WO2015021600A1 - Beta-amylase and methods of use - Google Patents

Abstract

The present teachings provide a beta amylase with maltogenic properties. Non-naturally occuring nucleic acids encoding the beta amylase and variants thereof, expression vectors, formulations, and host cells are also provided. Additional embodiments of the present teachings provide various methods of use and methods of manufacturing.

Description

Beta-Amylase and Methods of Use

Field of the Invention

The present teachings provide composition and methods relating to novel beta- amylases.

Background

Meeting the food demands of the earth's growing population is an increasing challenge. While the green evolution drastically improved the ability to harvest food calories from a given area of land, other means of increasing the planet's food supply are needed as the total acreage planted plateaus. Diversion of some acres away from food to biofuels can further complicate the picture. Sustainable approaches that employ biologically produced enzymes are a key element in meeting this need for increased food. One area amenable to such enzymatic approaches is the area of starch processing.

Starch is a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose is composed of 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.

Maltose, a di-saccharide composed of two D-glucopyranoses joined by an alpha 1 ,4'~glycosidic bond, has high commercial value in applications for the

food/frozen foods, baking, brewing and beverage industries. Maltose is also a substrate for production of the non-caloric sugar sweetener, maltitol. High purity maltose or pure maltose is an active component of intravenous injection liquids for diabetic patients. Commercial processes for the production of syrup containing different levels of maltose content, i.e. <50% maltose (high conversion or low maltose syrup), 50-55% maltose (high maltose syrup), 70-75% maltose (very high maltose) and >80% maltose (ultra high maltose) have been established depending on the applications. A common factor for these processes is that they involve a dual enzyme process with two different steps, i.e. liquefaction and saccharification. β-amyiase is a starch-degrading enzyme which hydroiyzes aipha-1 ,4 bonds. It is found e.g. in bacteria and plants and it breaks down starch mainly into maltose at the non-reducing end of the starch chain, β-amylase is abundant e.g. in grains, where it converts the nutrient store of the cereal, i.e. starch, into sugar. In cereals starch is mainly stored in the form of amylose and amy!opectin. β-amylase converts all of the amylose into maltose, whereas about 60% of the amylopectin is converted into maltose and the rest into dextrin. β-amylase is a commercially significant enzyme which is used e.g. in the starch industry to produce maltose. Products containing large amounts of maltose are used e.g. in the confectionery and food industry, β-amy!ase has been isolated both from bacteria and from plants. For example, it has been obtained from Bacillus bacteria (US 4 970 158 and JP 80 126 080) and from thermostable Clostridium bacteria (US 4 847 538). In addition to maltose, β-amylases derived from bacteria produce considerable amounts of maitotriose, whereas plant-based β-amyiases produce relatively more maltose and thus they are more suitable for processes where the purpose is to obtain as sweet and/or fermentable products as possible. Additionally, large-scale production of β-amyiase from bacteria is difficult. The β-amylase used in industry is plant-based, in which case usually cereal, particularly barley or wheat, but also soybeans are used as the enzyme source. β-amylase of barley has been studied thoroughly. This β-amyiase and its production are described e.g. in the following publications: D. E. Briggs, Barley, Chapman & Hail, London, 1978; Cook, Barley and Malt Academic Press, London, 1962; J.R.A. Pollock, Brewing Science, Academic Press, London, 1979. The systematic name of the enzyme is 1 ,4-aipha-D-glucan maltohydrolase (EC 3.2.1 .2). In the past the β-amylase of cereal was separated by first grinding or milling the grain and then extracting the β-amylase with water or a buffer. Purification of enzyme from extract of this kind is naturally difficult and laborious because in addition to the enzyme concerned, the extract contains several other soluble components of the grain. Attempts have been made to improve separation of β-amylase from a solution containing it e.g. by adsorbing the enzyme with polymer in the presence of ammonium sulphate (US 5 294 341 ). Release of β-amyiase from gluten has been experimented with protease (JP 63 079 590). β-amylase has also been isolated from the waste liquid of wheat starch production by adding sodium alginate and by recovering the coagulated enzyme (JP 80 027 383) or by forming a calcium phosphate gel to which the enzyme adsorbs and from which it is then recovered (JP 63 248 389). Waste liquid from starch production is not a good source of β-amylase because it is very dilute and contains large amounts of other components, which makes purification and concentration difficult and, as a result, the yield is low. To obtain a more pure raw extract and to avoid difficult downstream processing, it has been suggested that β-amy!ase be extracted from whole or partly husked grains. When e.g. barley grains are husked in such a manner that their endosperm does not break, the outmost layers of the endosperm function as a kind of filter which prevents access of insoluble substances to the steep water and restricts the access of soluble substances. It is preferable to carry out extraction in the presence of a reducing substance which releases β-amylase from other proteins of the grain (Fi 61 51 8 and US 4 675 296). Despite significant advances in the extraction of β-amyiases from grains (see for example US patent 4,875,269, US Patent 3,492,203, and EP1363999 B1 ), economic pressures for lower-cost enzymes produced in microbes continue to make recombinant β-amylase attractive.

Summary

In some embodiments, the present teachings provide a purified polypeptide, the amino acid sequence of which comprises a sequence at least 81 %, 82%, 83%, 84%, 85%, 87%, 90%, 92%, 95%, 98%, 99%, or 99.5% identical to SEQ ID NO:3.

In some embodiments, the present teachings provide a purified polypeptide comprising the amino acid sequence of SEQ ID NO:3, but with 0 to 20 conservative amino acid substitutions.

In some embodiment, the present teachings provide an expression vector comprising a non-natually occuring nucleic acid sequence, and, the nucleic acid sequence of SEQ ID NO:1 , and any nucleic acid 80%, 85%, 90%, 95%, 98%, or 99% to it, operably linked to an expression control sequence. In some embodiments, the present teachings contemplate a cultured cell comprising such an expression vector. In some embodiments, the present teachings provide a method of producing a protein, the method comprising culturing such a cell under conditions permitting expression of the polypeptide.

In some embodiments, the present teachings provide a method of using the polypeptides of the present teachings, the method comprising including the polypeptide in any of: starch liquefaction, starch saccharification, textile desizing, starch modification in the paper and pulp industry, brewing, baking, production of syrups for the food industry, production of feedstocks for fermentation processes, animal feed, and, removal of starchy soils and/or stains during dishwashing and/or laundry washing.

The present teachings also contemplate a composition comprising such

polypeptides, and at least one accessory enzyme selected from the group consisting of phytase, protease, pullulanase, an additional β-amylase, isoamylase, alpha amylase, glucoamylase, alpha-glucosidase, cellulase, xylanase, hemicellulase,

beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, choline oxidases, peroxidase/oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase, and/or a redox enzyme.

In some embodiments, the present teachings provide an isolated nucleic acid comprising at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , and, at least one non-naturally occuring DNA base at a position that allows encoding of a degenerate amino acid, wherein the polypeptide has starch hydrolysis activity.

In some embodiments, the present teachings provide an isolated nucleic acid comprising at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , and, at least one non-naturally occuring DNA base at a position that allows encoding of a conservative amino acid substitution, wherein the polypeptide has starch hydrolysis activity. In some embodiments, the present teachings provide an isolated nucleic acid comprising a sequence that hybridizes under stringent conditions to a hybridization probe the nucleotide sequence of which consists of SEQ ID NO: 1 , or the complement of SEQ ID NO: 1 , wherein the isolated nucleic acid comprises at least one non-naturally occuring DNA base at a position that allows encoding of a degenerate amino acid or allows encoding of a conservative amino acid substution. In some embodiments, such an isolated nucleic acid comprises a sequence at least 81 %, 82%, 83%, 84%, 85%, 87%, 90%, 92%, 95%, 98%, 99%, identical to SEQ ID NO: 1 . In some embodiments, such an isolated nucleic acid encodes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 1 -15, 1 -10, or 1 -5 degenerate amino acid positions, and the polypeptide has starch hydrolysis activity.

In some embodiments, such an isolated nucleic acid encodes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 1 -15, 1 -10, or 1 -5 conservative amino acid substitutions, and the polypeptide has starch hydrolysis activity,

These and other aspects and embodiments of the compositions and methods of the present teachings will be apparent from the present description and drawings.

Brief Description of the Drawings

Figure 1 depicts a plasmid map according to some embodiments of the present teachings. Figure 2 depicts a pH profile of PsPAmy9 according to some embodiments of the present teachings.

Figure 3 depicts a temperature profile according to some embodiments of the present teachings.

Figure 4 depicts a thermostability profile according to some embodiments of the present teachings.

Figure 5 depicts a sugar composition profile according to some embodiments of the present teachings. Figure 6 depicts a multiple sequence alignment according to some embodiments of the present teachings.

Figure 7 depicts a phylogenetic tree of PsPAmy9 according to some

embodiments of the present teachings.

Brief Description of the Sequences

SEQ ID NO:1 sets forth the full-length nucleotide sequence for PsPAmy9.

SEQ ID NO:2 sets forth the full length amino acid for the precursor PsPAmy9 protein, where the predicted native signal sequence is shown in italics.

SEQ ID NO:3 sets forth the full-length mature amino acid sequence for PsPAmy9.

SEQ ID NO:4 sets forth the nucleotide sequence of the synthesized PspAmy9 gene in plasmid p2JM784, with the oligonucleotide encoding the three residue addition (AGK) shown in bold.

SEQ ID NO:5 sets forth the amino acid sequence of the PsPAmy9 precursor protein expressed from plasmid p2JM784, where the predicted signal sequence is shown in italics, and the three residue addition (AGK) is shown in bold.

Detailed Description

Described are compositions and methods relating to beta amylase enzymes. This enzyme was discovered and analyzed by a combination of experimental approaches, as detailed in the Examples. Exemplary applications for the variant amylase enzymes are for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing {e.g., desizing), in animal feed for improving digestibility, and for baking and brewing. These and other aspects of the compositions and methods are described in detail, below.

All references cited herein are herein incorporated by reference in their entirety for all purposes. In order to further illustrate the compositions and methods, and advantages thereof, the following specific examples are given with the understanding that they are illustrative rather than limiting.

Prior to describing the various aspects and embodiments of the present compositions and methods, the following definitions and abbreviations are described.

Definitions and 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.

The present document is organized into a number of sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for different sections of the disclosure should not be construed as limiting.

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. The following terms are provided below. "β-Amylases" (EC 3.2.1 .2) are exo-acting beta amylases, which catalyze the hydrolysis of 1 ,4-a-glucosidic linkages in amylopectin and related glucose polymers, thereby releasing maltose. β-Amylases have been isolated from various plants and microorganisms. See Fogarty et al. (1979) in PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 112-115. As used herein, the term "Enzyme units" 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. As another example, beta amylase activity can be measured in degrees Diastatic Power (DP°) Units. This assay is based on a 30-min hydrolysis of a starch substrate at pH 4.6 and 20 °C. The reducing sugar groups produced on hydrolysis are measured in a titrimetric procedure using alkaline

ferricyanide. One unit of diastase activity, expressed as degrees DP (DP°), is defined as the amount of enzyme, contained in 0.1 ml of a 5% solution of the sample enzyme preparation, that will produce sufficient reducing sugars to reduce 5 ml_ of Fehling's solution when the sample is incubated with 100 ml_ of the substrate for 1 hour at 20 °C.

The term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula

(C₆HioO₅)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, sweet sorghum, 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 terms, "wild-type," "parental," or "reference," with respect to a polypeptide, refer 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, refer 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 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. 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.

The term "at least one non-naturally occuring DNA base at a position that allows encoding of a degenerate amino acid" refers to a normal DNA base (e.g. A,T,G, or C), or synthetic analog thereof (eg-PNA, LNA) that resides at the same position as a naturally occuring base (that is, one that exists in nature at that location in the sequence), but does not exist in nature itself in this sequence context, and which when transcribed and translated results in the same amino acid (ie-the degenerate amino acid) that would have arisen had the naturally-occuring DNA base actually been in the nucleic acid.

The term "at least one non-naturally occuring DNA base at a position that allows encoding of a conservative amino acid" refers to a normal DNA base (e.g. A,T,G, or C), or synthetic analog thereof (eg-PNA, LNA) that resides at the same position as a naturally occuring base (that is, one that exists in nature at that location in the sequence), but does not exist in nature itself in this sequence context, and which when transcribed and translated results in a conserved amino acid substitution (e.g. as provided in Table 1 herein) at that position.

In the case of the present beta amylases, "activity" refers to beta amylase 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. A vector comprising a nucleic acid encoding a beta- amylase is a recombinant vector.

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" polypeptides, thereof, includes, but is not limited to, a culture broth containing secreted polypeptide expressed in a heterologous host cell. 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 term "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.

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 a beta 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 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. 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). 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. "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 (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. Very stringent hybridization conditions involve 68 °C and 0.1 X SSC

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 {e.g., a beta amylase) has been introduced. Exemplary host strains are microorganism cells {e.g., bacteria, filamentous fungi, and yeast) or plants 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.

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 which 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.

"Biologically active" refers to a sequence having a specified biological activity, such an enzymatic activity.

The term "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.

As used herein, "water hardness" is a measure of the minerals {e.g., calcium and magnesium) present in water.

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. For amylases, 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. For example, 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. In another conceivable method, 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.

As used herein, "a cultured cell material comprising an amylase" or similar language, refers to a cell lysate or supernatant (including media) that includes a beta 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.

As used herein, "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: 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 termini are included. For example, a variant with five amino acid deletions of the C-terminus of the 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 two subject polypeptide sequences.

The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina, particularly Pezizomycotina species.

The term "degree of polymerization" (DP) refers to the number (n) of

anhydro-glucopyranose units in a given saccharide. Examples of DP1 are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose. 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.

The term "dry solids content" (ds) 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 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 an amylase, 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. An "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 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. Examples of beers include: full malted beer, beer brewed under the "Reinheitsgebot," ale, India pale ale, lager, pilsner, bitter, Happoshu (second beer), third beer, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock, dopplebock, stout, porter, 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 that 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. Additional mutations

In some embodiments, the present beta amylases further include one or more mutations that provide a further performance or stability benefit. Exemplary

performance benfits include but are not limited to increased hydrolysis of a starch substrate, increased grain, cereal or other starch substrate liquifaction performance, increased cleaning performance, increased thermal stability, increased storage stability, increased solubility, an altered pH profile, decreased calcium dependence, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, and increased expression. In some cases, the performance benefit is realized at a relatively low temperature. In some cases, the performance benefit is realized at relatively high temperature.

Furthermore, the present beta amylases may include any number of

conservative amino acid substitutions. Exemplary conservative amino acid

substitutions are listed in the following Table 1 . Table 1 . Conservative amino acid substitutions

The reader will appreciate that some of the above mentioned conservative mutations can be produced by genetic manipulation, while others are produced by introducing synthetic amino acids into a polypeptide by genetic or other means.

The present beta amylase 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 beta amylase polypeptides. The present beta amylase polypeptides may also be truncated to remove the N or C-termini, so long as the resulting polypeptides retain beta amylase activity.

The present beta amylase may be a "chimeric" or "hybrid" polypeptide, in that it includes at least a portion of a first beta amylase polypeptide, and at least a portion of a second beta amylase polypeptide (such chimeric amylases have recently been

"rediscovered" as domain-swap amylases). The present beta amylases 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.

Nucleotides encoding beta amylase polypeptides

In another aspect, nucleic acids encoding a beta amylase polypeptide are provided. The nucleic acid may encode a particular beta amylase polypeptide, or a beta amylase having a specified degree of amino acid sequence identity to the particular amylase.

In one example, the nucleic acid encodes a beta amylase having at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 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% identity to SEQ ID NO:1 . It will be appreciated that due to the degeneracy of the genetic code, a plurality of nucleic acids may encode the same polypeptide.

In another example, the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) a beta amylase having at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 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% identity to SEQ ID NO: 1 . Such stringent and very stringent hybridization conditions are described herein.

Nucleic acids may encode a "full-length" ("fl" or "FL") beta amylase, which includes a signal sequence, only the mature form of a beta amylase, which lacks the signal sequence, or a truncated form of a beta amylase, which lacks the N or C-terminus of the mature form.

A nucleic acid that encodes a beta amylase can be operably linked to various promoters and regulators in a vector suitable for expressing the beta amylase in host cells. Exemplary promoters are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and Streptomyces CelA. Such a nucleic acid can also be linked to other coding sequences, e.g., to encode a chimeric polypeptide.

Production of Variant Beta Amylases

The present beta amylases can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material {e.g., a whole-cell broth) comprising a beta amylase can be obtained following secretion of the beta amylase into the cell medium. Optionally, the beta amylase can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final beta amylase. A gene encoding a beta amylase 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 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 beta 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 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.

Vectors A DNA construct comprising a nucleic acid encoding beta amylases can be constructed to be expressed in a host cell. Representative nucleic acids that encode beta amylases include SEQ ID NO: 1 . Nucleic acids encoding beta amylases 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 beta amylase 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 beta amylase. 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, aiwww.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011 ) Applied Environ. Microbiol. 77: 391 6-22. pJG153can be modified with routine skill to comprise and express a nucleic acid encoding a beta amylase.

A nucleic acid encoding a beta amylase 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 a beta amylase, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA 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 a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. 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 beta amylase 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, 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 beta amylase 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. 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 variant beta amylase. 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, pUB110, 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 xxsC, 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 beta amylase for subsequent enrichment or purification. Extracellular secretion of amylase into the culture medium can also be used to make a cultured cell material comprising the isolated beta amylase.

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 beta amylase 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, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the beta amylase is operably linked to the control sequences in proper manner with respect to expression. The procedures used to ligate the DNA construct encoding a beta amylase, 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, 2^nd ed., Cold Spring Harbor, 1989, and 3^rd ed., 2001 ).

Transformation and Culture of Host Cells

An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of a beta amylase. 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.

Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, GeobaciHus (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. including Lactobacillus reuteri; Leuconostoc sp. ; Pediococcus sp. ; and Streptococcus sp. Alternatively, 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.

Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger,

Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, 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. In addition,

Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. A beta amylase 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 beta amylase. The type and/or degree of glycosylation may impart changes in enzymatic and/or biochemical properties.

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 beta amylase 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 ei 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 μΙ_ 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 beta amylase 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 a beta amylase. 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 beta amylase. 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 amylase 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 a beta amylase 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 a beta amylase. 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 75 °C {e.g., 30 °C to 45 °C), depending on the needs of the host and production of the desired beta amylase. 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 beta amylase. Methods for Enriching and Purifying Beta Amylases

Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated beta amylase 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 a beta amylase 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 beta amylase polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the enriched or 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 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 beta amylase 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 a beta amylase. 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 beta amylase 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 the 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 alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl 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 alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds.

Exemplary organic compounds are linear alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl 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, beta amylase 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 enriched or purified. Generally, the pH is adjusted at a level near the isoelectric point of the amylase. 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 an enriched or 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 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 enriched or 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 enriched or 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 enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enriched or 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 beta amylase polypeptide accumulates in the culture broth. For the isolation, enrichment, or purification of the desired beta amylase, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or 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 enrichment or purification, a conventional procedure such as ion exchange chromatography may be used.

Enriched or 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 more specific example of enrichment or purification, is described in Sumitani et al. (2000) "New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. 195 a-amylase contributes to starch binding and raw starch degrading," Biochem. J. 350: 477-484, and is briefly summarized here. The enzyme obtained from 4 liters of a Streptomyces lividans TK24 culture supernatant is treated with (NH₄)₂S0₄ at 80% saturation. The precipitate is recovered by centrifugation at 10,000 x g (20 min. and 4°C) and re-dissolved in 20 mM Tris/HCI buffer (pH 7.0) containing 5 mM CaCI₂. The solubilized precipitate is then dialyzed against the same buffer. The dialyzed sample is then applied to a Sephacryl S-200 column, which had previously been equilibrated with 20 mM Tris/HCI buffer, (pH 7.0), 5 mM CaCI₂, and eluted at a linear flow rate of 7 mL/hr with the same buffer. Fractions from the column are collected and assessed for activity as judged by enzyme assay and SDS-PAGE. The protein is further purified as follows. A Toyopearl HW55 column (Tosoh Bioscience, Montgomeryville, PA; Cat. No. 19812) is equilibrated with 20 mM Tris/HCI buffer (pH 7.0) containing 5 mM CaCI₂ and 1 .5 M (NH )₂S0₄. The enzyme is eluted with a linear gradient of 1 .5 to 0 M (NH₄)₂S0₄ in 20 mM Tris/HCL buffer, pH 7.0 containing 5 mM CaCI₂. The active fractions are collected, and the enzyme precipitated with (NH₄)₂S0₄ at 80% saturation. The precipitate is recovered, re-dissolved, and dialyzed as described above. The dialyzed sample is then applied to a Mono Q HR5/5 column (Amersham Pharmacia; Cat. No. 17-51 67-01 ) previously equilibrated with 20 mM Tris/HCI buffer (pH 7.0) containing 5 mM CaCI₂, at a flow rate of 60 mL/hour. The active fractions are collected and added to a 1 .5 M (NH₄)₂S0₄ solution. The active enzyme fractions are re-chromatographed on a Toyopearl HW55 column, as before, to yield a homogeneous enzyme as determined by SDS-PAGE. See Sumitani et al. (2000) Biochem. J. 350: 477-484, for general discussion of the method and variations thereon.

For production scale recovery, beta amylase polypeptides can be enriched or partially purified as generally described above by removing cells via flocculation with polymers. Alternatively, the enzyme can be enriched or purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, for some applications, 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.

Compositions and Uses of Beta Amylases

The beta amylases provided by the present teachings are useful for a variety of industrial applications. For example, beta 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. For example, 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; 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, and oxaloacetate); amino acids {e.g., serine and glycine); antibiotics;

antimicrobials; enzymes; vitamins; and hormones.

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). One skilled in the art is aware of various fermentation conditions that may be used in the production of these end-products. Variant beta amylases are also useful in compositions and methods of food preparation. These various uses of variant beta amylases are described in more detail below.

It will be appreciated by one of ordinary skill in the art that various accessory enzymes can be used with the beta enzymes of the present teachings, as will be the case in various applications and contexts.

In the field of grain processing to produce maltose syrups, the beta amylases can be employed in any of a variety of applications, including those described in US

Provisional Application 61 /61 6,990, filed March 28, 2012 and PCT 2013/30384, filed March 12, 2013.

Preparation of Starch Substrates from Plants

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, rice, peas, bean, banana, sweet sorghum, 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. 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 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.

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 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.

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(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 enrichment, 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 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 appropriately engineered fungal strain. Fungal host cells that express and secrete other enzymes, in addition to amylase, also can be used. Such cells may express glucoamylase, amylase, and/or a pullulanase, phytase, a/p/?a-glucosidase, isoamylase, beta-amylase, cellulase, xylanase, other hemicellulases, protease, £>efa-glucosidase, pectinase, esterase, redox enzymes, transferase, 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 Beta Amylases

The beta amylases of the present teachings may be combined with a

glucoamylase (EC 3.2.1 .3), e.g., a Trichoderma glucoamylase or variant thereof. An exemplary glucoamylase is 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.). 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. Beta amylases may advantageously increase the yield of glucose produced in a saccharification process catalyzed by TrGA.

Alternatively, the glucoamylase may be another glucoamylase derived from plants (including algae), fungi, or bacteria. For example, the glucoamylases may be Aspergillus niger ^ 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.)). Other contemplated 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). Other contemplated 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. Patent No. 4,587,215). Contemplated bacterial glucoamylases include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum {e.g., EP 135,1 38 (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 shown in SEQ ID NO:2 in WO

00/04136 (Novo Nordisk A/S). Also suitable are commercial glucoamylases, such as AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco US Inc.); AMIGASE™ and AMIGASE™ PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); and G-ZYME® G990 ZR {A. nigex

glucoamylase with a low protease content). Still other suitable glucoamylases include Aspergillus fumigatus glucoamylase, Talaromyces glucoamylase, Thielavia

glucoamylase, Trametes glucoamylase, Thermomyces glucoamylase, Athelia

glucoamylase, or Humicola glucoamylase {e.g., HgGA). Glucoamylases typically are added in an amount of about 0.1 - 2 glucoamylase units (GAU)/g ds, e.g., about 0.1 6 GAU/g ds, 0.23 GAU/g ds, or 0.33 GAU/g ds. Other suitable enzymes that can be used with the beta amylase of the present teachings include a phytase, protease, pullulanase, another β-amylase, isoamylase, an a-amylase, glucoamylase, alpha-glucosidase, cellulase, xylanase, other hemicellulases, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, or a combination thereof. For example, 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; and Trichoderma.

Compositions comprising the present beta amylases 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.

Compositions and Methods for Baking and Food Preparation The present teachings also relate to a "food composition," including but not limited to a food product, animal feed and/or food/feed additives, comprising a beta amylase, and methods for preparing such a food composition comprising mixing the beta amylase with one or more food ingredients, or uses thereof.

Furthermore, the present teachings relate to the use of a beta amylase in the preparation of a food composition, wherein the food composition is baked subsequent to the addition of the polypeptide of the present invention. As used herein the term "baking composition" means any composition and/or additive prepared in the process of providing a baked food product, including but not limited to bakers flour, a dough, a baking additive and/or a baked product. The food composition or additive may be liquid or solid.

As used herein, the term "flour" means milled or ground cereal grain. The term "flour" also may mean Sago or tuber products that have been ground or mashed. In some embodiments, flour may also contain components in addition to the milled or mashed cereal or plant matter. An example of an additional component, although not intended to be limiting, is a leavening agent. Cereal grains include wheat, oat, rye, and barley. Tuber products include tapioca flour, cassava flour, and custard powder. The term "flour" also includes ground corn flour, maize-meal, rice flour, whole-meal flour, self-rising flour, tapioca flour, cassava flour, ground rice, enriched flower, and custard powder.

For the commercial and home use of flour for baking and food production, it is important to maintain an appropriate level of a-amylase activity in the flour. A level of activity that is too high may result in a product that is sticky and/or doughy and therefore unmarketable. Flour with insufficient a-amylase activity may not contain enough sugar for proper yeast function, resulting in dry, crumbly bread, or baked products.

Accordingly, a beta amylase, by itself or in combination with an a-amylase(s), may be added to the flour to augment the level of endogenous α-amylase activity in flour.

A beta amylase can further be added alone or in a combination with other amylases to prevent or retard staling, i.e., crumb firming of baked products. The amount of anti-staling amylase will typically be in the range of 0.01 -10 mg of enzyme protein per kg of flour, e.g., 0.5 mg/kg ds. Additional anti-staling amylases that can be used in combination with an amylase include an endo-amylase, e.g., a bacterial endo-amylase from Bacillus. The additional amylase can be another beta-amylase (EC 3.2.1 .133), e.g., from Bacillus. Novamyl® is an exemplary maltogenic a-amylase from B. stearothermophilus strain NCIB 11837 and is described in Christophersen et al. (1997) Starch 50: 39-45. Other examples of anti-staling endo-amylases include bacterial a-amylases derived from Bacillus, such as B. licheniformis or B.

amyloliquefaciens. The anti-staling amylase may be an exo-amylase, such as β-amylase, e.g., from plant sources, such as soy bean, or from microbial sources, such as Bacillus.

The baking composition comprising a beta amylase further can comprise a phospholipase or enzyme with phospholipase activity. An enzyme with phospholipase activity has an activity that can be measured in Lipase Units (LU). The phospholipase may have A-i or A₂ activity to remove fatty acid from the phospholipids, forming a lysophospholipid. It may or may not have lipase activity, i.e., activity on triglyceride substrates. The phospholipase typically has a temperature optimum in the range of 30 °C -90°C, e.g., 30 °C -70°C. The added phospholipases can be of animal origin, for example, from pancreas, e.g., bovine or porcine pancreas, snake venom or bee venom. Alternatively, the phospholipase may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, for example.

The phospholipase is added in an amount that improves the softness of the bread during the initial period after baking, particularly the first 24 hours. The amount of phospholipase will typically be in the range of 0.01 -10 mg of enzyme protein per kg of flour, e.g., 0.1 -5 mg/kg. That is, phospholipase activity generally will be in the range of 20-1000 LU/kg of flour, where a Lipase Unit is defined as the amount of enzyme required to release 1 μηποΙ butyric acid per minute at 30 °C, pH 7.0, with gum arabic as emulsifier and tributyrin as substrate.

Compositions of dough generally comprise wheat meal or wheat flour and/or other types of meal, flour or starch such as corn flour, cornstarch, rye meal, rye flour, oat flour, oatmeal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch. The dough may be fresh, frozen or par-baked. The dough can be a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways, such as by adding chemical leavening agents, e.g., sodium bicarbonate or by adding a leaven, i.e., fermenting dough. Dough also may be leavened by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), e.g., a commercially available strain of S. cerevisiae.

The dough may also comprise other conventional dough ingredients, e.g., proteins, such as milk powder, gluten, and soy; eggs {e.g., whole eggs, egg yolks or egg whites); an oxidant, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L-cysteine; a sugar; or a salt, such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate. The dough further may comprise fat, e.g., triglyceride, such as granulated fat or shortening. The dough further may comprise an emulsifier such as mono- or diglycerides, diacetyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, or lysolecithin. In particular, the dough can be made without addition of emulsifiers. The dough product may be any processed dough product, including fried, deep fried, roasted, baked, steamed and boiled doughs, such as steamed bread and rice cakes. In one embodiment, the food product is a bakery product. Typical bakery (baked) products include bread - such as loaves, rolls, buns, bagels, pizza bases etc. pastry, pretzels, tortillas, cakes, cookies, biscuits, crackers etc. Optionally, an additional enzyme may be used together with the anti-staling amylase and the phospholipase. The additional enzyme may be a second beta amylase, such as an amyloglucosidase, an alpha amylase, a cyclodextrin

glucanotransferase, or the additional enzyme may be a peptidase, in particular an exopeptidase, a transglutaminase, a lipase, a cellulase, a xylanase, a protease, a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95/00636, for example, a glycosyltransferase, a branching enzyme (1 ,4-a-glucan branching enzyme), a 4-a-glucanotransferase (dextrin glycosyltransferase) or an oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a lipooxygenase, an L-amino acid oxidase or a carbohydrate oxidase. The additional enzyme(s) may be of any origin, including mammalian and plant, and particularly of microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art.

The xylanase is typically of microbial origin, e.g., derived from a bacterium or fungus, such as a strain of Aspergillus. Xylanases include Pentopan® and Novozym 384®, for example, which are commercially available xylanase preparations produced from Trichoderma reesei. The amyloglucosidase may be an A. niger amyloglucosidase (such as AMG®). Other useful amylase products include Grindamyl® A 1000 or A 5000 (Grindsted Products, Denmark) and Amylase® H or Amylase® P (DSM). The glucose oxidase may be a fungal glucose oxidase, in particular an Aspergillus niger glucose oxidase (such as Gluzyme®). An exemplary protease is Neutrase®.

The process may be used for any kind of baked product prepared from dough, either of a soft or a crisp character, either of a white, light or dark type. Examples are bread, particularly white, whole-meal or rye bread, typically in the form of loaves or rolls, such as, but not limited to, French baguette-type bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza and the like.

A beta amylase may be used in a pre-mix, comprising flour together with an anti-staling amylase, a phospholipase, and/or a phospholipid. The pre-mix may contain other dough-improving and/or bread-improving additives, e.g., any of the additives, including enzymes, mentioned above. A beta amylase can be a component of an enzyme preparation comprising an anti-staling amylase and a phospholipase, for use as a baking additive.

The enzyme preparation is optionally in the form of a granulate or agglomerated powder. The preparation can have a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 μηι.

Granulates and agglomerated powders may be prepared by conventional methods, e.g., by spraying an amylase onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g., a salt (such as NaCI or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.

Enveloped particles, i.e., beta amylase particles, can comprise a beta amylase. To prepare enveloped beta amylase particles, the enzyme is contacted with a food grade lipid in sufficient quantity to suspend all of the beta amylase particles. Food grade lipids, as used herein, may be any naturally organic compound that is insoluble in water but is soluble in non-polar organic solvents such as hydrocarbon or diethyl ether. Suitable food grade lipids include, but are not limited to, triglycerides either in the form of fats or oils that are either saturated or unsaturated. Examples of fatty acids and combinations thereof which make up the saturated triglycerides include, but are not limited to, butyric (derived from milk fat), palmitic (derived from animal and plant fat), and/or stearic (derived from animal and plant fat). Examples of fatty acids and combinations thereof which make up the unsaturated triglycerides include, but are not limited to, palmitoleic (derived from animal and plant fat), oleic (derived from animal and plant fat), linoleic (derived from plant oils), and/or linolenic (derived from linseed oil). Other suitable food grade lipids include, but are not limited to, monoglycerides and diglycerides derived from the triglycerides discussed above, phospholipids and glycolipids.

The food grade lipid, particularly in the liquid form, is contacted with a powdered form of the beta amylase particles in such a fashion that the lipid material covers at least a portion of the surface of at least a majority, e.g., 100% of the beta amylase particles. Thus, each beta amylase particle is individually enveloped in a lipid. For example, all or substantially all of the beta amylase particles are provided with a thin, continuous, enveloping film of lipid. This can be accomplished by first pouring a quantity of lipid into a container, and then slurrying the beta amylase particles so that the lipid thoroughly wets the surface of each beta amylase particle. After a short period of stirring, the enveloped beta amylase particles, carrying a substantial amount of the lipids on their surfaces, are recovered. The thickness of the coating so applied to the particles of beta amylase can be controlled by selection of the type of lipid used and by repeating the operation in order to build up a thicker film, when desired.

The storing, handling and incorporation of the loaded delivery vehicle can be accomplished by means of a packaged mix. The packaged mix can comprise the enveloped beta amylase. However, the packaged mix may further contain additional ingredients as required by the manufacturer or baker. After the enveloped beta amylase has been incorporated into the dough, the baker continues through the normal production process for that product.

The advantages of enveloping the beta amylase particles are two-fold. First, the food grade lipid protects the enzyme from thermal denaturation during the baking process for those enzymes that are heat labile. Consequently, while the beta amylase is stabilized and protected during the proving and baking stages, it is released from the protective coating in the final baked good product, where it hydrolyzes the glucosidic linkages in polyglucans. The loaded delivery vehicle also provides a sustained release of the active enzyme into the baked good. That is, followi ng the baking process, active beta amylase is continually released from the protective coating at a rate that counteracts, and therefore reduces the rate of, staling mechanisms.

In general, the amount of lipid applied to the beta amylase particles can vary from a few percent of the total weight of the beta amylase to many times that weight, depending upon the nature of the lipid, the manner in which it is applied to the beta amylase particles, the composition of the dough mixture to be treated, and the severity of the dough-mixing operation involved.

The loaded delivery vehicle, i.e., the lipid-enveloped enzyme, is added to the ingredients used to prepare a baked good in an effective amount to extend the shelf-life of the baked good. The baker computes the amount of enveloped beta amylase, prepared as discussed above, that will be required to achieve the desired anti-staling effect. The amount of the enveloped beta amylase required is calculated based on the concentration of enzyme enveloped and on the proportion of beta amylase to flour specified. A wide range of concentrations has been found to be effective, although, as has been discussed, observable improvements in anti-staling do not correspond linearly with the beta amylase concentration, but above certain minimal levels, large increases in beta amylase concentration produce little additional improvement. The beta amylase concentration actually used in a particular bakery production could be much higher than the minimum necessary to provide the baker with some insurance against inadvertent under-measurement errors by the baker. The lower limit of enzyme concentration is determined by the minimum anti-staling effect the baker wishes to achieve.

A method of preparing a baked good may comprise: a) preparing lipid-coated beta amylase particles, where substantially all of the beta amylase particles are coated; b) mixing a dough containing flour; c) adding the lipid-coated beta amylase to the dough before the mixing is complete and terminating the mixing before the lipid coating is removed from the beta amylase; d) proofing the dough; and e) baking the dough to provide the baked good, where the beta amylase is inactive during the mixing, proofing and baking stages and is active in the baked good.

The enveloped beta amylase can be added to the dough during the mix cycle, e.g., near the end of the mix cycle. The enveloped beta amylase is added at a point in the mixing stage that allows sufficient distribution of the enveloped beta amylase throughout the dough; however, the mixing stage is terminated before the protective coating becomes stripped from the beta amylase particle(s). Depending on the type and volume of dough, and mixer action and speed, anywhere from one to six minutes or more might be required to mix the enveloped beta amylase into the dough, but two to four minutes is average. Thus, several variables may determine the precise procedure. First, the quantity of enveloped beta amylase should have a total volume sufficient to allow the enveloped beta amylase to be spread throughout the dough mix. If the preparation of enveloped beta amylase is highly concentrated, additional oil may need to be added to the pre-mix before the enveloped beta amylase is added to the dough. Recipes and production processes may require specific modifications; however, good results generally can be achieved when 25% of the oil specified in a bread dough formula is held out of the dough and is used as a carrier for a concentrated enveloped a-amylase when added near the end of the mix cycle. In bread or other baked goods, particularly those having a low fat content, e.g., French-style breads, an enveloped beta amylase mixture of approximately 1 % of the dry flour weight is sufficient to admix the enveloped a-amylase properly with the dough. The range of suitable percentages is wide and depends on the formula, finished product, and production methodology requirements of the individual baker. Second, the enveloped beta amylase suspension should be added to the mix with sufficient time for complete mixture into the dough, but not for such a time that excessive mechanical action strips the protective lipid coating from the enveloped beta amylase particles.

In a further aspect of the invention, the food composition is an oil, meat, lard, composition comprising a beta amylase. In this context the term "oil/meat/lard" composition" means any composition, based on, made from and/or containing oil, meat or lard, respectively. Another aspect the invention relates to a method of preparing an oil or meat or lard composition and/or additive comprising a beta amylase, comprising mixing the polypeptide of the invention with a oil/meat/lard composition and/or additive ingredients. In a further aspect of the invention, the food composition is an animal feed composition, animal feed additive and/or pet food comprising a beta amylase and variants thereof. The present invention further relates to a method for preparing such an animal feed composition, animal feed additive composition and/or pet food

comprising mixing a beta amylase and variants thereof with one or more animal feed ingredients and/or animal feed additive ingredients and/or pet food ingredients.

Furthermore, the present invention relates to the use of a beta amylase in the

preparation of an animal feed composition and/or animal feed additive composition and/or pet food.

The term "animal" includes all non-ruminant and ruminant animals. In a particular embodiment, the animal is a non-ruminant animal, such as a horse and a mono-gastric animal. Examples of mono-gastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as shrimps and prawns. In a further embodiment the animal is a ruminant animal including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.

In the present context, it is intended that the term "pet food" is understood to mean a food for a household animal such as, but not limited to dogs, cats, gerbils, hamsters, chinchillas, fancy rats, guinea pigs; avian pets, such as canaries, parakeets, and parrots; reptile pets, such as turtles, lizards and snakes; and aquatic pets, such as tropical fish and frogs.

The terms "animal feed composition," "feedstuff' and "fodder" are used interchangeably and may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains {e.g., wheat, barley, rye, oats and

combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS)

(particularly corn based Distillers Dried Grain Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins. Textile Desizing Compositions and Use

Also contemplated are compositions and methods of treating fabrics {e.g., to desize a textile) using a beta amylase. Fabric-treating methods are well known in the art {see, e.g., U.S. Patent No. 6,077,31 6). For example, the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with a beta amylase in a solution. The fabric can be treated with the solution under pressure.

A beta amylase can be applied during or after the weaving of a textile, or during the desizing stage, or one or more additional fabric processing steps. During the weaving of textiles, the threads are exposed to considerable mechanical strain. Prior to weaving on mechanical looms, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and to prevent breaking. A beta amylase can be applied during or after the weaving to remove these sizing starch or starch derivatives. After weaving, a beta amylase can be used to remove the size coating before further processing the fabric to ensure a homogeneous and wash-proof result. A beta amylase can be used alone or with other desizing chemical reagents and/or desizing enzymes to desize fabrics, including cotton-containing fabrics, as detergent additives, e.g., in aqueous compositions. A beta amylase also can be used in compositions and methods for producing a stonewashed look on indigo-dyed denim fabric and garments. For the manufacture of clothes, the fabric can be cut and sewn into clothes or garments, which are afterwards finished. In particular, for the manufacture of denim jeans, different enzymatic finishing methods have been developed. The finishing of denim garment normally is initiated with an enzymatic desizing step, during which garments are subjected to the action of amylolytic enzymes to provide softness to the fabric and make the cotton more accessible to the subsequent enzymatic finishing steps. A beta amylase can be used in methods of finishing denim garments {e.g., a "bio-stoning process"), enzymatic desizing and providing softness to fabrics, and/or finishing process.

Cleaning Compositions An aspect of the present compositions and methods is a cleaning composition that includes a beta amylase as a component. A beta amylase polypeptide can be used as a component in detergent compositions for hand washing, laundry washing, dishwashing, and other hard-surface cleaning.

Overview of Cleaning Compositions Preferably, a beta amylase is incorporated into detergents at or near a concentration conventionally used for amylase in detergents. For example, a beta amylase polypeptide may be added in amount corresponding to 0.00001 - 1 mg

(calculated as pure enzyme protein) of beta amylase per liter of wash/dishwash liquor. Exemplary formulations are provided herein, as exemplified by the following: A beta amylase polypeptide may be a component of a detergent composition, as the only enzyme or with other enzymes including other amylolytic enzymes. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1 ,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in, for example, GB 1483591 . Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are known in the art. Protected enzymes may be prepared according to the method disclosed in for example EP 238 21 6. Polyols have long been recognized as stabilizers of proteins, as well as improving protein solubility.

The detergent composition may be in any useful form, e.g., as powders, granules, pastes, or liquid. A liquid detergent may be aqueous, typically containing up to about 70% of water and 0% to about 30% of organic solvent. It may also be in the form of a compact gel type containing only about 30% water.

The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic. The detergent will usually contain 0% to about 50% of anionic surfactant, such as linear alkylbenzenesulfonate (LAS);

a-olefinsulfonate (AOS); alkyl sulfate (fatty alcohol sulfate) (AS); alcohol ethoxysulfate (AEOS or AES); secondary alkanesulfonates (SAS); a-sulfo fatty acid methyl esters; alkyl- or alkenylsuccinic acid; or soap. The composition may also contain 0% to about 40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,

alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid

monoethanolamide, or polyhydroxy alkyl fatty acid amide (as described for example in WO 92/06154).

The detergent composition may additionally comprise one or more other enzymes, such as proteases, another amylolytic enzyme, cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase, peroxidase, and/or laccase in any combination.

The detergent may contain about 1 % to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates {e.g., SKS-6 from Hoechst). The detergent may also be unbuilt, i.e. essentially free of detergent builder. The enzymes can be used in any composition compatible with the stability of the enzyme. Enzymes generally can be protected against deleterious components by known forms of encapsulation, for example, by granulation or sequestration in hydro gels. Enzymes, and specifically maltoogenic amylases, either with or without starch binding domains, can be used in a variety of compositions including laundry and dishwashing applications, surface cleaners, as well as in compositions for ethanol production from starch or biomass.

The detergent may comprise one or more polymers. Examples include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), polyvinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂ source such as perborate or percarbonate, which may be combined with a

peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, the bleaching system may comprise peroxyacids {e.g., the amide, imide, or sulfone type peroxyacids). The bleaching system can also be an enzymatic bleaching system, for example,

perhydrolase, such as that described in International PCT Application WO 2005/056783.

The enzymes of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol; a sugar or sugar alcohol; lactic acid; boric acid or a boric acid derivative such as, e.g., an aromatic borate ester; and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g., fabric conditioners including clays, foam boosters, suds suppressors,

anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibiters, optical brighteners, or perfumes. The pH (measured in aqueous solution at use concentration) is usually neutral or alkaline, e.g., pH about 7.0 to about 11 .0.

Particular forms of detergent compositions for inclusion of the present beta amylase are described, below.

Heavy Duty Liquid (HDL) laundry detergent composition

Exemplary HDL laundry detergent compositions includes a detersive surfactant (10%-40% wt/wt), including an anionic detersive surfactant (selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof), and optionally non-ionic surfactant (selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohol, for example a C₈-Ci₈ alkyl ethoxylated alcohol and/or C₆-Ci₂ alkyl phenol alkoxylates), wherein the weight ratio of anionic detersive surfactant (with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersive surfactant is greater than 1 1 . Suitable detersive surfactants also include cationic detersive surfactants (selected from a group of alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulphonium compounds, and/or mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (selected from a group of alkanolamine sulpho-betaines); ampholytic surfactants; semi-polar non-ionic surfactants and mixtures thereof.

The composition may optionally include, a surfactancy boosting polymer consisting of amphiphilic alkoxylated grease cleaning polymers (selected from a group of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylenimines in the range of 0.05wt%-10wt%) and/or random graft polymers (typically comprising of hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated C-|-C₆ carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols such as glycerol, and mixtures thereof; and hydrophobic side chain(s) selected from the group consisting of: C₄-C₂5 alkyl group, polypropylene, polybutylene, vinyl ester of a saturated CrC₆ mono-carboxylic acid, CrC₆ alkyl ester of acrylic or methacrylic acid, and mixtures thereof.

The composition may include additional polymers such as soil release polymers (include anionically end-capped polyesters, for example SRP1 , polymers comprising at least one monomer unit selected from saccharide, dicarboxylic acid, polyol and combinations thereof, in random or block configuration, ethylene terephthalate-based polymers and co-polymers thereof in random or block configuration, for example Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL), anti-redeposition polymers (0.1 wt% to 10wt%, include carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof, vinylpyrrolidone homopolymer, and/or polyethylene glycol, molecular weight in the range of from 500 to 100,000 Da); cellulosic polymer (including those selected from alkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixures thereof) and polymeric carboxylate (such as maleate/acrylate random copolymer or polyacrylate homopolymer). The composition may further include saturated or unsaturated fatty acid, preferably saturated or unsaturated Ci₂-C₂₄ fatty acid (0 wt% to 10 wt%); deposition aids (examples for which include polysaccharides, preferably cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DAD MAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, in random or block configuration, cationic guar gum, cationic cellulose such as cationic hydoxyethyl cellulose, cationic starch, cationic polyacylamides, and mixtures thereof.

The composition may further include dye transfer inhibiting agents, examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof; chelating agents, examples of which include ethylene-diamine-tetraacetic acid (EDTA), diethylene triamine penta methylene phosphonic acid (DTPMP), hydroxy-ethane diphosphonic acid (HEDP), ethylenediamine Ν,Ν'-disuccinic acid (EDDS), methyl glycine diacetic acid (MGDA), diethylene triamine penta acetic acid (DTPA), propylene diamine tetracetic acid (PDT A), 2-hydroxypyridine-N-oxide (HPNO), or methyl glycine diacetic acid

(MGDA), glutamic acid Ν,Ν-diacetic acid (Ν,Ν-dicarboxymethyl glutamic acid

tetrasodium salt (GLDA), nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts thereof, N-hydroxyethylethylenediaminetri-acetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid

(EDTP), and derivatives thereof.

The composition preferably includes enzymes (generally about 0.01 wt% active enzyme to 0.03wt% active enzyme) selected from proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases,

perhydrolases, arylesterases, and any mixture thereof. The composition may include an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).

The composition optionally includes silicone or fatty-acid based suds

suppressors; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti-foam (0.001 wt% to about 4.0wt%), and/or structurant/thickener (0.01 wt% to 5wt%, selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof). The composition can be any liquid form, for example a liquid or gel form, or any combination thereof. The composition may be in any unit dose form, for example a pouch.

Heavy Duty Dry/Solid (HDD) laundry detergent composition Exemplary HDD laundry detergent compositions includes a detersive surfactant, including anionic detersive surfactants {e.g., linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and/or mixtures thereof), non-ionic detersive surfactant {e.g., linear or branched or random chain, substituted or unsubstituted C₈-Ci₈ alkyl ethoxylates, and/or C₆-Ci₂ alkyl phenol alkoxylates), cationic detersive surfactants {e.g., alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulphonium compounds, and mixtures thereof), zwitterionic and/or amphoteric detersive surfactants {e.g., alkanolamine sulpho-betaines), ampholytic surfactants, semi-polar non-ionic surfactants, and mixtures thereof; builders including phosphate free builders (for example zeolite builders examples which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0wt% to less than 10wt%), phosphate builders (for example sodium tri-polyphosphate in the range of 0wt% to less than 10wt%), citric acid, citrate salts and nitrilotriacetic acid, silicate salt {e.g., sodium or potassium silicate or sodium meta-silicate in the range of Owt% to less than 10wt%, or layered silicate (SKS-6)); carbonate salt {e.g., sodium carbonate and/or sodium bicarbonate in the range of 0 wt% to less than 80 wt%); and bleaching agents including photobleaches {e.g., sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines,

xanthenes dyes, and mixtures thereof) hydrophobic or hydrophilic bleach activators {e.g., dodecanoyl oxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl

oxybenzoic acid or salts thereof, 3,5,5-trimethy hexanoyl oxybenzene sulfonate, tetraacetyl ethylene diamine-TAED, nonanoyloxybenzene sulfonate-NOBS, nitrile quats, and mixtures thereof), sources of hydrogen peroxide {e.g., inorganic perhydrate salts examples of which include mono or tetra hydrate sodium salt of perborate, percarbonate, persulfate, perphosphate, or persilicate), preformed hydrophilic and/or hydrophobic peracids {e.g., percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof), and/or bleach catalysts {e.g., imine bleach boosters (examples of which include iminium cations and polyions), iminium zwitterions, modified amines, modified amine oxides, N-sulphonyl imines, N-phosphonyl imines, N-acyl imines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones, and mixtures thereof, and metal-containing bleach catalysts {e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations along with an auxiliary metal cations such as zinc or aluminum and a sequestrate such as ethylenediaminetetraacetic acid,

ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof).

The composition preferably includes enzymes, e.g., proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases,

mannanases, cutinases, laccases, phospholipases, lysophospholipases,

acyltransferase, perhydrolase, arylesterase, and any mixture thereof.

The composition may optionally include additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accord, hueing agents, additional polymers, including fabric integrity and cationic polymers, dye-lock ingredients, fabric-softening agents, brighteners (for example C.I. Fluorescent brighteners), flocculating agents, chelating agents, alkoxylated polyamines, fabric deposition aids, and/or cyclodextrin.

Automatic dishwashing (ADW) detergent composition

Exemplary ADW detergent composition includes non-ionic surfactants, including ethoxylated non-ionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols, or amine oxide surfactants present in amounts from 0 to 10% by weight; builders in the range of 5-60% including phosphate builders {e.g.,

mono-phosphates, di-phosphates, tri-polyphosphates, other oligomeric-poylphosphates, sodium tripolyphosphate-STPP) and phosphate-free builders {e.g., amino acid-based compounds including methyl-glycine-diacetic acid (MGDA) and salts and derivatives thereof, glutamic-N,N-diacetic acid (GLDA) and salts and derivatives thereof, iminodisuccinic acid (IDS) and salts and derivatives thereof, carboxy methyl inulin and salts and derivatives thereof, nitrilotriacetic acid (NTA), diethylene triamine penta acetic acid (DTPA), B-alaninediacetic acid (B-ADA) and their salts, homopolymers and copolymers of poly-carboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts in the range of 0.5% to 50% by weight; sulfonated/carboxylated polymers in the range of about 0.1 % to about 50% by weight to provide dimensional stability; drying aids in the range of about 0.1 % to about 1 0% by weight {e.g., polyesters, especially anionic polyesters, optionally together with further monomers with 3 to 6 functionalities - typically acid, alcohol or ester functionalities which are conducive to polycondensation, polycarbonate-, polyurethane- and/or polyurea-polyorganosiloxane compounds or precursor compounds, thereof, particularly of the reactive cyclic carbonate and urea type) ; silicates in the range from about 1 % to about 20% by weight (including sodium or potassium silicates for example sodium disilicate, sodium meta-silicate and crystalline phyllosilicates) ;

inorganic bleach (e.g., perhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate salts) and organic bleach (e.g., organic peroxyacids, including diacyl and tetraacylperoxides, especially diperoxydodecanedioc acid,

diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid); bleach activators (i.e., organic peracid precursors in the range from about 0.1 % to about 1 0% by weight); bleach catalysts (e.g., manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, and pentamine acetate cobalt(l l l) and related complexes); metal care agents in the range from about 0.1 % to 5% by weight (e.g., benzatriazoles, metal salts and complexes, and/or silicates) ; enzymes in the range from about 0.01 to 5.0 mg of active enzyme per gram of automatic

dishwashing detergent composition (e.g., proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferase, perhydrolase, arylesterase, and mixtures thereof) ; and enzyme stabilizer components (e.g.,

oligosaccharides, polysaccharides, and inorganic divalent metal salts). The present beta amylase polypeptide may be incorporated at a concentration conventionally employed in detergents. It is at present contemplated that, in the detergent composition, the enzyme may be added in an amount corresponding to 0.00001 -1 .0 mg (calculated as pure enzyme protein) of beta amylase polypeptide per liter of wash liquor.

The detergent composition may also contain other conventional detergent ingredients, e.g., deflocculant material, filler material, foam depressors, anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bactericides, fluorescers, thickeners, and perfumes.

The detergent composition may be formulated as a hand (manual) or machine (automatic) laundry detergent composition, including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener

composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for manual or automatic dishwashing operations.

Any of the cleaning compositions described, herein, may include any number of additional enzymes. In general the enzyme(s) should be compatible with the selected detergent, {e.g., with respect to pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, and the like), and the enzyme(s) should be present in effective amounts. The following enzymes are provided as examples.

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are included, as well as naturally processed proteins. The protease may be a serine protease or a metalloprotease, an alkaline microbial protease, a trypsin-like protease, or a

chymotrypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 1 68 {see, e.g., WO 89/06279). Examples of trypsin-like proteases are trypsin {e.g., of porcine or bovine origin), and Fusarium proteases {see, e.g., WO 89/06270 and WO 94/25583). Examples of useful proteases also include but are not limited to the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946. Commercially available protease enzymes include but are not limited to: ALCALASE®, SAVINASE®, PRIMASE™, DURALASE™, ESPERASE®, KANNASE™, and BLAZE™ (Novo Nordisk A/S and Novozymes A/S); MAXATASE®, MAXACAL™, MAXAPEM™, PROPERASE®, PURAFECT®, PURAFECT OXP™, FN2™, and FN3™ (Danisco US Inc.). Other exemplary proteases include NprE from Bacillus amyloliquifaciens and ASP from Cellulomonas sp. strain 69B4.

Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified, proteolytically modified, or protein engineered mutants are included. Examples of useful lipases include but are not limited to lipases from

Humicola (synonym Thermomyces), e.g., from H. lanuginosa {T. lanuginosus) {see e.g., EP 258068 and EP 305216), from H. insolens (see e.g., WO 96/13580); a

Pseudomonas lipase {e.g., from P. alcaligenes or P. pseudoalcaligenes; see, e.g., EP 218 272), P. cepacia {see e.g., EP 331 376), P. stutzeri {see e.g., GB 1 ,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 {see e.g., WO 95/06720 and WO

96/27002), P. wisconsinensis {see e.g., WO 96/12012); a Bacillus lipase {e.g., from B. subtiHs; see e.g., Dartois et al. Biochemica et Biophysica Acta, 1131 : 253-360 (1993)), B. stearothermophilus {see e.g., JP 64/744992), or B. pumilus {see e.g., WO 91 /16422). Additional lipase variants contemplated for use in the formulations include those described for example in: WO 92/05249, WO 94/01541 , WO 95/35381 , WO

96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, EP 407225, and EP 260105. Some commercially available lipase enzymes include LIPOLASE® and LIPOLASE ULTRA™ (Novo Nordisk A/S and

Novozymes A/S). Poly esterases: Suitable polyesterases can be included in the composition, such as those described in, for example, WO 01 /34899, WO 01 /14629, and US6933140.

Amylases: The compositions can be combined with other amylases, such as non-production enhanced amylase. These can include commercially available amylases, such as but not limited to STAINZYME®, NATALASE®, DURAMYL®, TERMAMYL®, FUNGAMYL® and BAN™ (Novo Nordisk A/S and Novozymes A/S); RAP I DAS E®, POWERASE®, and PURASTAR® (from Danisco US Inc.).

Cellulases: Cellulases can be added to the compositions. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed for example in U.S. Patent Nos. 4,435,307; 5,648,263; 5,691 ,178; 5,776,757; and WO 89/09259. Exemplary cellulases contemplated for use are those having color care benefit for the textile. Examples of such cellulases are cellulases described in for example EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, and WO 98/08940. Other examples are cellulase variants, such as those described in WO 94/07998; WO 98/12307; WO 95/24471 ; PCT/DK98/00299; EP 531315; U.S. Patent Nos. 5,457,046; 5,686,593; and 5,763,254. Commercially available cellulases include CELLUZYME® and CAREZYME® (Novo Nordisk A/S and Novozymes A/S); CLAZINASE® and PURADAX HA® (Danisco US Inc.); and

KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases contemplated for use in the compositions include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include for example GUARDZYME™ (Novo Nordisk A/S and Novozymes A/S). The detergent composition can also comprise 2,6- -D-fructan hydrolase, which is effective for removal/cleaning of biofilm present on household and/or industrial textile/laundry.

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive, i.e. a separate additive or a combined additive, can be formulated e.g., as a granulate, a liquid, a slurry, and the like. Exemplary detergent additive formulations include but are not limited to

granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products {e.g., polyethyleneglycol, PEG) with mean molar weights of 1 ,000 to 20,000; ethoxylated nonylphenols having from 1 6 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in, for example, GB 1483591 . Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,21 6.

The detergent composition may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste, or a liquid. A liquid detergent may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. Compact detergent gels containing about 30% or less water are also contemplated. The detergent composition can optionally comprise one or more surfactants, which may be non-ionic, including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants can be present in a wide range, from about 0.1 % to about 60% by weight. When included therein the detergent will typically contain from about 1 % to about 40% of an anionic surfactant, such as linear alkylbenzenesulfonate,

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

The detergent may contain 0% to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA),

diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g.,SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Exemplary polymers include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG), polyvinyl alcohol) (PVA), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates e.g., polyacrylates, maleic/acrylic acid copolymers), and lauryl methacrylate/acrylic acid copolymers.

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

It is contemplated that in the detergent compositions, in particular the beta amylase of the present teachings, may be added in an amount corresponding to about 0.01 to about 100 mg of enzyme protein per liter of wash liquor {e.g., about 0.05 to about 5.0 mg of enzyme protein per liter of wash liquor or 0.1 to about 1 .0 mg of enzyme protein per liter of wash liquor). Methods of Assessing Amylase Activity in Detergent Compositions

Numerous amylase cleaning assays are known in the art, including swatch and micro-swatch assays.

Brewing Compositions The present beta amylase may be a component of a brewing composition used in a process of brewing, i.e., making a fermented malt beverage. Non-fermentable carbohydrates form the majority of the dissolved solids in the final beer. This residue remains because of the inability of malt amylases to hydrolyze the alpha-1 ,6-linkages of the starch. The non-fermentable carbohydrates contribute about 50 calories per 12 ounces of beer. An amylase, in combination with a glucoamylase and optionally a pullulanase and/or isoamylase, assist in converting the starch into dextrins and fermentable sugars, lowering the residual non-fermentable carbohydrates in the final beer.

The principal raw materials used in making these beverages are water, hops and malt. In addition, adjuncts such as 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, and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like may be used as a source of starch. For a number of reasons, the malt, which is produced principally from selected varieties of barley, has the greatest effect on the overall character and quality of the beer. First, the malt is the primary flavoring agent in beer. Second, the malt provides the major portion of the fermentable sugar. Third, the malt provides the proteins, which will contribute to the body and foam character of the beer. Fourth, the malt provides the necessary enzymatic activity during mashing. Hops also contribute significantly to beer quality, including flavoring. In particular, hops (or hops constituents) add desirable bittering substances to the beer. In addition, the hops act as protein precipitants, establish preservative agents and aid in foam formation and stabilization. Grains, such as barley, oats, wheat, as well as plant components, such as corn, hops, and rice, also are used for brewing, both in industry and for home brewing. The components used in brewing may be unmalted or may be malted, i.e., partially germinated, resulting in an increase in the levels of enzymes, including amylases. For successful brewing, adequate levels of a-amylase enzyme activity are necessary to ensure the appropriate levels of sugars for fermentation. An amylase, by itself or in combination with another a-amylase(s), accordingly may be added to the components used for brewing.

As used herein, the term "stock" means grains and plant components that are crushed or broken. For example, barley used in beer production is a grain that has been coarsely ground or crushed to yield a consistency appropriate for producing a mash for fermentation. As used herein, the term "stock" includes any of the

aforementioned types of plants and grains in crushed or coarsely ground forms. The methods described herein may be used to determine beta amylase activity levels in both flours and stock.

Processes for making beer are well known in the art. See, e.g., Wolfgang Kunze (2004) "Technology Brewing and Malting," Research and Teaching Institute of Brewing, Berlin (VLB), 3rd edition. Briefly, the process involves: (a) preparing a mash, (b) filtering the mash to prepare a wort, and (c) fermenting the wort to obtain a fermented beverage, such as beer. Typically, milled or crushed malt is mixed with water and held for a period of time under controlled temperatures to permit the enzymes present in the malt to convert the starch present in the malt into fermentable sugars. The mash is then transferred to a mash filter where the liquid is separated from the grain residue. This sweet liquid is called "wort," and the left over grain residue is called "spent grain." The mash is typically subjected to an extraction, which involves adding water to the mash in order to recover the residual soluble extract from the spent grain. The wort is then boiled vigorously to sterilize the wort and help develop the color, flavor and odor. Hops are added at some point during the boiling. The wort is cooled and transferred to a fermentor. The wort is then contacted in a fermentor with yeast. The fermentor may be chilled to stop fermentation. The yeast flocculates and is removed. Finally, the beer is cooled and stored for a period of time, during which the beer clarifies and its flavor develops, and any material that might impair the appearance, flavor and shelf life of the beer settles out. The beer usually contains from about 2% to about 10% v/v alcohol, although beer with a higher alcohol content, e.g., 18% v/v, may be obtained. Prior to packaging, the beer is carbonated and, optionally, filtered and pasteurized.

The brewing composition comprising a beta amylase, in combination with a glucoamylase and optionally a pullulanase and/or isoamylase and/or alpha amylase, may be added to the mash of step (a) above, i.e., during the preparation of the mash. Alternatively, or in addition, the brewing composition may be added to the mash of step (b) above, i.e., during the filtration of the mash. Alternatively, or in addition, the brewing composition may be added to the wort of step (c) above, i.e., during the fermenting of the wort. A fermented beverage, such as a beer, can be produced by one of the methods above. The fermented beverage can be a beer, such as 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.

Reduction of Iodine-Positive Starch The beta amylases of the present teachings may reduce the iodine-positive starch (IPS), when used in a method of liquefaction and/or saccharification. One source of IPS is from amylose that escapes hydrolysis and/or from retrograded starch polymer. Starch retrogradation occurs spontaneously in a starch paste, or gel on aging, because of the tendency of starch molecules to bind to one another followed by an increase in crystallinity. Solutions of low concentration become increasingly cloudy due to the progressive association of starch molecules into larger articles. Spontaneous precipitation takes place and the precipitated starch appears to be reverting to its original condition of cold-water insolubility. Pastes of higher concentration on cooling set to a gel, which on aging becomes steadily firmer due to the increasing association of the starch molecules. This arises because of the strong tendency for hydrogen bond formation between hydroxy groups on adjacent starch molecules. See J. A. Radley, ed., STARCH AND ITS DERIVATIVES 194-201 (Chapman and Hall, London (1968)).

The presence of IPS in saccharide liquor negatively affects final product quality and represents a major issue with downstream processing. IPS plugs or slows filtration system, and fouls the carbon columns used for purification. When IPS reaches sufficiently high levels, it may leak through the carbon columns and decrease production efficiency. Additionally, it may results in hazy final product upon storage, which is unacceptable for final product quality. The amount of IPS can be reduced by isolating the saccharification tank and blending the contents back. IPS nevertheless will accumulate in carbon columns and filter systems, among other things. The use of the beta amylases of the present teachings is expected to improve overall process performance by reducing the amount of IPS.

Examples

A putative novel β amylase was identified from the sequenced genome of a

Paenibacillus sp, as described below. The resulting enzyme was then characterized. Protein sequence analysis indicates that PsPAmy9 β amylase shows no more than 80% sequence identity to known proteins in the public NCBI database. EXAMPLE 1

Cloning of beta-amylase PspAmy9 from Paenibacillus sp

Paenibacillus sp strain DSM 34, obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), was selected as a potential source for enzymes that may be useful for various industrial applications. To identify these enzymes and the genes that encode these enzymes, the entire genome of Paenibacillus sp strain DSM 34 was sequenced by lllumina's sequencing technology. Genomic DNA was isolated and used for genome sequencing. Genomic DNA for sequencing was obtained by first growing the strain on Heart Infusion agar plates (Difco) at 37 °C for 24 h. Cell material was scraped from the plates and used to prepare genomic DNA with the ZF Fungal/Bacterial DNA miniprep kit from Zymo (Cat No.

D6005). Genome sequencing and assembly of the sequence data was performed by BaseClear (Leiden, The Netherlands). Contigs were annotated by BioXpr (Namur, Belgium). One of the genes identified this way in Paenibacillus sp strain DSM 34 encodes a beta-amylase, called PspAmy9, and is depicted in SEQ ID NO:1 . The corresponding protein encoded by the PspAmy9 gene is depicted in SEQ ID NO:2. At the N-terminus, the protein has a signal peptide with a length of 37 amino acids as predicted by SignalP-4.0 (Nordahl Petersen et al. (2011 ) Nature Methods, 8:785-786). The presence of a signal sequence suggests that PspAmy9 is a secreted enzyme. The predicted sequence of the fully processed mature chain of PspAmy9 is depicted in SEQ ID NO: 3.

The nucleotide sequence of the PspAmy9 gene isolated from Paenibacillus sp is set forth as SEQ ID NO:1 :

ATGTTTCAGGCCATGCGGTCATGGATTAACCGAATTCGTATGTACGCAGCCGCTTCA

CTCACGGTCTTTCTCGCAGTCTCCATCGTGTTTCCGGCGTCTCAAGCCGAGGCAT

CCAGCATTCGCAGTGATTATAAGGCTTATGTCATGGCACCGCTGACGAAGATCACA

GATTGGAATGCCTTTCGCAATCAACTGGTGACGCTGAAGAATAACAACGTGTACGC

CCTCACAACCGACGTGTGGTGGGGAGATGTGGAGCAGGGCGGCGACAACGTATT

TGACTGGTCGTATTACAAAACCTATGCCGACACGGTGCGCGCTGCGGGCCTGAAA

TGGATTCCCATCCTTTCGACGCATCAATGCGGCGGCAATGTCGGTGACGATTGCAA

TATTAAGCTGCCTTCGTGGCTCTGGAGCAAAGGTACGGCGGACCAGATGACCATG

CGCAGCGAAACGGGCTATTCCGATTACGAAACGCTGTCTCCGTGGTGGACAGGAA

CGGCGACGCAATATGATGAGCTGTATGCCTCATTTGCTTCAAATTTTAGCGGGTACA

AGGATATTATCGCCAAAATCTATCTCTCCGGCGGCCCGGCGGGCGAACTCCGATTC

CCGTCCTATAATGCGGCCGACGGCTGGAGCTATCCGGGGCGCGGCAAGCTGCAG GCATACACCGATACGGCGAAGCTGGATTTTCAAAATGCCATGAAAACCAAATACAGC

ACGCTCGCCGCGCTCAATAGCGCTTGGGGGACGACTCTCACCGCTTTCTCGCAAG

TAAATCCGCCAAGCGACGGCGACAACTTTTTCATTAACGGCGTTAAGGGAACGTAT

GGCAAGGATTTCATGACCTGGTATCAAGGCGTGCTCACAAAGCATTTAAGTACCATT

GCTGCCAAAGCGCATAGCCGTTTTGATGCTGTTTTTGGCGTTCCGATTGGGGCAAA

GGTAGCTGGGGTTCATTGGCAAATGAACAATCCATCAATGCCGCATGCAGCTGAGT

ACGCCTCAGGCTACTACAACTACAGTACACTGCTGGATCAGTTTAAAACCTCGAATC

TCGATCTCACCTTTACCTGCCTGGAAATGACCGATAGCCAAGCTTCTACGAGCCCT

TATTACTCTGCGCCAAAATCGCTAGTGATTCAAGTGGCCAATCTGGCGAACCAGAA

GGGCATTCGCATTAATGGCGAGAACGCACTGCCGATCAGCGACGGAAGCGGCTAT

CAGAACGTGGCCGAAATGCTGTTCAACTATAATTTTGGCGGTTTTACCCTGCTTCG

GATGTCCAGCCTCGTCAATACGGACGGCTCAAAAACAGGCTTGATGGACGCCTAC

CGGGATGCTATCGCGATGACGCCGATTCCTGTTACGTTTATCGTGAAGAACGCGCC

GACCGTAACAGGGGACACCGTGTATGTGACGGGGAGCCGTTGGGAACTGGGAAA

CTGGACGACCGGCGTGTATCCGTTCAAGCTGACGTACAACAGCTCCACAGCTGAC

TGGCGCGGCACGGCCTATATCGGAGCCTCTCGCTATTATGAGTTCAAGGCAATCAT

TAAAGATAGCGGAGGCAATTTGAAAAGTTGGGAACCGGGCGCCAACAATACGTGG

ACGACGCCTGCGGCAGGGACGAATTATACGATAACCTGGThe amino acid sequence of the PspAmy9 precursor protein is set forth as SEQ ID NO: 2. The predicted native signal peptide is shown in italics

MFQAMRS WINRIRMYAAASL TVFLA VSIVFPASQAEASS I RS D YKA YVM AP LTKITDW N

AFRNQLVTLKNNNVYALTTDVWWGDVEQGGDNVFDWSYYKTYADTVRAAGLKWIPIL

STHQCGGNVGDDCNIKLPSWLWSKGTADQMTMRSETGYSDYETLSPWWTGTATQY

DELYASFASNFSGYKDIIAKIYLSGGPAGELRFPSYNAADGWSYPGRGKLQAYTDTAK

LDFQNAMKTKYSTLAALNSAWGTTLTAFSQVNPPSDGDNFFINGVKGTYGKDFMTWY

QGVLTKHLSTIAAKAHSRFDAVFGVPIGAKVAGVHWQMNNPSMPHAAEYASGYYNYS

TLLDQFKTSNLDLTFTCLEMTDSQASTSPYYSAPKSLVIQVANLANQKGIRINGENALPI

SDGSGYQNVAEMLFNYNFGGFTLLRMSSLVNTDGSKTGLMDAYRDAIAMTPIPVTFIV

KNAPTVTGDTVYVTGSRWELGNWTTGVYPFKLTYNSSTADWRGTAYIGASRYYEFKA

IIKDSGGNLKSWEPGANNTWTTPAAGTNYTITW The amino acid sequence of the predicted mature form of PspAmy9 is set forth as SEQ ID NO:3:

SSIRSDYKAYVMAPLTKITDWNAFRNQLVTLKNNNVYALTTDVWWGDVEQGGDNVFD

WSYYKTYADTVRAAGLKWIPILSTHQCGGNVGDDCNIKLPSWLWSKGTADQMTMRS

ETGYSDYETLSPWWTGTATQYDELYASFASNFSGYKDIIAKIYLSGGPAGELRFPSYN

AADGWSYPGRGKLQAYTDTAKLDFQNAMKTKYSTLAALNSAWGTTLTAFSQVNPPS

DGDNFFINGVKGTYGKDFMTWYQGVLTKHLSTIAAKAHSRFDAVFGVPIGAKVAGVH

WQMNNPSMPHAAEYASGYYNYSTLLDQFKTSNLDLTFTCLEMTDSQASTSPYYSAPK

SLVIQVANLANQKGIRINGENALPISDGSGYQNVAEMLFNYNFGGFTLLRMSSLVNTD

GSKTGLMDAYRDAIAMTPIPVTFIVKNAPTVTGDTVYVTGSRWELGNWTTGVYPFKLT

YNSSTADWRGTAYIGASRYYEFKAIIKDSGGNLKSWEPGANNTWTTPAAGTNYTITW

EXAMPLE 2

Expression of beta-amylase PspAmy9

The DNA sequence of the mature form of PspAmy9 was synthesized and inserted into the Bacillus subtilis expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif, 55:40-52, 2007) by Generay (Shanghai, China), resulting in plasmid p2JM784 (aprE- PspAmy9) (Figure 1 ). Ligation of this gene encoding the PspAmy9 protein into the digested vector resulted in the addition of three codons (Ala-Gly-Lys) between the 3' end of the Bacillus subtilis AprE signal sequence and the 5' end of the predicted PspAmy9 native propeptide. The gene has an alternative start codon (GTG). As shown in Figure 1 , p2JM784 (aprE- PspAmy9) contains an aprE promoter, an aprE signal sequence used to direct target protein secretion in B. subtilis, and mature region of PspAmy9 (SEQ ID NO: 4).

The p2JM784 (aprE- PspAmy9) plasmid was then transformed into B. subtilis cells {degU^Hy32, AscoC) and the transformed cells were spread on Luria Agar plates supplemented with 5ppm Chloramphenicol and transformants on the plates were selected and subjected to fermentation in a 250 mL shake flask with MBD medium (a MOPS based defined medium, supplemented with additional 5mM CaCI₂).

The nucleotide sequence of the synthesized PspAmy9 gene in plasmid p2JM784 (aprE- PspAmy9) is depicted in SEQ ID NO:4. The oligonucleotide encoding the three residue addition (AGK) is shown in bold:

GTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAATCTTTACG ATGGCGTTCAGCAACATGAGCGCGCAGGCTGCTGGAAAATCATCAATCAGAAGCG ATTACAAGGCATACGTTATGGCACCGCTGACGAAGATTACAGACTGGAACGCATTC AGAAACCAACTGGTTACGCTGAAGAACAACAATGTCTACGCGCTTACAACGGACGT TTGGTGGGGAGATGTGGAGCAAGGCGGCGATAACGTGTTTGATTGGAGCTACTAC AAAACGTACGCGGACACGGTTAGAGCAGCCGGCCTGAAATGGATTCCGATTCTGT CAACGCACCAATGCGGAGGCAATGTTGGCGATGATTGCAACATTAAACTGCCTAGC TGGCTGTGGTCAAAAGGCACGGCTGACCAGATGACAATGAGATCAGAGACGGGCT ACTCAGATTACGAAACACTGAGCCCGTGGTGGACAGGCACAGCGACGCAGTACGA CGAACTGTACGCCTCATTCGCTAGCAACTTTAGCGGCTACAAGGACATCATTGCCA AGATCTACCTGTCAGGAGGACCTGCCGGAGAGCTTAGATTCCCGAGCTATAATGCT GCAGACGGCTGGTCATATCCGGGCAGAGGCAAGCTGCAAGCCTACACGGATACGG CCAAGCTGGACTTCCAAAATGCCATGAAGACGAAATATTCAACACTGGCTGCGCTT AATTCAGCATGGGGAACGACACTGACAGCATTCTCACAGGTCAATCCGCCGTCAG ACGGAGACAATTTCTTCATCAACGGCGTCAAGGGCACATATGGCAAGGATTTTATGA CATGGTATCAGGGCGTCCTTACAAAGCACCTGAGCACAATCGCAGCGAAAGCACA CAGCAGATTCGATGCAGTTTTTGGCGTGCCGATTGGCGCGAAAGTGGCAGGCGTT CATTGGCAGATGAATAATCCGTCAATGCCGCACGCCGCTGAATATGCGTCAGGATA CTATAACTACAGCACGCTGCTGGACCAGTTCAAGACGAGCAACCTGGACCTGACG TTCACGTGCCTGGAAATGACGGACAGCCAAGCGAGCACGTCACCGTATTACTCAG CACCGAAAAGCCTGGTTATTCAGGTCGCGAACCTGGCGAACCAGAAAGGCATTAG AATTAATGGCGAGAACGCGCTGCCTATTAGCGACGGCAGCGGATACCAGAACGTG GCCGAAATGCTTTTCAATTACAACTTCGGCGGCTTCACACTGCTTAGAATGTCATCA CTGGTGAACACGGACGGATCAAAGACAGGACTGATGGATGCATATAGAGATGCTAT CGCAATGACACCGATCCCGGTCACATTCATCGTTAAAAACGCTCCTACAGTGACGG GCGATACGGTCTACGTGACAGGCTCAAGATGGGAACTGGGCAATTGGACAACGGG

CGTTTACCCTTTCAAACTGACGTACAATAGCAGCACGGCCGACTGGAGAGGAACA

GCTTATATCGGCGCGAGCAGATATTATGAGTTTAAGGCGATCATCAAGGATAGCGGC

GGAAACCTTAAAAGCTGGGAACCGGGAGCAAATAACACATGGACGACGCCGGCAG

CTGGCACAAACTATACAATTACGTGG

The amino acid sequence of the PspAmy9 precursor protein expressed from plasmid p2JM784 (aprE- PspAmy9) is depicted in SEQ ID NO:5. The predicted signal sequence is shown in italics, the three residue addition (AGK) is shown in bold.

MRSKKL WISLLFALTL IFTMA FSNMSA QAAG KSS I RS D YKAYV M A P LTK IT D W N A F R N Q

LVTLKNNNVYALTTDVWWGDVEQGGDNVFDWSYYKTYADTVRAAGLKWIPILSTHQC

GGNVGDDCNIKLPSWLWSKGTADQMTMRSETGYSDYETLSPWWTGTATQYDELYAS

FASNFSGYKDIIAKIYLSGGPAGELRFPSYNAADGWSYPGRGKLQAYTDTAKLDFQNA

MKTKYSTLAALNSAWGTTLTAFSQVNPPSDGDNFFINGVKGTYGKDFMTWYQGVLTK

HLSTIAAKAHSRFDAVFGVPIGAKVAGVHWQMNNPSMPHAAEYASGYYNYSTLLDQF

KTSNLDLTFTCLEMTDSQASTSPYYSAPKSLVIQVANLANQKGIRINGENALPISDGSGY

QNVAEMLFNYNFGGFTLLRMSSLVNTDGSKTGLMDAYRDAIAMTPIPVTFIVKNAPTVT

GDTVYVTGSRWELGNWTTGVYPFKLTYNSSTADWRGTAYIGASRYYEFKAIIKDSGGN

LKSWEPGANNTWTTPAAGTNYTITW

EXAMPLE 3

Purification of beta-amylase PspAmy9

PspAmy9 was purified via the beta-cyclodextrin coupled Sepharose 6 affinty chromatography, taking advantage of its carbohydrate binding domain. The 700 ml_ crude broth from the shake flask was concentrated by ultrafiltration and buffer

exchanged to 20 mM Tris pH 8. The solution was then loaded onto a 30 ml_

beta-cyclodextrin coupled Sepharose 6 column pre-equilibrated with 20 mM Tris pH 8 (buffer A). The column was applied with a gradient of 0-100% buffer A with 10 mM alpha-cyclodextrin (buffer B) in 2 column volumes, followed by 2 column volumes of 100% buffer B. The target protein was eluted in the gradient step with high purity. The fractions from the beta-cyclodextrin column were pooled and concentrated using an Amicon Ultra-15 device with 10 K MWCO. The purified sample is above 98% pure and stored in 40% glycerol at -80 °C until usage.

EXAMPLE 4 Beta-amylase PspAmy9 activity assay

Beta-amylase activity 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. 1 0118), 1 mL of 0.5 M buffer (pH 5.0 sodium acetate or pH 8.0 HEPES), 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 into 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 was 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. Beta-amylase activity can be quantified using the below equation, and the results are shown in Table 1 :

Specific Activity (U/mg) = Slope (enzyme) / slope (std) ^* 100

Define: 1 U = 1 μηποΙ glucose equivalent/min

Table 1. Specificity activities of beta-amylases

Diazymebb | 1360.6 | 655.8

EXAMPLE 5

Effect of pH on beta-amylase PspAmy9 activity The effect of pH (from 3.0 to 10.0) on beta-amylase activity was monitored using the PAHBAH assay protocol as described above. Buffer working solutions consisted of the combination of glycine/sodium acetate/HEPES (250 mM), with pH varying from 3.0 to 10.0. Substrate solutions were prepared by mixing 896 μΙ_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 10118), 100 μΙ_ of 250 mM buffer working solution (pH from 3.0 to 10.0), 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 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 100%. The optimal pH range was defined as containing equal or higher than 70% of activity (Figure 2 and Table 2).

Table 2. pH profile of PspAmy9

EXAMPLE 6

Effect of temperature on beta-amylase PspAmy9 activity

The effect of temperature (from 30 to 95 °C) on beta-amylase activity was monitored using the PAHBAH assay protocol as described above. Substrate solutions were prepared by mixing 3.6 ml_ of 1 % (w/w, in water) potato amylopectin (Sigma, Cat. No. 10118), 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 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%. The optimal temperature range was defined as containing equal to or higher than 70% of activity (Figure 3 and Table 3).

Table 3. Temperature profile of PspAmy9

EXAMPLE 7

Thermostability of beta-amylase PspAmy9

Thermostability of beta-amylase PspAmy9 was determined by measuring the enzyme activity before and after enzyme samples pre-incubated at temperatures from 40 to 95 °C for 2 h. Enzyme was diluted in 50 mM of sodium acetate buffer (pH5.0) containing 2 mM of CaCI₂ to appropriate concentration (showing signal within linear range as per dose response curve) and 40 μΙ_ was aliquot to PCR strip tubes. The tubes were transferred to a PCR machine at the desired temperature from 40 to 95 °C. After 2 h-preincubation, the residual activity of the enzyme after heat stress was assayed using the amylopectin/ PAHBAH method as described above. The residual activities were converted to percentages of relative activity, by defining the activity of the sample kept on ice as 100%. The thermostability was defined as the temperature at which the sample remains 50% of activity (Figure 4 and Table 4).

Table 4. Thermostability of PspAmy9

Gene

Thermostability, °C

name PspAmy9 57

EXAMPLE 8

Maltose production of liquefied starch using beta-amylase PspAmy9 Starch liquefact (34%ds, DE10) was obtained from a grain application lab. The substrate was prepared to have 32% dry solids by diluting with water and pH was adjusted to 5.2-5.5 prior to incubation. The reaction mixture was composed of 25 g of starch liquefact (32%ds, pH 5.4) and 0.1 mg/gds of PspAmy9. The incubations were done at 70 °C and samples were taken at different intervals of time. The reaction was stopped by heating the samples at 100 °C for 15 min and centrifuged for 10 min at 14,000 rpm. The supernatant was diluted by a factor of 20 using M ill iQ water, and 10 μΙ_ was loaded onto an HPLC column (Aminex HPX-42A, 300 mm^*7.8 mm) equipped with a refractive index detector. The mobile phase was MilliQ water, and the flow rate was 0.6 mL/min at 85 °C. Figure 5 showed that beta-amylase PspAmy9 was able to produce around 60% of DP2 after 24-h incubation with starch liquefact at 70 °C.

EXAMPLE 9

Homology Identification of PspAmy9

The PspAmy9 mature protein sequence (514 residues) was subjected to a BLASTP search, and 15 protein sequences were identified with 59% or greater identity. Figure 6 shows an alignment of PspAmy9 with similar beta-amylase sequences generated with Vector NTI (Invitrogen). Table 5 shows the percent identities and NCBI Accession Nos. of the aligned sequences: YP_004639885.1 , YP_005311823.1 , YP_006188614.1 , P19584.1 , YP_001885981 .1 , ZP_02996654.1 , YP_001780827.1 , ZP_02617719.1 , YP_002862039.1 , YP_002803561 .1 , YP_001786594.1 , YP_005677827.1 ,

YP_001390552.1 ,YP_001253727.1 , ZP_02613286.1 . The aligned sequences were used to prepare a phylogenetic tree using program MEGA 5. Figure 7 shows the phylogenic tree for PsPAmy9 and its homologs.

Table 5. Percent Identity Shared by PsPAmy9 similar Sequences Homolog % IDENTITY

(PID)

PspAmy9 100

YP_004639885.1 80

YP_005311823.1 80

YP_006188614.1 81

P19584.1 61

YP_001885981 .1 59

ZP_02996654.1 62

YP_001780827.1 62

ZP_02617719.1 62

YP_002862039.1 62

YP_002803561 .1 63

YP_001786594.1 62

YP_005677827.1 62

YP_001390552.1 63

YP_001253727.1 62

ZP_02613286.1 62

Other Uses for PspAmy9 and other Beta Amylases In some embodiments, the beta amylases provided herein, including

PspAmy9, 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 1 2, 2013, claiming priority to US Provisional 61 /61 6990, filed March 28, 2012.

Claims

Claims

1 . A purified polypeptide, the amino acid sequence of which comprises a sequence at least 81 %, 82%, 83%, 84%, 85%, 87%, 90%, 92%, 95%, 98%, 99%, or 99.5% identical to SEQ ID NO:3.

2. A purified polypeptide comprising the amino acid sequence of SEQ ID NO:3, but with 0 to 20 conservative amino acid substitutions.

3. An expression vector comprising a non-natually occuring nucleic acid sequence, and, the nucleic acid sequence of SEQ ID NO:1 , and any nucleic acid 80%, 85%, 90%, 95%, 98%, or 99% to it, operably linked to an expression control sequence.

4. A cultured cell comprising the vector of claim 3.

5. A method of producing a protein, the method comprising culturing the cell of claim 4 under conditions permitting expression of the polypeptide.

6. A method of using the polypeptide of any of claims 1 or 2, the method comprising including the polypeptide in any of: starch liquefaction, starch saccharification, textile desizing, starch modification in the paper and pulp industry, brewing, baking, production of syrups for the food industry, production of feedstocks for fermentation processes, animal feed, and, removal of starchy soils and/or stains during dishwashing and/or laundry washing.

7. A composition comprising the polypeptide of any of claims 1 or 2, and at least one accessory enzyme selected from the group consisting of phytase, protease, pullulanase, an additional β-amylase, isoamylase, alpha amylase, glucoamylase,

alpha-glucosidase, cellulase, xylanase, hemicellulase, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, choline oxidases, peroxidase/oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase,

acyltransferase, perhydrolase, arylesterase, and/or a redox enzyme.

8. An isolated nucleic acid comprising at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:1 , and, at least one non-naturally occuring DNA base at a position that allows encoding of a degenerate amino acid, wherein the polypeptide has starch hydrolysis activity.

9. An isolated nucleic acid comprising at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:1 , and, at least one non-naturally occuring DNA base at a position that allows encoding of a conservative amino acid substitution, wherein the polypeptide has starch hydrolysis activity.

10. An isolated nucleic acid comprising a sequence that hybridizes under stringent conditions to a hybridization probe the nucleotide sequence of which consists of SEQ ID NO:1 , or the complement of SEQ ID NO:1 , wherein the isolated nucleic acid comprises at least one non-naturally occuring DNA base at a position that allows encoding of a degenerate amino acid or allows encoding of a conservative amino acid substution.

11 . An isolated nucleic acid of claim 10 comprising a sequence at least 81 %, 82%, 83%, 84%, 85%, 87%, 90%, 92%, 95%, 98%, 99%, identical to SEQ ID NO:1 .

12. An isolated nucleic acid of claim 8 encoding 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 1-15, 1 -10, or 1 -5 degenerate amino acid positions, wherein the polypeptide has starch hydrolysis activity.

13. An isolated nucleic acid of claim 9 encoding 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 1-15, 1-10, or 1-5 conservative amino acid substitutions, wherein the polypeptide has starch hydrolysis activity.