FIELD OF INVENTION

[001] The present compositions and methods relate to the use of low concentrations of betaine to increase the activity and/or stability of enzymes in stressful environments, such as in starch liquefaction and sachharification.

BACKGROUND

[002] 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 g/mole. 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 g/mole.

[003] Sugars from starch, in the form of concentrated dextrose syrups, are currently produced by an enzyme-catalyzed process involving: (1) gelatinization and liquefaction (or viscosity reduction) of solid 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 glucose 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 also 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.

[004] Currently, the carbohydrate and fermentation alcohol industry carries out liquefaction at elevated temperature (i.e., 80 -120°C). Under such stressful conditions, the inactivation rate of even relatively thermostable enzymes is high, requiring the addition of a large amount of carbohydrate-processing enzymes to achieve desired performance.

[005] Other stressful industrial applications include biomass processing, as in the case of cellulosic ethanol production. Enzymatic cleaning is also a stressful environment as it may be desirable to use high temperatures and high concentrations of surfactant to achieve a required level of cleaning.

[006] While the careful selection of enzymes and protein engineering can reduce the stress- inactivation of enzymes, the need exists for other ways to promote enzyme stability to reduce enzyme costs and allow the use of a wider range of enzymes for industrial processes.

SUMMARY

[007] The present compositions and methods relate to improving the stability and/or productivity of enzymes using low concentrations of betaine. Aspects and embodiments of the present compositions and methods are summarized in the following separately-numbered paragraphs:

1. In one aspect, a method for increasing the stability and/or productivity of an enzyme in a high-stress environment is provided, comprising, adding to an aqueous solution or suspension comprising the enzyme and a substrate for the enzyme an amount of betaine sufficient to increase the stability and/or activity of the enzyme on the substrate compared the stability and/or activity under equivalent enzymatic conditions but in the absence of betaine.

2. In some embodiments of the method of paragraph 1, the high stress environment has a temperature greater than about 50°C, greater than about 60°C, greater than about 70°C, greater than about 80°C, greater than about 90°C, or even greater than about 100°C.

3. In some embodiments of the method of any of the preceding paragraphs, the increased amount of stability and/or activity is not due a beneficial effect on a

microorganism.

4. In some embodiments of the method of any of the preceding paragraphs, betaine further provides an additional beneficial effect on a microorganism.

6. In some embodiments of the method of any of the preceding paragraphs, the amount of betaine is between about 0.02 g/L and about 0.5 g/L aqueous solution or suspension.

7. In some embodiments of the method of any of the preceding paragraphs, the high stress environment is a starch hydrolysis process, a biomass hydrolysis process, or a cleaning or disinfecting process. 8. In another aspect, a method for increasing the stability or productivity of an enzyme in a starch hydrolysis process is provided, comprising, adding to a starch slurry during enzymatic liquefaction and/or saccharification an amount of betaine sufficient to increase the amount of reducing sugar produced in the slurry over time compared to the amount generated under equivalent enzymatic conditions but in the absence of betaine.

9. In some embodiments of the method of paragraph 8, saccharification is part of simultaneous saccharification and fermentation.

10. In some embodiments of the method of paragraph 8 or 9, the amount of betaine is between about 0.02 g/L and about 0.5 g/L slurry.

11. In some embodiments of the method of any of paragraphs 8-10, the increased amount of reducing sugar produced in the slurry in the presence of betaine is not due a beneficial effect on a microorganism present in the slurry.

12. In some embodiments of the method of any of paragraphs 8-11, at least some of the increase in the amount of reducing sugar produced in the slurry in the presence of betaine occurs prior to introducing a fermenting organism to the slurry.

13. In some embodiments of the method of any of paragraphs 8-12, at least some of the increase in the amount of reducing sugar produced in the slurry in the presence of betaine occurs prior to fermenting the slurry.

14. In some embodiments of the method of any of paragraphs 8-13, betaine further provides a benefit to a fermenting organism.

15. In some embodiments of the method of any of paragraphs 8-14, the enzyme is an a-amylase and/or a glucoamylase.

16. In another aspect, a method for stabilizing an enzyme composition is provided, comprising adding to the liquid enzyme composition about 0.02 g/L to about 0.5 g/L betaine.

17. In some embodiments of the method of any of the preceding paragraphs, the betaine is natural betaine.

18. In some embodiments of the method of any of the preceding paragraphs, the betaine is synthetic betaine. 19. In another aspect, a composition is provided, comprising an enzyme and a sufficient amount of betaine such that, upon dilution of the composition in an aqueous solution or suspension, the final concentration of betaine is about 0.02-0.5 g/L.

20. In some embodiments of the composition of paragraph 19, the enzyme is an a- amylase and/or a glucoamylase and the aqueous solution or suspension is a liquefaction or saccharification slurry.

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

DETAILED DESCRIPTION

I. Definitions and Abbreviations

[009] 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 abbreviations and terms terms are defined, below.

1.1. Abbreviations and Acronyms

[0010] The following abbreviations/acronyms have the following meanings unless otherwise specified:

°C degrees Centigrade

CAZy Carbohydrate-Active Enzymes database

DE dextrose equivalent

dH ₂ 0 or DI deionized water

dIH ₂ 0 deionized water, Milli-Q filtration

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

EC Enzyme Commission

EOF end of fermentation

eq. equivalents

EtOH ethanol

g or gm grams

AA a-amylase

GA glucoamylase

H ₂ 0 water

HFCS high fructose corn syrup

hr hour

hr(s) hour/hours

kg kilograms

mg milligrams

min minute mL and ml milliliters

mM millimolar

MW molecular weight

N normal

sp. species

SSF simultaneous saccharification and fermentation

SSU/g solid soluble starch unit/gram dry solids

T metric tonnes

temp temperature

U units

v/v volume/volume

w/v weight/volume

w/w weight/weight

wt weight

wt% weight percent

1.2. Definitions

[0011] As used herein, the term "betaine" refers to trimethyl glycine, trimethyl glycine monohydrate or active derivatives thereof. Active derivatives refer to organic salts of trimethyl glycine, such as citrates, acetates, formates and other derivatives. Betaine is usually either derived from natural sources, for instance extracted from sugar beet or obtained by biochemical processes. Such betaine is referred to as "natural betaine" or "synthetic betaine," respectively.

[0012] As used herein, the term, "hydrolysis of starch" refers to the cleavage of glucosidic bonds with the addition of water molecules.

[0013] As used herein, 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 (1 -4)-a-linked D- glucose units. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1 .2; a-D-(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 themaltohexaosidases (EC 3.2.1 .98) can produce malto-oligosaccharides of a specific length or enriched syrups of specific

maltooligosaccharides.

[0014] As used herein, the term "glucoamylase" (EC 3.2.1 .3) (otherwise known as glucan 1,4-a-glucosidase; glucoamylase; amyloglucosidase; γ-amylase; lysosomal a-glucosidase; acid maltase; exo- 1,4-a-glucosidase; glucose amylase; y-l,4-glucan glucohydrolase; acid maltase; 1,4-a-D-glucan glucohydrolase; or 4-a-D-glucan glucohydrolase) refers to a class of enzymes that catalyze the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides. These are exo-acting enzymes, which release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules. The enzymes also hydrolyze a-1,6 and a -1,3 linkages although at much slower rates than a-1,4 linkages.

[0015] As used herein, the term, "pullulanase" (E.C. 3.2.1 .41)" refers to a class of enzymes that are capable of hydrolyzing a-1,6 glucosidic linkages in an amylopectin molecule. The term "β-amylase" (E.C. 3.2.1 .2) refers to a class of enzymes that hydrolyze the a-l,4-glucan bonds in amylosaccahride chains from non-reducing ends and generate a di-saccharide maltose, linked by a-1,4 glucosidic linkages two glucose residues, β-amylases are well characterized in higher plants and microbial sources.

[0016] As used herein, the term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C6HioOs)x, wherein X can be any number. The term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, milo, potato, sweet potato, and tapioca. 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.

[0017] As used herein, "biomass" refers to cellulose, hemicellulose and lignin from plants.

[0018] As used herein, the term "liquefaction" or "liquefy" refers to a process by which starch is converted to less viscous and shorter chain dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of an a- amylase, although additional liquefaction-inducing enzymes optionally may be added.

Liquefaction can be performed above, at, or below the gelatinization of starch, depending on the processes and enzymes used.

[0019] As used herein, the term "saccharification" refers to a process by which shorter chain dextrins produced by liquefaction are further hydrolysed to glucose monomers. The process generally required a glucoamylase. [0020] As used herein, 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.

[0021] As used herein, 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.

[0022] As used herein, the term "purified" refers to material (e.g., an isolated polypeptide, polynucleotide, or betaine) 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.

[0023] As used herein, 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.

[0024] As used herein, the term "substrate" refers to a molecule that is acted upon by an enzyme. While proteases are capable of autodigestion, the present usage of the term substrate does not include the enzyme, itself.

[0025] As used herein, the terms "thermostable" and "thermostability," with reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (ti/2) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual a- amylase activity following exposure to (i.e., challenge by) an elevated temperature.

[0026] As used herein, a "pH range," with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.

[0027] As used herein, the terms "pH stable" and "pH stability," with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour). [0028] As used herein, "stress" refers to elevated temperature, extremes in pH, the presence of surfactants and chelants, and other conditions that adversely affect enzyme stability.

[0029] As used herein, the term "biologically active" refer to a sequence having a specified biological activity, such an enzymatic activity.

[0030] As used herein, 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.

[0031] As used herein, "water hardness" is a measure of the minerals (e.g., calcium and magnesium) present in water.

[0032] The term "degree of polymerization" (DP) refers to the number (n) of anhydro- glucopyranose units in a given saccharide. Examples of DPI are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose. An examples of DP3 is the trisaccharide maltotriose.

[0033] As used herein, 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] As used herein, the term "dry solids content" (ds) refers to the total solids of a slurry in a dry weight percent basis.

[0035] As used herein, the term "slurry" refers to an aqueous mixture containing insoluble solids.

[0036] As used herein, the term "ethanologenic microorganism" refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.

[0037] As used herein, 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.

[0038] As used herein, the term "malt" refers to any malted cereal grain, such as malted barley or wheat.

[0039] As used herein, 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.

[0040] As used herein, the term "about" refers to ± 15% to the referenced value. [0041] As used herein, 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.

[0042] 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.

[0043] All cited references are hereby incorporated by reference.

II. Use of Betaine to Increase the Stability or Activity of Enzymes

[0044] Betaine, i.e., trimethyl glycine, and in some embodiments its derivatives, is a substance that can be derived from natural sources, for example, extracted from sugar beet, or obtained by biochemical syntheses. Betaine obtained from natural sources is often referred to as "natural betaine" and sometimes has properties that are different from "synthetic betaine," which is typically a salt. In biological systems, betaine is known to serve as an organic osmolyte that protects cells from osmotic stress, such as drought, high salinity, and high temperature. Betaine is also known to protect proteins from heat and other stresses, typically at molar concentrations (see, e.g. Paleg, L.G. et al. (1984) Plant Physiol ^' . 75:974-78;

Dionisio-Seese, M L et al. (1999) Photosynetica 36:55"/ '-563); and Ohtake, S. et al. (2011) Adv. DrugDeliv. 63 : 1053-73.

[0045] The present compositions and methods are based on the surprising observation that the presence of much more moderate amounts of betaine significantly increase the productivity of enzymes in stressful environments, as exemplified by industrial-scale starch liquefaction and saccharification as it pertains to carbohydrate processing and ethanol fermentation. While betaine may reasonably be expected to protect living cells, such as ethanologens and other microorganisms used to produce commercially-valuable products from starch hydrolates, from the stresses of industrial processes; the present methods relate to benefits of betaine that are not tied to living cells. The present methods relate to the effect of betaine on the carbohydrate-processing enzymes themselves.

[0046] As evidenced by the data provided, herein, the increase in productivity of

carbohydrate processing enzymes is significant. Betaine is shown to increase the

productivity of such enzymes up to about 20%, e.g. at least about 5%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%) and even at least about 20% in a starch hydrolysate slurry. To provide such a benefit, the final concentration of betaine in a liquefaction and/or saccharification process need only be about 0.01 g/L slurry of higher, e.g., 0.02-0.5, 0.02-0.3, or 0.05-0.2 g/L. Experiments described herein suggest that a betaine dose of 0.2 g/L can in some case produces a similar benefit as nearly doubling the amount of enzyme used. 0.2 g/L corresponds to only about 1.7 mM betaine (MW=177.14 g/mole), which is far less than amounts previously described to stabilize proteins. Accordingly, amounts of, e.g., 0.2-5 mM are useful for practicing the present compositions and methods, with a practical amount in the range of about 1-2 mM.

[0047] The benefits of betaine can be observed upon analyzing the enzymatic hydrolysates of liquefaction and/or saccharification prior to contact with fermenting microorganisms. While fermentation organisms, including ethanolagens), may also benefit from the presence of betaine in the fermentation medium {i.e., mash or wort), the present methods involve benefits of betaine on enzyme performance that are independent, separate and distinct from benefits relating to the osmoprotection of a microorganism. Nonetheless, the present methods are fully compatible with simultaneous saccharification and fermentation (SSF), in which case the benefits of betaine in saccharification may be difficult to separate from the benefits in fermentation. In such cases, the benefits of betaine in saccharification, independent of fermentaion (and the presence of microorganisms) may be confirmed simply by delaying the introduction of microorganism to a saccharifide medium and analysing its sugar profile.

III. Embodiments of the present methods

1. Preparation of Starch Substrates

[0048] Numerous methods may be used to prepare starch substrates for use according to the present methods. Useful starch substrates may be obtained from tubers, roots, stems, legumes, cereals or whole grain. 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.

[0049] 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.

2. Gelatinization and Liquefaction of Starch

[0050] The starch substrate prepared as described above may be slurried with water. The starch slurry may contain starch as a weight percent of dry solids of about 10-55%, about 20- 45%, about 30-45%), about 30-40%>, or about 30-35%. a-amylase may be added to the slurry. The α-amylase is usually supplied, for example, at about 1,500 units per kg dry matter of starch. To optimize α-amylase stability and activity, the pH of the slurry typically is adjusted to about pH 4.5-6.5 and about 1 mM of calcium (about 40 ppm free calcium ions) can also be added, depending upon the properties of the amylase used.

[0051] The slurry of starch plus the α-amylase may be pumped continuously through a jet cooker, which is steam heated to 105°C. Gelatinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate. The residence time in the jet cooker is brief. The partly gelatinized starch may be passed into a series of holding tubes maintained at 105-110°C to complete the gelatinization process (i.e., "primary liquefaction"). The slurry may then be allowed to cool to room temperature. The liquefied starch typically is in the form of a slurry having a dry solids content (w/w) of about 10-50%>; about 10-45%>; about 15-40%>; about 20- 40%; about 25-40%; or about 25-35%.

3. Saccharification

[0052] The liquefied starch can be saccharified into a syrup that is rich in lower DP (e.g., DPI + DP2) saccharides, using variant amylases, optionally in the presence of other enzymes. The exact composition of the products of saccharification depends on the combination of enzymes used, as well as the type of granular starch processed.

[0053] Whereas liquefaction is generally run as a continuous process, saccharification is often conducted as a batch process. Saccharification typically is most effective at

temperatures of about 60-65°C and a pH of about 4.0-4.5, e.g., pH 4.3, necessitating cooling and adjusting the pH of the liquefied starch. The temperature and pH range can vary depending upon the properties of the enzymes. Saccharification may be performed, for example, at a temperature between about 40°C, about 50°C, or about 55°C to about 60°C or about 65°C. Saccharification is normally conducted in stirred tanks, which may take several hours to fill or empty. Enzymes typically are added either at a fixed ratio to dried solids as the tanks are filled or added as a single dose at the commencement of the filling stage. When a maximum or desired DE has been attained, the reaction is stopped by heating to 85°C for 5 min., for example. Saccharification may be conducted over a pH range of about pH 3 to about pH 7, e.g., pH 3.0-7.5, pH 3.5-5.5, pH 3.5, pH 3.8, or pH 4.5.

[0054] An a-amylase may be added to the slurry as an isolated enzyme solution. For example, an α-amylase can be added in the form of a cultured cell material produced by host cells expressing an amylase. An α-amylase may also be secreted by a host cell into the reaction medium during the fermentation or SSF process, such that the enzyme is provided continuously into the reaction. The host cell producing and secreting amylase may also express an additional enzyme, such as a glucoamylase. For example, U.S. Patent No.

5,422,267 discloses the use of a glucoamylase in yeast for production of alcoholic beverages. For example, a host cell, e.g., Trichoderma reesei ox Aspergillus niger, may be engineered to co-express an α-amylase and a glucoamylase, e.g., HgGA, TrGA, or a TrGA variant, during saccharification. The host cell can be genetically modified so as not to express its endogenous glucoamylase and/or other enzymes, proteins or other materials. The host cell can be engineered to express a broad spectrum of various saccharolytic enzymes. For example, the recombinant yeast host cell can comprise nucleic acids encoding a

glucoamylase, an alpha-glucosidase, an enzyme that utilizes pentose sugar, an a-amylase, a pullulanase, an isoamylase, and/or an isopullulanase (see, e.g., WO 2011/153516 A2).

4. Isomerization

[0055] Soluble starch hydrolysate produced by treatment with carbohydrate-processing enzymes can be converted into high fructose starch-based syrup (FIFSS), such as high fructose corn syrup (FIFCS). This conversion can be achieved using a glucose isomerase, particularly a glucose isomerase immobilized on a solid support. The pH is increased to about 6.0 to about 8.0, e.g., pH 7.5 (depending on the isomerase), and Ca ²⁺ is removed by ion exchange.

5. Fermentation

[0056] Soluble starch hydrolysate, particularly glucose 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.

[0057] Ethanologenic microorganisms include yeast, such as Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis.

[0058] 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, CO2 generated by fermentation may be collected with a CO2 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.

6. Compositions Comprising Betaine

[0059] Betaine may be combined with enzymes to increase their stability during storage and/or to improve their performance in a final application. Enzymes for use in liquefaction and saccharification are well known and include a-amylase, glucoamylase, pullulanase, β-amylase, and phytase, as well as isoamylase, protease, cellulase, xylanase, β-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, and a combination thereof. Enzymes for use in biomass processing include a vast array of cellulases and hemicellulases. Enzymes used for cleaning are mainly proteases but can include amylases, mannanases, lipases, pectinases, perhydrolases, and the like.

[0060] Exemplary a-amylases contemplated for use in with betaine include GC 358, SPEZYME® XTRA, SPEZYME® HP A, SPEZYME® RSL, SPEZYME® FRED,

MAXALIG™ ONE, and STARGEN® (Danisco US Inc.), LIQUOZYME® SC,

LIQUOZYME® SC DS, TERMAMYL® 120-L, and SUPRA® (Novozymes A/S),

FUELZYME® LF (Verenium Corp.), and KLEISTASE® E5NC, T10S (Amano).

Exemplary glucoamylases include G-ZYME® 480, G-ZYME® 480 ETHANOL, G-ZYME® G990 ZR, GC 147, DISTILLASE®, and FERMENZYME®, OPTIDEX® 300, and

OPTIDEX L-400 (Danisco US Inc.). SPIRIZYME® Fuel, SPIRIZYME® PLUS, and SPIRIZYME® ULTRA, AMG 200L, AMG 300 L, SAN™ SUPER and AMG™ E (Novozymes A/S, Denmark), and AMIGASE™ and AMIGASE™ PLUS (DSM). Exemplary pullulanases include DIAZYME® (Danisco US Inc.), PROMOZYME® (Novozymes A/S), and KLEISTASE® PLF3 (Amano). Exemplary β-amylases include, SPEZYME® BBA 1500, SPEZYME® DBA, OPTIMAL T™ ME, OPTIMALT™ BBA (Danisco US Inc.), NOVOZYM™ WBA (Novozymes A/S) AND β-AMYLASE F (Amano). Exemplary phytases include PHZYME (Danisco A/S, Diversa), NATUPHOS (BASF), RONOZYME P (Novozymes A/S), and FINASE (AB Enzymes).

[0061] Other enzymes are described in, e.g., Boel et al. {\9M) EMBO J. 3 : 1097-02, WO 92/00381, WO 00/04136, WO 84/02921, WO 99/28448, WO 86/01831, WO 00/04136, EP 135,138, US4587215, USRE32153, Chen et al. (1996) Prot. Eng. 9: 499-505), Chen et al.

(1995) Prot. Eng. 8: 575-582), Chen et al. (1994) Biochem. J. 301 : 275-281, Fierobe et al.

(1996) Biochemistry, 35: 8698-8704, and Li et al. (1997) Protein Eng. 10: 1199-1204.

[0062] Compositions comprising the present 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.

[0063] Betaine is ideally present in an enzyme composition in a sufficient amount such that, upon dilution of the enzyme composition in a liquefaction or saccharification process, the final concentration of betaine is about 0.02-0.5, 0.02-0.3, or 0.05-0.2 g/L. These values correspond to about 1-5 mM, a range which can reasonably be achieved in an industrial scale process. Even about 2 mM betaine appears to almost double the activity of carbohydrate- processing enzymes. Alternatively, where betaine is present primarily to stabilize an enzyme in storage (as opposed to in the final application), it is ideally present in an enzyme composition at about 0.02-0.5, 0.02-0.3, or 0.05-0.2 g/L. Again, these values correspond to about 1-5 mM.

[0064] The compositions and methods disclosed herein are illustrated by, but not limited to, the following examples. EXAMPLES Example 1. General materials and methods

Materials:

[0065] Corn starch was obtained from Sigma Aldrich (Catalogue # S4126). Natural betaine was obtained from DuPont (i.e. , Betafin BP 20, anhydrous from Beta vulgaris). Synthetic betaine (betaine hydrochloride) was obtained from Sigma Aldrich (Catalogue # B3501). The enzymes used in the example were (i) SPEZYME® ALPHA™ (a variant of the a-amylase from Bacillus stearothermophilus), (ii) OPTFMAX® 4060VHP (a variant of the glucoamylase from Aspergillus niger plus a variant of pullulanase from Bacillus licheniformis) and (iii) FvGA (a glucoamylase from Fusarium verticilloides), all from Danisco US, Inc.

Analytical methods:

[0066] Amounts of reducing sugars were determined using the Fehling method, wherein a sample is titrated with methylene blue as indicator. The Fehling's solution was standardised using 1% w/v glucose solution.

[0067] The sugar composition of samples was measured using a high pressure liquid chromatographic method. Briefly, 20 iL of appropriately diluted samples were loaded onto an Aminex Column HPX-87H (Bio-Rad, Cat. No. 1250140) maintained at 60°C using 0.01 N sulfuric acid as a mobile phase at a flow rate of 0.7 mL/min. Detection was carried out using a refractive index detector (RID). Elution profiles were obtained over 15 minutes. The distribution of saccharides and the amount of each saccharide were determined from previously run standards.

Improvement calculations:

[0068] Relative improvement, e.g. , in the amount of reducing sugars produced during liquefaction and/or saccharification in the presence of betaine, were calculated as a ratio compared to the control, which was arbitrarily set at " 1."

Example 2. Effect of betaine addition on corn starch liquefaction at 32% DS

[0069] Starch slurry was prepared by adding 64 g corn starch (Sigma Aldrich Catalogue # S4126) to 132 g of water in a 500 mL Erlenmeyer flask. The pH of the resulting slurry was adjusted to 5.60 ± 0.10 using 1 N HC1, followed by addition of SPEZYME® ALPHA™ in an amount of 0.20 or 0.40 g/T of starch. Natural betaine at a concentration ranging from 0.00- 0.20 g/1 was added to the flasks and the final slurry weights were adjusted with water to 200 g. Liquefaction was carried out at 93°C with continuous mixing at 350 rpm for 2 hours 30 minutes in a water bath. The liquefact was cooled to room temperature and the liquefact weights were adjusted to 200 g using water. Thereafter, the mixed samples from each flask were diluted with water and analyzed for amounts of reducing sugars. The results are shown in Table 1.

Table 1. Effect of betaine on reducing sugar production in corn starch liquefaction

[0070] Addition of betaine in starch liquefaction resulted in increased reducing sugar production as compared to liquefaction carried out without addition. The effect of betaines was dosage-dependent. The maximum observed improvement using 0.2 g/L betaine approached the benefit of using double the amount of a-amylase in the absence of betaine.

Example 3. Effect of betaine addition on corn starch liquefaction at different pH

[0071] Starch slurry was prepared by adding 64 g corn starch (Sigma Aldrich Catalogue # S4126) to 132 g of water in a 500 mL Erlenmeyer flask. The pH of the resulting slurry was adjusted to adjusted to 5.60 ± 0.10, 5.00 ± 0.10 or 4.50 ± 0.10, using 1 N HCl, followed by addition of SPEZYME ^® ALPHA™ at 0.20 and 0.40 kg/T of starch. Natural betaine at concentrations ranging from 0.00- 0.20 g/1 was added to the flasks and the final slurry weights were adjusted with water to 200 g. Liquefaction was carried out at 93°C with continuous mixing at 350 rpm for 2.5 hr in a water bath. The liquefact was cooled to room temperature and the weight was adjusted to 200 g with water. Thereafter, the mixed samples from each flask were diluted with water and analyzed for amounts of reducing sugars. The results are shown in Tables 2-4. Table 2. Effect of betaine on reducing sugar production in corn starch liquefaction at pH 5.5

Table 3. Effect of betaine on reducing sugar production in corn starch liquefaction at pH 5.0

Table 4. Effect of betaine on reducing sugar production in corn starch liquefaction at pH 4.5 [0072] Addition of betaine to the starch liquefaction resulted in increased amounts of reducing sugar production across the tested pH range of 4.50 to 5.50 as compared to liquefaction carried out without addition.

Example 4. Effect of betaine addition on corn starch liquefaction at high DS

[0073] Starch slurry was prepared by adding 73.34 g corn starch (Sigma Aldrich Catalogue # S4126) to 122 g of water in a 500 mL Erlenmeyer flask. The pH of the slurry was adjusted to 5.60 ± 0.10 using 1 N HC1, followed by addition of SPEZYME ^® ALPHA™ at 0.20 and 0.40 kg/T of starch. Natural betaine at concentrations ranging from 0.00-2.00 g/1 was added to the flasks and the final slurry weights was adjusted with water to 200 g. Liquefaction was carried out at 93°C with continuous mixing at 350 rpm for 2 hours 30 minutes in a water bath. The liquefact was cooled to room temperature and the weight was adjusted to 200 g using water. Thereafter, the well mixed samples from each flask were diluted with water and analyzed for amounts of reducing sugar. The results are shown in Table 5.

Table 5. Effect on betaine on reducing sugar production in corn starch liquefaction at 36.7% DS

[0074] The addition of betaine to the starch liquefaction resulted in increased reducing sugar production as compared to liquefaction carried out without addition. Example 5. Effect of natural betaine addition in corn flour liquefaction

[0075] Corn flour slurry was prepared by weighing 84 g corn starch (Local Market) followed by addition of 210 g of water into 500 mL Erlenmeyer flask. The pH of the slurry was adjusted to 5.60 ± 0.10 using 1 N HCl, followed by addition of SPEZYME ^® ALPHA™ at 0.20 and 0.40 Kg/T of starch. Natural Betaine at a concentration ranging from 0.00-0.50 g/1 was added to the flasks and the final slurry weights was adjusted with water to 300 g.

Liquefaction was carried out at 93°C with continuous mixing at 350 rpm for 2 hours 30 minutes in a water bath. The Liquefact was cooled to room temperature and the weight was adjusted to 300 g using water. Thereafter, the mixed samples from each flask were diluted with water and analyzed for amounts of reducing sugar. The results are shown in Table 6.

Effect on betaine on reducing sugar production in corn flour liquefaction at 27%

[0076] The addition of betaine to the starch liquefaction resulted in increased reducing sugar production as compared to liquefaction carried out without addition of natural betaine.

Example 6. Effect of natural and synthetic betaine addition in corn starch liquefaction

[0077] Starch slurry was prepared by adding 64 g corn starch (Dry Basis, Sigma Aldrich Catalogue # S4126) to 132 g of water in a 500 mL Erlenmeyer flask. The slurry of the pH was adjusted to 5.60 ± 0.10 using 1 N HCl, followed by addition of SPEZYME ^® ALPHA™ at 0.20 and 0.40 Kg/T of starch. Natural betaine at a concentration of 0.06 g/1 or synthetic betaine at a concentration ranging from 0.00-0.10 g/1 was added to the flasks and the final slurry weights was adjusted with water to 200 g. Liquefaction was carried out at 93°C with continuous mixing at 350 rpm for 2 hours 30 minutes in a water bath. The liquefact was cooled to room temperature and the weight was adjusted to 200 g using water. Thereafter, the mixed samples from each flask were diluted with water and analyzed for amounts of reducing sugar. The results are shown in Table 7.

Table 7. Effect of natural and synthetic betaine on reducing sugar production in corn starch liquefaction at 32% DS

[0078] The addition of synthetic betaine to the starch liquefaction resulted in increased reducing sugar production as compared to liquefaction carried out without addition of betaine. The improvement in the reducing sugar with synthetic betaine is similar to natural betaine.

Example 7. Effect of natural and synthetic betaine addition in corn starch liquefaction and saccharification on glucose production

[0079] Starch slurry was prepared by weighing 64 g corn starch (Dry Basis, Sigma Aldrich Catalogue # S4126) followed by addition of 132 g of water into 500 mL Erlenmeyer flask. Slurry pH was adjusted to 5.60 ± 0.10 using 1 N HC1, followed by addition of SPEZYME ^® ALPHA™ at 0.20 kg/T of starch. No betaine or natural betaine or synthetic betaine at a concentrations of 0.10 g/1 was added and the final slurry weights was adjusted with water to 200 g. Liquefaction was carried out at 93°C with continuous mixing at 350 rpm for 2 hours 30 minutes in a water bath. The Liquefact was cooled to 60C after adjusting pH to 4.50 with IN HC1. The liquefact weight was adjusted to 200 g with water. No betaine or natural betaine or synthetic betaine at a concentration of 0.10 g/1 was added to the flask.

Saccharification was initiated by addition of OPTIMAX ^® 4060VHP at 0.70 Kg/T of starch followed by incubation at 60°C with continuous mixing for 72 hours. The flasks were sampled at 0, 24, 48 and 72 hours for determination of DPI (glucose) using HPLC. The results are shown in Table 8.

Table 8. Effect on betaine on DPI production on corn starch liquefaction and saccharification at 32% DS

[0080] The addition of natural or synthetic betaine to the starch liquefaction and/or saccharification resulted in increased DPI production as compared to liquefaction and saccharification carried out without addition of betaine. The improvement in the DPI was more pronounced when the betaine was added at both liquefaction and saccharification.

Example 8. Effect of natural betaine addition on glucose production at elevated

saccharification temperature and pH 4.50

[0081] A 32% w/w maltodextrin solution was prepared by adding 640 g of maltodextrin powder (Sigma Aldrich Catalogue # 419680) to 1300 g of water in a 5 L glass beaker with continuous mixing. The pH of the maltodextrin solution was adjusted to 4.50 ± 0.10 using 5 N HC1 and the final weight adjusted to 2000 g with water. No betaine, or natural betaine at a concentration of 0.10 g/1, was added to 250 mL Erlenmeyer flasks containing 100 g of maltodextrin solution. Saccharification was initiated by adding OPTEVIAX ^® 6040VHP at 0.70 kg/T of starch followed by incubation at 65°C with continuous mixing at 200 rpm for 72 hours. The flasks were sampled at 24, 48 and 72 hours for determination of DPI (glucose) by HPLC. The results are shown in Table 9.

Table 9. Effect of betaine on glucose production at elevated saccharification temperature and low pH

[0082] The addition of natural betaine to the saccharification at higher temperature resulted in increased DPI (glucose) production as compared to saccharification carried out without addition of natural betaine.

Example 9. Effect of betaine addition on glucose production at elevated saccharification temperature and pH 5.2

[0083] A 30% w/w maltodextrin solution was prepared by adding 600 g of maltodextrin powder (Shangdong luzhou) to 1350 g of water in a 5000 mL glass beaker with continuous mixing. The pH of the maltodextrin solution pH was adjusted to 5.2 ± 0.10 using 5 N HC1 and the final weight adjusted to 2000 g with water. No betaine or natural betaine at a concentration of 0.10 kg/T of starch was added to 250 mL Erlenmeyer flask containing 100 g of maltodextrin solution. Saccharification was initiated by adding FvGA at 0.25 kg/T of starch followed by incubation at 65°C with continuous mixing at 200 rpm for 72 hours. The flasks were sampled at 19, 28 and 45 hours for determination of DPI (glucose) by HPLC. The results are shown in Table 10. Table 10: Effect of natural betaine on glucose production at elevated saccharification temperature and pH 5.20

[0084] The addition of betaine to the saccharification at higher temperature results in increased DPI production as compared to saccharification carried out without addition of natural betaine.

Example 10. Effect of natural betaine on improving the storage stability of enzyme at elevated temperature

[0085] The commercially-available SPEZYME® ALPHA™ formulation was used to prepare the three additional formulations shown in Table 11. All four formulations were stored in 15 mL falcon tube at 50°C for 1 week in Thermo HEREAUS static incubator. The performance of these formulations were assessed at Day "0" and "7," by carrying out corn starch liquefaction as outlined in example 1 using a dose of 0.20 kg/T of starch. The results are shown in Table 12.

Table 11. Formulation containing betaine for assessing storage stability at 50°C

Table 12. Effect of natural betaine on improving the storage stability

[0086] The dilution of the original formulation led to substantial loss of performance of AA activity when stored at 50°C. Addition of betaine to the diluted formulation resulted in improved stability at 50°C compared to the diluted formulation without betaine. The improvement in the stability is more pronounced at a higher betaine concentration.