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WO2015066667A1 - Protéases utilisées dans le traitement du blé - Google Patents

Protéases utilisées dans le traitement du blé Download PDF

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Publication number
WO2015066667A1
WO2015066667A1 PCT/US2014/063791 US2014063791W WO2015066667A1 WO 2015066667 A1 WO2015066667 A1 WO 2015066667A1 US 2014063791 W US2014063791 W US 2014063791W WO 2015066667 A1 WO2015066667 A1 WO 2015066667A1
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WIPO (PCT)
Prior art keywords
enzyme
slurry
protease
wheat
starch
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PCT/US2014/063791
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English (en)
Inventor
Vivek Sharma
Paula Johanna Maria Teunissen
Regina Chin
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Danisco Us Inc.
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Filing date
Publication date
Application filed by Danisco Us Inc. filed Critical Danisco Us Inc.
Publication of WO2015066667A1 publication Critical patent/WO2015066667A1/fr

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Classifications

    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Alcohol fermentation production processes and particularly ethanol production processes are generally characterized as wet milling or dry milling processes. Reference is made to Bothast et al., 2005,Appl. Microbiol. Biotechnol. 67: 19-25 and THE ALCOHOL TEXTBOOK, 5th Ed, W.M. Ingledew et al. Eds, 2009, Nottingham University Press, UK for a review of these processes.
  • Wet milling process involves a series of soaking (steeping) steps to soften the cereal grain wherein soluble starch is removed followed by recovery of the germ, fiber (bran) and gluten (protein). The remaining starch is further processed by drying, chemical and/or enzyme treatments. The starch may then be used for alcohol production, high fructose corn syrup or commercial pure grade starch or other fermentration products.
  • Dry grain milling involves a number of basic steps, which include: grinding, cooking, liquefaction, saccharification, fermentation and separation of liquid and solids to produce alcohol and other co-products.
  • whole cereal such as corn cereal
  • a slurry tank is subjected to high temperatures in a jet cooker along with liquefying enzymes (e.g. alpha-amylases) to solubilize and hydrolyze the starch in the cereal to dextrins.
  • liquefying enzymes e.g. alpha-amylases
  • saccharifying enzymes e.g. glucoamylases
  • the mash containing glucose is then fermented for approximately 24 to 120 hours in the presence of ethanol producing microorganisms.
  • the solids in the mash are separated from the liquid phase and ethanol and useful co-products such as distillers' grains are obtained.
  • Increased ethanol production may result by use of the simultaneous saccharification and fermentation processes.
  • Wheat is a primary feedstock for grain processing in many parts of the world for the production of fuel alcohol and starch. Wheat grain is similar to other grains in total proximate composition but differs in processing characteristics. Wheat processing in the dry grind process suffers with higher viscosity during liquefaction due to the presence of glucans and
  • arabinoxylans Other similar grains include, but are not limited to, triticale, barley, rye, etc. Due to the high viscosity, processing plants may need to add other enzymes and use lower solids concentrations to avoid processing problems.
  • Wheat processing also suffers with high amount of foaming characteristic during the fermentation process for fuel alcohol. Perhaps, the reduced amount of oil in wheat and presence of specific soluble proteins lead to high amounts of surface foaming during fermentation. To combat the foaming problems, plant owners may add anti-foaming chemicals. In addition, fermentation tanks are only partially filled to compensate for the extra head space that the foam may take up.
  • the present invention relates to, among other things, use of proteases that may help reduce the viscosity during liquefaction and the foaming characteristic during fermentation of wheat processing. Also the proteases may enhance the wheat, and other similar grains' ethanol fermentation rates.
  • the invention relates to use of proteases during the process of conversion of starch containing material, e.g., wheat or other similar grains, to fermentation products, such as alcohol, providing benefits such as (1) reducing the foaming characteristics of the grain slurry during liquifaction and fermentation and (2) reducing the viscosity during liquefaction unit operation and 3) enhancing the rate and/or yield of the fermentation.
  • benefits such as (1) reducing the foaming characteristics of the grain slurry during liquifaction and fermentation and (2) reducing the viscosity during liquefaction unit operation and 3) enhancing the rate and/or yield of the fermentation.
  • the invention comprises a method for producing a fermentation product from wheat or similar grain, comprising: liquifying the wheat-like grain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 65°C using: an alpha amylase, a protease; and optionally a beta-glucanase; saccharifying and fermenting the slurry using enzymes, including a glucoamylase and a fermenting organism to produce the
  • the invention comprises a method for increasing the yield and/or rate of ethanol production from wheat or similar grain, comprising: liquifying the wheat-like grain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 65°C using: an alpha amylase, a protease; and optionally a beta-glucanase; saccharifying and fermenting the slurry using enzymes, including a glucoamylase and a fermenting organism to produce ethanol, wherein the yield and/or rate of ethanol production is greater than the rate observed witout the addition of protease.
  • the invention comprises a method for reducing the viscosity of the wheat-like grain slurry during a fermentation, comprising: liquifying the wheat-like grain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 65°C using: an alpha amylase, a protease; and optionally a beta-glucanase; saccharifying and fermenting the slurry using enzymes, including a glucoamylase and a fermenting organism to produce the fermentation product, wherein the viscosity of the slurry is reduced by at least 20% compared to a slurry not treated with a protease.
  • the viscosity is reduced by 30%, 50% or 60%.
  • the invention comprises a method for reducing the foam in the wheatlike grain slurry during a fermentation, comprising: liquifying the wheat-like grain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 65°C using: an alpha amylase, a protease; and optionally a beta-glucanase; saccharifying and fermenting the slurry using enzymes, including a glucoamylase and a fermenting organism to produce the fermentation product; wherein the foam in the slurry is reduced by at least 20% compared to a slurry not treated with a protease.
  • the foam is reduced by 40%, 60%, or 80%.
  • the invention comprises a method for reducing the viscosity of the wheat-like grain slurry during a liquefaction process, comprising: liquifying the wheat-like gain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 65°C using an alpha amylase, a protease; and optionally a beta-glucanase; wherein the viscosity of the slurry is reduced by at least 20% compared to a slurry not treated with a protease.
  • the viscosity is reduced by 30%, 50% or 60%.
  • the invention comprises a method for reducing the foam in the wheatlike grain slurry during a liquefaction process, comprising: liquifying the wheat-like grain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 55°C using: an alpha amylase, a protease; and optionally a beta-glucanase; wherein the foam in the slurry is reduced by at least 20% compared to a slurry not treated with a protease.
  • the foam is reduced by 40%, 60%, or 80%.
  • the invention comprises a method for reducing the viscosity of the wheat-like grain slurry during a liquefaction process, comprising: liquifying the wheat-like gain slurry at a pH of about 4.0 to about 7.0 at a temperature in the range from about 25°C to about 90°C using: an alpha amylase, a protease; and optionally a beta-glucanase; wherein the viscosity of the slurry is reduced by at least 20% compared to a slurry not treated with a protease.
  • the viscosity is reduced by 30%, 50% or 60%.
  • the protease is a metallopro tease.
  • the metalloprotease is a thermolysin or Proteinase T.
  • the protease is a variant thermolysin with one or more substitutions.
  • the protease is NprE.
  • the protease is an NprE varaint with one or more substitutions.
  • the protease is Fermgen, an acid fungal protease from These substitutions are disclosed in various patent applications, for example, WO 2007/044993, WO 2009/058679, WO 2009/058661, WO 2009/058518, WO 2009/058303, and US provisional application 61/722,660 filed on 05
  • Figure 1 Effect of Proteinase T treatment on viscosity reduction.
  • Figure 3 Thermostability of Proteinase T variants and wild type enzyme.
  • the invention relates to use of proteases during the process of conversion of starch containing material, e.g., wheat or other similar grains, to fermentation products, such as alcohol, providing benefits such as (1) reducing the foaming characteristics of the grain slurry during liquifaction and fermentation and (2) reducing the viscosity during liquefaction unit operation and 3) enhancing the rate and/or yield of the fermentation.
  • benefits such as (1) reducing the foaming characteristics of the grain slurry during liquifaction and fermentation and (2) reducing the viscosity during liquefaction unit operation and 3) enhancing the rate and/or yield of the fermentation.
  • DPn degree of saccharide polymerization having n subunits
  • ds or DS dry solids
  • DTMPA diethylenetriaminepentaacetic acid
  • EC Enzyme Commission
  • EDTA ethylenediaminetetraacetic acid
  • EO ethylene oxide (polymer fragment); EOF: End of
  • GA glucoamylase
  • GAU/g ds glucoamylase activity unit/gram dry solids
  • HFCS high fructose corn syrup
  • HgGA Humicola grisea glucoamylase
  • IPTG isopropyl ⁇ -D- thiogalactoside
  • IRS insoluble residual starch
  • kDa kiloDalton
  • LAS linear
  • RNA relative centrifugal/centripetal force (i.e., x gravity);
  • SAS alkanesulfonate
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • SSF simultaneous saccharification and fermentation
  • SSU/g solid soluble starch unit/gram dry solids
  • TAED tetraacetylethylenediamine
  • Tm melting temperature
  • TrGA Trichoderma reesei glucoamylase
  • w/v weight/volume
  • w/w
  • Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • mS/cm milli- Siemens/cm
  • CV column volumes.
  • protease and “proteinase” refer to an enzyme protein that has the ability to break down proteins or peptides. A protease has the ability to conduct
  • proteolysis which begins protein catabolism by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein.
  • proteolytic activity This activity of a protease as a protein-digesting enzyme is referred to as "proteolytic activity.”
  • proteolytic activity Many well known procedures exist for measuring proteolytic activity (See e.g., Kalisz, "Microbial Proteinases,” In: Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology, (1988)). For example, proteolytic activity may be ascertained by comparative assays which analyze the respective protease' s ability to hydrolyze a commercially available substrate.
  • Exemplary substrates useful in the analysis of protease or proteolytic activity include, but are not limited to, di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111). Colorimetric assays utilizing these substrates are well known in the art (See e.g., WO 99/34011 and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference). The pNA assay (See e.g., Del Mar et ah, Anal. Biochem.
  • thermolysin refers any member of the M4 protease family as described in MEROPS - The Peptidase Data base (See, Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34 Database issue, D270-272 [2006]), of which thermolysin (TLN; EC 3.4.24.27) is the prototype.
  • TNN The amino acid sequence of thermolysin, (EC 3.4.24.27) the neutral metallo endo-peptidase secreted from Bacillus thermoproteolyticus was first reported by Titani et al (Titani et al, (1972), Amino-acid sequence of thermolysin.
  • the only differences between the protein sequences reported by Titani et al and O'Donohue et al are the confirmation of Asn at position 37 (instead of Asp) and Gin at position 119 (instead of Glu).
  • thermolysin As such the terms “thermolysin,” “stearolysin”, “bacillolysin,” “proteinase-T”, “PrT”, “Thermolysin-like protease”, and “TLPs”, are used interchangeably herein to refer to the neutral metalloprotease enzyme of Bacillus thermoproteolyticus.
  • 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-(l ⁇ 4) O-glycosidic linkages in starch.
  • a-amylases (EC 3.2.1.1; a-D-(l ⁇ 4)- glucan glucanohydrolase) are defined as endo-acting enzymes cleaving a-D-(l ⁇ 4) O-glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (l-4)-a-linked D-glucose units.
  • the exo-acting amylolytic enzymes such as ⁇ -amylases (EC 3.2.1.2; a-D-(l ⁇ 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, ⁇ -amylases, a-glucosidases (EC 3.2.1.20; a-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; a-D-(l ⁇ 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.
  • Enzyme units herein refer 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
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C 6 H 10 O5) x , wherein X can be any number.
  • the term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, milo, 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.
  • wild-type refers to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions.
  • 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 encompasses any polynucleotide encoding the wild- type, parental, or reference polypeptide.
  • references to the wild-type polypeptide is understood to include the mature form of the polypeptide.
  • a "mature" polypeptide or variant, thereof, is one in which a signal sequence is absent, for example, cleaved from an immature form of the polypeptide during or following 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 a-amylase activity, which can be measured as described, herein.
  • 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.
  • 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.
  • a vector comprising a nucleic acid encoding an amylase is a recombinant vector.
  • 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 polypeptides includes, but is not limited to, a culture broth containing secreted polypeptide 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.
  • enriched refers to material (e.g., an isolated polypeptide or polynucleotide) that is in about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.
  • thermostability refers to the ability of the enzyme to retain activity after exposure to an elevated temperature.
  • the thermostability of an enzyme is measured by its half-life (t 1/2 ) 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 a-amylase 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.
  • 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 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 be 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.
  • transformed 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 non-native sequence may be endogenous sequence.
  • 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 (e.g., an amylase) has been introduced.
  • exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) 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.
  • 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.
  • 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 which 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.
  • Bioly active refer to a sequence having a specified biological activity, such an enzymatic activity.
  • specific activity refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U)/mg of protein.
  • water hardness is a measure of the minerals (e.g., calcium and magnesium) present in water.
  • an "effective amount of amylase,” or similar expressions refers to an amount of amylase sufficient to produce a visible, or otherwise measurable amount of starch hydrolysis in an particular application.
  • Starch hydrolysis may result in, e.g., a visible cleaning of fabrics or dishware, reduced viscosity of a starch slurry or mash, and the like.
  • a "swatch" is a piece of material such as a fabric that has a stain applied thereto.
  • the material can be, for example, fabrics made of cotton, polyester or mixtures of natural and synthetic fibers.
  • the swatch can further be paper, such as filter paper or nitrocellulose, or a piece of a hard material such as ceramic, metal, or glass.
  • the stain is starch based, but can include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of these compounds.
  • a "smaller swatch" is a section of the swatch that has been cut with a single hole punch device, or has been cut with a custom manufactured 96-hole punch device, where the pattern of the multi-hole punch is matched to standard 96-well microtiter plates, or the section has been otherwise removed from the swatch.
  • the swatch can be of textile, paper, metal, or other suitable material.
  • the smaller swatch can have the stain affixed either before or after it is placed into the well of a 24-, 48- or 96-well microtiter plate.
  • the smaller swatch can also be made by applying a stain to a small piece of material.
  • the smaller swatch can be a stained piece of fabric 5/8" or 0.25" in diameter.
  • the custom manufactured punch is designed in such a manner that it delivers 96 swatches simultaneously to all wells of a 96-well plate.
  • the device allows delivery of more than one swatch per well by simply loading the same 96-well plate multiple times.
  • Multi-hole punch devices can be conceived of to deliver simultaneously swatches to any format plate, including but not limited to 24-well, 48-well, and 96-well plates.
  • the soiled test platform can be a bead made of metal, plastic, glass, ceramic, or another suitable material that is coated with the soil substrate. The one or more coated beads are then placed into wells of 96-, 48-, or 24-well plates or larger formats, containing suitable buffer and enzyme.
  • a cultured cell material comprising an amylase refers to a cell lysate or supernatant (including media) that includes an amylase as a component.
  • the cell material may be from a heterologous host that is grown in culture for the purpose of producing the amylase.
  • Percent sequence identity means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified 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: G, P, S, N, D, Q, E, K, R; 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 500-amino acid residue polypeptide with a deletion of five amino acid residues from the C-terminus would have a percent sequence identity of 99% (495/500 identical residues x 100) relative to the parent polypeptide. Such a variant would be encompassed by the language, "a variant having at least 99% sequence identity to the parent.”
  • fused polypeptide sequences are connected, i.e. , operably linked, via a peptide bond between two subject polypeptide sequences.
  • filamentous fungi refers to all filamentous forms of the subdivision
  • Eumycotina particularly Pezizomycotina species.
  • degree of polymerization refers to the number (n) of anhydro- glucopyranose units in a given saccharide.
  • DPI the monosaccharides glucose and fructose.
  • DP2 the disaccharides maltose and sucrose.
  • 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.
  • 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.
  • SSF saccharification and fermentation
  • An "ethanologenic or fermenting 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 fungal fermentation.
  • a fermentation process such as a microbial fermentation, e.g., a bacterial and/or fungal 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.
  • malt refers to any malted cereal grain, such as malted barley or wheat.
  • adjunct refers to any starch and/or sugar containing plant material that 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.
  • starch 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.
  • 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.”
  • starch retro gradation refers to changes that occur spontaneously in a starch paste or gel on ageing.
  • wheat and “wheat-like” refer to various grains that contain higher amounts of glucans and arabinoxylans, then majority starch-containing grains such as corn and rice.
  • Proteases are currently classified into six broad groups: Serine proteases, Threonine proteases, Cysteine proteases, Aspartate proteases, Glutamic proteases, Metallopro teases.
  • the mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine (proteases) or a water molecule (aspartic acid, metallo- and glutamic acid proteases) nucleophilic so that it can attack the peptide carboxyl group.
  • One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile.
  • proteases of the invention may be wild type proteases or a variant thereof. Any suitable protease may be adapted to perform in the methods of the invention.
  • the protease is a metallopro tease.
  • the metalloprotease is a thermolysin or Proteinase T.
  • the protease is a variant thermolysin with one or more substitutions.
  • the protease is NprE.
  • the protease is an NprE varaint with one or more substitutions.
  • PCT/CN13/076386 PCT/CN13/076387, PCT/CN 13/076390, PCT/CN 13/076401,
  • PCT/CN13/076383 PCT/CN 13/076406, PCT/CN13/076414, PCT/CN13/076384,
  • the present enzymes have a defined degree of amino acid sequence identity to other enzymes, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity.
  • the present enzymes are derived from a parental enzyme having a defined degree of amino acid sequence identity, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity.
  • the present enzymes comprise conservative substitution of one or several amino acid residues relative to the amino acid sequence of a parent enzyme.
  • Exemplary conservative amino acid substitutions are listed in the Table 1. Some conservative mutations can be produced by genetic manpulation, while others are produced by introducing synthetic amino acids into a polypeptide other means. Table 1. Conservative amino acid substitutions
  • Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln
  • Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D- Met, lie, D-Ile, Orn, D-Orn
  • the present enzymes comprises a deletion, substitution, insertion, or addition of one or a few amino acid residues.
  • the present enzymes are derived from the amino acid sequence of the parent enzyme by conservative substitution of one or several amino acid residues.
  • the present enzymes are derived from the amino acid sequence of the parent enzyme by deletion, substitution, insertion, or addition of one or a few amino acid residues. In all cases, the expression "one or a few amino acid residues" refers to 10 or less, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acid residues.
  • the present enzymes are encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid sequence that is complementary to a nucleic acid that encodes the parent enzyme.
  • the present enzymes 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. Mature forms of the polypeptides are generally the most useful. Unless otherwise noted, the amino acid residue numbering used herein refers to the mature forms of the respective amylase
  • amylase polypeptides may also be truncated to remove the N or C- termini, so long as the resulting polypeptides retain enzyme activity.
  • the present enzyme may be a "chimeric" or “hybrid” polypeptide, in that it includes at least a portion of a first enzyme polypeptide, and at least a portion of a second enzyme polypeptide (such chimeric enzymes have recently been “rediscovered” as domain-swap enzymes).
  • the present enzymes may further include heterologous signal sequence, an epitope to allow tracking or purification, or the like.
  • Exemplary heterologous signal sequences are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and Streptomyces CelA.
  • nucleic acids encoding an enzyme polypeptide may encode the enzyme having the amino acid sequence having a specified degree of amino acid sequence identity.
  • the nucleic acid encodes an enzyme having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity to the enzyme.
  • the nucleic acid has at least 80%, at least 85%, at least 90%, at least 95%, or even at least 98% nucleotide sequence identity to parental enzyme.
  • the present compositions and methods include nucleic acids that encode an enzyme having deletions, insertions, or substitutions, such as those mentioned, above. It will be appreciated that due to the degeneracy of the genetic code, a plurality of nucleic acids may encode the same polypeptide.
  • the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid complementary to a nucleic acid encoding an enzyme having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity to a parent enzyme.
  • the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid complementary to a nucleic acid having the sequence of a parent enzyme. Such hybridization conditions are described herein but are also well known in the art.
  • Nucleic acids may encode a "full-length" (“fl” or “FL”) amylase, which includes a signal sequence, only the mature form of an enzyme, which lacks the signal sequence, or a truncated form of an enzyme, which lacks the N or C-terminus of the mature form.
  • the nucleic acids are of sufficient length to encode an active enzyme.
  • a nucleic acid that encodes an enzyme can be operably linked to various promoters and regulators in a vector suitable for expressing the enzyme in host cells.
  • exemplary promoters are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and Streptomyces CelA.
  • LAT B. licheniformis amylase
  • B. subtilis AmyE or AprE
  • Streptomyces CelA Such a nucleic acid can also be linked to other coding sequences, e.g., to encode a chimeric polypeptide.
  • the present enzymes can be produced in host cells, for example, by secretion or intracellular expression.
  • a cultured cell material ⁇ e.g., a whole-cell broth) comprising an enzyme can be obtained following secretion of the enzyme into the cell medium.
  • the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme.
  • a gene encoding an enzyme can be cloned and expressed according to methods well known in the art.
  • Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei.
  • Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis, as well as Streptomyces.
  • the host cell further may express a nucleic acid encoding a homologous or heterologous glucoamylase, i.e., a glucoamylase that is not the same species as the host cell, or one or more other enzymes.
  • the glucoamylase may be a variant glucoamylase, such as one of the glucoamylase variants disclosed in U.S. Patent No. 8,058,033 (Danisco US Inc.), for example.
  • 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 various 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 an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, different 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 enzymes 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 an enzyme 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 enzyme.
  • Host cells that serve as expression hosts can include filamentous fungi, for example.
  • 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, at www.fgsc.net (last modified January 17, 2007).
  • pJG153 a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (2011) Applied Environ. Microbiol. 77:3916-22.
  • pJG153 can be modified with routine skill to comprise and express a nucleic acid encoding an enzyme variant.
  • a nucleic acid encoding an enzyme 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.
  • Exemplary promoters for directing the transcription of the DNA sequence encoding an enzyme, especially in a bacterial host, are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dag A or celA promoters the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens ⁇ -amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • 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.
  • TAKA amylase Rhizomucor miehei aspartic proteinase
  • Aspergillus niger neutral a-amylase A. niger acid stable a-amylase
  • A. niger glucoamylase Rhizomucor miehei lipase
  • Rhizomucor miehei lipase Rhizomucor miehe
  • a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter.
  • 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 AOXl or AOX2 promoters
  • cbhl is an endogenous, inducible promoter from T. reesei. See 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 enzyme gene to be expressed or from a different Genus or species.
  • 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 an enzyme. 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, pACYClW, pUBl lO, pE194, pAMBl, and pIJ702.
  • 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 xxsC, 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 enzyme for subsequent enrichment or purification.
  • Extracellular secretion of enzyme into the culture medium can also be used to make a cultured cell material comprising the isolated enzyme.
  • 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 enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment.
  • a targeting sequence includes but is not limited to the sequence, SKL.
  • the nucleic acid sequence of the enzyme is operably linked to the control sequences in proper manner with respect to expression.
  • An isolated cell is advantageously used as a host cell in the recombinant production of an enzyme.
  • 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.
  • suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis; Lactobacillus sp.
  • Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
  • strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.
  • a suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species.
  • a strain of the methylotrophic yeast species, Pichia pastoris can be used as the host organism.
  • the host organism can be a Hansenula species.
  • Suitable host organisms among filamentous fungi include species of Aspergillus, e.g.,
  • Aspergillus nidulans strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species.
  • Trichoderma sp. such as T. reesei can be used as a host.
  • a suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023.
  • An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety.
  • the glycosylation pattern can be the same or different as present in the wild-type enzyme.
  • the type and/or degree of glycosylation may impart changes in enzymatic and/or biochemical properties.
  • genes from expression hosts where the gene deficiency may 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, egll, 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 an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.
  • the preparation of 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.
  • 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 CaCl 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. [00116] Usually transformation of Trichoderma sp.
  • protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 10 5 to 107 /mL, particularly 2xl0 6 /mL.
  • a volume of 100 of these protoplasts or cells in an appropriate solution e.g., 1.2 M sorbitol and 50 mM CaCl 2
  • an appropriate solution e.g., 1.2 M sorbitol and 50 mM CaCl 2
  • 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.
  • a method of producing an enzyme may comprise cultivating a 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.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an enzyme. 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 an enzyme. 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 enzyme 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 an enzyme 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 an enzyme.
  • Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate 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 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 75°C ⁇ e.g., 30°C to 45°C), depending on the needs of the host and production of the desired enzyme. 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 an enzyme.
  • assays can measure the expressed protein, corresponding mRNA, or enzyme activity.
  • 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 enzyme activity in a sample, for example, by assays directly measuring products in the culture media. For example, glucose concentration may be determined using glucose reagent kit No. 15-UV (Sigma Chemical Co.) or an instrument, such as Technicon Autoanalyzer.
  • a- Amylase activity also may be measured by any known method, such as the PAHBAH or ABTS assays, described below. Assays are also known in the art to measure other enzyme activities. 3.5. Methods for Enriching and Purifying Enzymes of the Invention
  • Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated enzyme 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.
  • 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 enrichment and 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 enzyme polypeptide-containing solution is at a desired level.
  • the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or 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.
  • enzyme polypeptides can be enriched or partially purified as generally described above by removing cells via flocculation with polymers.
  • the enzyme can be enriched or purified by microfiltration followed by
  • the enzyme does not need to be enriched or purified, and whole broth culture can be lysed and used without further treatment.
  • the enzyme can then be processed, for example, into granules.
  • the present proteases are useful for a variety of industrial applications.
  • a-amylases are useful in a starch conversion process, particularly in a saccharification process of a starch that has undergone liquefaction.
  • the desired end-product may be any product that may be produced by the enzymatic conversion of the starch substrate.
  • the desired product may be a syrup rich in glucose and maltose, which can be used in other processes, such as the preparation of HFCS, or which can be converted into a number of other useful products, such as ascorbic acid intermediates (e.g.
  • gluconate gluconate; 2-keto-L-gulonic acid; 5- keto-gluconate; and 2,5-diketogluconate); 1,3-propanediol; aromatic amino acids (e.g. , tyrosine, phenylalanine and tryptophan); organic acids (e.g. , lactate, pyruvate, succinate, isocitrate, gluconic acid, and oxaloacetate); amino acids (e.g. , serine, lysine, glutamic acid, and glycine); antibiotics; antimicrobials; enzymes; vitamins; and hormones.
  • aromatic amino acids e.g. , tyrosine, phenylalanine and tryptophan
  • organic acids e.g. , lactate, pyruvate, succinate, isocitrate, gluconic acid, and oxaloacetate
  • amino acids e.g. , serine, lysine,
  • the starch conversion process may be a precursor to, or simultaneous with, a fermentation process designed to produce alcohol for fuel or drinking (i.e. , potable alcohol).
  • a-amylases are also useful in compositions and methods of food preparation. These various uses of ⁇ -amylases are described in more detail below.
  • 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, 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 also be highly refined raw starch or feedstock from starch refinery processes.
  • 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 thus 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. 4.2. Gelatinization and Liquefaction of Starch
  • the term "liquefaction” or “liquefy” means a process by which starch is converted to less viscous and shorter chain dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of an a-amylase, although additional liquefaction-inducing enzymes optionally may be added.
  • the starch substrate prepared as described above is slurried with water.
  • the starch slurry may contain starch as a weight percent of dry solids of about 10-55%, about 20-45%, about 30-45%, about 30-40%, or about 30-35%.
  • a-Amylase (EC 3.2.1.1) may be added to the slurry, with a metering pump, for example.
  • the a-amylase typically used for this application is a thermally stable, bacterial a-amylase, such as a Geobacillus stearothermophilus a-amylase.
  • the ⁇ -amylase is usually supplied, for example, at about 1500 units per kg dry matter of starch.
  • the pH of the slurry typically is adjusted to about pH 5.5-6.5 and about 1 mM of calcium (about 40 ppm free calcium ions) can also be added.
  • Geobacillus stearothermophilus variants or other a- amylases may require different conditions.
  • Bacterial ⁇ -amylase remaining in the slurry following liquefaction may be deactivated via a number of methods, including lowering the pH in a subsequent reaction step or by removing calcium from the slurry in cases where the enzyme is dependent upon calcium.
  • the slurry of starch plus the ⁇ -amylase may be pumped continuously through a jet cooker, which is steam heated to 105°C. Gelatinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate.
  • the residence time in the jet cooker is brief.
  • the partly gelatinized starch may be passed into a series of holding tubes maintained at 105-110°C and held for 5-8 min. to complete the gelatinization process ("primary liquefaction").
  • Hydrolysis to the required DE is completed in holding tanks at 85-95°C or higher temperatures for about 1 to 2 hours (“secondary liquefaction"). These tanks may contain baffles to discourage back mixing.
  • minutes of secondary liquefaction refers to the time that has elapsed from the start of secondary liquefaction to the time that the Dextrose Equivalent (DE) is measured.
  • the slurry is then allowed to cool to room temperature. This cooling step can be 30 minutes to 180 minutes, or more.
  • the liquefied starch typically is in the form of a slurry having a dry solids content (w/w) of about 10-50%; about 10-45%; about 15-40%; about 20-40%; about 25-40%; or about 25-35%.
  • Liquefaction with a-amylases advantageously can be conducted at low pH, eliminating the requirement to adjust the pH to about pH 5.5-6.5.
  • ⁇ -amylases can be used for liquefaction at a pH range of 2 to 7, e.g., pH 3.0 - 7.5, pH 4.0 - 6.0, or pH 4.5 - 5.8.
  • a-amylases can maintain liquefying activity at a temperature range of about 85°C - 95°C, e.g., 85°C, 90°C, or 95°C.
  • liquefaction can be conducted with 800 ⁇ g an amylase in a solution of 25% DS corn starch for 10 min at pH 5.8 and 85°C, or pH 4.5 and 95°C, for example.
  • Liquefying activity can be assayed using any of a number of known viscosity assays in the art.
  • starch liquefaction is performed at a temperature range of 90-115°C, for the purpose of producing high-purity glucose syrups, HFCS, maltodextrins, etc.
  • the liquefied starch can be saccharified into a syrup rich in lower DP (e.g., DPI + DP2) saccharides, using ⁇ -amylases, optionally in the presence of other enzyme(s).
  • DP e.g., DPI + DP2
  • ⁇ -amylases optionally in the presence of other enzyme(s).
  • the exact composition of the products of saccharification depends on the combination of enzymes used, as well as the type of granular starch processed.
  • the syrup obtainable using the provided ⁇ -amylases may contain a weight percent of DP2 of the total oligosaccharides in the saccharified starch exceeding 30%, e.g., 45% - 65% or 55% - 65%.
  • the weight percent of (DPI + DP2) in the saccharified starch may exceed about 70%, e.g., 75% - 85% or 80% - 85%.
  • the present amylases also produce a relatively high yield of glucose, e.g., DPI > 20%, in the syrup product.
  • Saccharification typically is most effective at temperatures of about 60-65°C and a pH of about 4.0-4.5, e.g., pH 4.3, necessitating cooling and adjusting the pH of the liquefied starch.
  • Saccharification may be performed, for example, at a temperature between about 40°C, about 50°C, or about 55°C to about 60°C or about 65°C. Saccharification is normally conducted in stirred tanks, which may take several hours to fill or empty. Enzymes typically are added either at a fixed ratio to dried solids as the tanks are filled or added as a single dose at the commencement of the filling stage. A saccharification reaction to make a syrup typically is run over about 24-72 hours, for example, 24-48 hours. When a maximum or desired DE has been attained, the reaction is stopped by heating to 85°C for 5 min., for example.
  • saccharification optimally is conducted at a temperature range of about 30°C to about 75°C, e.g., 45°C - 75°C or 47°C - 74°C.
  • the saccharifying may be conducted over a pH range of about pH 3 to about pH 7, e.g., pH 3.0 - pH 7.5, pH 3.5 - pH 5.5, pH 3.5, pH 3.8, or pH 4.5.
  • An amylase may be added to the slurry in the form of a composition.
  • Amylase can be added to a slurry of a granular starch substrate in an amount of about 0.6 - 10 ppm ds, e.g., 2 ppm ds.
  • An amylase can be added as a whole broth, clarified, enriched, partially purified, or purified enzyme.
  • the specific activity of the amylase may be about 300 U/mg of enzyme, for example, measured with the PAHBAH assay.
  • the amylase also can be added as a whole broth product.
  • An amylase may be added to the slurry as an isolated enzyme solution.
  • an amylase can be added in the form of a cultured cell material produced by host cells expressing an amylase.
  • An amylase may also be secreted by a host cell into the reaction medium during the fermentation or SSF process, such that the enzyme is provided continuously into the reaction.
  • the host cell producing and secreting amylase may also express an additional enzyme, such as a glucoamylase.
  • U.S. Patent No. 5,422,267 discloses the use of a glucoamylase in yeast for production of alcoholic beverages.
  • a host cell e.g., Trichoderma reesei or Aspergillus niger
  • a host cell may be engineered to co-express an amylase and a glucoamylase, e.g., HgGA, TrGA, or a TrGA variant, during saccharification.
  • the host cell can be genetically modified so as not to express its endogenous glucoamylase and/or other enzymes, proteins or other materials.
  • the host cell can be engineered to express a broad spectrum of various saccharolytic enzymes.
  • the recombinant yeast host cell can comprise nucleic acids encoding a glucoamylase, an alpha-glucosidase, an enzyme that utilizes pentose sugar, an a-amylase, a pullulanase, an isoamylase, and/or an isopullulanase. See, e.g., WO 2011/153516 A2.
  • 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 for alcohol-producing yeast.
  • a fermenting organism typically at a temperature around 32°C, such as from 30°C to 35°C for alcohol-producing yeast.
  • the temperature and pH of the fermentation will depend upon the fermenting organism.
  • 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.
  • 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 mobilis, 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. (2011) Sheng Wu Gong Cheng Xue Bao 27: 1049-56.
  • yeast Commercial sources include ETHANOL RED® (LeSaffre); THERMOSACC®
  • 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) Biotechnol. Adv. 25:244-63; John et al. (2009) Biotechnol. Adv. 27: 145-52.
  • the saccharification and fermentation processes may be carried out as an SSF process. Fermentation may comprise subsequent enrichment, purification, and recovery of ethanol, for example.
  • 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.
  • 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 that express and secrete amylase continuously throughout SSF.
  • the fungal cells expressing amylase 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 amylase so that less or no enzyme has to be added exogenously.
  • the fungal host cell can be from an
  • Fungal host cells that express and secrete other enzymes, in addition to amylase, also can be used. Such cells may express one or more glucoamylase and/or a pullulanase, phytase, ⁇ / ⁇ -glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, beto-glucosidase, pectinase, esterase, redox enzymes, transferase, or other enzyme.
  • glucoamylase and/or a pullulanase phytase, ⁇ / ⁇ -glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, beto-glucosidase, pectinase, esterase, redox enzymes, transferase
  • 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.
  • Proteases may be combined with a glucoamylase (EC 3.2.1.3), e.g., a
  • Trichoderma glucoamylase or variant thereof Trichoderma reesei glucoamylase (TrGA) and variants thereof that possess superior specific activity and thermal stability. See U.S. Published Applications Nos. 2006/0094080, 2007/0004018, and 2007/0015266 (Danisco US Inc.).
  • TrGA Trichoderma reesei glucoamylase
  • Suitable variants of TrGA include those with glucoamylase activity and at least 80%, at least 90%, or at least 95% sequence identity to wild-type TrGA.
  • a- amylases advantageously increase the yield of glucose produced in a saccharification process catalyzed by TrGA.
  • the glucoamylase may be another glucoamylase derived from plants (including algae), fungi, or bacteria.
  • the glucoamylases may be Aspergillus niger Gl or G2 glucoamylase or its variants (e.g., Boel et al. (1984) EMBO J. 3: 1097-1102; WO 92/00381; WO 00/04136 (Novo Nordisk A/S)); and A awamori glucoamylase (e.g., WO 84/02921 (Cetus Corp.)).
  • Aspergillus glucoamylase include variants with enhanced thermal stability, e.g., G137A and G139A (Chen et al. (1996) Prot. Eng. 9:499-505); D257E and D293E/Q (Chen et al. (1995) Prot. Eng. 8:575-582); N182 (Chen et al. (1994) Biochem. J. 301:275-281); A246C (Fierobe et al. (1996) Biochemistry, 35: 8698-8704); and variants with Pro residues in positions A435 and S436 (Li et al. (1997) Protein Eng. 10: 1199- 1204).
  • glucoamylases include Talaromyces glucoamylases, in particular derived from T. emersonii (e.g., WO 99/28448 (Novo Nordisk A/S), T. leycettanus (e.g., U.S. Patent No. RE 32,153 (CPC International, Inc.)), T. duponti, or T. thermophilus (e.g., U.S.
  • Contemplated bacterial glucoamylases include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (e.g., EP 135138 (CPC International, Inc.) and C. thermohydrosulfuricum (e.g., WO 86/01831 (Michigan Biotechnology Institute)).
  • Suitable glucoamylases include the glucoamylases derived from Aspergillus oryzae, such as a glucoamylase disclosed in WO 00/04136 (Novo Nordisk A/S).
  • glucoamylases such as AMG 200L; AMG 300 L; SANTM SUPER and AMGTM E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco US Inc.); AMIGASETM and AMIGASETM PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); and G-ZYME® G990 ZR (A. niger glucoamylase with a low protease content). Still other suitable glucoamylases include
  • Glucoamylases typically are added in an amount of about 0.1 - 2 glucoamylase units (GAU)/g ds, e.g., about 0.16 GAU/g ds, 0.23 GAU/g ds, or 0.33 GAU/g ds.
  • GAU glucoamylase units
  • OPTIMASHTM BG may be used which is an enzyme preparation intended for the fuel alcohol industry. This product is capable of reducing viscosity of barley and wheat mashes.
  • OPTIMASHTM BG enzyme contains a combination of enzymes, including but not limited to, a beta glucanase and a xylanase, which effectively modify and digest non- starch carbohydrates, the structural material of plant cells.
  • OPTIMASHTM BG is produced by submerged fermentation of a genetically modified strain of Trichoderma reesei.
  • OPTIMASHTM TBG may be used, which is an enzyme blend that is heat stable, food grade preparation of the enzyme cellulase, EC 3.2, 1.4.
  • the product has been specifically formulated for use in the fermentation ethanol and starch processing industries for the breakdown of the non-starch polysaccharides of barley and wheat. It is produced by the fermentation of a non-genetically modified strain of Geosmithia emersonii, also known as Talaromyces emersonii.
  • the major enzyme activity of OPTIMASHTM TBG enzyme is a component, endo- 1,3(4)- ⁇ -glucanase (systematic name: (l ,3-l ,3;L4) ⁇ a-D ⁇ glucan 3(4)- glucanohydrolase), which catalyses the endohydrolysis of 1 ,3- or 1 ,4-linkages in B-D-glucans.
  • Suitable enzymes include a phytase, protease, pullulanase, ⁇ -amylase, isoamylase, a different a-amylase, alpha-glucosidase, cellulase, xylanase, other hemicellulases, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, or a combination thereof.
  • a debranching enzyme such as an isoamylase (EC 3.2.1.68), may be added in effective amounts well known to the person skilled in the art.
  • a pullulanase (EC 3.2.1.41), e.g., PROMOZYME®, is also suitable. Pullulanase typically is added at 100 U/kg ds.
  • Further suitable enzymes include proteases, such as fungal and bacterial proteases. Fungal proteases include those obtained from Aspergillus, such as A. niger, A.
  • awamori A. oryzae
  • Mucor e.g., M. miehei
  • Rhizopus Rhizopus
  • Trichoderma Trichoderma
  • ⁇ -Amylases (EC 3.2.1.2) are exo-acting maltogenic amylases, which catalyze the hydrolysis of 1,4-a-glucosidic linkages into amylopectin and related glucose polymers, thereby releasing maltose.
  • ⁇ -Amylases have been isolated from various plants and microorganisms. See Fogarty et al. (1979) in PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 112-115. These ⁇ -Amylases have optimum temperatures in the range from 40°C to 65°C and optimum pH in the range from about 4.5 to about 7.0.
  • Contemplated ⁇ -amylases include, but are not limited to, ⁇ -amylases from barley SPEZYME® BBA 1500, SPEZYME® DBA, OPTIMALTTM ME, OPTEVIALTTM BBA (Danisco US Inc.); and NOVOZYMTM WBA (Novozymes A/S).
  • compositions comprising the present proteases may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further comprise any one or more of the additional enzymes listed, herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, and the like.
  • Such compositions may work in combination with endogenous enzymes or other ingredients already present in a slurry, water bath, washing machine, food or drink product, etc., for example, endogenous plant (including algal) enzymes, residual enzymes from a prior processing step, and the like.
  • the proteases were added as such 1) no protease 2) Proteinase T at 0.05mg/g ds, 3) FNA at 0.05mg/g ds and 4) NprE at 0.05mg/g ds.
  • the flasks were incubated at 50°C for 2 hours with constant mixing. After the initial incubation the pH was adjusted to pH 4.2 using 4N sulfuric acid. ADY at 3.57 mg/g ds, urea at 600 ppm and GA at 0.8 GAUs /g ds were added to each flask. 50 g of slurry was thereafter transferred to 50 ml glass graduated cylinders for fermentation. The cylinders were incubated at room temperatures for 48 hrs. The foaming measurements were performed at 24 and 48 hrs incubation times. At 24 hrs, foaming measurements were performed without disturbing the slurry. It was then subsequently mixed and the foam was monitored and measured at 1 and 1.5 hrs after mixing.
  • thermostable variants of thermolysin (Proteinase)
  • thermo stability/heat stress stability was analyzed as described below.
  • Heat Stress Diluted the wt. to 1.36 ppm and the variants to 2 ppm in the stress buffer (50mM Potassium Acetate, O. lmM CaCl 2 , pH5.8). 50 samples were added to PCR strips (1 well / temperature). An unstressed sample of all enzymes was immediately put on ice. The PCR was set on a temperature gradient that ranged from 65°C - 85°C. Samples were stressed for 15 min and then put on ice to cool to RT.
  • stress buffer 50mM Potassium Acetate, O. lmM CaCl 2 , pH5.8
  • 50 samples were added to PCR strips (1 well / temperature). An unstressed sample of all enzymes was immediately put on ice. The PCR was set on a temperature gradient that ranged from 65°C - 85°C. Samples were stressed for 15 min and then put on ice to cool to RT.
  • AGLA Assay Reaction was initiated by transferring 5 ⁇ 1 of enzyme samples to a black flat-bottom 96-well plate containing 195 uL of Assay Solution per well (50mM MES, 2.5mM CaCl 2 , 0.005% Tween-80, 5% DMF, 2.4mM Abz-AGLA-Nba pH 6.5). Immediately transfer assay microplate to microplate spectrofluorometer and begin recording fluorescence measurements at excitation of 350 nm and emission of 415 nm. The spectrofluorometer software should calculate the reaction rates of the increase in fluorescence for each well to a linearly regressed line of RFU / sec. The thermostability is calculated based on enzyme activity after the heat incubation divided by enzyme activity before the heat incubation, and is expressed as a ratio of remaining activity. mes and Doses:
  • control cylinder had 60 mm foam + spill over, while the proteinase T and its variants exhibited only 2 mm foam. These data show that proteinase T variants were effective at reducing the foaming at higher temperatures. Along with the contribution of a protease activity, these lower numbers may also reflect the precipitation of some proteins/protein fragments at higher temperature tested.
  • a 33 g wheat slurry sample was weighed in to the RVA aluminum cans and dosed with 1.0 AAUs of SPEZYME® CL and/or 0.025 kg/MT OPTIMASH® TBG and/or various dosages of Protienase T and NprE protease (table below).
  • a double skirted paddle was placed in the can, and the can was placed in the RVA. The viscosity was continuously analyzed by the RVA over the entire 48 minute test.
  • OPTIMASH® TBG is generally used in the industry to reduce the wheat slurry viscosity in liquefaction.
  • Table 3 Effect of Proteinase T and NprE addition on the wheat slurry peak viscosity during the liquefaction process
  • proteases were added as such 1) no protease 2) Proteinase T at 0.05mg/g ds, 3) FNA at 0.05mg/g ds and 4) NprE at 0.05mg/g ds.
  • the metal beakers were fitted back into the LABOMAT for liquefaction unit operation at 60°C for 45 min followed by 85°C for 90 min with clockwise and counter-clockwise constant mixing at 60rpm.

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Abstract

La présente invention concerne la réduction de la viscosité d'une suspension de grains du type blé pendant la liquéfaction et/ou la fermentation. L'invention concerne également l'augmentation de la production et/ou du rendement de produits de fermentation, y compris de l'éthanol à partir de grains du type blé.
PCT/US2014/063791 2013-11-04 2014-11-04 Protéases utilisées dans le traitement du blé WO2015066667A1 (fr)

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CN107937291B (zh) * 2017-12-22 2021-06-04 广州南沙珠江啤酒有限公司 一种用于酵母扩培的麦汁及其制备工艺

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