WO2016036834A1 - Dp5-enriched syrups - Google Patents

Abstract

Disclosed are compositions and methods relating to DP5-enriched specialty syrups prepared from starch substrates using α-amylases. The syrups have application in the food and beverage industry.

Description

DP5 -ENRICHED SYRUPS

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of priority from US provisional application USSN 62/044,583, filed September 2, 2014, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[002] Disclosed are compositions and methods relating to DP5-enriched specialty syrups prepared from starch substrates using a-amylases. The syrups have applications in the food and beverage industry.

BACKGROUND

[003] Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose consists of linear chains of a-l,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.

[004] Sugars from starch, usually in the form of concentrated dextrose (glucose) syrups, are currently produced by an enzyme catalyzed process involving: (1) liquefaction (dissolving and hydrolysis of starch with an a-amylase into dextrins having an average degree of polymerization of about 7-10, and (2) saccharification of the resulting liquefied starch (i.e. starch hydrolysate) with amyloglucosidase (also called glucoamylase or GA). The resulting syrup has a high dextrose content. Much of the glucose syrup that is commercially produced is subsequently enzymatically isomerized to a dextrose/fructose mixture known as isosyrup. The resulting syrup may be fermented with microorganisms, such as yeast, to produce commercial products including ethanol, citric acid, lactic acid, succinic acid, itaconic acid, monosodium glutamate, gluconates, lysine, other organic acids, other amino acids, and other biochemicals, for example. Fermentation and saccharification can be conducted

simultaneously (i.e., an SSF process) to achieve greater economy and efficiency. In some cases, the syrup produced following incubation with a-amylases is itself a valuable product, and it is not desirable to fully process the syrup to glucose or to otherwise enzymatically process or ferment the syrup. [005] α-amylases are endo-acting enzymes and have a minimum starch substrate length requirement. As a result, a-amylases tend to produce a population of oligosaccharides of varying length and little glucose. These oligosaccharides are identified by the number of glucose residues they contain, also referred to as their degree of polymerization (DP).

Different α-amylases produce a different population of products. Some oligosaccharides are more desirable than others. For example, oligosaccharides containing five glucose residues (i.e., G5 or DP5) have been used as a nutrients for patients with renal failure and those in a state of calorie-deprivation. DP5 is also used a substrate in commercially available a- amylase assays.

[006] While α-amylases that produce DP5 as a significant proportion of total

oligosaccharides products have been described (e.g., Okemoto, H. et al. (1986) Appl.

Microbiol. Biotechnol. 25: 137-42; Saito, N. (1973) Arch. Biochem. Biophys. 155:290-98; Morgan, F. J. and Priest, F.G. (1981) /. Appl. Bacteriol. 50: 107-14; Shida, O. et al. (1992) Biosci. Biotech. Biochem. 56:76-80), the need exists for amylases that can produce even higher amounts of DP5, preferably with reduced amounts of similar sized products, such as DP6.

SUMMARY

[007] The present compositions and methods relate to DP5-enriched specialty syrups and a- amylases used to produce them. Aspects and embodiments of the compositions and methods are illustrated by the following separately- numbered paragraphs:

1. In one aspect, a non-naturally-occurring syrup produced from a starch substrate by incubation with an α-amylase is provided, comprising >30 DP5 and <10 DP6 as fractions of total reaction products or DP6/DP5 <0.30, DP3/DP5 <0.70, and DP2/DP5 <0.50.

2. In some embodiments, the syrup of paragraph 1 has DP6/DP5 <0.30.

3. In some embodiments, the syrup of paragraph 1 has DP6/DP5 <0.20.

4. In some embodiments, the syrup of paragraph 1 has DP6/DP5 <0.15.

5. In some embodiments, the syrup of any of paragraphs 1-4 is produced by incubating a starch substrate in the presence of CspAmy2 α-amylase or a variant, thereof.

6. In some embodiments, the syrup of paragraph 5 is produced by an a-amylase variant of CspAmy2 comprising a deletion of residues 178 and 179 or 180 and 181, using SEQ ID NO: 3 for numbering.

7. In another aspect, a method for producing a syrup from a starch substrate is provided, comprising incubating the starch substrate in the presence of an α-amylase capable of producing >30% DP5 and <10% DP6 as fractions of total reaction products, or DP6/DP5 <0.30, DP3/DP5 <0.70, and DP2/DP5 <0.50, in a period of 30 hours at pH 5.2 and 60°C.

8. In some embodiments of the method of paragraph 7, the a-amylase is capable of producing a reaction product having a DP6/DP5 <0.30 in a period of 30 hours at pH 5.2 and 60°C.

9. In some embodiments of the method of paragraph 8, the α-amylase is capable of producing a reaction product having a DP6/DP5 <0.20 in a period of 30 hours at pH 5.2 and 60°C.

10. In some embodiments of the method of paragraph 9, the α-amylase is capable of producing a reaction product having a DP6/DP5 <0.15 in a period of 30 hours at pH 5.2 and 60°C.

11. In some embodiments of the method of any of paragraphs 7-10, the starch substrate is a corn, cereal, grain, or tuber liquefact.

12. In some embodiments of the method of any of paragraphs 7-11, the starch substrate comprises from about 10% to about 40% dissolved solids.

13. In some embodiments of the method of any of paragraphs 7-12, the amount of a- amylase used is from about 0.001 to about 0.05 mg/g dissolved solids.

14. In some embodiments of the method of any of paragraphs 7-12, incubating the starch substrate in the presence of α-amylase is performed without the addition of calcium to the starch substrate.

15. In some embodiments of the method of any of paragraphs 7-13, incubating the starch substrate in the presence of CspAmy2 α-amylase or a variant, thereof, is performed at a pH <7.0.

16. In some embodiments of the method of any of paragraphs 7-14, incubating the starch substrate in the presence of α-amylase is performed at a pH <6.0.

17. In some embodiments of the method of any of paragraphs 7-15, the starch substrate is incubated in the presence of CspAmy2 α-amylase or a variant, thereof.

18. In some embodiments of the method of paragraph 17, the α-amylase is a variant of CspAmy2 comprising a deletion of residues 178 and 179 or 180 and 181, using SEQ ID NO: 3 for numbering.

19. In another aspect, a syrup produced by the method of any of paragraphs 7-18 is provided.

[008] These and other aspects and embodiments of the compositions and methods will be apparent from the present description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[009] Figure 1 is a graph showing the relative amounts of oligosaccharides produced over time following incubation of a starch substrate with CspAmy2.

[0010] Figure 2 is a graph showing the relative amounts of oligosaccharides produced over time following incubation of a starch substrate with SPEZYME XTRA®.

[0011] Figure 3 is a graph showing the relative amounts of oligosaccharides produced over time following incubation of a starch substrate with SPEZYME ALPHA®.

[0012] Figure 4 is a graph showing the relative amounts of oligosaccharides produced over time following incubation of a starch substrate with SPEZYME FRED®.

[0013] Figure 5 is a graph showing the relative amounts of oligosaccharides produced over time following incubation of a starch substrate with OPTEVIASE® AA 56L.

[0014] Figure 6 is a graph showing the relative amounts of oligosaccharides produced over time following incubation of a starch substrate with PURASTAR® HP Am 5000L.

[0015] Figure 7 is a graph showing the relative amounts of DP5 produced over time following incubation of a starch substrate with CspAmy2, OPTEVIASE® AA 56L, or

PURASTAR® HP Am 5000L.

[0016] Figure 8 is a graph showing the relative amounts of DP5, DP6, DP3 and DP2 produced over time following incubation with different amounts of CspAmy2.

DETAILED DESCRIPTION

1. Definitions and Abbreviations

[0017] 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.

[0018] 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.

[0019] 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. Selected abbreviations and terms are defined, below. 1.1. Abbreviations and Acronyms

[0020] The following abbreviations/acronyms have the following meanings unless otherwise specified:

DE dextrose equivalent

DPn degree of saccharide polymerization having n subunits ds or DS dry solids

EC Enzyme Commission

HFCS high fructose corn syrup

MT metric ton

kg/MT DS kilograms per metric ton dry solid

MW molecular weight

ppm parts per million, e.g., μg protein per gram dry solid

SSF simultaneous saccharification and fermentation

sp. species

w/v weight/volume

w/w weight/weight

v/v volume/volume

wt weight percent

°C degrees Centigrade

RI refractive index

H₂0 water

dH₂0 or DI deionized water

dIH₂0 deionized water, Milli-Q filtration

g or gm grams

μg micrograms

mg milligrams

mg/g DS milligrams per gram of dry solid

kg kilograms

μL· and μΐ microliters

mL and ml milliliters

mm millimeters

μιη micrometer

M molar

mM millimolar

μΜ micromolar

U units

sec seconds

min(s) minute/minutes

hr(s) hour/hours

eq. equivalents

N normal

> greater than

< less than

> greather than or equal to

< less than or equal to 1.2. Definitions

[0021] The terms "amylase" or "amylolytic enzyme" refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch, a-amylases are hydrolases that cleave the a-D-(l→4) O-glycosidic linkages in starch. Generally, a-amylases (EC 3.2.1.1; a-D- (l→4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving a-D-(l→4) O- glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (l-4)-a-linked D-glucose units. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1.2; a-D-(l→4)-glucan maltohydrolase) and some product- specific amylases like maltogenic a-amylase (EC 3.2.1.133) cleave the polysaccharide molecule from the non-reducing end of the substrate, β-amylases, a- glucosidases (EC 3.2.1.20; a-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; a-D- (l→4)-glucan glucohydrolase), and product- specific amylases like the maltotetraosidases (EC 3.2.1.60) and the maltohexaosidases (EC 3.2.1.98) can produce malto-oligosaccharides of a specific length or enriched syrups of specific maltooligosaccharides.

[0022] The term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin. The term includes plant- based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, milo, potato, sweet potato, and tapioca. The term "starch" includes granular starch.

[0023] The term "granular starch" refers to raw, i.e., uncooked starch, e.g., starch that has not been subject to gelatinization.

[0024] The term "monosaccharide" refers to a carbohydrate that cannot be hydrolyzed to produce more simple sugars. Monosaccharides are also referred to as monosaccharide units, glucopyranose units, monomers, and residues.

[0025] The term "oligosaccharide" refers to a carbohydrate consisting of two to ten monosaccharide units.

[0026] The term "polysaccharide" refers to a carbohydrate consisting of more than 10 linked monosaccharide units.

[0027] A "syrup" is a liquid containing dissolved monosaccharides, oligosaccharides, and soluble polysaccharides.

[0028] The term "total soluble sugars" refers to all dissolved monosaccharides,

oligosaccharides, and soluble polysaccharides in a syrup. Where the syrup is produced by incubating a starch substrate with an a-amylase, total soluble sugars is synonymous with the term "total reaction products."

[0029] A "non-naturally-occurring syrup" is a liquid containing dissolved monosaccharides, oligosaccharides, and soluble polysaccharides that is produced by the intentional addition of a-amylase to a starch composition. Non-naturally-occurring syrups exclude syrups that occur in nature and do not involve the hand of man.

[0030] A "specialty syrup" is a syrup containing a specified relative amount of dissolved monosaccharide, oligosaccharide, and soluble polysaccharide components, for example, >30 DP5 relative to total soluble sugars, <10 DP6 relative to total soluble sugars, or a specified ratio of DP5 to DP6, DP5 to DP3, and the like.

[0031] The term "degree of polymerization" (DP) refers to the number (n) of glucopyranose units in a given saccharide. Examples of DPI are the monosaccharides glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose.

[0032] The term DP>10 refers to all soluble saccharides longer than DP 10 that can be measured by HPLC using the column and assay method specified in the Examples.

[0033] 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.

[0034] The term "dry solids content" (ds) refers to the total solids of a slurry in a dry weight percent basis.

[0035] The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0036] The term "minutes of secondary liquefaction" refers to the elapsed time of from the start of secondary liquefaction.

[0037] 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. Reference to the wild-type polypeptide is understood to include the mature form of the polypeptide.

[0038] 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.

[0039] 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. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context. [0040] 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.

[0041] The term "performance benefit" refers to an improvement in a desirable property of a molecule. Exemplary performance benefits include, but are not limited to, increased hydrolysis of a starch substrate, increased grain, cereal or other starch substrate liquefaction, improved oligosaccharide reaction product profile, improved viscosity reduction, 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.

[0042] 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 non-native gene encoding an amylase is a recombinant vector.

[0043] The terms "recovered," "isolated," and "separated," refer to a compound, protein (polypeptide), 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.

[0044] The term "purified" refers to material (e.g. , an isolated polypeptide, DP5, etc.) 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.

[0045] The terms "thermostable" and "thermostability," with reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (tl/2) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual a- amylase activity following exposure to (i.e. , challenge by) an elevated temperature.

[0046] 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).

[0047] 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.

[0048] As used herein, "water hardness" is a measure of the minerals (e.g. , calcium and magnesium) present in water.

[0049] "A cultured cell material comprising an amylase" or similar language, refers to a cell lysate or supernatant (including media) that includes an amylase as a component. The cell material may be from a heterologous host that is grown in culture for the purpose of producing the amylase.

[0050] "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

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.

[0051] 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 CspAmy2 polypeptide of SEQ ID NO: 1 would have a percent sequence identity of 99% (612 / 617 identical residues x 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 amylase polypeptide.

[0052] The term "about" refers to + 15% to the referenced value.

2. DP5-enriched syrups

[0053] An aspect of the present compositions and methods are non-naturally-occuring specialty syrups prepared from starch substrates using a-amylases. These syrups are rich in DP5 and contain reduced amounts of other oligosaccharides, such as a DP6, DP3, and DP2 compared to typical syrups. Such syrups have applications in the food and beverage industry as sweeteners. In addition, the syrups are useful for preparing purified DP5, which has been used as a nutrient for patients with renal failure and in a state of calorie-deprivation. DP5 is also used a substrate in commercially available a-amylase assays.

[0054] The syrups can be defined by the amount of DP5 they contain as a fraction of total reaction products (i.e. , total soluble sugars) after incubation for a given period of time (e.g. , 30 hours) with a-amylase. In some embodiments, the amount of DP5 as a fraction of total reaction products is >30%, >35%, or even >40%. The syrups can be defined by the amount of DP6 they contain as a fraction of total reaction products after incubation for a given period of time (e.g. , 30 hours) with a-amylase. In some embodiments, the amount of DP6 as a fraction of total reaction products is <10%, <7%, or even <5%. The syrups can also be defined by the amount of DP5 they contain relative to the amount of a different

oligosaccharide, such as DP6, DP3, DP2, or DPI, after incubation for a given period of time (e.g. , 30 hours) with a-amylase. In some embodiments, the DP6/DP5 is <0.50, <0.45, <0.40, <0.35, <0.30, <0.25, <0.20, <0.15, or even <0.10. In some embodiments, DP3/DP5 is <0.70, <0.65, or even <0.60. In some embodiments, DP2/DP5 is <0.50, <0.45, <0.40, or even <0.35. In some embodiments, DP1/DP5 is >0.20, or even >0.25.

[0055] The syrups are particularly useful as a source of DP5. However, they can also be used for the preparation of HFCS or 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. [0056] Alternatively, the syrups may be a precursor to, or may be prepared simultaneous with, a fermentation process designed to produce alcohol for fuel or drinking (i.e., potable alcohol).

3. a-Amylases for producing DP5-enriched syrups

[0057] The DP5-enriched syrups are produced by contacting a starch substrate with a suitable a- amylase, as exemplified by an a- amylase from a Cytophaga sp., herein refered to as CspAmy2. The oligosaccharides product profile of CspAmy2 is unusual in that the enzyme produces large amounts of DP5 and small amounts of other oligosaccharides to produce the syrup described, above. While other a-amylases, such as Bacillus licheniformis a-amylase, also produces a significant amount of DP5, they do not produce syrups with, for example, such a high DP5/DP6 ratio.

[0058] CspAmy2, which was previously described by Jeang, C-L. et al. ((2002) Applied and Environmental Microbiology, 68:3651-54). The amino acid sequence of the mature form of the CspAmy2 a-amylase polypeptide is shown below as SEQ ID NO: 1 :

AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV WTPPAYKGTS

QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD

VVMNHKAGAD YTENVTAVEV NPSNRNQETS GEYNIQAWTG FNFPGRGTTY

SNFKWQWFHF DGTDWDQSRS LSRIFKFRGT GKAWDWEVSS ENGNYDYLMY

ADIDYDHPDV VNEMKKWGVW YANEVGLDGY RLDAVKHIKF SFLKDWVDNA

RAATGKEMFT VGEYWQNDLG ALNNYLAKVN YNQSLFDAPL HYNFYAASTG

GGYYDMR IL NNTLVASNPT KAVTLVENHD TQPGQSLEST VQPWFKPLAY

AFILTRSGGY PSVFYGDMYG TKGTTTREIP ALKSKIEPLL KARKDYAYGT

QRDYIDNPDV IGWTREGDST KAKSGLATVI TDGPGGSKRM YVGTSNAGEI

WYDLTGNRTD KITIGSDGYA TFPVNGGSVS VWVQQ

[0059] In SEQ ID NO: 1, R178 and G179 are underlined. A variant of the Cytophaga sp. a- amylase having a deletion of both R178 and G179 (herein, "CspAmy2-vl") has also been described (Shiau, R-J. et al. (2003) Applied and Environmental Microbiology , 69:2383-85). The amino acid sequence of the mature CspAmy2-vl a-amylase polypeptide is shown below as SEQ ID NO: 2:

AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD VVMNHKAGAD YTENVTAVEV NPSNRNQETS GEYNIQAWTG FNFPGRGTTY SNFKWQWFHF DGTDWDQSRS LSRIFKFTGK AWDWEVSSEN GNYDYLMYAD IDYDHPDVVN EMKKWGVWYA NEVGLDGYRL DAVKHIKFSF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLAKVNYN QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGTQR DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI TIGSDGYATF PVNGGSVSVW VQQ

[0060] CspAmy2 and CspAmy2-vl are expected to have the same oligosaccharide product profiles; however, CspAmy2-vl is more stable (data not shown). Another variant of

CspAmy2 expected to have similar thermostability to CspAmy2-vl is a CspAmy2 variant in which residues T180 and G181 are deleted. The amino acid sequence of the mature form of this variant of the CspAmy2 a-amylase polypeptide is shown below as SEQ ID NO: 3:

AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD VVMNHKAGAD YTENVTAVEV NPSNRNQETS GEYNIQAWTG FNFPGRGTTY SNFKWQWFHF DGTDWDQSRS LSRIFKFRGK AWDWEVSSEN GNYDYLMYAD IDYDHPDVVN EMKKWGVWYA NEVGLDGYRL DAVKHIKFSF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLAKVNYN QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGTQR DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI TIGSDGYATF PVNGGSVSVW VQQ

[0061] All three of the above polypeptides, as well as additional CspAmy2 variants, are useful for practicing the present compositions and methods.

[0062] In some embodiments, the present a-amylase variants have indicated combinations of mutations and a defined degree of amino acid sequence homology/identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, for example, at least 60%, at least 65%, 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% amino acid sequence

homology/identity.

[0063] In some embodiments, the present α-amylase variants have indicated combinations of mutations and are derived from a parental amylase having a defined degree of amino acid sequence homology/identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, for example, at least 60%, at least 65%, 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% amino acid sequence homology/identity.

[0064] In some embodiments, in addition to the mutations described above, the present a- amylases further include one or more mutations that provide a further performance benefit. These include mutation that have been previously described in a-amylases having at least 60%, at least 65%, 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% amino acid sequence homology/identity to SEQ ID NO: 1.

[0065] Based on experimental data described elsewhere, embodiments of the present variant a-amylases include variants having a mutation at an amino acid position corresponding to E187 or S241, optionally in combination with at least one mutation at an amino acid position corresponding to a position selected from N126, Y150, F153, L171, T180, and 1203 (using SEQ ID NO: 1 for numbering).

[0066] Exemplary mutations at amino acid position E187 include E187V and E187P.

Exemplary mutations at amino acid position S241 include S241Q and S241A. Exemplary mutations at amino acid position N126 includes N126Y. Exemplary mutations at amino acid position Y150 include Y150F, Y150H, and Y150W. Exemplary mutations at amino acid position F153 include F153H, F153W, and F153Y. Exemplary mutations at amino acid position L171 include L171F, L171G, L171I, L171M, L171R, L171V, L171W, L171Y, L171H, L171K, L171N, L171Q, and L171S. Exemplary mutations at amino acid position T180 include T180D and T180H. Exemplary mutations at amino acid position 1203 includes I203C, I203V, I203F, I203L, I203M, and I203Y.

[0067] In some embodiments, the variant α-amylases include a mutation in amino acid residues corresponding to E132, Q167, A277, and/or T400, using SEQ ID NO: 1 for numbering. Exemplary mutations at amino acid position E132 include E132A, E132C, E132D, E132F, E132G, E132H, E132I, E132K, E132L, E132M, E132N, E132P, E132Q, E132R, E132V, and E132W. Exemplary mutations at amino acid position Q167 include Q167A, Q167D, Q167E, Q167G, Q167H, Q167K, Q167M, Q167N, Q167P, Q167S, Q167T, and Q167V. Exemplary mutations at amino acid position A277 include A277C, A277D, A277E, A277F, A277G, A277I, A277K, A277L, A277M, A277N, A277Q, A277R, A277S, A277T, A277V, A277W, and A277Y. Exemplary mutations at amino acid position T400 include T400A, T400C, T400D, T400F, T400G, T400I, T400K, T400L, T400M, T400N, T400Q, T400R, T400W, and T400Y.

[0068] In some embodiments, the variant a-amylases include a mutation in an amino acid residue corresponding to G476, using SEQ ID NO: 1 for numbering. Exemplary mutations at amino acid position G476 include G476A, G476C, G476H, G476K, G476N, G476P, G476Q, G476R, G476S, G476T, G476V, and G476Y.

[0069] In some embodiments, the variant a-amylases are those that include mutations in both amino acid residues corresponding to G476 and G477, using SEQ ID NO: 1 for numbering.

[0070] In some embodiments, the variant α-amylases include mutations in amino acid residues corresponding to R458, T459, and/or D460. Exemplary mutations are R458N, T459S, and D460T, respectively.

[0071] In some embodiments, the variant α-amylases include mutations at positions corresponding to E132 and/or T180 (using SEQ ID NO: 1 for numbering), in combination with an RG-deletion or a TG-deletion (or equivalent deletion based on the sequence of the parent a-amylases), such that a stabilizing interaction can occur between the remaining non-G residue in the XiG/SiXiGi motif and the residue at position 132. In some embodiments, the residue at position 132 is negatively charged (i.e., D or E) and the remaining non-G residue is positively charged (i.e., H, R, or K). In some embodiments, the residue at position 132 is positively charged (i.e., H, R, or K) and the remaining non-G residue is negatively charged (i.e., D or E).

[0072] Exemplary combinations of mutation (using SEQ ID NO: 1 for numbering) are shown, below:

E187P + I203Y + G476K;

E187P + 1203 Y + G476K + R458N + T459S + D460T;

T180D + E187P + I203Y + G476K;

N126Y + T180D + E187P + I203Y + G476K;

N126Y + T180D + E187P + I203Y + Y303D + G476T + G477E;

N126Y + T180D + E187P + I203Y + Y303D + N475E + G477Q;

N126Y + T180D + E187P + I203Y + Y303R + N475E + G476T + G477R;

T038N + N88H + N126Y + T129I + N134M + F153W + L171R + T180D + E187P + I203Y

+ G476K + G477E;

N126Y + E132H + T180D + E187P + I203Y + Y303D + G476T + G477E; N126Y + E187P + I203Y;

N126Y + I203Y + S241Q;

N126Y + T180H + E187P + I203Y;

N126Y + T180H + I203Y + S241Q;

N126Y + F153W + T180H + E187P + I203Y;

N126Y + F153W + T180H + I203Y + S241Q;

N126Y +Y150H + F153W + L171N + E187P + I203Y;

N126Y +Y150H + F153W + L171N + I203Y + S241Q;

N126Y +Y150H + F153W + L171N + T180H + E187P + I203Y;

N126Y +Y150H + F153W + L171N + T180H + I203Y + S241Q;

N126Y + F153W + T180D + I203Y + S241Q;

N126Y + E132H + F153W + T180D + I203Y + S241Q + A277F;

N126Y + E132H + F153W + Q167E + T180D + I203Y + S241Q + A277F; and

N126Y + E132H + F153W + Q167E + T180D + I203Y + S241Q + A277F + T400K.

[0073] All the above combinations of mutation are contemplated for use in conjunction with the aforementioned deletions at positions corresponding to R178, G179, T180, and/or G181.

Additional mutations experimentally determined to provide at least one performance advantage when combined with the aforementioned variants include mutations at positions corresponding to 6, 7, 8, 11, 14, 15, 20, 21, 23, 26, 27, 28, 37, 38, 39, 40, 42, 45, 46, 48, 49,

50, 51, 52, 53, 54, 58, 61, 62, 68, 70, 71, 72, 73, 79, 80, 81, 82, 84, 85, 87, 88, 89, 92, 93, 94,

95, 96, 97, 98, 101, 108, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 126,

127, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 142, 143, 144, 147, 148, 149, 150,

151, 152, 153, 154, 155, 156, 158, 159, 165, 167, 168, 170, 171, 172, 175, 176, 177, 180,

181, 182, 187, 190, 191, 193, 199, 200, 201, 203, 206, 208, 210, 211, 212, 214, 215, 216,

219, 221, 223, 225, 226, 227, 235, 238, 239, 240, 241, 242, 243, 245, 246, 247, 248, 249,

250, 252, 253, 254, 256, 257, 258, 260, 261, 262, 266, 267, 268, 269, 270, 271, 273, 276,

277, 279, 280, 282, 284, 285, 286, 288, 296, 299, 300, 301, 302, 303, 304, 307, 308, 310,

311, 312, 313, 316, 317, 318, 320, 321, 325, 327, 335, 338, 342, 348, 349, 352, 356, 357,

360, 362, 363, 368, 369, 377, 381, 382, 383, 384, 385, 388, 390, 392, 394, 395, 396, 397,

398, 400, 401, 402, 403, 404, 405, 407, 408, 410, 414, 415, 416, 418, 419, 420, 421, 422,

423, 424, 426, 428, 429, 430, 431, 434, 435, 436, 439, 441, 442, 444, 445, 446, 447, 448,

449, 450, 451, 454, 455, 457, 460, 461, 462, 463, 464, 465, 466, 467, 469, 470, 471, 473,

474, 475, 476, 477, 479, 480, 481, 482, 483, and 484, using SEQ ID NO: 1 for numbering.

Specific mutations are T6A, T6D, T6E, T6G, T6K, T6M, T6N, T6Q, T6S, M7A, M7V, M8C, M8F, M8I, M8L, M8Y, F11V, Y14A, Y14I, Y14Q, Y14T, Y14V, V15C, V15D, V15I, V15N, V15T, Q20A, Q20C, Q20D, Q20H, Q20K, Q20M, Q20N, Q20R, Q20S, Q20Y, Q21F, Q21W, N23A, N23C, N23D, N23E, N23H, N23K, N23Q, N23R, N23S, N23T, N23V, R26C, R26E, R26G, R26K, R26M, R26S, R26T, T27A, T27C, T27D, T27E, T27F, T27H, T27I, T27K, T27L, T27M, T27N, T27Q, T27R, T27S, T27V, T27Y, D28A, D28C, D28T, I37F, I37V, T38D, T38N, A39L, V40A, V40C, V40D, V40G, V40H, V40I, V40K, V40M, V40P, V40Q, V40R, V40S, V40T, V40W, V40Y, T42A, T42C, T42I, T42M, T42V, A45C, A45G, Y46F, G48A, T49I, S50A, S50C, S50E, S50G, S50K, S50M, S50N, S50Q, S50R, S50T, S50Y, Q51C, Q51D, Q51E, Q51S, Q51V, A52C, A52D, A52E, A52F, A52G, A52H, A52K, A52L, A52M, A52R, A52S, A52T, A52V, A52Y, D53E, V54N, V54T, P58A, P58C, P58H, P58I, P58S, P58T, P58V, L61M, L61V, Y62A, Y62C, Y62D, Y62F, Y62G, Y62H, Y62I, Y62K, Y62L, Y62M, Y62N, Y62P, Y62Q, Y62R, Y62S, Y62V, N68C, N68D, N68E, N68F, N68H, N68L, N68P, N68Q, N68R, N68S, N68V, N68W, N68Y, K70R, G71A, G71C, G71D, G71E, G71K, G71R, G71S, T72G, T72S, V73S, T79F, T79I, T79L, T79M, T79N, T79S, T79Y, K80A, K80C, K80D, K80F, K80H, K80I, K80M, K80N, K80Q, K80R, K80S, K80T, K80V, K80Y, G81A, G81D, G81E, G81F, G81H, G81I, G81K, G81N, G81P, G81R, G81S, G81T, E82A, E82D, E82M, E82Q, K84A, K84C, K84E, K84I, K84Q, K84R, K84S, K84T, K84Y, S85A, S85C, S85D, S85E, S85G, S85H, S85I, S85L, S85M, S85N, S85Q, S85R, S85T, S85V, S85Y, V87I, V87T, N88C, N88D, N88E, N88G, N88H, N88I, N88K, N88L, N88M, N88Q, N88R, N88S, N88T, N88V, N88W, N88Y, T89A, T89D, T89E, T89F, T89H, T89I, T89K, T89L, T89M, T89N, T89Q, T89R, T89S, T89Y, S92A, S92C, S92D, S92E, S92F, S92G, S92H, S92L, S92M, S92N, S92Q, S92R, S92T, S92W, S92Y, N93A, N93C, N93E, N93F, N93H, N93I, N93K, N93L, N93Q, N93S, N93T, N93Y, G94A, G94C, G94N, I95M, Q96A, Q96E, Q96H, Q96I, Q96K, Q96L, Q96M, Q96N, Q96R, Q96V, Q96Y, V97I, V97T, Y98F, Y98I, Y98L, Y98V, V101C, V101T, G108A, G108S, Y111D, Y111E, Yl 11L, Yl 1 IN, Yl 1 IS, Yl 1 IT, Yl 1 IV, Tl 12A, Tl 12F, Tl 12H, Tl 121, Tl 12K, Tl 12L, T112M, T112N, T112P, T112R, T112V, T112W, E113D, E113N, E113Q, E113T, N114A, N114C, N114D, N114E, N114F, N114G, N114H, N114I, N114L, N114P, N114Q, N114R, N114S, N114T, N114V, N114W, N114Y, V115A, VI 151, T116A, T116C, T116D, T116G, T116H, T116I, T116K, T116N, T116P, T116Q, T116R, T116S, A117C, A117I, A117S, A117V, V118A, V118C, V118E, V118F, V118H, V118I, V118L, V118M, V118N, V118Q, V118R, V118S, V118W, V118Y, V120A, V120C, V120I, V120M, V120T, P122A, P122D, P122E, P122G, P122H, P122N, P122R, P122S, P122T, P122W, S123A, S123C, S123G, S123K, S123L, N124A, N124D, N124F, N124G, N124L, N124R, N124S, N124V, Y126A, Y126C, Y126D, Y126E, Y126G, Y126H, Y126I, Y126K, Y126L, Y126M, Y126N, Y126Q, Y126R, Y126S, Y126T, Y126V, Y126W, Q127A, Q127C, Q127D, Q127E, Q127F, Q127H, Q127I, Q127K, Q127L, Q127M, Q127N, Q127R, Q127S, Q127T, Q127V, Q127W, Q127Y, T129A, T129C, T129D, T129E, T129F, T129G, T129I, T129K, T129L, T129M, T129N, T129Q, T129R, T129S, T129V, T129W, T129Y, S130A, S130G, S130I, S130K, S130L, S130M, S130N, S130P, S130R, S130T, S130V, S130W, G131A, G131C, G131D, G131F, G131H, G131I, G131K, G131L, G131M, G131P, G131Q, G131V, G131W, G131Y, E132A, E132C, E132D, E132F, E132G, E132H, E132I, E132K, E132L, E132M, E132N, E132P, E132Q, E132R, E132V, E132W, Y133A, Y133D, Y133E, Y133G, Y133K, Y133L, Y133M, Y133R, Y133S, Y133T, Y133W, N134A, N134D, N134F, N134G, N134H, N134I, N134K, N134L, N134M, N134R, N134S, N134V, N134W, N134Y, Q136A, Q136C, Q136D, Q136E, Q136F, Q136H, Q136K, Q136L, Q136M, Q136N, Q136R, Q136S, Q136T, Q136V, Q136W, Q136Y, A137S, A137T, A137V, W138F, W138Y, G140A, G140M, N142F, N142K, N142L, N142M, N142P, N142V, N142W, N142Y, F143H, F143Y, P144C, P144D, P144E, P144G, P144H, P144I, P144K, P144L, P144M, P144N, P144Q, P144S, P144T, P144Y, G147A, G147C, G147H, G147K, G147L, G147M, G147N, G147Q, G147R, T148A, T148D, T148E, T148F, T148I, T148K, T148L, T148R, T148S, T148V, T148W, T149A, T149C, T149D, T149E, T149F, T149H, T149K, T149L, T149M, T149N, T149Q, T149V, T149W, T149Y, Y150H, S151A, S151E, S151F, S151H, S151I, S151K, S151L, S151M, S151Q, S151R, S151V, N152A, N152C, N152D, N152E, N152G, N152H, N152K, N152M, N152P, N152Q, N152R, N152S, N152T, W153F, W153H, W153Q, W153R, W153T, W153Y, K154A, K154C, K154D, K154E, K154G, K154I, K154L, K154M, K154N, K154R, K154T, K154Y, W155P, Q156A, Q156D, Q156E, Q156F, Q156G, Q156H, Q156I, Q156L, Q156M, Q156N, Q156R, Q156S, Q156Y, F158C, F158D, F158K, F158L, F158M, F158N, F158P, F158Q, F158R, F158S, F158V, H159M, H159Y, W165C, W165D, W165E, W165F, W165H, W165I, W165K, W165L, W165M, W165Q, W165R, W165T, W165Y, Q167A, Q167D, Q167E, Q167G, Q167H, Q167K, Q167M, Q167N, Q167P, Q167S, Q167T, Q167V, S168A, S168D, S168F, S168H, S168I, S168K, S168M, S168N, S168Q, S168R, S168T, S168V, S168W, S168Y, S170A, S170C, S170D, S170E, S170F, S170L, S170M, S170N, S170Q, S170R, S170T, L171A, L171C, L171F, L171G, L171H, L171I, L171N, L171Q, L171R, L171T, L171V, L171W, L171Y, S172A, S172D, S172H, S172R, S172T, F175M, F175Y, K176I, K176T, F177L, F177V, F177W, D180A, D180C, D180F, D180G, D180H, D180I, D180L, D180M, D180N, D180Q, D180R, D180S, D180T, D180V, D180W, D180Y, G181A, G181C, G181D, G181E, G181F, G181H, G181K, G181L, G181M, G181N, G181Q, G181R, G181S, G181T, G181V, G181Y, K182A, K182P, E187D, E187I, E187K, E187M, E187N, E187P, E187R, E187S, E187T, E187V, E187Y, S190A, S190C, S190D, S190F, S190L, S190N, S190P, S190Q, E191A, E191C, E191F, E191G, E191H, E191I, E191K, E191N, E191Q, E191R, E191S, E191T, E191W, E191Y, G193A, G193C, G193D, G193E, G193F, G193H, G193K, G193L, G193M, G193N, G193Q, G193R, G193S, G193T, G193V, G193W, M199L, Y200F, A201M, Y203I, Y203L, Y203V, D206A, D206E, D206G, D206H, D206M, D206N, D206Q, D206R, D206S, D206T, P208A, P208E, P208F, P208I, P208K, P208L, P208T, P208V, P208Y, V210A, V210E, V210H, V210K, V210N, V210Q, V210R, V210S, V210T, V211A, V211E, V211H, V211I, V211Q, V211R, N212E, N212F, N212G, N212L, N212M, N212R, N212V, M214I, M214L, K215C, K215E, K215F, K215M, K215N, K215R, K215Y, K216F, V219I, V219T, Y221F, Y221I, Y221L, N223C, N223E, N223I, N223K, N223Q, N223R, N223S, N223T, N223V, N223W, N223Y, V225A, V225I, V225L, V225M, G226D, G226M, G226Q, G226R, G226S, L227F, L227I, L227W, L227Y, V235A, I238A, I238L, I238M, K239D, K239E, K239P, K239Q, K239R, K239S, K239T, F240K, F240L, F240M, F240Q, F240R, Q241A, Q241C, Q241D, Q241E, Q241G, Q241H, Q241K, Q241L, Q241M, Q241N, Q241P, Q241R, Q241S, Q241T, Q241V, Q241W, Q241Y, F242V, L243C, L243Y, D245A, D245C, D245E, D245G, D245L, D245M, D245N, W246F, V247I, V247L, D248E, D248H, D248N, D248T, D248V, N249A, N249E, N249G, N249H, N249Q, N249Y, A250M, A250S, A250V, A252C, A252D, A252E, A252G, A252H, A252I, A252K, A252L, A252M, A252N, A252Q, A252S, A252V, A252W, A252Y, A253E, A253I, A253K, A253L, A253M, A253Q, A253S, A253T, A253V, A253Y, T254F, T254K, T254S, K256A, K256M, K256N, K256S, E257Q, E257S, M258L, T260A, T260C, T260S, T260V, V261I, V261W, G262A, Q266A, Q266D, Q266E, Q266H, Q266I, Q266M, Q266N, Q266S, Q266T, Q266V, Q266Y, N267H, N267I, N267Q, N267R, N267S, N267T, N267V, N267Y, D268G, D268N, L269C, L269D, L269I, L269K, L269Q, L269S, L269T, L269Y, G270A, G270D, G270E, G270F, G270H, G270I, G270L, G270M, G270Q, G270T, G270V, G270Y, A271C, A271D, A271E, A271H, A271K, A271M, A271Q, A271R, A271S, A271T, A271V, A271Y, N273C, N273G, N273H, N273I, N273K, N273R, L276I, L276M, A277C, A277D, A277E, A277F, A277G, A277I, A277K, A277L, A277M, A277N, A277Q, A277R, A277S, A277T, A277V, A277W, A277Y, V279C, V279T, N280A, N282S, N282T, S284T, S284Y, L285A, L285C, L285I, L285V, F286M, A288C, A296C, A296D, A296E, A296F, A296G, A296H, A296I, A296L, A296M, A296N, A296Q, A296R, A296S, A296V, A296W, A296Y, T299A, T299D, T299E, T299F, T299G, T299K, T299L, T299M, T299R, T299S, T299V, T299W, G300A, G300C, G300D, G300E, G300F, G300H, G300K, G300M, G300Q, G300R, G300V, G300W, G301A, G301C, G301D, G301E, G301F, G301H, G301K, G301L, G301M, G301Q, G301R, G301S, G301T, G301V, G301W, G301Y, G302S, Y303A, Y303C, Y303D, Y303E, Y303F, Y303G, Y303H, Y303I, Y303K, Y303L, Y303M, Y303N, Y303Q, Y303R, Y303S, Y303T, Y303V, Y303W, Y304F, Y304K, Y304W, R307A, R307C, R307E, R307G, R307H, R307K, R307M, R307N, R307Q, R307S, R307T, N308C, N308D, N308E, N308G, N308L, N308M, N308T, N308V, L310A, L310C, L310D, L310E, L310H, L310I, L310M, L310P, L310W, L310Y, N311C, N311E, N311G, N311H, N311K, N311Q, N311R, N311S, N311V, N311W, N311Y, N312D, N312F, N312G, N312H, N312K, N312Q, N312R, T313A, T313S, A316D, A316E, A316G, A316H, A316K, A316Q, A316R, A316Y, S317C, S317D, S317G, S317H, S317K, S317L, S317N, S317Q, S317R, S317T, S317W, S317Y, N318A, N318C, N318F, N318G, N318I, N318K, N318L, N318M, N318Q, N318R, N318S, N318T, N318V, N318W, T320A, T320C, T320D, T320E, T320G, T320H, T320I, T320K, T320N, T320P, T320Q, T320R, T320V, T320W, T320Y, K321C, K321F, K321H, K321N, K321S, K321Y, L325A, L325C, L325F, L325I, L325M, L325Q, L325V, E327D, E327L, Q335C, Q335E, E338A, E338D, E338F, E338G, E338H, E338I, E338K, E338P, E338Q, E338R, E338T, E338V, E338Y, Q342A, Q342C, Q342G, Q342L, Q342M, Q342R, Q342S, Q342T, Q342V, Q342W, L348A, L348C, L348H, L348I, L348M, L348Q, L348S, L348T, A349G, A349R, F352I, F352L, F352M, F352T, F352V, R356Q, S357A, S357C, S357D, S357E, S357F, S357H, S357I, S357K, S357L, S357N, S357Q, S357T, S357V, S357W, S357Y, Y360F, Y360I, Y360L, Y360M, Y360V, S362A, S362C, S362E, S362I, S362T, S362V, V363I, V363L, M368F, M368I, M368L, M368Y, Y369A, Y369E, Y369I, Y369L, Y369N, Y369V, R377A, R377C, R377D, R377E, R377F, R377G, R377H, R377I, R377K, R377L, R377N, R377Q, R377S, R377T, R377V, R377W, R377Y, A381C, A381E, A381G, A381H, A381K, A381L, A381M, A381N, A381P, A381Q, A381V, A381W, A381Y, L382F, L382H, L382Q, L382S, K383A, K383C, K383D, K383E, K383H, K383I, K383L, K383M, K383N, K383Q, K383R, K383S, K383W, K383Y, S384A, S384C, S384D, S384F, S384G, S384H, S384I, S384L, S384N, S384R, S384V, S384Y, K385A, K385D, K385E, K385F, K385G, K385H, K385L, K385M, K385Q, K385R, K385T, K385V, K385Y, P388A, P388C, P388D, P388I, P388L, P388N, P388R, P388S, P388T, P388V, L390M, L390V, A392C, A392S, K394A, K394C, K394E, K394F, K394G, K394H, K394I, K394L, K394Q, K394R, K394S, K394T, K394V, K394W, K394Y, D395A, D395E, D395N, D395S, D395T, Y396A, Y396D, Y396F, Y396K, Y396M, Y396N, Y396Q, Y396T, Y396V, Y396W, A397D, A397E, A397H, A397K, A397M, A397N, A397Q, A397V, Y398A, Y398C, Y398F, Y398H, Y398I, Y398L, Y398W, T400A, T400C, T400D, T400F, T400G, T400I, T400K, T400L, T400M, T400N, T400Q, T400R, T400W, T400Y, Q401A, Q401C, Q401F, Q401H, Q401I, Q401L, Q401M, Q401N, R402C, R402D, R402F, R402K, R402L, R402M, R402N, R402Q, R402S, R402W, D403E, D403N, D403S, Y404E, Y404G, Y404K, Y404M, Y404N, Y404R, Y404W, I405C, I405F, I405L, I405M, I405V, N407A, N407C, N407D, N407E, N407G, N407K, N407M, N407Q, N407R, P408A, P408C, P408D, P408I, P408K, P408L, P408M, P408N, P408Q, P408V, P408W, V410A, V410C, V410D, V410E, V410F, V410H, V410I, V410L, V410M, V410N, V410Q, V410S, V410T, T414A, T414I, T414S, T414V, R415M, E416C, E416D, E416F, E416H, E416I, E416K, E416L, E416M, E416N, E416Q, E416R, E416T, E416V, E416W, E416Y, D418A, D418E, D418G, D418H, D418I, D418K, D418L, D418M, D418N, D418Q, D418S, D418T, D418V, D418W, S419A, S419C, S419E, S419G, S419L, S419M, S419N, S419R, S419V, S419W, S419Y, T420A, T420C, T420D, T420E, T420G, T420H, T420I, T420K, T420M, T420P, T420S, T420V, T420W, T420Y, K421A, K421D, K421E, K421H, K421I, K421L, K421M, K421N, K421P, K421Q, K421R, K421T, K421V, K421W, K421Y, A422C, A422D, A422E, A422F, A422G, A422I, A422L, A422N, A422P, A422Q, A422R, A422S, A422Y, K423A, K423D, K423E, K423F, K423H, K423I, K423L, K423M, K423N, K423Q, K423R, K423S, K423T, K423V, K423W, K423Y, S424A, S424C, S424G, S424K, S424N, S424Q, S424R, S424T, L426S, L426T, L426V, T428G, T428V, V429A, V429C, V429I, V429L, I430C, I430G, I430L, I430M, I430Q, I430V, T431A, T431C, T431S, P434A, P434C, P434D, P434E, P434F, P434H, P434I, P434K, P434L, P434M, P434N, P434Q, P434R, P434S, P434V, P434Y, G435A, G435C, G435D, G435E, G435F, G435H, G435I, G435K, G435M, G435N, G435P, G435Q, G435R, G435S, G435T, G435W, G436F, G436I, G436M, G436N, G436Q, G436S, G436V, R439A, R439D, R439G, R439H, R439K, R439M, R439N, R439P, R439Q, R439S, R439V, R439W, R439Y, Y441A, Y441C, Y441D, Y441F, Y441G, Y441H, Y441K, Y441L, Y441M, Y441N, Y441P, Y441R, Y441S, Y441T, Y441W, V442A, V442C, V442I, V442T, T444C, T444D, T444E, T444F, T444G, T444H, T444I, T444K, T444L, T444M, T444N, T444P, T444R, T444S, T444W, S445A, S445C, S445E, S445G, S445H, S445K, S445L, S445M, S445N, S445T, S445V, N446A, N446C, N446H, N446K, A447C, A447D, A447F, A447H, A447L, A447M, A447N, A447Q, A447R, A447S, A447Y, G448A, G448C, G448D, G448E, G448H, G448K, G448L, G448M, G448N, G448Q, G448R, G448S, G448T, G448W, E449D, E449H, E449K, E449T, I450A, I450C, I450D, I450E, I450G, I450K, I450L, I450M, I450N, I450Q, I450S, I450T, I450W, I450Y, W451Y, L454A, L454I, L454K, L454M, L454W, T455A, T455I, T455L, T455S, N457H, N457K, N457R, N457T, N457V, N457Y, D460A, D460E, D460G, D460M, D460N, D460Q, D460S, D460V, K461C, K461H, K461L, K461M, K461N, K461Q, K461T, K461Y, I462A, I462L, I462M, I462Q, I462T, I462V, T463D, T463E, T463H, T463P, T463Q, T463R, T463V, T463Y, I464P, I464T, G465A, G465C, G465D, G465E, G465K, G465L, G465M, G465N, G465Q, G465W, G465Y, S466A, S466C, S466D, S466G, S466H, S466K, S466L, S466M, S466N, S466T, S466W, S466Y, D467E, D467G, D467L, Y469A, Y469D, Y469E, Y469I, Y469M, Y469N, Y469R, Y469S, Y469T, Y469V, Y469W, A470S, A470V, T471A, T471C, T471E, T471G, T471H, T471I, T471L, T471M, T471N, T471S, T471V, T471W, P473C, P473D, P473E, P473G, P473I, P473K, P473L, P473R, P473T, P473W, V474A, V474C, V474L, V474S, N475A, N475C, N475E, N475F, N475H, N475K, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, G476A, G476D, G476E, G476F, G476H, G476I, G476L, G476M, G476N, G476P, G476Q, G476R, G476S, G476T, G476V, G476W, G476Y, G477A, G477D, G477F, G477H, G477I, G477K, G477L, G477M, G477Q, G477S, G477T, G477V, G477W, G477Y, V479C, V479D, V479E, V479F, V479H, V479I, V479N, V479P, V479Y, S480A, S480C, S480H, V481A, V481C, V481N, W482Y, V483A, V483G, V483I, V483K, V483L, V483M, V483R, V483Y, Q484A, Q484C, Q484F, Q484G, Q484H, Q484K, Q484L, Q484M, Q484P, Q484R, Q484T, and Q484Y.

[0074] In all cases, corresponding amino acid positions in other a-amylases be identified by amino acid sequence alignment with CspAmy2 (SEQ ID NO: 1) using Clustal W with default parameters. Preferred CspAmy2 variants produce at least as much DP5, in absolute or relative amounts, as the parental CspAmy2 a-amylase, and/or demonstrate a further performance benefit, as described, herein. In some embodiments, the CspAmy2 variants produce more DP5, in absolute or relative amounts, compared to the parental CspAmy2 a- amylase.

[0075] The present amylases may include any number of conservative amino acid

substitutions. Exemplary conservative amino acid substitutions are listed in Table 1.

Table 1. Conservative amino acid substitutions

[0076] 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.

[0077] The present 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 amylase polypeptides. The present amylase polypeptides may also be truncated to remove the N or C-termini, so long as the resulting polypeptides retain amylase activity.

[0078] The present amylase may be a "chimeric" or "hybrid" polypeptide, in that it includes at least a portion of a first amylase polypeptide, and at least a portion of a second amylase polypeptide (such chimeric amylases have recently been "rediscovered" as domain-swap amylases). The present 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.

4. Starch Substrates

[0079] The following section describes the selection and preparation of starch substrates. 4.1. Selection of Starch Substrates

[0080] 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 e.g., corn, cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes. Corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch; wheat contains about 60-65% starch; and polished rice contains 70-72% starch. Specifically contemplated starch substrates are corn starch and wheat starch. The starch from a grain may be ground or whole and includes corn solids, such as kernels, bran and/or cobs. The starch may also be highly refined raw starch or feedstock from starch refinery processes. 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).

[0081] 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 substrate can also be from a direct starch to sugar process, a raw or granular starch hydrolysis process. The starch substrate can also be a hydrolyzed form of starch, such as maltodextrin. The starch substrate to be processed may optionally be refined and may be, for example, at least 90%, at least 95%, at least 97%, or at least 99.5% pure.

4.2. Gelatinization, Liquefaction and Saccharification of Starch

[0082] Liquefaction refers to a process by which starch is converted to less viscous and shorter chain carbohydrates. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of a-amylase, although additional enzymes may optionally be added to affect liquefaction. In some embodiments, the starch substrate as described above is slurried with water. The starch slurry may contain starch at a dry solid content of about 10-55%, about 20-45%, about 30-45%, about 30-40%, or about 30-35%. <x- amylase may be added to the slurry, with a metering pump, for example. To optimize a- amylase stability and activity, the pH of the slurry typically is adjusted to about pH 5.5 - 6.5 and about 1 mM of calcium (about 40 ppm free calcium ions) can also be added. Bacterial a- amylase remaining in the slurry following liquefaction may be deactivated via a number of methods known in the art.

[0083] The slurry of starch plus the a-amylase may be pumped continuously through a jet cooker, which is steam heated to about 105-110°C. Gelatinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate. The residence time in the jet cooker is brief. The partly gelatinized starch may be passed into a series of holding tubes maintained at about 105- 110°C and held for 5-8 min. to complete the gelatinization process (i.e., "primary

liquefaction"). Secondary liquefactoin is performed in holding tanks at 85-95°C or higher temperatures for about 1 to 2 hours . These tanks may contain baffles to discourage back mixing. The slurry is then allowed to cool, typically to about 60-64° or even to room temperature. Starch processing may in some cases also be conducted entirely below the gelatinizing temperature of starch. The liquefied starch typically is in the form of a slurry having a dry solids content (w/w) of about 10-50%; about 10-45%; about 15-40%; about 20- 40%; about 25-40%; or about 28-38%.

[0084] Liquefaction with a-amylases advantageously can be conducted at low pH, eliminating the requirement to adjust the pH to about pH 5.5 - 6.5. a-amylases can be used for liquefaction at a pH range of 2.0 - 7.0, e.g., pH 3.0 - 7.5, pH 4.0 - 6.0, or pH 4.5 - 5.8. <x- amylases can maintain liquefying activity at a temperature range of about 85°C - 95°C, e.g., 85°C, 90°C, or 95°C. For example, secondary liquefaction can be conducted with a solution of 25% DS corn starch for 10 min at pH 5.8 and 85°C, or pH 4.5 and 95°C. Liquefying activity can be assayed using any of a number of known assays in the art.

[0085] Saccharification refers to a process by which a starch substrate produced in liquefaction, also called liquefact, or a starch substrate generated through granular starch hydrolysis, is further hydrolysed into a specialty syrup or into glucose. Those skilled in the production of syrups and glucose are able to optimize the conditions of saccharification depending on the desired end-product. Enzymes used for saccharification include a- amylases, β-amylases, glucoamylases, pullulanases, and maltogenic amylases, depending on the desired end product. The pH may vary between 4.0 - 6.0 and the temperature between 55 - 70°C, for example, 58 - 64°. The process is typically carried out for at least 6, at least 12, at least 24, at least 36, at least 48, at least 60, at least 72, at least 84, or even at least 96 hours.

[0086] Although the conditions of saccharification greatly affect the characteristics of the final syrup, the properties of the enzymes are also important. Particularly where it is desirable for the end product to be a specialty syrup containing a large amount of particular oligosaccharides, such as DP5 (as opposed to mainly glucose), the enzymatic properties of the a-amylase have a significant effect on the final syrup.

[0087] 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.

EXAMPLES

Example 1

[0088] In a bench-scale saccharification experiment duplicate samples of 20 g of corn starch liquefact with a DS of 30% and DE of 11.6 were incubated in the presence of α-amylase at 60°C, pH 5.2, for up to 30 hours. The α-amylase used was either a commercially available Bacillus α-amylase product {i.e., OPTIMASE® AA 56L, PURASTAR® HP Am 5000L, SPEZYME® XTRA, SPEZYME® ALPHA PF, or SPEZYME® FRED (all from DuPont Industrial Biosciences (Palo Alto, California, USA) or CspAmy2-vl (SEQ ID NO: 2). The commercially available products were dosed at 0.2 kg/MT DS. CspAmy2-vl was dosed at 0.006 mg/g DS. The target amount of enzyme in the corn starch liquefact was typically about 0.006 mg/g DS. Since the size distribution of starch hydrolysis products is primarily a feature of the α-amylase rather than the concentration of the a-amylase, minor differences in the final concentration of α-amylase were not considered to be important. The concentrations of each α-amylase used are shown in Table 2. Table 2. α-amylases used for proces

[0089] Samples of the syrup were taken over time during the 30 hrs incubation. The samples were lOx diluted with demineralized water and boiled for 10 minutes to kill enzyme activity and were filtered with a 0.22 μιη filter before measuring them with a HPLC system

(Shimadzu) using a column specialized for DPI- 10 sugars, namely Aminex HPx-42A (Bio- Rad) as described in Messaoud, E.B. et al. (2004) Enzyme and Microbial Technology 34:662-666. The following analysis conditions were used: column temperature 85°C, ultrapure water as eluens, flow rate of 0.6 ml/min, injection volume of 20 μΐ, and a RI detector with cell temperature of 40°C. The oligosaccharide profile produced by the various tested a-amylases over time are shown in the graphs in Figures 1-6. The amount of DP5 produced over time by CspAmy2, PURASTAR® HP Am 5000L, and OPTIMASE® AA 56L is shown in more detail in Figure 7.

[0090] In general, all the a-amylases tested produced DP2, DP3, DP5, and DP6 as the main oligosaccharide products. However, the oligosaccharide profile produced by CspAmy2 was unique. In particular, after 30 hours incubation:

(i) DP6/DP5 was 0.11 using CspAmy2 while being >0.49 using the other tested a- amylases;

(ii) DP4/DP5 was 0.21 using CspAmy2 while being >0.28 using the other tested a- amylases;

(iii) DP3/DP5 was 0.61 using CspAmy2 while being >0.71 using the other tested a- amylases;

(iv) DP2/DP5 was 0.36 using CspAmy2 while being >0.51 using the other tested a- amylases;

(v) DP1/DP5 was 0.24 using CspAmy2 while being 0.12 - 0.18 using the other tested a- amylases; and

(vi) the DPI concentration was higher using CspAmy2 than using the other tested a- amylases (which could be explained by the hydrolysis of DP6 into DP5 and DPI). [0091] Overall, CspAmy2 produced a much purer DP5 syrup than any of the other tested a- amylases, with >30 DP5 and <10 DP6 as fractions of total reaction products.

Example 2

[0092] In a similar experiment, the amounts of DP6, DP5, DP3, and DP2 were measured after treating maltodextrin with a DE of 7.4 (Sigma Aldrich) in a 30% DS solution with varying amounts of CspAmy2-vl (i.e., 3, 6, or 9 μg/g DS). As shown in the graph in Figure 8, while starch hydrolysis is slower using decreasing amounts of enzyme, the signature oligosaccharide profile produced by CspAmy2 remains the same.

[0093] Although the foregoing compositions and methods have been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

[0094] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Claims

CLAIMS What is claimed is:

1. A non-naturally-occurring syrup produced from a starch substrate by incubation with an a-amylase, comprising >30 DP5 and <10% DP6 as fractions of total reaction products or DP6/DP5 <0.30, DP3/DP5 <0.70, and DP2/DP5 <0.50.

2. The syrup of claim 1, having a concentration ratio of DP6/DP5 <0.30.

3. The syrup of claim 2, having a concentration ratio of DP6/DP5 <0.20.

4. The syrup of claim 3, having a concentration ratio of DP6/DP5 <0.15.

5. The syrup of any of claims 1-4, produced by incubating a starch substrate in the presence of CspAmy2 a-amylase or a variant, thereof.

6. The syrup of claim 5, wherein the α-amylase is a variant of CspAmy2 comprising a deletion of residues 178 and 179 or 180 and 181, using SEQ ID NO: 3 for numbering.

7. A method for producing a syrup from a starch substrate, comprising incubating the starch substrate in the presence of an α-amylase capable of producing >30 DP5 and <10% DP6 as fractions of total reaction products, or DP6/DP5 <0.30, DP3/DP5 <0.70, and

DP2/DP5 <0.50, in a period of 30 hours at pH 5.2 and 60°C.

8. The method of claim 7, wherein the α-amylase is capable of producing a reaction product having a concentration ratio of DP6/DP5 <0.30 in a period of 30 hours at pH 5.2 and 60°C.

9. The method of claim 8, wherein the α-amylase is capable of producing a reaction product having a concentration ratio of DP6/DP5 <0.20 in a period of 30 hours at pH 5.2 and 60°C.

10. The method of claim 9, wherein the α-amylase is capable of producing a reaction product having a concentration ratio of DP6/DP5 <0.15 in a period of 30 hours at pH 5.2 and 60°C.

11. The method of any of claims 7-10, wherein the starch substrate is a corn, cereal, grain, or tuber liquifact.

12. The method of any of claims 7-11, wherein the starch substrate comprises from about 10% to about 40% dissolved solids.

13. The method of any of claims 7-12, wherein the amount of a-amylase used is from about 0.001 to about 0.05 mg/g dissolved solids.

14. The method of any of claims 7-13, wherein incubating the starch substrate in the presence of α-amylase is performed without the addition of calcium to the starch substrate.

15. The method of any of claims 7-14, wherein incubating the starch substrate in the presence of CspAmy2 α-amylase or a variant, thereof, is performed at a pH <7.0.

16. The method of any of claims 7-15, wherein incubating the starch substrate in the presence of α-amylase is performed at a pH <6.0.

17. The method of any of claims 7-15, wherein the starch substrate is incubated in the presence of CspAmy2 α-amylase or a variant, thereof.

18. The method of claim 17, wherein the α-amylase is a variant of CspAmy2 comprising a deletion of residues 178 and 179 or 180 and 181, using SEQ ID NO: 3 for numbering.

19. A syrup produced by the method of any of claims 7-18.