MXPA97002933A - A method for the utilization of improved raw material in fermentac processes - Google Patents

Abstract

Raw materials such as glucose syrups, cane molasses and beet molasses used in fermentative production processes usually contain saccharides which can not be used by the microorganisms used in the fermentation processes. The present invention describes the use of enzymatic preparations capable of hydrolyzing non-fermentable saccharides in fermentable saccharides to improve the yield of the fermentative production process. Enzymatic preparations can be applied at a point in the process where the total carbohydrate concentration is less than 20% to avoid reversion reactions. The enzyme preparations are preferably applied in immobilized form

Description

A METHOD FOR THE USE OF RAW MATERIAL. IMPROVED IN FERMENTATION PROCESSES FIELD OF THE INVENTION The present invention relates to the enzymatic hydrolysis of non-fermentable carbohydrates into fermentable carbohydrates. More specifically, the invention provides a method for producing fermentable monosaccharides from non-fermentable saccharides, present in, for example, liquefied and / or saccharified starch, beet molasses and cane molasses, to improve the use of the raw material in fermentation processes such as the fermentative production of ethanol.

BACKGROUND OF THE INVENTION The performance is a crucial aspect in the processes of fermentative production. The yield is of particular importance in the production of primary metabolites such as ethanol, glycerol and lactic acid due to the low profit margins provided by these products. As a result, a significant effort has been focused on improving performance to facilitate achieving high levels of production from the REF: 24571 raw materials used. In the field of fermentative ethanol production, the yield of the product has been improved by reducing the amount of the by-products produced and also improving the utilization of the raw material. For example, yeast recycling systems (reuse of yeast) have been used to reduce sugar consumption by the production of yeast biomass. Additionally, simultaneous saccharification and fermentation to keep the free glucose level to a minimum, and thus prevent high levels of infection, has been shown to result in improved yields. In addition, the application of cellulase and semicellulase to release additional fermentable carbohydrates from the cellulose fiber material and is icelulose (Patents 85DD-274453, 78SU698402 and 78DD-210143) has been successful in increasing yield as the application of a fermenting yeast cellobiose (US Patent 5,100,791). Fermentation with immobilized yeast has been effective in reducing carbohydrate consumption for biomass production. Similarly, coinmobilized enzymes and yeasts have been advantageously used to achieve saccharification and simultaneous fermentation which results in reduced carbohydrate consumption for biomass production (EP-B1-0 222 462). Although these attempts to increase ethanol yield have had varying degrees of success, new methods to increase yield are the subject of such research. The presence of certain non-fermentable sugars based on non-cellulosic and non-cellulosic raw materials in the broth at the end of the fermentation has been recognized in the art. These non-fermentable sugars originate from the raw material itself or arise as by-products during the processing of raw materials, for example, maceration, liquefaction, saccharification or isomerization of the starch stream. Examples of non-fermentable sugars ("residual sugars") which exist in the starch processing streams include: 1. Maltulose: α-D-glucose (1,4) -a-fructose. High temperatures are used during the starch liquefaction process (hydrolysis of the starch in dextrin). These high temperatures, in combination with the applied pH, stimulate the isomerization of the glucose unit at the reducing end of a dextrin fructose molecule. 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). The maltulose, however, is not hydrolyzed by commonly used starch processing enzymes such as amyloglucosidase, pullulanase or acid amylase. Depending on the conditions used during liquefaction, the concentration of maltulose in the liquefied product can be up to 2%. 2. Stachyose: α-D-galactosyl (1, 6) -a-D-galacto-sil (1,6) -a-D-glucose (1,2) -β-D-fructose. This non-fermentable sugar is found in, among others, sugarcane molasses. Porter et al., Biotech. Bioeng. vol 35, pp 15-22 (1990) report the hydrolysis of stachyose with a soy a-galactosidase preparation. 3. Raffinose: a-D-galactosyl (1, 6) -a-D-glucose- (1, 2) -β-D-fructose. This sugar is found in, among others, beet molasses. Raffinose is also a product of the partial hydrolysis of stachyose. Porter et al., Describe the hydrolysis of raffinose. 4. Melibiose: α-D-galactosyl (1, 6) -a-D-glucose. This disaccharide, found in, among others, beet molasses, is a product of the partial hydrolysis of both stachyose and raffinose. It has been reported that melibiose is hydrolyzed by the α-galactosidase of Aspergillus niger, Kane or et al., Agrie. Biol. Chem., Vol 55, no. 1, pp 109-115 (1991), and exo-a-galactosidase from Azotobacter vionelandii, ong. Appl. of Environ. Microb., Vol. 56, no. 7 pp 2271-2273 (1990). The enzymatic hydrolysis of these non-fermentable carbohydrates in this way is far from being applied to improve the utilization of the raw material during the production of primary metabolites such as ethanol. The use of such a process has probably been ignored in the industry due to the expectation in the field that the hydrolysis of those non-fermentable carbohydrates, before fermering, (when carbohydrate concentrations are high) and using an immobilized enzyme or enzyme mixture, result in the production of large quantities of non-fermentable reduction products, and thus, would only make the situation more difficult. In addition, the fractions of non-fermentable carbohydrates consist of a mixture of carbohydrates and as a consequence require a mixture of enzymes for hydrolysis. This situation is further complicated when enzymatic mixtures are used. Thus, there is a need in the art for a convenient method for producing fermentable carbohydrates from non-fermentable residual sugars produced during the hydrolysis process of the starch.

BRIEF DESCRIPTION OF THE INVENTION An object of the invention is to provide an economically feasible method to increase the yield of the raw material product (improved utilization) by providing the hydrolysis of non-fermentable sugars based on non-cellulose and non-cellulose raw materials into fermentable sugars (mainly onosaccharides) in the fermentative production processes. According to the present invention, there is provided a fermentative production process for the hydrolysis of saccharides based on non-cellulose raw materials and non-fermentable non-cellulosic raw materials using an enzyme preparation that hydrolyzes residual sugar capable thereof. Preferably, the enzyme preparation is applied at a point in the process where the total carbohydrate concentration is less than 20% w / v. According to a compositional modality, enzymes are provided for the hydrolysis of non-fermentable saccharides in fermentative production processes. Enzymes are preferably used in mixtures of enzymes and / or are used in immobilized form. The invention further describes plants for the fermentative production of ethanol, which comprise enzymatic reactors for the hydrolysis of non-fermentable saccharides based on non-cellulose and non-cellulosic raw materials.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A. Schematic representation of an ethanol plant by "wet milling".

Fig IB. Schematic representation of an ethanol plant by "wet milling" that includes an enzymatic reactor for the hydrolysis of non-fermentable saccharides. Figure 2A. Schematic representation of a plant for the batch production of ethanol, which includes an enzymatic reactor for the hydrolysis of non-fermentable saccharides. Figure 3A. CLAP chromatogram of beet from ethanol production by "wet milling". Figure 3B. CLAP chromatogram of beet from ethanol production by "wet milling" after treatment with a-galactosidase (SUMIZYME AGS). Figure 3C. CLAP chromatogram of beet from ethanol production by "wet milling" after treatment with an enzymatic cocktail to hydrolyze non-fermentable saccharides. 3D figure. CLAP chromatogram of beet from ethanol production by "wet milling" after treatment with an enzymatic cocktail to hydrolyze non-fermentable saccharides, followed by the addition of yeast. Figure 4. Chromatogram of a-galactosidase gel fation on Sephacryl 5200 HR OD (at OD 280) vs. elution time.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for increasing the yield of a fermentative production process by increasing the amount of fermentable sugars used as raw material in such processes through the enzymatic hydrolysis of saccharides based on non-cellulose raw materials and non-fermentable non-cellulosic raw materials. The enzymatic composition can be derived from the fermentation broth of a microorganism that produces the enzyme. The enzyme that hydrolyzes the residual sugar of the invention comprises any enzyme capable of hydrolyzing the residual sugars present in the carbohydrate raw material streams which are not fermentable by organisms that ferment sugar and, especially, the glucose syrup or precursors thereof. of the starch processing streams. Such enzymes that hydrolyze the residual sugar should preferably have as their main activity the hydrolysis of one or more residual sugars. Preferably, the enzyme composition is derived from a fungal source. More preferably Aspergillus or Trichoderma. In a more preferred embodiment of the invention, the enzyme composition is derived from A. niger and comprises a mantulose hydrolyzing activity and / or a residual sugar hydrolyzing activity having a molecular weight of approximately 132 kD and 120 kD, respectively, as measured by gel filtration. The place of the enzymatic composition includes an enzyme commonly used in the preparation of sugar standards for fermentative production processes, for example, amyloglucosidase, pullulanase or acid amylase, it is preferable to enrich the composition to include more of the enzyme that hydrolyzes sugar residual that exists in the natural composition. A "enriched" residual sugar hydrolyzing enzyme according to the present invention is a preparation which is derived from a fermentation broth produced by the fermentation of a microorganism found in nature, which produces the hydrolyzing enzyme of the residual sugar and whose preparation includes a higher concentration of residual sugar hydrolyzing enzyme than could be naturally found 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, from a natural microorganism genetically added to "enrich" the residual sugar hydrolyzing enzyme in relation to the contaminants removed. Similarly, the purified residual sugar hydrolyzing enzyme can be added to an enzyme mixture found in nature that contains, for example, pullulanase, or acid amylase, in a higher concentration than that which exists in the fermentation of organisms that they are found in nature from which the enzyme mixture is desired. Additionally, an enriched residual sugar hydrolyzing enzyme preparation can be derived from the fermentation of a genetically modified microorganism, which has been subjected to recombinant techniques to amplify the expression of the residual sugar hydrolyzing enzyme in a fermentation broth. The enzyme preparation applied to the present invention for the hydrolysis of saccharides based on non-cellulosic and non-cellulosic raw materials can comprise the enzyme maltulase (described herein in the Application for Copending patent (Serial No..) No. of Applicant's file GC319) entitled "A method for increasing the levels of monosaccharides in starch saccharification"), or any other enzyme capable of hydrolyzing saccharides based on non-cellulose and non-cellulosic raw materials, as well as combinations thereof.

The enzyme preparation is applied at an appropriate step during a fermentative production process. Preferably, at the point at which the enzyme is added to the process, the total carbohydrate concentration is less than 20% weight / volume, more preferably less than 10% weight / volume, and more preferably less than 5% by weight / volume, in order to reduce to a minimum the reduction reactions which could result in other and / or new non-fermentable sugars. When immobilized enzyme systems are used, the total carbohydrate content is preferably lower and, i.e., less than 10% w / v. However, the addition of the enzyme preparation can be modified to be added in a step in the process that is less disruptive of the sugar preparation process and that is more suitable for the optimum conditions under which the enzyme acts. In general, an advantageous use of the enzymes and processes according to the present invention will be to further treat the starch which has been subjected to liquefaction and saccharification. In a preferred embodiment the residual sugar hydrolyzing enzyme is hydrolysed to hydrolyse residual sugars in a solution of liquefied starch. Thus, for example, a residual sugar hydrolyzing enzyme can be added to the liquefied starch produced by jet starch liquefaction with α-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 glucoamylase to further increase the DX value of the saccharified starch or during the current fermentation process to increase the fermented substrate. Finally, isomerized fructose / glucose syrups can be treated with the maltulose enzyme to further increase the glucose and fructose concentration and reduce the maltulose content. Each of these variations will benefit the enzyme of the present invention through the increased production of 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. Those skilled in the art would be able to easily find which variation is optimal with a given starch processing method. The use of the residual sugar hydrolyzing enzyme according to the present invention will be modified to take more advantage of the kinetics of the specific enzyme selected. Such modification of the process is within the experience of the technique. Where the activity of the residual sugar hydrolyzing enzyme is isolated or derived from A. niger, the temperature of the hydrolysis step of the residual sugar is from about 15 ° C to about 70 ° C, more preferably from about 20 ° C to about 60 ° C; the pH is preferably about 4-8 and more preferably about 4.5-7. This embodiment of the invention has proved especially successful in the hydrolysis of maltulose and isomaltose present in sugar syrup derived from corn starch. It is believed that the residual sugar content increases with the pH increase of the liquefaction step of the hydrolysis of the starch. Similarly, the isomaltose content is isomerized with the increase in DS content during saccharification. In this way, the present invention will be especially useful in the fermentative production process, which implies a starch product which has been produced by liquefaction at a pH between 5-7, or having a DS content greater than 20% by weight / volume during saccharification. The concentration of residual sugar hydrolyzing enzyme used in a particular process will depend on the specific process in use. However, given the discussion of the present, one skilled in the art would be able to easily find the appropriate concentration. For example, in the case of the hydrolysis of maltulose in a sugar solution with 20% dry solids containing 2% maltulose, the maltulose will be present in an amount of about 4 g / kg d.s. sugar. In this way, where one unit is equal to the hydrolysis of 1 μmol of maltulose / minute, 43 U / kg of syrup will be necessary to hydrolyse the maltulose in solution in 10 hours. Preferably, the hydrolyzing activity of added residual sugar, in this case maltulose, is about 10 U / kg of sugar ds, more preferably between 20 and 5000 U7kg of sugar ds, and more preferably of between 25 and 1000 U / kg of sugar ds In the present invention, the term "fermentative fermentation process" is defined as any production process comprising the cultivation of a microorganism to produce a desired product. Although the present invention is demonstrated with examples from the field of fermentative ethanol production, it should be appreciated that the invention is not limited to ethanol production, but can also be applied to other fermentative production processes where raw material-based saccharides are present. non-cellulosic and non-fermentable semi-cellulosic. Possible examples of such processes are the fermentative production of primary metabolites (such as ethanol and glycerol), organic acids (eg, lactic acid, acetic acid, succinic acid, etc.), amino acids, antibiotics (eg penicillin), yeasts, biomass to be used as a simple cellular protein, proteins (such as enzymes), vitamins, inks and steroids. In the present invention, the term "saccharide based on non-fermentable non-cellulosic and non-cellulosic raw materials" refers to any saccharide which does not originate from cellulose or semicellulose and which is not fermentable. Examples of such saccharides include isomaltose, maltulose, stachyose, raffinose, and melibiose. In this respect, the term "fermentable" refers to the capacity of the microorganism used in a fermentative production process (for example the use of the yeast S. cerevisiae to produce ethanol from glucose) to use those saccharides. The term "residual sugars" means the non-fermentable sugars present in the carbohydrate-based fermentation medium including isomaltose, maltulose, stachyose, raffinose, and melibiose. In the wet and dry milling process, for example, corn is used as the starting material, the residual sugars consist mainly of isomaltose and maltulose. A further aspect of the present invention ensures that the method for hydrolyzing non-fermentable saccharides is applied in an economically attractive form. The application of the enzymatic preparation for hydrolysis into non-fermentable saccharides in an immobilized form results in a drastic reduction in the amount of enzymes required as compared to the use of soluble enzymes. Suitable means of immobilizing enzymes are known in the art and include, for example, inclusion, binding, fixation in, para, or on carrier materials. The present invention contemplates the incorporation of a reaction with immobilized enzyme in the ethanol production process. The outline of the process is presented in Figures 1A and IB for the continuous production of ethanol by wet milling and in Figures 2A and 2B for batch ethanol production. In the production of ethanol of the type of wet milling, the method of the invention is advantageously used in a step having a low carbohydrate concentration to allow the efficient conversion of the residual sugars to glucose without the production of reversion products. In the production of ethanol of the batch type, the invention should also be used at low concentrations of carbohydrate, for example, at the end of fermentation. Thus, advantageously the present invention is used as a separate reactor passage during the fermentation process or as a combined step by applying the residual sugar hydrolyzing enzyme to the fermenter microorganism vessel. Additionally, the fermentation broth can be recycled from the fermenter and subjected to the method of the invention by returning the product stream back to the fermenter for the fermentation of the fermentable products released. The following examples are illustrative of the invention and should not be construed as limiting thereof.EXAMPLES Example 1 Detection of Maltulose Hydrolysis Activity The analysis of carbohydrates can be carried out by the CLAP method under the following conditions: Column: carbohydrate column (Waters Corp. part nr.84038) Eluent: Acetonitrile / water (80/20 for the separation of the different mono- and disaccharides or 65 / 35 for the separation of the oligosaccharides). Flow: 2 ml / min. Room temperature. Detection: detection of the IR (refractive index). * (A) Preparation of Maltulose The maltulose is a disaccharide α-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 (aD-glucopyranosyl, 4-aglucopyranose) as follows: 2 g of aluminum oxide were mixed with 100 ml of a 40% maltose solution ( weight / volume). The pH was adjusted to pH 11.5 using sodium hydroxide. The reaction mixture was maintained at 60 ° C for 24 hours. The pH was then adjusted to pH 4.5 and 5 g of baking yeast were added to ferment the maltose and other fermentable sugars resulting from the alkaline incubation conditions (the maltulose is not fermented by the yeast). Finally, the reaction mixture is filtered to obtain a clear solution, which was concentrated under vacuum to remove the ethanol (resulting from the fermentation) and to obtain a solution of maltulose with a high content of dry solids.

(B) Enzymatic hydrolysis of maltulose The enzymatic hydrolysis of maltulose was investigated with several commercially available enzyme preparations. The enzymes were mixed with a solution of 5% maltulose in distilled water under the preferred conditions for the specific enzyme preparation. For each of the enzymes, 5 mg of enzyme was added to 5 ml of maltulose solution. The mixtures were incubated under the conditions listed in Table 1. The reaction mixtures were analyzed using CLAP as described above. The results of those analyzes are shown in Table 1 The results show that only T. reesei cellulase preparations and A. niger a-galactosidase contain a maltulose hydrolyzing activity.

Example 2 Enzymatic Hydrolysis of Stachyose A solution of 1% stachyose was incubated with a commercially available preparation of a-galactosidase and with a mixture of α-galactosidase and invertase to investigate enzymatic hydrolysis of stachyose by these preparations. The incubation was carried out at 55 ° C and pH = 5.5. Samples were taken 2 hours after the incubation time and analyzed by means of CLAP. Results are shown in table 2.

Table 2 The results show that stachyose can be hydrolyzed in fermentable monosaccharides.

Example 3 The Enzymatic Hydrolysis of Raffinose A 1% raffinose solution was incubated with a commercially available preparation of α-galactosidase and with a mixture of commercially available preparations of α-galactosidase and invertase, to investigate the enzymatic hydrolysis of raffinose by these preparations. The incubation was carried out at 55 ° C and pH = 5.5. Samples were taken 2 hours after the incubation time and analyzed by means of CLAP. The results are shown in Table 3.

Table 3 The results demonstrate that raffinose can be hydrolyzed in fermentable monosaccharides.

Example 4 The Enzymatic Hydrolysis of Melibiose A 1% melibose solution was incubated with a commercially available preparation of α-galactosidase to investigate the enzymatic hydrolysis of melibiose by this preparation. The incubation was carried out at 55 ° C and pH = 5.5.

Samples were taken 3 hours after the incubation time and analyzed by means of CLAP. The results are shown in Table 4. Table 4 The results show that melibiose can be hydrolyzed in fermentable monosaccharides.

Example 5 The Enzymatic Hydrolysis of Residual Sugars in the Beer of Ethanol Production by Wet Milling (Starch as Matera Prima) The beer resulting from fermentation in the ethanol production process by wet milling was incubated with different commercially available enzyme preparations to investigate whether enzymatic hydrolysis appears to be a general feature of many preparations and does not specify any of the major components in the process. mixture. The mixture was incubated at 33 ° C and pH = 4.0. The analysis was carried out through CLAP (column: HPX-87H from Bio-rad, eluent: H2S04 0.005 M, flow: 0.6 ml / min, temperature: 65 ° C, IR detection). Representative chromatograms of the starting material (Figure 3A) and after the enzyme treatment (α-galactosidase; SUMIZYME AGS) (Figure 3B) can be seen in the attached Figures. The products of the incubation are shown in Table 5. Table 5 The results shown in Table 5 demonstrate that beer from the production of ethanol by wet milling contains residues (non-fermentable) that can not be hydrolyzed by the α-glucosidase into fermentable monosaccharides. In contrast, other enziotic preparations are capable of hydrolyzing some of these monosaccharide residues.

Example 6 The Enzymatic Hydrolysis of Residual Sugars in the Beer of Ethanol Production by Wet Grinding and Fermentation of the Derived Monosaccharides Aliquots of beer were incubated from the ethanol production process by wet milling at 33 ° C and pH = 4.0 with a mixture of amyloglucosidase (AMIGASE from Gist-brocades), xylanase (LYXASAN from Gist-brocades), pectinase (RAPIDASE C80 by Gist-brocades), and two different α-galactosidases (SUMIZYME AGS and AC, both from Shin Nihon) to hydrolyse the residual sugars (oligosaccharides). A quantity of yeast was subsequently added to investigate whether the released monosaccharides had been fermented. The results are shown in Table 6.

The representative chromatograms of the beer, treated with the enzymatic cocktail (Figure 3C) and subsequently treated with yeast (Figure 3D) can be seen in the attached Figures.

Table 6 The data in Table 6 demonstrate that the enzyme mixture released a significant amount of monosaccharides from the residual sugar. Surprisingly, the ethanol level also increased. However, this can be explained by the fact that often beer could contain small yeast cells that are capable of converting monosaccharides to ethanol as soon as those monosaccharides are produced. The addition of yeast to the enzymatic treatment often results in a higher ethanol level, however, the fermentation time was too short to ferment all the monosaccharides present.

Example 7 The Enzymatic Hydrolysis of Residual Sugars in the Beer of Ethanol Production by Wet Grinding Using an Immobilized Enzyme Mixture and Fermentation of Released Monosaccharides Α-Galactosidase (SUMIZYME AGS, Shin Nihon) was immobilized using the procedure described in US Patent No. 3,838,007. 10 g of this immobilized enzyme were incubated with 100 ml of beer from the production of ethanol by wet grinding and incubated for 24 hours at 33 ° C and pH 4.0. The reaction mixture was then filtered and 5 g of yeast was added to the filtrate to ferment the liberated monosaccharides. The results are shown in Table 7.

Table 7 The results demonstrate that using an immobilized enzyme system, an effect similar to that compared to the use of a soluble enzyme can be achieved (see Example 6).

Example 8 Purification of the Hydrolyzing Activity of Maltulose and Residual Sugar of SUMIZYME AGS and Measurement of Molecular Weight The a-galactosidase preparation (SUMIZYME AGS, Shin Nihon, Japan) was partially purified using gel filtration chromatography. The procedure is described later. The fractions collected were selected for the presence of different activities.

The fractions of interest were gathered to determine the specific activity. Chromatographic procedure: Column: 58 x 2.5 cm. Support material: Sephacryl S 200 HR. Elution buffer: 50 mM acetate buffer, pH = 4.5 including 0.02% sodium azide. Flow rate: 2 ml / min. Detection: UV 280 nm. Collection of the fraction: fractions of 2 minutes. Sample: 4 ml of SUMIZYME AGS (lot 60902-02) 30 mg / ml in elution buffer.

Activity detection (selection of fractions): 1. Detection of a-galactosidase. 100 μl of 1 mM paranitrophenyl-α-D-galactose in 50 mM acetate buffer pH 5.5 was incubated with 100 ml of the collected fraction. After 3 minutes of incubation at room temperature, 100 μl of 0.0625 M borax buffer pH 9.7 was added to stop the reaction. The yellow color, resulting from paranitrophenol, was a measure of the activity of a-galactosidase. The results were judged visually.

Detection of the hydrolyzing activity of maltulose.

The maltulose preparation was diluted 4 times with distilled water. 100 μl of this solution was mixed with 200 μl of a fraction having maltulose hydrolyzing activity and 700 μl of distilled water. This mixture was incubated for 3 hours at 33 ° C. The mixtures were then placed in a boiling water bath to inactivate the enzyme. Finally, the mixtures were analyzed on CLAP using the Bio-Rad HPX 87C column. The increase in the area of the fructose peak was a direct measure of activity.

Detection of the residual hydrolyzing activity of the residual sugar. 50 μl of a fraction having residual sugar hydrolyzing activity was mixed with 50 μl of beer (from the production of fuel ethanol by wet milling, from Pekin Energy, Pekin, Illinois) and incubated for 16 hours at 33 ° C. Next, 900 μl of 0.006 N sulfuric acid was added and the reaction mixture was centrifuged in an Eppendorf centrifuge. The supernatant was analyzed by means of CLAP on a BIO-Rad HPX 87H column. The decrease in residual sugar peak was a direct measure of activity.

Specific activity test: The specific activity was expressed as an activity value per mg protein per minute reaction time. 1. Determination of protein content.

The protein content was determined using the BCA method with bovine serum albumin as standard. 2. The activity of a-galactosidase.

A solution of p-nitrophenol in 10 mM was made in 50 mM sodium acetate buffer pH 5.5. this solution was diluted to 240-160-80-40 mM. 1 ml of these solutions was added to 2 ml of 0.80 mM p-nitrophenyl-α-D-galactopyranoside in acetate buffer. To this mixture was added 5 ml of 625 mM borax buffer pH 9.7 (stop reagent). The OD of these solutions was measured at 405 nm against water (standard curve). Incubation of the enzyme: 1 ml of diluted enzyme solution in place of p-nitrophenol. The mixture was incubated for 15 minutes at 37 ° C. The reaction was stopped by adding 5 ml of borax solution. The OD was measured as mentioned above. Definition of activity: an α-galactosidase unit is the amount of enzymes that hydrolyzes 1 μmol of p-NPGal / minute under standard conditions. The hydrolyzing activity of maltulose: 100 μl of maltulose solution was mixed with 200 μl of enzyme solution and 700 μl of distilled water. The mixture was incubated at 33 ° C. Samples were taken at different incubation times and analyzed by CLAP to determine the amount of hydrolyzed maltulose. The hydrolyzing activity of the residual sugar 100 μl of beer from the production of wet-produced ethanol was mixed with 200 μl of enzyme solution and incubated at 33 ° C. 700 μl of 8 mM phosphoric acid was added to the samples taken at different incubation times. The samples were analyzed by CLAP to determine the amount of residual hydrolyzed sugar.

Definitions of the specific activity: c, 1. a-galactosidase: units of a-gal per mg of protein. 2. Hydrolyzing activity of maltulose: μg of hydrolyzed maltulose per mg of protein per minute. 3. Hydrolyzing activity of residual sugar: μg of 0 residual sugars hydrolyzed per mg of protein per minute.

Determination of molecular weight: standard commercially available protein mixtures (Bio-Rad) were used as markers of known molecular weight: Tyroglobulin (MW = 670 kD), gappta-qlobulin (MW = 158 kD), ovalbumin (MW = 44 kD), myoglobin (MW = 17 kD), vitamin B12 (MW = 13.5 kD). Gel filtration of the markers with the subsequent comparison with the maltulase and the residual sugar hydrolyzing enzyme resulted in an approximate molecular weight of about 132 kD and 120 kD respectively.

Results: The chromatogram resulting from the gel filtration of the α-galactosidase preparation is presented in Figure 4. The results of testing the fractions in the presence of a-galactosidase activity, the hydrolyzing activity of maltulose and the hydrolyzing activity of the residual sugar are presented in the following Table 8. The results were used for the collected fractions. The pooled a-galactosidase was contained in fractions 8-10 (elution time = 55-60 minutes). The hydrolyzing activity of the residues and the assembled maltulose was contained in fractions 12-14 (elution time = 63-68 minutes). The pooled fractions and the starting material were tested for the specific activity. The results are shown in Table 9.

Table 8 Table 9 Discussion / conclusions: Tables 8 and 9 show that the hydrolyzing activity of maltulose and residual sugar are side activities in the preparation of a-galactosidase and are not due to a-galactosidase itself. Furthermore, it seems that the specific activity of both enzymes can be significantly increased by means of a simple purification step.

Example 9 Thermal inactivation of a-galactosidase activity A solution of α-galactosidase was heated for 30 minutes at 65 ° C. Next, the starting material and the thermally treated solution were tested to determine the specific activity. The results of this experiment are listed in Table 10.

Table 10 The results show that the activity of α-galactosidase and the hydrolyzing activity of residual and maltulose sugar come from different enzymes.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (10)

1. A fermentative production process, characterized in that it comprises the step of hydrolyzing saccharides based on non-cellulose raw materials and non-fermentable non-cellulosic raw materials using an enzyme preparation capable of the same.

2. The fermentative production process according to claim 1, characterized in that at least one of the saccharides based on non-cellulose raw materials and non-fermentable non-cellulosic raw materials is selected from the group consisting of maltulose, stachyose, raffinose, melibose and isomaltose.

3. The fermentative production process according to claim 1, characterized in that the fermentative process is for the production of primary metabolites.

4. The process according to claim 3, characterized in that the primary metabolite is ethanol.

5. The process according to claim 1, characterized in that the enzyme preparation comprises a maltulose hydrolyzing activity or a residual sugar hydrolyzing activity.

6. The process according to claim 1, characterized in that the enzyme preparation is applied in immobilized form.

7. The process according to claim 5, characterized in that the enzyme preparation further comprises at least one enzyme selected from the group consisting of pectinase, glucoamylase, cellulase, α-galactosidase, xylanase (semicellulase), fungal amylase and phytase.

8. The process according to claim 1, characterized in that it comprises an enzyme that exhibits maltulose hydrolyzing activity or residual sugar hydrolyzing activity, which is an enzyme derived from a fungal source.

9. A wet grinding ethanol plant, characterized in that it comprises a fermentor, and further comprises, an enzymatic reactor for the enzymatic hydrolysis of saccharides based on non-cellulose and non-cellulosic raw materials, wherein the hydrolyzed saccharides are recycled or fed to the fermenter.

10. A fermentation plant for the production of batch ethanol, characterized in that it comprises an enzymatic reactor for the hydrolysis of saccharides based on non-cellulose and non-cellulosic raw materials, and where the fermentation broth is recycled through the enzymatic reactor during fermentation .