CA2203811A1

CA2203811A1 - A method for improved raw material utilization in fermentation processes

Info

Publication number: CA2203811A1
Application number: CA002203811A
Authority: CA
Inventors: Bertus Noordam
Original assignee: Individual
Current assignee: Danisco US Inc
Priority date: 1994-10-27
Filing date: 1995-10-26
Publication date: 1996-05-09
Also published as: EP0788551A1; WO1996013600A1; MX9702933A; FI971781A; FI971781A0; AU4013295A

Abstract

Raw materials such as glucose syrups, cane or beet molasses used in fermentative production processes usually contain saccharides which cannot be utilized by the microorganism employed in the fermentative process. The present invention discloses the use of enzyme preparations capable of hydrolysing the unfermentable saccharides into fermentable saccharides in order to improve the yield of the fermentative production process. The enzyme preparations may be applied at a point in the process where the overall carbohydrate concentration is less than 20 % in order to avoid reversion reactions. The enzyme preparations are preferably applied in an immobilized form.

Description

CA 02203811 1997-04-2~

A METHOD FOR IMPROVED RAW MATERIAL UTILIZATION
IN FERMENTATION PROCF-SSF-S

Field of the invention The present invention relates to the enzymatic hydrolysis of unfermentable carbohydrates into fermentable carbohydrates. More specifically, the invention provides a method to produce fermentable monosaccharides from unfermentable saccharides, present in, for example, liquefied and/or saccharified starch, beet mol~-sses and cane molasses, in order to improve the raw material utilization in fermentation processes 10 such as the fermentative production of ethanol.

Back~round of the invention Yield is a crucial issue in fermentative production processes. Yield is of particular importance in the production of primary metabolites such as ethanol, glycerol 15 and lactic acid due to the low profit margins these products provide. As a result, significant effort has been focused on the improvement of yield to facilitate achieving higher levels of product from the raw materials used. In the field of fermentative ethanol production, product yield has been improved by reducing the amount of byproducts produced and also by improving the utilization of the raw material. For 20 example, yeast recycle systems (yeast re-use) have been used to reduce sugar consumption for yeast biomass production. Additionally, simultaneous saccharification and fermentation to maintain the free glucose level at a minimum, and thereby prevent high infection levels, has been shown to result in improved yields. Further, theapplication of cellulase and hemicellulase to release additional fermentable 25 carbohydrates from cellulose and hemicellulose fibre material (patents 85DD-274453, 78SU698402 and 78DD-210143) has been successful in increasing yield as has the application of a cellobiose fermenting yeast (U.S. Patent 5,100,791). Fermentation with immobilized yeast has been effective to reduce carbohydrate consumption for biomass production. Similarly, co-immobilized enzyme(s) and yeast have been advantageously 30 used to achieve simultaneous saccharification and fermentation resulting in reduced carbohydrate consumption for biomass production (EP-B1-0 222 4~2).
c While these attempts to increase ethanol yield have met with varying degrees of success, new methods for increasing yield are the subject of much investigation. The presence of certain noncellulose and nonhemicellulose based unfermentable sugars in 3s broth at the end of fermentations has been recognized in the art. These unfermentable sugars originate from the raw material itself or arise as byproducts during the CA 02203811 1997-04-2~

processing of the raw-materials, e.g. from steeping, liquefaction, saccharification or isomerization of the starch stream. Examples of unfermentable sugars ("residual sugars") which exist in starch processing streams include:
1. Maltulose; a-D-glucose(1,4)-a-fructose. High temperatures are used during 5 the starch liquefaction process (the hydrolysis of starch into dextrins). These high temperatures, in combination with the applied pH, stimulate the isomerization of the glucose unit at the reducing end of a dextrin molecule into fructose. Hydrolysis of these dextrins, including the isomerized glucose unit (fructose) at the reducing end results in free glucose and a disaccharide, called maltulose (glucose-a-1,4-fructose). Maltulose, lO however, is not hydrolysed by commonly used starch processing enzymes such asamyloglucosidase, pullulanase or acid amylase. Depending on the conditions used during liquefaction, the concentration of maltulose in the liquefied product can be as high as 2%.

2. Stachyose; a-D-galactosyl(1,6)-a-D-galactosyl(1,6)-a-D-glucose(1,2)-,B-D-fructose. This unfermentable sugar is found in, among others, sugar cane molasses.
Porter et al., Biotech. Bioeng. vol 35, pp 15-22 (1990) report the hydrolysis of stachyose with a soybean a-galactosidase preparation.

3. Raffinose; a-D-galactosyl(1,6)-a-D-glucose(1,2)-,B-D-fructose. This sugar is found in, among others, beet molasses. Raffinose is also a product of partial hydrolysis of stachyose. Porter et al., disclose hydrolysis of raffinose.

4. Melibiose; a-D-galactosyl(1,6)-a-D-glucose. This disaccharide, found in, among others, beet molasses, is a product of partial hydrolysis of both stachyose and raffinose. Melibiose has been reportedly hydrolyzed by Aspergillus niger a-galactosidase, Kaneko et al., Agric. Biol. Chem., vol 55, no.1, pp 109-115 (1991), and Azofobacfer vionelandii exo-a-galactosidase, Wong, Appl. of Environ. Microb., vol 56, no. 7 pp 2271-2273 (1990).
Enzymatic hydrolysis of these unfermentable carbohydrates has thus far not been applied to improve raw material utilization during production of primary metabolites such as ethanol. The utilization of such a process has likely been ignored in the industry due to an expectation in the field that hydrolysis of these unfermentable carbohydrates, prior to fermentation (when the carbohydrate concentrations are high) and using an immobilized enzyme or enzyme mixture, will result in production of large amounts of unfermentable reversion products and, thus, will only make the situation worse. Moreover, the unfermentable carbohydrate fraction consists of a mixture of carbohydrates and as a consequence requires a mixture of enzymes for hydrolysis.

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W O 96/13600 PCTrUS95/13876 This situation is further complicated when using enzyme mixtures. Thus, a need exists in the art for a convenient method of producing fermentable carbohydrates from unfermentable residual sugars produced during the starch hydrolysis process.

s summarY of the invention It is an object of the invention to provide an economically feasible method for increasing product output from raw material (improved utilization) by providing for the hydrolysis of noncellulose and nonhemicellulose based unfermentable sugars into fermentable sugars (mostly monosaccharides) in fermentative production processes.
According to the present invention, a fermentative production process is provided for the hydrolysis of unfermentable noncellulose and nonhemicellulose based saccharides using a residual sugar hydrolyzing enzyme preparation capable thereof.
Preferably, the enzyme preparation is applied at a point in the process where the overall carbohydrate concentration is below 20% w/v.
According to a composition embodiment, enzymes for the hydrolysis of unfermentabie saccharides in fermentative production processes are provided. Theenzymes are preferably used in mixtures of enzymes and/or are used in an immobilized form.
The invention further discloses plants for the fermentative production of ethanol which comprise enzyme reactors for the hydrolysis of noncellulose and nonhemicellulose based unfermentable saccharides.

Brief description of the fi~ures Figure 1A. Schematic representation of a "wet milling" ethanol plant.
Figure 1B. Schematic representation of a "wet milling" ethanol plant including an enzyme reactor for the hydrolysis of unfermentable saccharides.
Figure 2A. Schematic representation of a plant for the batchwise production of ethanol, including an enzyme reactor for the hydrolysis of unfermentable saccharides.
Figure 3A. HPLC chromatogram of beer from "wet milling" ethanol production.
30 Figure 3B. HPLC chromatogram of beer from "wet milling" ethanol production after treatment with a-galactosidase (SUMIZYME AGS).
Figure 3C. HPLC chromatogram of beer from "wet milling" ethanol production aftertreatment with an enzyme cocktail to hydrolyse unfermentable saccharides .
Figure 3D. HPLC chromatogram of beer from "wet milling" ethanol production after3s treatment with an enzyme cocktail to hydrolyse unfermentable saccharides, followed by the addition of yeast.

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W O96/13600 PCT~US95113876 Figure 4. Chromatogram of gel filtration of a-galactosidase on Sephacryl 5200 HR OD
(at OD 280) vs. elution time.

Description of the invention I
The present invention provides a method to increase the yield of a fermentative production process by increasing the amount of fermentable sugars used as a starting material in such processes through the enzymatic hydrolysis of unfermentable noncellulose and nonhemicellulose based saccharides. The enzyme composition may be derived from the fermentation broth of a microorganism which produces the enzyme.
The residual sugar hydrolyzing enzyme of the invention comprises any enzyme capable of hydrolyzing residual sugars present in carbohydrate raw material streams which are unfermentable by sugar fermenting organisms and, especially, in glucose syrup or precursors thereof derived from starch processing streams. Such residual sugar hydrolyzing enzymes will preferably possess as their major activity the hydrolysis l~ of one or more residual sugars. Preferably, the enzyme composition is derived from a fungal source, more preferably from Aspergillus or Trichoderma. In a most preferred embodiment of the invention the enzyme composition is derived from A. niger and comprises a maltulose hydrolyzing activity and/or a residual sugar hydrolyzing activity having a molecular weight of approximately 132 kl:) and 120 kD, respectively, asmeasured by gel filtration. Where the enzyme composition includes an enzyme commonly used in preparing sugar stocks for fermentative production processes, e.g., amyloglucosidase, pullulanse or acid amylase, it is preferable to enrich the composition to include more of the residual sugar hydrolyzing enzyme than exists in the natural composition.
An "enriched" residual sugar hydrolyzing enzyme preparation according to the present invention is a preparation which is derived from a fermentation broth produced by the fermentation of a naturally occurring microorganism which produces residual sugar hydrolyzing enzyme and which preparation includes a higher concentration of residual sugar hydrolyzing enzyme than would be found naturally due to the fermentation of the microorganism. Alternatively, an enriched residual sugar hydrolyzing enzyme preparation can be prepared by purifying the residual sugar hydrolyzing enzyme from the fermentation broth of a natural or genetically engineered microorganism so as "enrich" the residual sugar hydrolyzing enzyme relative to the removed contaminants. Similarly, the purified residual sugar hydrolyzing enzyme can be added to a naturally occurring enzyme mixture containing, for example, pullulanase, or acid amylase, in a concentration greater than exists in the naturally occurring CA 02203811 1997-04-2~

fermentation of the organism(s) from which the enzyme mixture is desired. Additionally, an enriched residual sugar hydrolyzing enzyme preparation may be derived from the fermentation of a genetically modified microorganism which has been subject to recombinant techniques so as to amplify expression of residual sugar hydrolyzings enzyme in a fermentation broth.
The enzyme preparation applied in the present invention for the hydrolysis of the noncellulose and nonhemicellulose based saccharides may comprise the enzyme maltulase (described herein in and co-pending Patent Application (Serial No.
Applicants Docket No. GC319) entitled "A method for increasing monosaccharide levels in the saccharification of starch"), or any other enzyme capable of the hydrolysis of a noncellulose and nonhemicellulose based saccharide, as well as combinations thereof.
The enzyme preparation is applied at a suitable step.during the fermentative production process. Preferably, at the point which the enzyme is added to the process, the overall carbohydrate concentration is below 20% w/v, more preferably below 10%
w/v, and most preferably below 5% w/v, in order to minimize reversion reactions which will result in other and/or new unfermentable sugars. When using immobilized enzyme systems, the overall carbohydrate content is preferably low, i.e., below 10% w/v.
However, the addition of the enzyme preparation can be modified so as to be added at a step in the process which is least disruptive of the sugar preparation process and will be most suitable for the optimal conditions under which the enzyme acts. In general, an advantageous use of the enzymes and processes according to the present inventionwill be in further treating starch which has been subjected to liquefaction and saccharification. In a preferred embodiment, the residual sugar hydrolyzing enzyme is utilized to hydrolyze residual sugars in a liquefied starch solution. Thus, for example, the residual sugar hydrolyzing enzyme can be added to the liquefied starch produced by jet liquefaction of starch with a-amylase. Alternatively, the residual sugar hydrolyzing enzyme can be added simultaneously with glucoamylase in the saccharification step. In yet another variation, the residual sugar hydrolyzing enzyme can be added after the liquefied starch has been treated with glucoa-mylase to further 30 increase the DX value of the saccharified starch or during the actual fermentation process to increase fermentable substrate. Finally, isomerized fructose/glucose syrups may be treated with the maltulase enzyme to further increase the concentration of glucose and fructose and reduce maltulose content. Each of these variations willbenefit from the enzyme of the present invention through the increased production of 3s fermentable sugars. However, the choice of which variation to use in a given process will depend on the specific parameters under which the process at hand is operated.

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W O96/13600 PCTrUS95/13876 Those of skill in the art would be able to easily ascertain which variation is optimal with a given starch processing method.
The use of residual sugar hydrolyzing enzyme according to the present invention will be modified to best take advantage of the kinetics of the specific enzyme 5 selected. Such process modification is well within the skill in the art. Where the residual sugar hydrolyzing enzyme activity is isolated or derived from A. niger, the temperature of the residual sugar hydrolysis step is from about 15 to about 70C, more preferably from about 20C to about 60C; the pH is preferably from about 4-8 and more preferably from about 4.5-7. This embodiment of the invention has proven to be lO especially successful in the hydrolysis of maltulose and isomaltose present in corn starch derived sugar syrup. It is believed that residual sugar content increases with the increasing pH of the liquefaction step of starch hydrolysis. Similarly the isomaltose content isomerases with increasing DS content during saccharification. Thus, thepresent invention will be especially useful in fermentative production processes which lS involve a starch product which was produced by liquefaction at a pH of between 5-7, or which has a DS content of greater than 20% w/v during saccharification.
The concentration of residual sugar hydrolyzing enzyme used in a particular process will be dependent on the specific process in use. However, given the disclosure herein, one of ordinary skill in the art would be able to easily ascertain the 20 appropriate concentration. For example, in the case of maltulose hydrolysis in a 20%
dry solids sugar solution containing 2% maltulose, maltulose will be present in a quantity of approximately 4 g/kg of d.s. sugar. Thus, where 1 unit equals the hydrolysis of 1!1mole of maltulose/minute, 43 U/kg of syrup will be needed to hydrolyze themaltulose in solution in 10 hours. Preferably, added residual sugar hydrolyzing activity, 2s in this case maltulase, is greater than about 10 U/kg sugar d.s., more preferably between 20 and 5000 U/kg sugar d.s., and most preferably between 25 and 1000 U/kg sugar d.s.
In the present invention, the term "fermentative production process" is defined as any production process which comprises the culturing of a microorganism to produce 30 a desired product. Although the present invention is demonstrated with examples from the field of fermentative production of ethanol, it should be appreciated that the invention is not limited to ethanol production, but can also be applied in otherfermentative production processes where unfermentable noncellulose and nonhemicellulose based saccharides are present. Possible examples of such 3s processes are the fermentative production of primary metabolites (such as ethanol, and glycerol), organic acids (e.g. Iactic acid, acetic acid, succinic acid, etc), amino acids, CA 02203811 1997-04-2~

W O 96/13600 . PCTAUS9~/13876 antibiotics (e.g. penicillin), yeast, biomass to be used as single cell protein, proteins (such as enzymes), vitamins, dyes, and steroids.
In the present invention, the term "unfermentable noncellulose and nonhemicellulose based saccharide" refers to any saccharide which does not originate 5 from cellulose or hemicellulose and which is not fermentable. Examples of suchsaccharides include isomaltose, maltulose, stachyose, raffinose, and melibiose. In this respect the term fermentable refers to the ability of the microorganism employed in a fermentative production process (e.g. the use of yeast S. cerevisiae to produce ethanol from glucose) to utilize these saccharides.
lo The term "residual sugars" means unfermentable sugars present in carbohydrate based fermentation media including isomaltose, maltulose, stachyose, raffinose and melibiose. In wet and dry milling processes using, for example, corn as a starting material, the residual sugars consist mainly of isomaltose and maltulose.
A further aspect of the present invention ensures that the method to hydrolyze the unfermentable saccharides is applied in an economically attractive way. The application of the enzyme preparation for the hydrolysis in the unfermentable saccharides in an immobilized form results in a drastic reduction in the amount of enzymes required compared to the use of soluble enzymes. Suitable means of immobilization of enzymes are known in the art and include, e.g., inclusion, attachment, fixation in, to, or on carrier materials.
The present invention contemplates incorporation of an immobilized enzyme reactor in ethanol production processes. Process outlines thereof are presented in Figures 1A and 1B forwet milling continuous ethanol production and in Figures 2A and 2B for batchwise ethanol production. In wet milling type ethanol production, the method of the invention is advantageously used at a step having a low carbohydrate concentration to allow efficient conversion of residual sugars to glucose without the production of reversion products. In batch type ethanol production, the invention should also be used at low carbohydrate concentrations, for example, at the end of the fermentation. Thus, advantageously the present invention is utilized as a separate reactor step during the fermentation process or as a combined step by applying residual sugar hydrolyzing enzyme to the fermenting microorganism vessel. Additionally, the fermentation broth may be recycled from the fermenter and subjected to the inventive method with the product stream sent back to the fermenter for fermentation of released fermentables.
The following examples are illustrative of the invention and should not be interpreted as limiting thereof.

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EXAMPLES

Example 1 5Detection of Maltulose HYdrolYsis Activity Carbohydrate analysis may be performed by the HPLC method under the following conditions:
Column: carbohydrate column (Waters Corp. part nr. 84038) Eluent: Acetonitrile/water (80/20 for separation of different mono- and disaccharides or lo 65/35 for separation of oligo-saccharides).
Flow: 2 ml/min.
Temperature: Ambient.
Detection: Rl (Refraction Index) detection.
(A) Preparation of Maltulose Maltulose is the disaccharide a-D-glucopyranosyl-1,4-a-fructofuranose. This disaccharide can be prepared by alkaline isomerization of the glucose residue at the reducing end of the disaccharide maltose (a-D-glucopyranosyl1~4-aglucopyranose) as follows:
2 9 of aluminiumoxide is mixed with 100 ml of a 40% (w/v) maltose solution.
20 The pH is adjusted to pH 11.5 using sodium hydroxide. The reaction mixture is kept at 60C for 24 hours. Next, the pH is adjusted to pH 4.5 and 5 g of baker's yeast are added in order to ferment maltose and other fermentable sugars resulting from the alkaline incubation conditions (maltulose is not fermented by the yeast). Finally, the reaction mixture is filtered to obtain a clear solution, which is concentrated under 25 vacuum to remove the ethanol (resulting from the fermentation) and to obtain a high dry solids solution of maltulose.

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W O96/13600 PCT~US95/13876 (B) Enzymatic hydrolysis of maltulose Enzymatic maltulose hydrolysis was investigated with several commercially available enzyme preparations. The enzymes were mixed with a 5% maltulose solution in distilled water under conditions suggested for the specific enzyme preparation. For s each of the enzymes, 5 mg of enzyme were added to 5 ml of maltulose solution. The mixtures were incubated under conditions listed in Table 1.
The reaction mixtures were analyzed using HPLC as described above. The results of these analyses are shown in Table 1.
Table 1 Series Enzyme ,b~ dti~Temp. pH ~ Fructose Glucose ~ ~Others in oc time h. % % % %
Starting material 0.0 0.0 87.0 13.0 1 T. reesei cellulase; 50 4.5 40 38.0 29.4 24.0 8.6 MAXAZYME CL 2,000 (Gist-brocades) 1 Kluveromyces lactis 37 6.5 40 0.0 0.0 86.9 13.1 ~-galactosidase;
MAXILACT LX5,000 (Gist-brocades) 1 Yeast Invertase; 50 4.5 40 0.0 0.0 91.5 8.5 MAXINVERT L 10,000 (Gist-brocades) 2 Starting material 0.0 0.0 56.2 43.8 series 2 2 A. niger~- 60 4.2 4 15.5 14.6 11.5 58.4 galactosidase;
SUMIZYME AGS
(Shin Nihon) The results demonstrate that only the T. reesei cellulase and A. niger a-galactosidase preparations contain a maltulose hydrolysing activity.

Example 2 The Enzymatic Hvdrolysis of Stachvose A 1% stachyose solution was incubated with a commercially available preparation of a-galactosidase and with a mixture of o~-galactosidase and invertase to investigate enzymatic hydrolysis of stachyose by these preparations. The incubation was carried out at 55C and pH = 5.5. Samples were taken after 2 hours incubation 20 time and analyzed by means of HPLC. The results are shown in Table 2.

Wo 96/13600 PCT/US95/13876 Table 2 Enzyme StachyoseRamnoseMelibiose Glucose Fructose t;~ t~se % % ~/~ % % %
Starting material 100.0 0.0 0.0 0.0 0.0 -A. nigera~ e; 5.6 12.2 8.9 11.1 22.2 40.0 SUMIZYME AGS
(Shin Nihon) A. ni9er yeast a4~ t~ c 0 0 4 ~i 2.3 20.5 25.0 47.7 SUMIZYME AGS (Shin Nihon) plus Invertase (MAXINVERT
(Gist-brocades) The results demonstrate that stachyose can be hydrolysed into fermentable monosaccharides.
S ExamPle 3 The EnzYmatic Hydrolysis of Raffinose A 1% raffinose solution was incubated with a commercially available preparation of a-galactosidase and with a mixture of commercially available preparations of -galactosidase and invertase, to investigate enzymatic hydrolysis of raffinose by these lo preparations. The incubation was carried out at 55C and pH = 5.5. Samples were taken after 2 hours incubation time and analyzed by means of HPLC. The results are shown in Table 3.
Table 3 Enzyme Raffinose DP2GlucoseFructose ~ se % % % % %
Starting material 100.0 0.0 0.0 0.0 0.0 A. nigeru-G;~ tu~ . SUMIZYME 6.513.9 24.1 29.6 25.9 AGS (Shin Nihon) A. niger~-G~ t~,.;.l~c~, SUMIZYME 0.0 0.0 33.3 33.3 33.3 AGS (Shin Nihon) plus yeast invertase MAXINVERT (Gist-brocades) The results demonstrate that raffinose can be hydrolysed into fermentable monosaccharides.

Example 4 The Enzymatic HYdrolysis of Melibiose A 1% melibiose solution was incubated with a commercially available preparation of a-galactosidase to investigate enzymatic hydrolysis of melibiose by this preparation. The incubation was carried out at 55C and pH 5.5. Samples were taken after 3 hours incubation time and analyzed by means of HPLC. The results are shown 25 in Table 4.

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Table 4 EnzymeMelibiose %Glucose %~;ala~ se %
Starting material 100.0 0.0 0.0 A. nigera-G~Iactusicl~se, 0.0 50.0 50.0 SUMIZYME AGS (Shin Nihon) The results demonstrate that melibiose can be hydrolysed into fermentable monosaccharides.

Example 5 The Enzymatic HYdrolysis of Residual Suqars in the Beer From Wet Millinq Ethanol Production (Starch as Raw Material) lo Beer resulting from fermentation in a wet milling ethanol production process was incubated with different commercially available enzyme preparations to investigate whether enzymatic hydrolysis appears to be a general feature of many preparations and not specific to any of the major components in the mixture. The incubation was carried out at 33C and pH = 4Ø Analysis done by means of HPLC (column: HPX-87H
1S from Bio-rad, eluent: 0.005 M H2S04, flow: 0.6 ml/min, temperature: 65C, detection Rl).
Representative chromatograms of the starting material (Figure 3A) and after enzyme (a-galactosidase; SUMIZYME AGS) treatment (Figure 3B) can be seen in the added figures.
The incubation effects are shown in Table 5.
Table 5 r7 ,a,i.le peak Oli_ ' i~- peak at O; ~ peak Enzyme Pltpd~dti~)n at 9.48/9.84 minutes in7.64 minutes in mglml at 6.94 minutes in mg/ml mg/ml Starting material 1.03 3.39 0.42 A. nigerAmyloglucosid~e; 1.19 3.27 0.43 AMIGASE (Gist-brocades) A. nigerXylanase LYXASAN 4.11 0.86 0.00 (Gist-brocades) A. nigerPectinase RAPIDASE 6.14 0.00 0.38 C80 (Gist-brocades) T. reesei Cellulase MAXAZYME 3.38 1.81 0.45 CL 2000 (Gist- brocades) A. niger-Galactosidase;6.76 0.00 0.00 SUMIZYME AGS (Shin Nihon) A. niger-Galactosidase;6.69 0.15 0.28 SUMIZYME AC (Shin Nihon) - A. niger Fungal amylase 3.22 1.65 0.40 MYCOLASE (Gist-brocades) A. fcuum Phytase NATUPHOS 2.20 2.35 0.32 (Gist-brocades) =
CA 022038ll l997-04-25 W O96/13600 PCTrUS95/13876 The results shown in Table 5 demonstrate that ~he beer from wet milling ethanol production contains residuals (unfermentables) that cannot be hydrolysed by amyloglucosidase into fermentable monosaccharides. In conLr~sl, other enzyme preparations are capable of hydrolysis of some of these residuals into s monosaccharides.

Example 6 The Enzymatic Hydrolysis of Residual Suqars in the Beer From Wet Millinq EthanolProduction and Fermentation of the Released Monosaccharides Aliquots of beer from a wet milling ethanol production process were incubated at33C and pH=4.0 with a mixture of amyloglucosidase (AMIGASE from Gist-brocades),xylanase (LYXASAN from Gist-brocades), pectinase (RAPIDASE C80 from Gist-brocades), and two different a-galactosidases (SUMIZYME AGS and AC, both from 15 Shin Nihon) in order to hydrolyse residual sugars (oligosaccharides). An amount of yeast was subsequently added in order to investigate whether the released monosaccharides were fermented. The results are shown in Table 6.
Representative chromatograms of the beer, treated with an enzyme cocktail (Figure 3C) and subsequently treated with yeast (Figure 3D) can be seen in the added figures.
Table 6 Sample Cl;~ le ~ a 'lo~ le Ethanol in peak at 6.94peak at 7.64peaks at mg/ml minutes inminutes in mglml9.64/9.84/10.04 r g~ i rninutes nmg/ml S~arting material ~ a ~ .
Ater enzyme treatment . I . . ~, .
Ater yeast treatment .. . 'n' The data in Table 6 demonstrates that the enzyme mixture released a siyni~icanl amount of monosaccharides from residual sugar. Surprisingly, the ethanol level also increased. However, this can be explained by the fact that often beer will contain a few yeast cells which are capable of converting monosaccharides into ethanol as soon as these monosaccharides are produced. Addition of yeast often the enzyme treatmentresulted in a higher ethanol level, however, fermentation time was too short to ferment all monosaccharides present.

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Example 7 The Enzymatic Hydrolysis of Residual Su~ars in the Beer From Wet Millinq Ethanol Production Usinq an Immobilized EnzYme Mixture and Fermentation of the Released Monosaccharides a-Galactosidase (SUMIZYME AGS, Shin Nihon) was immobilized using the procedure described in U.S. Patent No. 3,838,007. 10 9 of this immo~ ed enzyme were incubated with 100 ml of beer from wet milling ethanol production and incubated for 24 hours at 33C and pH 4Ø Next the reaction mixture was filtered and 5 9 of yeast lo were added to the filtrate in order to ferment the released monosaccharides. The results are shown in Table 7.
Table 7 Sample Composition C~ e~ 'c ~ ltsEthanolmg/ml mglml mg/ml Starting material 3.76 0.99 81.99 Aftertreatmentwith 0.11 6.40 83.50 immobilized enzyme After f~r")er,l~ion 0.10 2.1 85.61 The results demonstrate that using an immobilized enzyme system, a similar 15 effect can be achieved as compared to using a soluble enzyme (see Example 6).
Example 8 Purification of the Maltulose and Residual Suqar HvdrolYsinq Activity from SUMIZYME AGS and Measurement of Molecular Weiqht The a-g~lar,tosid~se preparation (SUMIZYME AGS, Shin Nihon, Japan) was partially purified using gel filtration chromatography. The procedure is described below. Collected fractions were screened for the presence of different activities.
Interesting fractions were pooled for determination of the specific activity.
Chromatographic procedure:
25 Column: 58 x 2.5 cm.
Support material: Sephacryl S 200 HR.
Elution buffer: 50 mM acetate buffer, pH = 4.5 including 0.02% sodiumazide.
Flowrate: 2 ml/min.
Detection: UV 280 nm.
30 Fraction coller,tion: 2 minute fractions.
Sample: 4 ml of 30 mg/ml SUMIZYME AGS (lot 60902-02) in elution buffer.

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W O96/13600 PCTrUS95/13876 Activity detection (screening of fractions):
1. a-~~'nctn~sidase activity detection.
100 ~11 1 mM pa,~ ,ophenyl-a-D-g~'~cl~se in 50 mM acetate bufferpH 5.5was inu~hated with 100 ml of co!lcctPd fraction. After a 3 minute inulh~tion at rooms temperature 100 ~1 of 0 0625 M borax buffer pH 9.7 was added to stop the ,t:a~.lion.
The yellow colour, resulting from pa,d, '.ophenol, was a measure for a~J~'~cll~cid~ce activity. The result was judged visually.
2. M ~It~ S e hydrolysing activity det~ction.
The m~itl~'ose pr~pa,c,Lion was diluted 4 times with distilled water. 100 ~l of this solution were mixed with 200 ~11 of a fraction having m~it~ se hydrolyzing activity and 700 ,ul of distilled water. This mixture was inc~h~t~d for 3 hours at 33C. Next, the mixtures were placed in a boiling water bath in order to inactivate the enzyme. Finally, the mixtures were analyzed on HPLC using the Bio-Rad HPX 87C column. The increase in the fructose peak area was a direct measure for the activity.
15 3. Residual sugar hydrolysing activity detection.
50 1ll of a fraction having residual sugar hydrolyzing activity was mixed with 50 ,ul beer (from wet milling fuel ethanol production, from Pekin Energy, Pekin, Illinois) and incubated for 16 hours at 33C. Next, 900 ,ul 0.006 N sulphuric acid was added and the reaction mixture was centrifuged in an Eppendorf centrifuge. The supe"~alanl wasanalyzed by means of HPLC on a BlO-Rad HPX 87H column. The decrease of the residual sugar peak was a direct measure for the activity.
Specific activity assay:
The specific activity is expressed as an activity value per mg of protein per minute of reaction time.
1. Detemmination of the protein content.
The protein content is detemmined using the BCA method with bovine serum albumin as standard.
2. The a-g~lactosidase activity.
A solution is made of 10 mM p-nitrophenol in 50 mM sodium acetate buffer pH 5.5.This solution is diluted to 240-160-80-40 mM. 1 ml of these solutions is added to 2 ml of 0.80 mM p-nitrophenyl-a-D-g~iactnpyranoside in acetate buffer. To this mixture 5 ml 625 mM borax buffer pH 9.7 is added (stop reagent). The OD of these solutions ismeasured at 405 nm against water (standard curve).

CA 022038ll l997-04-2~

W O96/13600 PCT~US95/13876 .
Enzyme incl~hation: 1 ml of diluted enzyme solution in stead of p-nitrophenol. The mixture is incllhated for 15 minutes at 37C. The reaction is stopped by adding 5 ml borax solution. The OD is measured as mentioned before.
Activity derinition: one Unit of a-g-l-^tosid~ce is the amount of enzyme which S hydrolyses 1 ,umol of p-NPGal/minute under the standard cohditions.
3. The maltulose hydrolysing activity.
100 ~11 of m~lh~'osc solution is mixed with 200 ~11 of enzyme solution and 700 ~11 of distilled water. The mixture is incllhatPd at 33C. Samples were taken at cJirrt:r~nL
inu Ih~tion times and analyzed by HPLC in order to determine the amount of maltulose hydrolysed.
4. The residual sugar hydrolysing activity.
100 ,ul of beer from wet milling ethanol production is mixed with 200 ~11 of enzyme solution and incubated at 33C. 700 ~l of 8 mM sulphuric acid was added to sam,~'os taken at different incubation times. The samples are analyzed by HPLC in order to detemmine the amount of residual sugar hydrolysed.
Deffnitions of specific activity:
1. a-g~'actosidase: units a-gal per mg protein.
2. Maltulose hydrolysing activity: ,ug m~lt~ ~'ose hydrolysed per mg of protein per minute.
3. Residual sugar hydrolysing activity: llg residual sugars hydrolysed per mg of protein per minute.
Molecular Weight Determination:
St~nd~l.l commercially available protein mixtures (Bio-Rad) were used as molecular weight markers as follows: Thyroglobulin (MW = 670 kD), gamma-globulin (MW = 158 kD), ovalbumin (MW = 44 kD), myoglobin (MW = 17 kD), vitamin B12 (MW = 13.5 kD). Gel 2s filtration of the markers with subsequent comparison to the rr~lt~ se and residual sugars hydrolyzing enzyme resulted in an approximate mc'ecl 1'-~ weight of about 132 kD and 120 kD respectively.
Results:
The chromatogram resulting from gel filtration of the a-galactosidase preparation is presented in Figure 4. The results of assaying of fractions on the presence of a-g~lactosidase activity, maltulose hydrolysing activity and residual sugar hydrolysing activity are presented in the following Table 8. The results were used to pool fractions. The a-g~'actosidase pool was contained in fraction 8-10 (elution time 55-60 minutes). The residu~ls- and m~ltl ~ se hydrolysing activity pool was Wo 96/13600 PCTIUS95113876 contained in rlaclions 12-14 (elution time = 63 - 68 minutes). The pooled fractions and starting "~dlerial were assayed for specific activity. The results are shown in Table 9.
Table 8 Fraction Fractiona ~ - t.. ~ Maltulose l~esidualwgar numberelution timeactivity h~ u~h,. ol~. D
(yellow colour)activityactivity (peak ,.Ju~lion) 4 47-48 + 2 6 51-52 ++ 3 11 8 55-56 +++ 33 37 59-60 +++ 100 63 12 63-64 ++ 93 100 14 67-68 + 91 100 CA 02203811 l997-04-25 Table 9 Specific Specific Speclhc a- ' e residual MaterialuancjttiSJimy9 Ratio activity mg hJd~ùly g Imglmin . ~
mg/min Starting 8100 4.82 0.52 material Fractions184000 22.787.0 18.1 12.9 24.8 Fractions 16900 2.1163.8 34.0 28.4 54.6 s Discussion/conclusions:
Tables 8 and 9 demonstrate that the malt~'nse and residual sugar hydrolysing activity are side activities in the a-g~'actncidase prepa,~lion and are not due to the a-g^'act~.sid~.se itself. In addition it appears that the specific activity of both enzymes can be significantly increased by a single purification step.

Example 9 Heat inactivation of the a-r~alactosidase activity A solution of a-g~lactocid~ce was heat treated for 30 minutes at 65C. Next the lS starting " ,aLerial and the heat treated solution were assayed for specific activity.

The result of this experiment is listed in Table 10.

Table 10 Specific c~-Specihc, - Specific residual J~ I"r. ùly;.i.. g sugar Material activity Ratio activity Ratio l.y~l~uly;.i--g activity Rabo unitslmg mg ,.. ~ ~' mg mg/min ~ 3 starting 8100 4.82 0.52 material Heattreated 18.5 .002 4.19 0.9 0.42 0.8 materiai The results demonstrate that the a-g~lactr~sidase activity and the residual sugar and maltulose hydrolysing activity are coming from different enzymes.

Claims

1. A fermentative production process comprising the step of hydrolysing unfermentable noncellulose and nonhemicellulose based saccharides using an enzyme preparation capable thereof.

2. The fermentative production process according to claim 1, wherein at least one of the unfermentable noncellulose and nonhemicellulose based saccharides is selected from the group consisting of maltulose, stachyose, raffinose, melibiose and isomaltose.

3. A fermentative production process according to claim 1, wherein said fermentative process is for the production of primary metabolites.

4. A process according to claim 3, wherein the primary metabolite is ethanol.

5. A process according to claim 1, wherein the enzyme preparation comprises a maltulose hydrolyzing activity or a residual sugar hydrolysing activity.

6. A process according to claim 1, wherein the enzyme preparation is applied in an immobilized form.

7. A process according to claim 5, wherein the enzyme preparation further comprises at least one enzyme selected from the group consisting of pectinase, glucoamylase, cellulase, .alpha.-galactosidase, xylanase (hemicellulase), fungal amylase, and phytase.

8. A process according to claim 1, comprising an enzyme exhibiting maltulose hydrolyzing activity or residual sugar hydrolyzing activity which is an enzyme derived from a fungal source.

9. A wet milling ethanol plant comprising a fermenter, and further comprising anenzyme reactor for the enzymatic hydrolysis of unfermentable noncellulose and nonhemicellulose based saccharides, wherein said hydrolyzed saccharides are recycled or fed to said fermenter.

10. A fermentation plant for the batchwise production of ethanol, wherein said plant comprises an enzyme reactor for the enzymatic hydrolysis of unfermentable noncellulose and nonhemicellulose based saccharides, and wherein the fermentation-broth is recycled through the enzyme reactor during fermentation.