Increased availability of fermentable sugars during fermentation

Abstract

Previously, methods have been described for increasing the amount of fermentable sugars in a fermentation substrate by treating it with a combination of an enzyme having transglucosidase activity and an enzyme having glucoamylase activity to hydrolyze oligosaccharides and / or polysaccharides that are not hydrolyzed by glucoamylase alone during fermentation. This method is most effective using a fermentation substrate containing small amounts of maltose and maltotriose.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 437,556, filed January 6, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The method involves increasing the amount of fermentable sugars in a fermentation substrate by treating it with a combination of an enzyme having transglucosidase activity and an enzyme having glucoamylase activity to hydrolyze oligosaccharides and / or polysaccharides that are not conventionally hydrolyzed by glucoamylase alone during fermentation. The method is most effective using a fermentation substrate containing small amounts of maltose and maltotriose. [Background technology]

[0003] Fermentation is often limited by glucose as the primary carbon source for the fermenting organism. Examples of such fermentation include ethanol production by yeast, lactic acid production by lactic acid bacteria, and amino acid production by organisms such as Corynebacterium. In each case, the yield of the final product depends on the available glucose, which can be the product of starch-hydrolyzing enzymes on suitable starch-containing fermentation substrates. Such enzymes hydrolyze most of the starch but also produce oligosaccharides that cannot be utilized by commercially relevant fermentation microorganisms. Summary of the Invention [Problem to be solved by the invention]

[0004] What is needed is a method to reduce the production of non-fermentable oligosaccharides and increase the fermentable sugars in the fermentation substrate. [Means for solving the problem]

[0005] Previously, enzymatic methods have been described for increasing the amount of fermentable sugars in a fermentation substrate by treating it with a combination of an enzyme having transglucosidase activity and an enzyme having glucoamylase activity to hydrolyze oligosaccharides and / or polysaccharides that are not hydrolyzed by an enzyme having only glucoamylase activity during fermentation. Aspects and embodiments of this method are described in the independently numbered paragraphs below. 1. In a first aspect, there is provided a method for increasing the amount of glucose available for fermentation in a fermentation substrate, the method comprising contacting the fermentation substrate with an enzyme having transglucosidase activity and an enzyme having glucoamylase activity at a time when the combined amount of maltose and maltotriose in the fermentation substrate is less than a selected amount. 2. In some embodiments of the method described in paragraph 1, the selected amount is less than 0.5% w / v, less than 0.05% w / v, preferably less than 0.01% w / v. 3. In some embodiments of the method of paragraphs 1 or 2, the fermentation substrate after contact with the enzyme having transglucosidase activity has reduced levels of isomaltose, panose, and / or maltulose compared to an otherwise identical fermentation substrate that has not been contacted with the enzyme having transglucosidase activity. 4. In some embodiments of the method of any of paragraphs 1-3, the fermentation substrate after contact with the enzyme having transglucosidase activity does not have a significant increase in the amount of ethyl-glucoside compared to an otherwise identical fermentation substrate that has not been contacted with the enzyme having transglucosidase activity. 5. In some embodiments of the method of any of paragraphs 1-4, the fermentation substrate is contacted simultaneously with the enzyme having transglucosidase activity and the enzyme having glucoamylase activity. 6. In some embodiments of the method of any of paragraphs 1-4, the fermentation substrate is contacted sequentially with an enzyme having transglucosidase activity and an enzyme having glucoamylase activity. 7. In some embodiments of the method of any of paragraphs 1-6, the enzyme having transglucosidase activity is added after the start of fermentation. 8. In some embodiments of the method of any of paragraphs 1-7, the transglucosidase is added 32 hours, 40 hours, 48 ​​hours, 54 hours, or 64 hours after the start of fermentation. 9. In some embodiments of the method of any of paragraphs 1-8, the fermentation substrate is a high glucose fermentation substrate. 10. In some embodiments of the method of any of paragraphs 1-8, the fermentation substrate is thin stillage that is added as backset to the liquefaction. 11. In some embodiments of the method of any of paragraphs 1-8, the fermentation substrate is whole stillage, at least a portion of which is added to the liquefaction. 12. In some embodiments of the method of any of paragraphs 1-8, the enzyme substrate is starch liquefaction. 13. In some embodiments, the method of any of paragraphs 1-12 further includes fermenting the glucose made available for fermentation to ethanol, lactic acid, or amino acids. 14. In some embodiments, the method of any of paragraphs 1-13 further includes fermenting the glucose made available for fermentation using yeast and / or bacteria and a fermenting organism. 15. In some embodiments of the method of any of paragraphs 1-14, the amount of enzyme having transglucosidase activity contacted with the fermentation substrate is at least 0.017, at least 0.085, at least 0.170, at least 0.850, or at least 1.70 transglucosidase units per gram of dissolved solids of the substrate.

[0006] These and other aspects and embodiments of the present method will become apparent from the description and any accompanying drawings. DETAILED DESCRIPTION OF THE INVENTION

[0007] I. Definitions and Abbreviations Before describing the method in detail, the following terms are defined for clarity. Terms not defined should be accorded their ordinary meaning as used in the relevant technical field.

[0008] As used herein, the term "starch" refers to a starch of the formula (CH 10 O5) X "Finally, the term refers to any substance composed of a complex polysaccharide carbohydrate of a plant composed of amylose and / or amylopectin having the formula: X = 1, where X can be any number. In particular, the term refers to any plant-based substance, including, but not limited to, grains, grasses, tubers, and roots, more specifically wheat, barley, corn, rye, rice, sorghum, legumes, cassava, millet, potato, sweet potato, and tapioca. After the complex polysaccharide carbohydrate has been refined from other plant components, it is called "refined starch."

[0009] As used herein, "maltodextrin" generally refers to oligosaccharides produced from starch by partial chemical or enzymatic hydrolysis. The size of the polysaccharides generally ranges from DP3 to DP20, but can be longer.

[0010] As used herein, "degree of polymerization (DP)" refers to the number of anhydroglucopyranose units (n) in a given sugar. An example of a DP1 is the monosaccharide glucose. Examples of DP2 are the disaccharides maltose and isomaltose.

[0011] As used herein, the phrase "end of fermentation," abbreviated as "EoF," refers to the stage of fermentation at which the economic benefit of continuing the fermentation to produce small amounts of additional product is exceeded by the cost of continuing the fermentation in terms of fixed and variable costs. In a more general sense, "end of fermentation" refers to the point at which the fermentation will no longer produce significant amounts of additional product, i.e., about 1% or less of additional product.

[0012] As used herein, the term "high glucose" with respect to a fermentation substrate refers to a fermentation substrate in which greater than 70% of the sugar content is glucose.

[0013] As used herein, the term "easily / simply utilized / hydrolyzed substrate" refers to a substrate that is preferred as a donor molecule by TGL in transglucosidase and / or hydrolysis reactions. The small linear substrates maltose and maltotriose are preferred donor molecules for TGL and are utilized more quickly than other donor molecules.

[0014] The term "dry solids content" (DS) refers to the total solids of the slurry on a dry weight percent basis.

[0015] The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0016] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process for the production of biochemicals in which a microorganism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present in the same process step.

[0017] "Ethanol-producing microorganism" refers to a microorganism with the ability to convert sugars or oligosaccharides into ethanol.

[0018] As used herein, a "starch processing enzyme" is an enzyme that depolymerizes starch substrates (including maltodextrins). Exemplary starch processing enzymes are α-amylase, glucoamylase, β-amylase, pullulanase, α-glucosidase, and transglucosidase.

[0019] As used herein, "contacting" an enzyme with a substrate refers to bringing the enzyme and substrate together in a common aqueous environment, generally with mixing to achieve uniform distribution. The term "contacting" is used interchangeably with "treating."

[0020] As used herein, "producing" refers to producing a reaction product as a result of an enzymatic process and is synonymous with the term "producing."

[0021] As used herein, "total stillage" is the product of an ethanol production facility after distillation.

[0022] As used herein, "thin stillage" is the liquid portion of the total stillage after separation of the solid materials. As used herein, "thin stillage" is also referred to simply as "stillage" as well as "backset" and "recycle."

[0023] As used herein, "distillers grains (DG)" is the solid / slurry component of total distillation residue.

[0024] As used herein, "dried distillers grains (DDG)" is DG that has been dried.

[0025] As used herein, "dried distillers grains with solutes (DDGS)" is DG that has been dried with concentrated thin distiller's grains to add nutritional value.

[0026] As used herein, the term "performance benefit" refers to an improvement in a desirable biochemical property of a fermentation substrate or a product derived therefrom, including an improvement that increases the amount of fermentable sugars available to a fermenting organism.

[0027] As used herein, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. All references cited herein are incorporated by reference in their entirety.

[0028] The following abbreviations / acronyms have the following meanings unless otherwise specified: Not all abbreviations may be used. ℃ Celsius temperature DP Degree of Polymerization DP3+ DP3 or higher DPn value is unknown DS dry solid EoF End of fermentation g grams HPAE High Performance Anion Exchange Chromatography HPLC High Performance Liquid Chromatography hr time kg kilogram min ml milliliter mm millimeters MT Metric Ton NaAc sodium acetate NaOH Sodium hydroxide nC nanocoulomb nC·min nanocoulomb minute PAD Pulsed Amperometric Detection PU pullulanase rpm Revolutions per minute TG or TGL transglucosidase U or u units w / v weight / volume

[0029] II. Enzymes with transglucosidase activity to increase fermentable sugars A. Introduction The presence of an enzyme with transglucosidase (TGL) activity along with an enzyme with glucoamylase (GA) activity has been found to make additional glucose available to the fermenting organism under selected fermentation conditions. This additional glucose represents carbon that, under the best circumstances, would previously be lost in the form of non-fermentable oligosaccharides. During fermentation, the enzyme with TGL activity appears to hydrolyze oligosaccharides and / or polysaccharides that are not hydrolyzed solely by the enzyme with GA activity, thereby liberating additional glucose substrate for utilization by the fermenting organism. Notably, in addition to transferring glucose units, TGL can also hydrolyze substrates, especially when the concentration of acceptors other than water is low.

[0030] B. Related enzyme activities Enzymes with TGL activity, primarily referred to herein as transglucosidases (TGLs), transfer glucosyl groups from donor molecules to acceptor molecules, most often resulting in an α1-6 linkage. TGLs also hydrolyze linear and branched maltooligosaccharides into smaller maltooligosaccharides and glucose, which are readily utilized by fermenting organisms. When the acceptor oligosaccharide concentration is low, the hydrolysis reaction (transfer to water) is favored over the transfer reaction to the organic hydroxyl-containing donor molecule. Exemplary enzymes are described herein.

[0031] The preferred substrates for TGL are those that are most easily utilized as donor molecules. Short, linear malto-oligosaccharides are particularly good donor molecules. Maltose and maltotriose are the most abundant TGL substrates in starch-based fermentation, and their amounts over the course of fermentation represent a good proxy for the total amount of TGL substrates that can be used as donor molecules by TGL. Branched malto-oligosaccharides are less preferred as donor molecules for TGL compared to linear malto-oligosaccharides.

[0032] TGL production depends on the population of available acceptor molecules: any molecule with a hydroxyl group can be an acceptor molecule, including water, alcohols, and most (if not essentially all) oligosaccharides present in the fermentation substrate.

[0033] C. Timing of enzyme addition The method is based in part on the observation that the timing of TGL addition in a fermentation is important for increasing production of a desired product: the time of addition during the fermentation determines the population of substrate donor molecules available at the time of TGL addition.

[0034] Early in the fermentation process, significant amounts of linear maltooligosaccharides are present, which are readily hydrolyzed by GA to become suitable donor molecules for enzymes with TGL activity. Early in the fermentation process, potential acceptor molecules are also abundant, including glucose, maltose, maltotriose, linear and branched oligosaccharides, and countless other molecules. As noted above, any molecule with a hydroxyl group, including water and alcohols, can potentially be an acceptor.

[0035] In such a donor- and acceptor-rich environment, transglycosylation produces some products that can be easily hydrolyzed by GA (or TGL) to glucose and then fermented to the desired product. However, other TGL products are not good GA substrates and cannot be easily hydrolyzed by GA to fermentable sugars. For example, branched maltooligosaccharides (whether present at the beginning of fermentation or produced after TGL addition) are much less favorable GA substrates than linear maltooligosaccharides. As a result, TGL activity can actually convert substrates that are easily hydrolyzed by GA into substrates that are much more difficult to hydrolyze. In fact, adding TGL early in fermentation can negatively affect product yield, resulting in more DPn and, potentially, more DP3 and DP2.

[0036] TGL can hydrolyze some of the branched oligosaccharides that GA cannot easily hydrolyze, and some benefit is expected from adding TGL early in the fermentation. However, in general, the negative effects outweigh the positive effects.

[0037] During the later stages of fermentation, including the end of fermentation (EoF), there are fewer simple donor and acceptor molecules overall, primarily branched oligosaccharides. If TGL is added at a later time, it can hydrolyze some of the branched molecules that GA cannot, thereby producing more fermentable glucose that can be converted to valuable products.

[0038] III. Exemplary Enzymes for Use in the Method A. Enzymes with transglucosidase activity Preferred enzymes with transglycosylation activity readily utilize short, linear oligosaccharides as donors and transfer glucose from such donors to an acceptor, which can be any molecule with a hydroxyl group, including water, alcohol, and any oligosaccharides present in the fermentation substrate.

[0039] Enzymes with transglycosylation activity include, but are not limited to, enzymes previously described as transglucosidases or annotated transglucosidases and enzymes with previously unknown activities or side activities characteristic of transglucosidases. Transglucosidases (TGLs), also known as α-glucosidases and α-D-glucoside glucohydrolases, are enzymes classified as EC 3.2.1.20 and have been identified in many organisms. GenBank contains over 400 entries for transglucosidases.

[0040] The enzyme exemplified herein is derived from Aspergillis niger and expressed in Trichoderma reesei. Although this enzyme is expressed at high levels, it is not recognized as having unique properties compared to other transglucosidases studied. Therefore, numerous transglucosidases from many organisms are believed to be suitable for use in this method.

[0041] Transglucosidase is preferably added when the combined concentration of maltose and maltotriose is less than about 0.5% w / v. This can be at the beginning of the fermentation or after about 10, 20, 30, 40, 50, or 60 hours of fermentation, depending on the fermentation substrate. The required TGL dosage depends on the remaining fermentation time and can be between 0.01 and 1.0 kg / MT DS.

[0042] An exemplary enzyme is commercially available as TRANSGLUCOSIDASE L2000® (IFF), which has an activity of 1,700 transglucosidase units (TGU) / g. One TGU is defined as the amount of enzyme required to produce 1 micromole of panose per minute under assay conditions. Typically, at least 0.1 kg / MT TRANSGLUCOSIDASE L2000® / MT DS is used. A range of 0.01 to 1.0 kg / MT DS was used in all work described herein. Representative amounts of transglucosidase are about 0.017 to 1.70 TGU / g DS, e.g., about 0.017, 0.085, 0.170, 0.850, and 1.70 TGU / g DS.

[0043] Although the time and dosage of addition are important to the method, what is most important is the relative amount of short, linear malto-oligosaccharides, which are particularly good donor molecules for TGL. Preferably, at the time of TGL addition, the fermentation substrate contains a combined amount of maltose and maltotriose in the fermentation substrate that is less than 0.5%, less than 0.05%, or even less than 0.01% w / v. In some embodiments, the fermentation substrate contains less than 0.5%, less than 0.05%, or even less than 0.01% w / v of maltose. In some embodiments, the fermentation substrate contains less than 0.5%, less than 0.05%, or even less than 0.01% w / v of maltotriose.

[0044] B. Enzymes with glucoamylase activity Preferred enzymes having glucoamylase activity include, but are not limited to, enzymes previously described as "glucoamylases" or annotated as "glucoamylases" and enzymes having previously unknown activities or side activities characteristic of glucoamylases.

[0045] Glucoamylases are enzymes classified as EC 3.2.1.3 and have been identified in many organisms. GenBank contains well over 2,000 entries for glucoamylases. Exemplary glucoamylases are Trichoderma reesei glucoamylase (TrGA) and its variants, which have excellent specific activity and thermostability. See U.S. Patent Application Publication Nos. 2006 / 0094080, 2007 / 0004018, and 2007 / 0015266 (Danisco US Inc.).

[0046] Alternatively, the glucoamylase can be derived from, for example, the genera Aspergillus, Talaromyces, Clostridium, Fusarium, Thielavia, Thermomyces, Athelia, Humicola, Penicillium, Artomyces, Gloeophyllum, Pycnoporus, Steccherinum, or Trametes. Suitable commercially available glucoamylases include AMG 200L; AMG 300L; SAN™ SUPER and AMG™ E (Novozymes); OPTIDEX® 300, OPTIDEX L-400, DISTILLASE® CX and DISTILLASE® PLUS (IFF); AMIGASE™ and AMIGASE™ PLUS (DSM); G-ZYME® G900 and G-ZYME® G990 ZR (Enzyme Bio-Systems).

[0047] Glucoamylase is typically added in an amount of about 0.1 to 2.0 glucoamylase units / g dissolved solids (ie, GAU / g DS), such as about 0.16 GAU / g DS, 0.23 GAU / g DS, 0.33 GAU / g DS, etc.

[0048] IV. No significant increase in ethyl glucoside An advantage of this method is that no significant amounts of ethyl-glucosides are produced in the fermentation substrate, which would reflect a direct loss of product production and would be unacceptable.

[0049] It has now been shown that TGL activity, when added early in fermentation, actually increases the amount of glyceryl-glucoside and ethyl-glucoside. The increase in these products suggests that both ethanol and glycerol, respectively, can act as acceptors in transglycosylation reactions. However, these undesired products are largely avoided by adding an enzyme with TGL activity under the described late fermentation conditions.

[0050] V. Mode of Addition of Enzymes with Transglucosidase and Glucoamylase Activities If the fermentation substrate is initially low in maltose and maltotriose, TGL can be added from the beginning of fermentation without any adverse effects being observed. In such fermentation substrates, the amount of short linear maltooligosaccharides is low enough to avoid the formation of indigestible branched maltooligosaccharides, which are not good substrates for glucoamylase.

[0051] If the fermentation substrate is initially high in maltose and maltotriose, enzymes with TGL activity are preferably added toward or present at EoF, or may be added to the fermentation substrate forming only a portion of the liquefact or only a portion of the final fermentation medium. For example, TGL and GA can be added to the total stillage, including any fraction of the total stillage, where the TGL and GA can liberate glucose for inclusion as backset recycle in related or unrelated fermentation substrates, including later stages of sequential fermentations and subsequent or different fermentations.

[0052] VI. Fermentation Substrate The method is not limited to a particular subset of fermentation substrates, nor to a particular fermentation product or fermentation microorganism. As described, TGL will liberate more glucose for fermentation as long as it is added to or present in the fermentation at the appropriate time.

[0053] Exemplary fermentation substrates include those from dry-grind fuel ethanol facilities and wet-mill carbohydrate processing facilities and variations thereof, as well as those used to produce other useful biochemical products such as lactic acid and amino acids.

[0054] These and other aspects and embodiments of the present methods and compositions resulting therefrom will be apparent to those skilled in the art in light of the present description. The following examples further illustrate, but do not limit, the compositions and methods. [Example]

[0055] In the examples, the following enzymes were used: SPEZYME® HT: A thermostable α-amylase for starch liquefaction DISTILLASE® CX Blend of Trichoderma GA, Mutant Aspergillus α-Amylase, and Trehalase DISTILLASE® PLUS Blend of Trichoderma GA, Aspergillus α-Amylase, Trehalase, and Protease Same as above, but unclear: DISTILLASE® PLUS WB FERMGEN® 2.5X Protease

[0056] Example 1. Treatment of stillage with transglucosidase Corn liquefaction was prepared by combining 673.1 g of ground corn with 1,326.9 g of water. The mixture was brought to pH 5.2 with 4 N sulfuric acid and heated to 60°C in a water bath with continuous stirring. 134.6 μg of SPEZYME® HT α-amylase was added. The mixture was held at 60°C for 30 minutes, heated to 90°C for 90 minutes, and then cooled to 32°C for use in subsequent fermentations.

[0057] 2,000 g of the resulting liquefact was placed in a beaker and adjusted to pH 4.5 with 4N sulfuric acid. 600 ppm urea, 200 ppm silicone antifoam (Sigma), and 12.3 μl of FERMGEN® 2.5x were added. 100 g of this fermentation material was placed in each of two 200 ml Erlenmeyer flasks equipped with foam stoppers. Fermentation was initiated by adding 20.8 μl of DISTILLASE® PLUS and 0.1% w / w hydrated active dry yeast (ETHANOL RED®, Fermentis, FR) to the first flask and 13.1 μl of DISTILLASE® CX and the same 0.1% w / w hydrated active dry yeast to the second flask.

[0058] The flasks were kept in a water bath at 32°C for 66 hours. The ethanol was evaporated at 85°C for 1.5 hours. The remaining stillage was homogenized in a blender (Braun Multiquick 5) at maximum speed for 128 seconds, with a 1-minute pause between each of three cycles. The pH was adjusted to 3.9-4.3 (using 4N NaOH), and the pH-adjusted stillage was used to screen the effects of various enzyme activities.

[0059] Screening was performed in 96-well microtiter plates. Each well received 0.19 ml of prepared distillation residue at 5% DS. Some wells received only GA at a dose of 0.1 kg / MT DS. The GA® used was DISTILLASE® CX or DISTILLASE® PLUS. Other wells received both GA at a dose of 0.1 kg / MT and TRANSGLUCOSIDASE® L2000 TGL (TGL) at a dose of 1.0 or 5.0 kg / MT.

[0060] The reaction was incubated at 32°C for 18 hours in a 220 rpm shaker. After 18 hours, the reaction was stopped by adding 0.15 ml of 0.1 N sulfuric acid, and glucose release was measured spectrophotometrically using the GOPOD assay. Five μL of filtered supernatant from the quenched sample was mixed with 80 μL of GOPOD reagent (Megazyme) and incubated at 50°C for 20 minutes. Absorbance was measured at 510 nm, where OD corresponds to the amount of glucose released. The performance index (PI) is the amount of glucose released in the presence of TGL divided by the amount of glucose released with GA alone. If this PI is significantly greater than 1, TGL releases additional glucose in the stillage compared to GA alone. Table 1 below provides the PI numbers for the screening experiments. [Table 1]

[0061] Table 1 shows that for both stillage samples prepared from fermentation with either DISTILLASE® CX or DISTILLASE® PLUS, the combination of TRANSGLUCOSIDASE® L2000 and glucoamylase releases significantly more glucose from stillage than glucoamylase alone. For both GAs used in screening, the combination with TGL releases at least 1.4 times more glucose. These results suggest that TGL hydrolyzes oligosaccharides and / or polysaccharides that are not hydrolyzed by glucoamylase during fermentation.

[0062] Example 2. Analysis of isosugars during fermentation The sugar isoforms present during fermentation were analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD), which can separate isomers such as maltotriose, panose, and isomaltotriose. For this analysis, 100 μl of fermentation sample was collected, diluted 1,000-fold, boiled for 10 minutes, and filtered. A 10 μl sample was injected onto a CarboPac PA200 column (3 mm × 250 mm) equipped with a guard column at a flow rate of 0.5 ml / min and a temperature of 30°C. PAD was performed at a cell temperature of 25°C. During the 60-minute chromatography run, the following conditions were used: (i) Prior to sample injection, the column was equilibrated with 10% 1 M NaOH and 10% 500 mM NaOAc in 80% MilliQ water for 10 minutes. Sugar separation was achieved by eluting with a constant 10% 1 M sodium hydroxide and 90% MilliQ water for 5 minutes. A gradient of 500 mM NaOAc was initiated where the % NaOAc in the mobile phase increased from 0 to 8% and the % MilliQ water decreased from 90 to 82% over the next 5 minutes. Over the next 50 minutes, the gradient changed, with the % MilliQ in the mobile phase decreasing from 82% to 0% and the % NaOAc increasing from 8 to 90%. The gradient is shown in Table 2. [Table 2]

[0063] With the appropriate calibration curves, the HPAEX-PAD chromatogram can be used to calculate the content of each of these isosaccharides in % w / v. Commercially available reference sugars are available for glucose, fructose, isomaltose (IM2), maltulose, isomaltotriose (IM3), maltose (M2), isomaltotetraose (IM4), panose, isomaltopentaose (IM5), maltotriose (M3), isomaltohexaose (IM6), isomaltoheptaose (IM7), maltotetraose (M4), maltopentaose (M5), maltohexaose (M6), and maltoheptaose (M7).

[0064] In addition to the peaks corresponding to the reference sugars, the HPAEX-PAD chromatogram shows other peaks that most likely correspond to branched oligosaccharides. Because there are no commercially available reference sugars for these branched oligosaccharides, they were labeled using defined standards. The unknown peak between maltotriose and maltotetraose was designated branched maltotetraose (B4). The first peak following maltotriose was designated B4-a, the second B4-b, and so on. The peaks between maltotetraose and maltopentaose were designated B5-a, B5-b, etc. The peak areas of all branched DP4-a, b, c, d, e, etc. were added together and designated branched DP4. The same was done for DP5-a, b, c, etc., DP6-a, b, c, etc., and DP7-a, b, c, etc. Considering the absence of reference standards, the peak areas in the chromatogram cannot be converted to concentrations. For these peaks, the chromatogram peak area (units: nC min) is used for comparison between fermentation times and runs.

[0065] Example 3. Addition of transglucosidase during fermentation Corn liquefaction was prepared as in Example 1 using SPEZYME® HT. The pH was adjusted to 4.5 with sulfuric acid and 600 ppm urea, and 200 ppm silicone antifoam (Sigma) and 0.02 kg / MT DS FERMGEN® 2.5x were added. 100 g of liquefaction was placed in several 200 ml Erlenmeyer flasks and incubated with stirring in a 32°C water bath. A water lock was placed over each flask. Fermentation was initiated by adding 0.01 or 0.1 kg / MT TRANSGLUCOSIDASE L2000 and 0.9 kg / MT DS DISTILLASE® PLUS WB with or without 0.1% w / w hydrated active dry yeast (as above). Fermentations were performed in triplicate. Samples were taken at 72 hours of fermentation and analyzed for ethanol by HPLC. The average results are shown in Table 3. [Table 3]

[0066] Addition of TGL to the fermentation resulted in faster ethanol formation due to higher ethanol levels in the middle of the fermentation. At higher TGL doses, the ethanol production rate was higher than at lower doses. However, at the end of fermentation (EoF), the lowest dose of TGL did not result in higher ethanol production, and the highest dose appeared to negatively affect ethanol levels.

[0067] Example 4. Stepwise administration of transglucosidase during fermentation As shown in Example 3, adding TGL at the beginning of fermentation can reduce ethanol yield. Next, the addition of TGL at different time points after the start of fermentation (i.e., 40-64 hours) was investigated. Higher dosages were added to compensate for the shorter time the enzyme was present during fermentation. The experiment described in Example 3 was repeated, except that TRANSGLUCOSIDASE® L2000 was added at dosages of 0.05 and 0.25 kg / MT DS after 40 hours of fermentation or at dosages of 0.1 and 0.5 kg / MT DS after 64 hours. Results averaged over three experiments are shown in Table 4. [Table 4]

[0068] The results show that when TGL is added late in the fermentation, higher ethanol yields are obtained than when TGL is not added to the fermentation.

[0069] Example 5. Timing of transglucosidase administration during fermentation As shown in the previous examples, adding TGL early in the fermentation negatively impacts ethanol yield, while adding it later positively impacts ethanol yield. TGL was then added during the fermentation over a wider time range to identify the best dosing point. The tested doses and timing are summarized in Table 5. Sample analysis was as described in Examples 2 and 3. Tables 6, 7, and 8 show the relative ethanol, DPn, and DP2 values ​​(percent), respectively, compared to a reference fermentation in which no TGL was added. [Table 5] [Table 6]

[0070] The data in Table 6 confirm that the addition of TGL early in the fermentation had a negative effect on ethanol yield. There was a clear dose response, with the effect being greater at higher TGL doses. When TGL was added before 48 hours, higher doses resulted in lower ethanol yields. Comparing the results at equal doses taking into account the time of addition, it is clear that the addition of TGL later in the fermentation resulted in higher ethanol yields. Addition of TGL after 56 hours or more had a positive effect on ethanol yield (i.e., relative ethanol values ​​greater than 100%) compared to no TGL addition. [Table 7]

[0071] As shown in Table 7, the timing of TGL addition had a significant effect on the DPn (malto-oligosaccharides with DP4 or higher) content at EoF. When TGL was added early in the fermentation, the DPn at EoF was up to 15% higher than that of the reference. The residual DPn at EoF correlated with the TGL dosage and addition time. The earlier the TGL was added, the higher the DPn at EoF, and the higher the dosage, the higher the DPn at EoF. [Table 8]

[0072] The addition of TGL also resulted in changes in the DP2 and DP3 contents at EoF. Table 8 shows the relative DP2 values. At lower doses of TGL, DP2 was higher than the reference when added early in the fermentation. At higher doses or when added late in the fermentation, the effect of TGL addition was smaller, even inverse. Similar results were observed for DP3, although the difference between the presence and absence of TGL was smaller. At doses of 0.5 kg / MT and above, DP2 content was approximately 40–43% lower than the reference, and DP3 content was approximately 5–7% lower.

[0073] A subset of the above fermentations was analyzed using HPAEX-PAD chromatography as described in Example 2 (data not shown). Analysis showed no significant difference in glucose levels during fermentations with or without TGL addition. For the isosugars maltose, maltotriose, and maltotetraose, analysis showed that the content of these linear sugars decreased more rapidly when TGL was added to the fermentation. At EoF, the content of these isosugars was the same as when TGL was not added. The faster decrease in isosugars was consistent with TGL using these substrates as donors and / or acceptors.

[0074] Specifically, based on this analysis, it was found that (i) the isomaltose and panose contents initially increased when TGL was added during fermentation and then decreased near EoF; (ii) less isomaltose remained at EoF when TGL was added; and (iii) the panose content decreased to below detection at EoF regardless of TGL addition. The increase in isomaltose and panose contents indicated that TGL uses glucose and maltose as acceptor molecules. The analysis further showed that the addition of TGL resulted in greater amounts of branched DP4, DP5, and DP6 oligosaccharides at EoF. These oligosaccharides were highest when TGL was added early in the fermentation and were nearly equal when added 40 to 48 hours later. This analysis led to the conclusion that the addition of TGL early in the fermentation negatively affected ethanol yield.

[0075] The concentrations of maltose and maltotriose measured in fermentations without added TGL are shown in Table 9. The maltose concentrations in fermentations without TGL were 0.67%, 0.033%, and 0.013% w / v after 30, 40, and 48 hours of fermentation, respectively. For maltotriose, the concentrations were 0.57% w / v after 8 hours and 0.044% w / v after 24 hours. In some cases, maltose and maltotriose were below the detection limit, indicated by dashes. [Table 9]

[0076] HPLC analysis identified two additional peaks in samples from fermentations using TGL. These peaks were identified as glyceryl-glucoside and ethyl-glucoside, which were produced especially when TGL was added at the beginning of fermentation. The production of these glucosides suggests that both ethanol and glycerol were acting as acceptor molecules for the transglycosylation reaction.

[0077] When TGL was added at the beginning of fermentation, glyceryl glucoside was formed immediately, reaching a maximum around 24–40 h. As fermentation progressed, the amount of glyceryl glucoside decreased and was nominally present by 72 h. This suggested that the formed glyceryl glucoside was hydrolyzed, possibly by one or more enzymes remaining or present later in fermentation. When TGL was added 32–40 h after the start of fermentation, the glyceryl glucoside content was the same as in the reference fermentation performed without TGL addition.

[0078] When TGL is added at the beginning of the fermentation, ethyl glucoside is also formed and continues to form throughout the fermentation. The formation of ethyl glucoside may explain why adding TGL early in the fermentation results in lower ethanol yields. When TGL was added 32-40 hours after the start of the fermentation, little or no ethyl glucoside was formed.

[0079] Example 6 Addition of transglucosylase to high glucose fermentation The preceding examples generally describe fermentation conditions found in dry-mill, starch-consuming, ethanol production plants, which are well known in the industrial field. Other ethanol plants produce ethanol from fermentable, high-glucose streams, such as glucose and fructose syrup, greens, and / or raffinates. Such rich fermentation feedstocks may be rich in sugars larger than glucose and may contain significant amounts of isomaltose, panose, and / or higher-branched sugars. At the same time, they may be low in linear maltose, maltotriose, maltotetraose, and the like.

[0080] To determine whether adding TGL at the beginning of a fermentation of a glucose-rich syrup containing small amounts of maltose and / or maltotriose enhanced the fermentation, an exemplary glucose fermentation feedstock was obtained from a wet milling plant. The substrate was a mixture of 70% glucose syrup and 30% corn steep liquor containing 14% w / v glucose, 0.8% w / v fructose, 0.9% w / v disaccharides, and 1.4% w / v DP3 or higher (DP3+) oligosaccharides. The maltose concentration in this substrate was 0.594% w / v, and no maltotriose was detected. Because this fermentation substrate is low in maltose and maltotriose, adding TGL to a fermentation containing low amounts of maltose and maltotriose is well suited to ensure that no undesirable effects are observed.

[0081] The fermentation substrate had a pH of 4.0-4.5 and was not adjusted. 400 ppm silicone antifoam (Sigma) was added, and 100 g of the fermentation material was distributed into 200 ml Erlenmeyer flasks equipped with foam stoppers. These flasks were placed in a water bath at 32°C and stirred at 150 rpm. At the start of fermentation, 0.26 kg / MT DS of OPTIMAX® 4060VHP was added to each flask, followed by 0.1% w / w of hydrated active dry yeast (as described above). After 0, 4, 8, and 24 hours of fermentation, TRANSGLUCOSIDASE® L2000 was added to the various flasks as shown in Table 10. The experiment was performed in triplicate. [Table 10]

[0082] At appropriate time intervals, samples were taken for HPLC analysis as described in Examples 2 and 3. For each sample, the relative difference compared to the reference fermentation (number 1) without added transglucosidase was calculated. For each sample time point, the average amount of ethanol produced was divided by the average amount (expressed as a percentage) produced in the reference sample. A positive impact by transglucosidase was indicated by a value greater than 100%, and a negative impact by a value less than 100%. DPn, DP3, DP2, and DP1 sugar content were analyzed in the same way. Table 11 shows the relative ethanol amount compared to the reference fermentation without added transglucosidase. [Table 11]

[0083] As shown in Table 11, in all fermentations where TGL was added, the ethanol yield at 54 hours of fermentation was higher than in the reference fermentation without TGL. The relationship between TGL and relative ethanol yield was dose-dependent. There did not appear to be a correlation between TGL addition time and relative ethanol yield. As seen in the previous example, the addition of TGL resulted in a significant decrease in DP3 and DP2 values. In contrast to the previous example, the addition of TGL to this high-glucose substrate also decreased DPn values. DP2 values ​​were up to 80% lower with TGL addition compared to the reference. DP3 values ​​were up to 75% lower, and DPn values ​​were up to 7% lower. The lower DPn, DP3, and DP2 were dependent on the TGL dose and not on the addition time.

[0084] For one of the fermentations in which TGL was added after 24 hours, the end-of-fermentation samples were analyzed for isosugars as described in Example 2. The results of the HPAE-PAD analysis, shown in Table 12, demonstrated that isomaltose, maltulose, and panose were efficiently degraded by the addition of TGL during the fermentation of high-glucose substrates. Therefore, TGL can efficiently hydrolyze substrates that cannot be hydrolyzed by GA alone, potentially resulting in higher ethanol yields. [Table 12]

[0085] Example 7: Corn fermentation with GA and TGL treated stillage Thin stillage (5.82% DS) from a European corn dry mill ethanol production facility was processed with and without 0.25 Kg / MT TGL + 0.25 Kg / MT DISTILLASE® PLUS WB.

[0086] Incubation of the treated thin stillage was carried out at 45°C and pH 4.65 for 18 hours. Enzyme was added at 5.82% DS on a dry solids basis. This treated thin stillage was then added to a standard corn liquefaction at 35% w / w. The liquefaction was carried out at 84°C, 32% DS corn, and pH 5.1-5.2 for 2.5 hours. After the addition of the thin stillage, the liquefaction DS was 34.04%. At the start of the liquefaction, SPEZYME® HT was added at a dose of 0.19 kg / MT corn DS. The control was the liquefaction to which untreated thin stillage was added.

[0087] When liquefaction was performed on stillage treated with GA and TGL, the DPn content was 0.25% lower than that of the control, DP3 and DP2 were almost unchanged, and DP1 was 0.28% higher than that of the control. TGL and GA hydrolyzed DPn sugars mainly to DP1.

[0088] The corn liquefact was then adjusted to pH 4.8 with sulfuric acid. 1,000 ppm urea and 400 ppm silicone antifoam (Sigma) were added, and 100 g of the liquefact was transferred to a 100 ml Erlenmeyer flask for incubation at 32°C. A needle-pierced rubber stopper was placed on top of each flask. Fermentation was initiated by adding 0.93 kg / MT DS of DISTILLASE® PLUS WB and 0.1% w / w hydrated active dry yeast (as described above). Fermentations were run six times, and samples were taken at 65 hours for analysis by HPLC as described above. The relative ethanol content during fermentations of the TGL+GA-treated thin stillage-containing liquefact was 100.6% compared to fermentations of the untreated thin stillage-containing liquefact (data not shown).

[0089] EoF samples were analyzed on a Dionex CarboPac PA200 column as described in Example 2. The contents of isomaltose and maltulose in fermentations containing TGL+GA-treated thin stillage were lower than in fermentations containing untreated thin stillage.

[0090] The results demonstrated that treating stillage with TGL+GA before being recycled to liquefaction can result in a reduction in isomaltose and maltulose content and an increase in ethanol yield.

Claims

1. 1. A method for increasing the amount of glucose available for fermentation in a fermentation substrate, the method comprising contacting the fermentation substrate with an enzyme having transglucosidase activity and an enzyme having glucoamylase activity at a time when the combined amount of maltose and maltotriose in the fermentation substrate is less than a selected amount.

2. 2. The method of claim 1, wherein the selected amount is less than 0.5% w / v, less than 0.05% w / v, preferably less than 0.01% w / v.

3. 3. The method of claim 1 or 2, wherein the fermentation substrate after contact with the enzyme having transglucosidase activity has reduced levels of isomaltose, panose and / or maltulose compared to an otherwise identical fermentation substrate that has not been contacted with the enzyme having transglucosidase activity.

4. 4. The method of any one of claims 1 to 3, wherein the fermentation substrate after contact with the enzyme having transglucosidase activity does not have a significant increase in the amount of ethyl-glucoside compared to an otherwise identical fermentation substrate that has not been contacted with the enzyme having transglucosidase activity.

5. 5. The method of claim 1, wherein the fermentation substrate is contacted simultaneously with the enzyme having transglucosidase activity and the enzyme having glucoamylase activity.

6. 5. The method according to any one of claims 1 to 4, wherein the fermentation substrate is contacted sequentially with the enzyme having transglucosidase activity and the enzyme having glucoamylase activity.

7. The method according to any one of claims 1 to 6, wherein the enzyme having transglucosidase activity is added after the start of fermentation.

8. The method according to any one of claims 1 to 7, wherein the transglucosidase is added 32 hours, 40 hours, 48 ​​hours, 54 hours or 64 hours after the start of fermentation.

9. The method of any one of claims 1 to 8, wherein the fermentation substrate is a high glucose fermentation substrate.

10. 10. The method of any one of claims 1 to 9, wherein the fermentation substrate is thin stillage that is added as backset to the liquefaction.

11. 11. The method according to any one of claims 1 to 10, wherein the fermentation substrate is whole stillage, at least a portion of which is added to the liquefaction.

12. The method according to any one of claims 1 to 11, wherein the fermentation substrate is a starch liquefaction product.

13. 13. The method of any one of claims 1 to 12, further comprising fermenting the glucose made available for fermentation to ethanol, lactic acid or amino acids.

14. 14. The method of any one of claims 1 to 13, further comprising fermenting the glucose made available for fermentation using yeast and / or bacteria and fermenting organisms.

15. 15. The method of any one of claims 1 to 14, wherein the amount of enzyme having transglucosidase activity contacted with the fermentation substrate is at least 0.017, at least 0.085, at least 0.170, at least 0.850, or at least 1.70 transglucosidase units per gram of dissolved solids of substrate.

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