 5.5 1 .03 51.91 16.16 30.91 treatments
1A 7 1 .07 52.4 16.19 30.34
1B 7 1 .08 54.16 16.07 28.68
1C 7 1 .04 52.34 16.13 30.49
1A 22.5 1 .39 54.33 16.11 28.18
1B 22.5 1 .44 56.1 16.72 25.74
1C 22.5 1 .43 54.78 16.76 27.04
As shown in Table 9, the addition of pullulanase helps in decreasing the DP4+ amount from 28.18% to 25.74 to 27.04% with and without additional beta-amylase. The pullulanase in this study is not thermostable hence the temperature had to be reduced to 60 °C. TthAmyl Variant Characterization
As will be appreciated by one of skill in the art in light of the present teachings, variant TthAmyl polypeptides are those that retain beta-amylase activity. They may have a specific activity higher or lower than the wild-type TthAmyl polypeptide.
Additional characteristics of the TthAmyl variant include stability, pH range,
temperature profile, oxidation stability, and thermostability. For example, the variant may be pH stable for 24-60 hours from pH 3 to about pH 8, e.g., pH 3.0 - 7.8; e.g., pH 3.0 - 7.5; pH 3.5 - 7.0; p H 4.0 - 6.7; or pH 5.0. A TthAmyl variant can be expressed at higher levels than the wild-type TthAmyl , while retaining the performance
characteristics of the wild-type TthAmyl . TthAmyl variants also may have altered oxidation stability in comparison to the parent glucoamylase. For example, decreased oxidation stability may be advantageous in compositions for starch liquefaction. The variant TthAmyl , can have altered temperature profile compared to the wild-type beta amylase. Such TthAmyl variants are advantageous for use in baking or other processes that require elevated temperatures. Levels of expression and enzyme activity can be assessed using standard assays known to the artisan skilled in this field, including those disclosed below.
Production of TthAmyl and Variants Thereof
The TthAmyl or variant thereof can be isolated from a host cell, for example by secretion of the TthAmyl or variant from the host cell. A cultured cell material comprising TthAmyl or variant thereof can be obtained following secretion of the
TthAmyl or variant from the host cell. The TthAmyl or variant is optionally purified prior to use. The TthAmyl gene can be cloned and expressed according to methods well known in the art. Suitable host cells include bacterial, plant, or yeast cells, e.g., filamentous fungal cells. Particularly useful host cells include Trichoderma reesei and Bacillus subtilus. Others include Bacillus licheniformis, and yeasts. In some
embodiments, Trichoderma reesei and Bacillus subtilus host cells can express TthAmyl at higher, or at least comparable, levels to natively expressed TthAmyl . The host cell may further express a nucleic acid encoding a homologous or heterologous beta amylase, i.e., a beta amylase that is not the same species as the host cell, or one or more other enzymes. The beta amylase may be a variant amylase. Additionally, the host may express one or more accessory enzymes, proteins, peptides. These may benefit liquefaction, saccharification, fermentation, SSF, etc. processes. Furthermore, the host cell may produce biochemicals in addition to enzymes used to digest the carbon feedstock(s). Such host cells may be useful for fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need to add enzymes.
Vectors
A DNA construct comprising a nucleic acid encoding a TthAmyl or variant thereof can be constructed to be expressed in a host cell. Representative nucleic acids that encode TthAmyl include SEQ ID NO: 2. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding a TthAmyl or variant thereof can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below. The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding a TthAmyl or variant thereof can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional TthAmyl or variant thereof. Host cells that serve as expression hosts can include filamentous fungi. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, aiwww.fgsc.net (last modified January 17, 2007).
A nucleic acid encoding a TthAmyl or a variant thereof can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a- amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding a TthAmyl or variant thereof is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable
promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. Additional discussion of promoters can be found in Liu et al. (2008) "Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl ) promoter optimization," Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158- 65.
The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the TthAmyl gene to be expressed. For example, the DNA may encode the TthAmyl signal sequence of operably linked to a nucleic acid encoding a TthAmyl or a variant thereof. The DNA encodes a signal sequence from a species other than T.
thermosulfurigenes. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.
An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a TthAmyl or variant thereof. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYCI 77, pUB1 10, pE194, pAMB1 , and plJ702.
The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD, and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91 /17243.
Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of a TthAmyl or variant thereof for subsequent purification. Extracellular secretion of the TthAmy or variant thereof into the culture medium can also be used to make a cultured cell material comprising the isolated TthAmyl or variant thereof.
The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the TthAmyl or variant thereof to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence serine-lysine-leucine (SKL), which is a known peroxisome target signal. For expression under the direction of control sequences, the nucleic acid sequence of the TthAmyl or variant thereof is operably linked to the control sequences in proper manner with respect to expression. The procedures used to ligate the DNA construct encoding an TthAmyl or variant thereof, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ed., Cold Spring Harbor, 1 989, and 3RD ed., 2001 ).
Transformation and Culture of Host Cells
A Trichoderma reesei host cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of a TthAmyl or variant thereof. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell.
Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
It is advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene
inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2, egl1, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.
Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction;
transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001 ), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No.
6,022,725. Reference is also made to Cao et al. (2000) Science 9:991 -1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding a TthAmyl or variant thereof is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques. The preparation of Trichoderma sp. for transformation, for example, may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56. The mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1 .2 M, e.g., a 1 .2 M solution of sorbitol can be used in the suspension medium.
Uptake of DNA into the host Trichoderma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50 mM CaCI2 is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.
Usually transformation of Trichoderma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105 to 107/mL, particularly 2x106/ml_. A volume of 100 μί of these protoplasts or cells in an
appropriate solution (e.g., 1 .2 M sorbitol and 50 mM CaCI2) may be mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725.
Expression
A method of producing a TthAmyl or variant thereof may comprise cultivating a Trichoderma reesei host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium. Trichoderma reesei host cells express TthAmyl at higher, or at least comparable, levels to natively expressed TthAmyl .
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of a TthAmyl or variant thereof. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of a glucoamylase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the glucoamylase to be expressed or isolated. The term "spent whole fermentation broth" is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term "spent whole fermentation broth" also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity
chromatography, or the like.
The polynucleotide encoding TthAmyl or a variant thereof in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The control sequences may be modified, for example by the addition of further
transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.
Host cells may be cultured under suitable conditions that allow expression of the TthAmyl or variant thereof. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate
expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system.
An expression host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25 °C to about 78°C (e.g., 30 °C to 45 °C), depending on the needs of the host and production of the desired TthAmyl or variant thereof. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of a TthAmyl or variant thereof.
Identification of TthAmyl Activity To evaluate the expression of a TthAmyl or variant thereof in a host cell, assays can measure the expressed protein, corresponding mRNA, or maltose- producing activity. For example, suitable assays include Northern blotting, reverse transcriptase polymerase chain reaction, and in situ hybridization, using an
appropriately labeled hybridizing probe. Suitable assays also include measuring
TthAmyl activity in a sample, for example, by assays directly measuring reducing sugars such as maltose in the culture media.
Methods for Purifying TthAmyl and Variants Thereof
Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated TthAmyl or variant glucoamylase polypeptide-containing solution.
After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.
It is desirable to concentrate a TthAmyl or variant polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions can require increased incubation time in order to collect the purified enzyme precipitate. The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of purification include but are not limited to rotary vacuum filtration and/or ultrafiltration. The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated TthAmyl or variant polypeptide-containing solution is at a desired level.
Concentration may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent. Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides, and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide, and blends of two or more of these metal halides. The metal halide precipitation agent, sodium chloride, can also be used as a preservative.
The metal halide precipitation agent is used in an amount effective to precipitate the TthAmyl or variant thereof. The selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.
Generally, at least about 5% w/v (weight/volume) to about 25% w/v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w/v.
Generally, no more than about 25% w/v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w/v. The optimal concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific TthAmyl or variant polypeptide and on its concentration in the concentrated enzyme solution.
Another alternative way to precipitate the enzyme is to use organic
compounds. Exemplary organic compound precipitating agents include: 4- hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4- hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of said organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.
Generally, the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyi esters of 4-hydroxybenzoic acid, wherein the alkyi group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitation agents can be, for example, linear or branched alkyi esters of 4-hydroxybenzoic acid, wherein the alkyi group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds. Exemplary organic compounds are linear alkyi esters of 4-hydroxybenzoic acid, wherein the alkyi group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used. Additional organic compounds also include but are not limited to 4-hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both amylase preservative agents. For further descriptions, see, e.g., U.S. Patent No. 5,281 ,526. Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature,
TthAmyl or variant polypeptide concentration, precipitation agent concentration, and time of incubation.
The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing. Generally, at least about 0.01 % w/v of organic compound precipitation agent is added to the concentrated enzyme solution and usually at least about 0.02% w/v. Generally, no more than about 0.3% w/v of organic compound precipitation agent is added to the concentrated enzyme solution and usually no more than about 0.2% w/v.
The concentrated polypeptide solution, containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be purified. Generally, the pH is adjusted at a level near the isoelectric point of the glucoamylase. The pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pi) up to about 2.5 pH units above the isoelectric point.
The incubation time necessary to obtain a purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less than about 10 hours and in most cases even about 6 hours.
Generally, the temperature during incubation is between about 4°C and about 50 °C. Usually, the method is carried out at a temperature between about 10°C and about 45 °C {e.g., between about 20 °C and about 40 °C). The optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used.
The overall recovery of purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound. The agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique. After the incubation period, the purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further purification of the purified enzyme precipitate can be obtained by washing the precipitate with water. For example, the purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.
During fermentation, a TthAmyl or variant polypeptide accumulates in the culture broth. For the isolation and purification of the desired TthAmyl or variant, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme purification. In one embodiment, the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation- precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column, and eluted to recover the enzyme-active fraction. For further purification, a conventional procedure such as ion exchange chromatography may be used.
Purified enzymes, as well as partially purified enzymes, are useful for laundry and cleaning applications. For example, they can be used in laundry detergents and spot removers. They can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).
For production scale recovery, a TthAmyl or variant polypeptide can be partially purified as generally described above by removing cells via flocculation with polymers. Alternatively, the enzyme can be purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be purified, and whole broth culture can be lysed and used without further treatment; or the broth can be clarified, and optionally concentrated and/or further processed, and used. The enzyme can then be processed, for example, into granules. Compositions and Uses of TthAmyl and Variants Thereof
Preparation of Starch Substrates
Those of general skill in the art are well aware of available methods that may be used to prepare starch substrates for use in the processes disclosed herein. For example, a useful starch substrate may be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically, the granular starch may be obtained from corn, cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, sweet sorghum, rice, peas, bean, banana, or potatoes. Corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch;
wheat contains about 60-65% starch; and polished rice contains 70-72% starch.
Specifically contemplated starch substrates are corn starch and wheat starch. The starch from a grain may be ground or whole and includes corn solids, such as kernels, bran and/or cobs. The starch may be highly refined raw starch or feedstock from starch refinery processes. Various starches also are commercially available. For example, corn starch is available from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is available from Sigma; sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).
The starch substrate can be a crude starch from milled whole grain, which contains non-starch fractions, e.g., germ residues and fibers. Milling may comprise either wet milling or dry milling or grinding. In wet milling, whole grain is soaked in water or dilute acid to separate the grain into its component parts, e.g., starch, protein, germ, oil, kernel fibers. Wet milling efficiently separates the germ and meal (i.e., starch granules and protein) and is especially suitable for production of syrups. In dry milling or grinding, whole kernels are ground into a fine powder and often processed without fractionating the grain into its component parts. In some cases, oils from the kernels are recovered. Dry ground grain generally will comprise significant amounts of non- starch carbohydrate compounds, in addition to starch. Dry grinding of the starch substrate can be used for production of ethanol and other biochemicals. The starch to be processed may be a highly refined starch quality, for example, at least 90%, at least 95%, at least 97%, or at least 99.5% pure.
Fermentation The soluble starch hydrolysate, particularly a maltose rich syrup, can be fermented by contacting the starch hydrolysate with a fermenting organism typically at a temperature around 32°C, such as from 30 °C to 35 °C. EOF products include
metabolites, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, lysine and other amino acids, omega 3 fatty acid, butanol, isoprene, 1 ,3-propanediol and other
biomaterials.
Ethanologenic microorganisms include yeast, such as Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis, expressing alcohol dehydrogenase and pyruvate decarboxylase. The ethanologenic microorganism can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. Improved strains of ethanologenic microorganisms, which can withstand higher temperatures, for example, are known in the art and can be used. See Liu et al. (201 1 ) Sheng Wu Gong Cheng Xue Bao 27(7): 1049-56. Commercial sources of yeast include ETHANOL RED® (LeSaffre); Thermosacc® (Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); and SUPERSTART® (Alltech). Microorganisms that produce other metabolites, such as citric acid and lactic acid, by fermentation are also known in the art. See, e.g., Papagianni (2007) "Advances in citric acid fermentation by Aspergillus niger. biochemical aspects, membrane transport and modeling," Biotechnol. Adv. 25(3): 244- 63; John et al. (2009) "Direct lactic acid fermentation: focus on simultaneous
saccharification and lactic acid production," Biotechnol. Adv. 27(2): 145-52.
The saccharification and fermentation processes may be carried out as an SSF process. Fermentation may comprise subsequent purification and recovery of ethanol, for example. During the fermentation, the ethanol content of the broth or "beer" may reach about 8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to produce enriched, e.g., 96% pure, solutions of ethanol. Further, C02 generated by fermentation may be collected with a C02 scrubber, compressed, and marketed for other uses, e.g., carbonating beverage or dry ice production. Solid waste from the fermentation process may be used as protein-rich products, e.g., livestock feed. As mentioned above, an SSF process can be conducted with fungal cells, such as Trichoderma reesei, that express and secrete TthAmyl or its variants continuously throughout SSF. The fungal cells expressing TthAmyl or its variants also can be the fermenting microorganism, e.g., an ethanologenic microorganism. Ethanol production thus can be carried out using a fungal cell that expresses sufficient TthAmyl or its variants so that less or no enzyme has to be added exogenously. The fungal host cell can be from an appropriately engineered fungal strain. Fungal host cells that express and secrete other enzymes, in addition to TthAmyl or its variants, also can be used. Such cells may express a-amylase and/or a pullulanase, phytase, alpha- glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, £>efa-glucosidase, pectinase, esterase, redox enzymes, transferase, a glucoamylase other than TthAmy 1 or other enzyme.
A variation on this process is a "fed-batch fermentation" system, where the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. The actual substrate concentration in fed-batch systems is estimated by the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases, such as C02. Batch and fed-batch fermentations are common and well known in the art.
Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation permits modulation of cell growth and/or product concentration. For example, a limiting nutrient such as the carbon source or nitrogen source is maintained at a fixed rate and all other parameters are allowed to moderate. Because growth is maintained at a steady state, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of optimizing continuous fermentation processes and maximizing the rate of product formation are well known in the art of industrial microbiology.
Compositions Comprising TthAmyl or Variants Thereof
Other suitable enzymes that can be used with TthAmyl or its variants include a thermoresistant maltose-producing enzyme that is not TthAmyl , phytase, protease, pullulanase, β-amylase, isoamylase, a-amylase, alpha-glucosidase, cellulase, xylanase, hemicellulase, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, or a combination thereof.
Other Uses for TthAmyl and thermoresistant maltose-producing enzymes
In some embodiments, the thermoresistant maltose-producing enzymes provided herein, including TthAmyl and variants thereof, can be used in a direct starch to maltose process as described in US2013/30384, Method for Making High Maltose Syrup, filed March 12, 2013, claiming priority to US Provisional 61 /61 6990, filed March 28, 2012.

Claims

WHAT IS CLAIMED IS: . A recombinant host cell expressing a TthAmyl or variant thereof having at least 80% sequence identity to SEQ ID NO: 2.
2. The recombinant host cell of claim 1 wherein the host is Trichoderma reesei.
3. The recombinant host cell of claim 1 wherein the host is a Bacillus.
4. A recombinant TthAmyl or variant thereof, wherein said TthAmyl has at least 70% activity at a pH range of 5.1 -7.1 as assayed under standard conditions of 50 °C, wherein the TthAmyl has at least 80% identity to SEQ ID NO:3.
5. The recombinant TthAmyl or variant thereof of claim 4, wherein said TthAmyl has an optimum temperature of about 95 °C.
6. The recombinant TthAmyl of any one of claims 4-5 comprising an amino acid sequence with at least 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO:3.
7. The recombinant TthAmyl of any of claims 4-6 made in a T. reesei or Bacillus host cell.
8. The recombinant TthAmy 1 of any of claims 4-7 wherein the Bacillus host is Bacillus subtilis.
9. A method for producing a recombinant TthAmy or variant thereof, comprising:
(a) providing a T. reesei host cell or Bacillus host cell that expresses a recombinant TthAmyl or variant thereof having at least 80% sequence identity to SEQ ID NO: 2; and, (b) culturing said host cell under conditions which permit the production of said recombinant TthAmyl or variant thereof.
10. The method of claim 9, wherein said host cell further expresses and secretes an alpha-amylase.
1 1 . The method of any of claims 9-10, wherein said host cell further expresses and secretes a pullulanase and/or isoamylase.
12. The method of any one of claims 9-1 1 , wherein said composition comprising starch is contacted with said host cell.
13. A method of malto-saccharifying a composition comprising starch to produce a composition comprising maltose, wherein said method comprises:
(i) contacting a starch composition with a thermoresistant maltose-producing enzyme, and an alpha amylase; and
(ii) malto-saccharifying the starch composition to produce said composition comprising maltose.
14. The method of claim 13, wherein said composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch.
15. The method of any of claims 13-14, wherein the malto-saccharifying is conducted at a temperature range of about 65 °C to about 90 °C.
1 6. The method of any of claims 12-15 wherein said temperature range is 75 °C - 85 °C.
17. The method of any one of claims 13-1 6, wherein the malto-saccharifying is conducted over a pH range of pH 4.0 - pH 6.0.
18. The method of any of claims 13-17, wherein said pH range is pH 4.5 - pH 5.5.
19. The method of any of claims 13-18, wherein the alpha-amylase comprises AmyE.
20. The method of any of claims 13-19, wherein the alpha-amylase comprises SEQ ID NO:4.
21 . The method of any one of claims 13-20, wherein the method further comprises contacting a starch composition with a pullulanase.
22. The method of any one of claims 13-21 , further comprising fermenting the maltose composition to produce an End of Fermentation (EOF) product.
23. The method of any one of claims 13-22, further comprising adding an additional enzyme, wherein the additional enzyme is a glucoamylase, a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a protease, a pullulanase, an additional alpha amylase, an additional beta amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, an alpha-glucosidase, a beta- glucosidase, a lyase, a hydrolase, or a combination thereof, to said starch composition.
24. The method of any of claims 13-23, wherein said thermoresistant maltose- producing enzyme is added at a dosage of 5-100 TBAs/g dss.
25. The method of any of claims 13-24, wherein said thermoresistant maltose- producing enzyme is added at a dosage of 20-60TBAs/g dss.
26. The method of any one of claims 13-25, wherein said pullulanase is added at a dosage of 0.1 -10 kg/MT.
27. A composition comprising maltose produced by the method of any one of claims 1 3-26, wherein the composition comprises a DP2 level of at least 42%, and wherein at least 90% of the starch is solubilized, wherein the malto-saccarfiying occurs for between 2-1 2 hours.
28. A liquefied starch produced by the method of any one of claims 13-27.
29. A fermented beverage produced by the method of any one of claims 1 3-28.
30. Use of the TthAmyl or variant thereof of any of claims 1 3-29 in the production maltose.
31 . A method of producing a food composition, comprising combining
(i) one or more food ingredients, and
(ii) an isolated TthAmyl or variant thereof of claims 4-8,
wherein said pullulanase and said isolated TthAmyl or variant thereof catalyze the hydrolysis of starch components present in the food ingredients to produce maltose.
32. The method claim 31 , wherein the food composition is selected from the group consisting of a food product, a baking composition, a food additive, an animal food product, a feed product, a feed additive, an oil, a meat, and a lard.
33. The method of any one of claims 31 or 32 wherein the one or more food ingredients comprise a baking ingredient or an additive.
34. The method of any one of claims 31 -33, wherein said one or more food ingredients is selected from the group consisting of flour; an anti-staling amylase; a phospholipase; a phospholipid; a maltogenic alpha-amylase or a variant, homologue, or mutants thereof which has maltogenic alpha-amylase activity; a bakery xylanase (EC 3.2.1 .8) ; and a lipase.
35. The method claim 34, wherein said one or more food ingredients is selected from the group consisting of
(i) a maltogenic alpha-amylase from Bacillus stearothermophilus,
(ii) a bakery xylanase is from Bacillus, Aspergillus, Thermomyces or Trichoderma, (iii) a glycolipase from Fusarium heterosporum.
36. The method according to any of claims 13-35 wherein the starch composition comprises a starch slurry of 25 to 35% ds.
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