Method for producing disaccharides using beta-glucosidase and its cofactors, and composition for inducing enzyme production in Trichoderma sp. strains containing the produced disaccharides

Abstract

The present invention relates to a method for producing disaccharides using beta-glucosidase and its cofactors, and a composition for inducing enzyme production in a Trichoderma strain, which contains the produced disaccharides. Specifically, the method comprises inducing beta-glucosidase and a divalent metal ion, manganese ion (Mn ion), as a cofactor, on a glucose substrate. 2+ ), magnesium ion (Mg 2+ ), zinc ion (Zn 2+ ) or copper ions (Cu 2+ ), the disaccharide production rate can be increased and disaccharides can be obtained at a high concentration. In addition, since the composition with a high content of disaccharides produced by the production method of the present invention has an excellent effect of inducing enzyme production in Trichoderma strains, it is possible to improve the enzyme productivity of Trichoderma by applying a fermentation process that utilizes the produced disaccharides at a high concentration.

Description

[Technical Field]

[0001] [Cross-Citation of Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0168329 dated November 30, 2021 and Korean Patent Application No. 10-2021-0168330 dated November 30, 2021, and all contents disclosed in the documents of said Korean patent applications are incorporated herein by reference.

[0002] The present invention relates to a method for producing disaccharides using beta-glucosidase and its cofactors, and a composition for inducing enzyme production in Trichoderma strains, containing the produced disaccharides. Specifically, the present invention relates to a method for increasing the rate of production of disaccharides, such as gentiobiose or sophorose, from glucose by adding beta-glucosidase and a divalent metal ion as a cofactor, thereby obtaining disaccharides at a high concentration. The present invention also relates to a composition for promoting enzyme production in Trichoderma strains, which is rich in the produced disaccharides, and a method for producing the enzymes using the composition. [Background technology]

[0003] Sophorose is a disaccharide consisting of two molecules of D-glucopyranosyl 2-O-β bond, and is a type of maltose-type disaccharide.

[0004] Gentiobiose, also known as amygdalose, is a disaccharide consisting of two D-glucose molecules linked by a β-bond. It is a white crystalline solid that dissolves in water or methanol and can be hydrolyzed with acid or β-glucosidase to produce two D-glucose molecules.

[0005] Disaccharides such as sophorose and gentiobiose are known to be enzyme inducers in Trichoderma reesei.

[0006] One useful method for producing disaccharides is the use of enzymatic reverse hydrolysis. Beta-glucosidase is a typical enzyme used in methods for oligosaccharide synthesis. Beta-glucosidase hydrolyzes the beta-1,4-glycosidic bond between two glucose molecules or between a glucose and another substituted molecule to release glucose. It is a type of exocellulase that exhibits specificity for various beta D-glucoside substrates. However, enzymatic reverse hydrolysis has a problem in that the equilibrium is biased toward hydrolysis, resulting in low conversion efficiency from monosaccharides to disaccharides, limiting large-scale production.

[0007] Cellulases produced by many microorganisms are inducible enzymes, and cellulose, cellobiose, lactose, or sophorose have been reported as good inducers (Antonov et al. Microb Cell Fact (2016) 15:164).

[0008] The process of cellulolytic enzyme biosynthesis is known to be regulated by induction and catabolite repression. Specifically, sophorose, gentiobiose, and laminaribiose are known to induce cellulase synthesis in the Penicillium purpurogenum P-26 strain (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, January 1992, pp. 106-110), and trehalose is known to induce cellulase synthesis in the Clostridium papyrosolvens CFR-703 strain (Process Biochemistry 37 (2001) 241-245). In addition, the activity of cellulose-degrading enzymes is also regulated by end product inhibition: cellulase is inhibited by cellobiose, and in Thermonospora fusca and Trichoderma reesei, β-glucosidase is inhibited by glucose. As a result of these complex regulatory processes, the degree of synthesis and components of cellulose-degrading enzymes synthesized vary greatly depending on the type of microorganism and culture conditions. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent Application No. 10-2021-0168329 [Patent Document 2] Korean Patent Application No. 10-2021-0168330 [Non-patent literature]

[0010] [Non-Patent Document 1] Antonov et al.Microb Cell Fact(2016)15:164 [Non-patent document 2] APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Jan.1992,p.106-110 [Non-patent document 3] Process Biochemistry 37(2001)241-245 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a composition comprising 1-hydroxyglucitol-D-glucopyranoside or an isomer thereof.

[0012] Another object of the present invention is to 1) contacting glucose with beta-glucosidase; and 2) contacting the reactant of step 1 with a divalent metal ion; and a method for producing a composition for inducing enzyme production in a microorganism or increasing enzyme activity in a microorganism, the method comprising the steps of:

[0013] It is yet another object of the present invention to provide a composition for inducing enzyme production in a Trichoderma strain, which comprises the disaccharides trehalose, isomaltose, gentiobiose, cellobiose, sophorose, and maltose.

[0014] It is still another object of the present invention to provide a method for producing an enzyme from a Trichoderma strain, using a composition for inducing enzyme production from a Trichoderma strain, which comprises the disaccharide.

[0015] It is yet another object of the present invention to provide the use of a composition rich in the disaccharides trehalose, isomaltose, gentiobiose, cellobiose, sophorose, and maltose to induce enzyme production. [Means for solving the problem]

[0016] Each description and embodiment disclosed in the present invention also applies to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention are within the scope of the present invention. Furthermore, the specific descriptions set forth below are not to be construed as limiting the scope of the present invention. Furthermore, those skilled in the art will be able to recognize or ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. Furthermore, these equivalents are intended to be encompassed by the present invention.

[0017] In one aspect, the present invention provides 1) contacting glucose with beta-glucosidase; and 2) contacting glucose or the reactant of step 1) with a divalent metal ion.

[0018] The "glucose" in step 1) may be in the form of, but is not limited to, liquid glucose or an aqueous glucose solution. The glucose of the present invention may have a concentration of 65-77% w / w, 67-75% w / w, or 69-73% w / w.

[0019] The beta-glucosidase may be derived from the genus Trichoderma. Specifically, the beta-glucosidase of the present invention may be derived from Trichoderma reesei. More specifically, the beta-glucosidase of the present invention may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0020] The microorganism refers to a microorganism that produces an enzyme, and may be a microorganism that produces cellulase, xylanase, or phytase enzymes, or may be a microorganism that can produce all of cellulase, xylanase, and phytase enzymes, but is not limited thereto. In one specific example, the microorganism may be a Trichoderma strain, more specifically a Trichoderma reesei strain.

[0021] Beta-glucosidase may be, but is not limited to, 1,300 to 2,000 U / ml, 1,400 to 1,900 U / ml, 1,500 to 1,800 U / ml, or 1,550 to 1,740 U / ml.

[0022] The divalent metal ion is manganese ion (Mn 2+ ), magnesium ions (Mg 2+ ), zinc ions (Zn 2+ ) and copper ions (Cu 2+ ), and zinc ions (Zn 2+ ) and copper ions (Cu 2+ The compound may be, but is not limited to, one or more selected from the group consisting of:

[0023] The term "contacting step" in step 2) may be used herein in the same sense as the terms "standing step" or "reacting step."

[0024] The "contact" in step 2) may be performed at, but is not limited to, 20 to 50 rpm, 25 to 45 rpm, or 28 to 42 rpm.

[0025] The "contact" in step 2) may be performed at a pH of 3.0 to 7.0, pH 3.5 to 6.5, pH 4.0 to 6.0, or pH 4.5 to 5.5, but is not limited thereto.

[0026] The "contact" in step 2) may be carried out at a temperature of 50°C to 70°C, 55°C to 65°C, or 57°C to 63°C, but is not limited thereto.

[0027] The "contact" in step 2) may be carried out for 5 days or more, 6 days or more, 7 days or more, 5 to 22 days, or 6 to 21 days, but is not limited to these.

[0028] The produced composition may contain, but is not limited to, any one or more selected from the group consisting of trehalose, isomaltose, cellobiose, maltose, gentiobiose, sophorose, 1-hydroxyglucitol-D-glucopyranoside, and isomers thereof.

[0029] The compositions produced may include, but are not limited to, gentiobiose and sophorose.

[0030] The produced composition has an excellent effect of inducing enzyme production or fermentation activity of the produced enzyme from Trichoderma strains.

[0031] Additionally, the method of producing the composition of the present invention can further comprise the step of performing chromatography involving divalent cations.

[0032] The chromatography of the present invention may be simulated moving bed (SMB) chromatography.

[0033] In the chromatography of the present invention, the divalent cations may be contained in the form of a column packed with a cation exchange resin.

[0034] In the chromatography of the present invention, the divalent cation may be one or more of calcium ions, barium ions, and strontium ions.

[0035] The simulated moving bed chromatography of the present invention may include eight or more columns.

[0036] The simulated moving bed chromatography of the present invention may be one in which elution is carried out with water at 50° C. to 70° C. Specifically, the elution of the present invention can be carried out with water at 55° C. to 65° C. or 58° C. to 62° C.

[0037] In another aspect, the present invention provides (1S)-1-hydroxyglucitol-D-glucopyranoside having the structure of Chemical Formula 1 below:

[0038] [ka]

[0039] In another aspect, the present invention provides 1-epi-1-hydroxyglucitol-D-glucopyranoside having the structure of Chemical Formula 2 below.

[0040] [ka]

[0041] In yet another aspect, the present invention provides a composition containing 1-hydroxyglucitol-D-glucopyranoside or an isomer thereof.

[0042] The composition may induce the production of an enzyme in a microorganism and / or increase the activity of an enzyme produced by a microorganism.

[0043] Specifically, 1-hydroxyglucitol-D-glucopyranoside or its isomer may be (1S)-1-hydroxyglucitol-D-glucopyranoside or 1-epi-1-hydroxyglucitol-D-glucopyranoside.

[0044] In one aspect, the present invention provides a composition for inducing the production of a disaccharide-rich enzyme.

[0045] The disaccharide may be any one or more selected from the group consisting of trehalose, isomaltose, gentiobiose, cellobiose, sophorose, maltose, 1-hydroxyglucitol-D-glucopyranoside, and isomers thereof.

[0046] In one aspect, the present invention provides a composition for inducing the production of enzymes in microorganisms, including trehalose, isomaltose, gentiobiose, cellobiose, sophorose, and maltose.

[0047] As used herein, "induction of enzyme production" means inducing the amount of enzyme produced by a microorganism, and "induction of enzyme production" can be confirmed by measuring the fermentation activity of the produced enzyme.

[0048] The microorganism may be a Trichoderma strain, or a Trichoderma reesei strain, more specifically, a Trichoderma reesei QM6a strain, but is not limited thereto.

[0049] The enzyme may be an enzyme produced by a Trichoderma strain, or an enzyme produced by a Trichoderma reesei strain, and more specifically, may be an enzyme produced by a Trichoderma reesei QM6a strain, but is not limited thereto.

[0050] The enzyme may be a cellulase, a xylanase or a phytase, preferably a xylanase.

[0051] The composition for inducing enzyme production in a Trichoderma strain may further comprise 1-hydroxyglucitol-D-glucopyranoside or an isomer thereof.

[0052] 1-hydroxyglucitol-D-glucopyranoside or its isomer may be (1S)-1-hydroxyglucitol-D-glucopyranoside or 1-epi-1-hydroxyglucitol-D-glucopyranoside.

[0053] 1-hydroxyglucitol-D-glucopyranoside or its isomers are 1) contacting glucose with beta-glucosidase; and 2) contacting glucose or the reactant of step 1 with a divalent metal ion.

[0054] The composition may be low in monosaccharides and high in disaccharides.

[0055] The composition may have a higher disaccharide content than the monosaccharide content of the composition, and the increased disaccharide content may be achieved by simulated moving bed (SMB) chromatography.

[0056] The monosaccharide content in the composition may be 45% by weight or less, 40% by weight or less, 35% by weight or less, 30% by weight or less, 20% to 45% by weight, 25% to 45% by weight, 20% to 40% by weight, 25% to 40% by weight, 20% to 35% by weight, 25% to 35% by weight, 20% to 30% by weight, or 25% to 30% by weight, based on the total weight of the composition.

[0057] The content of disaccharides in the composition is 27% by weight or more, 30% by weight or more, 33% by weight or more, 36% by weight or more, 39% by weight or more, 27% to 50% by weight, 30% to 50% by weight, 33% to 50% by weight, 36% to 50% by weight, 39% to 50% by weight, 27% to 45% by weight, 30% to 45% by weight, 33% to 45% by weight, 45 ... It may be 5% by weight, 36% to 45% by weight, 39% to 45% by weight, 27% to 42% by weight, 30% to 42% by weight, 33% to 42% by weight, 36% to 42% by weight, 39% to 42% by weight, 27% to 40% by weight, 30% to 40% by weight, 33% to 40% by weight, 36% to 40% by weight, or 39% to 40% by weight.

[0058] The trehalose content of the composition may be 2.5% by weight or more, 3% by weight or more, 4% by weight or more, 2.5% to 6% by weight, 3% to 6% by weight, 4% to 6% by weight, 2.5% to 5% by weight, 3% to 5% by weight, or 4% to 5% by weight, based on the total weight of the composition.

[0059] The content of isomaltose in the composition may be 4.5% by weight or more, 5% by weight or more, 4.5% to 7% by weight, 5% to 7% by weight, 4.5% to 6% by weight, or 5% to 6% by weight, based on the total weight of the composition.

[0060] The content of gentiobiose in the composition may be 15% by weight or more, 17% by weight or more, 19% by weight or more, 21% by weight or more, 15% to 28% by weight, 17% to 28% by weight, 19% to 28% by weight, 21% to 28% by weight, 15% to 25% by weight, 17% to 25% by weight, 19% to 25% by weight, 21% to 25% by weight, 15% to 23% by weight, 17% to 23% by weight, 19% to 23% by weight, or 21% to 23% by weight, based on the total weight of the composition.

[0061] The content of cellobiose in the composition may be 1.9 wt % or more, 2.0 wt % or more, 2.1 wt % or more, 2.2 wt % or more, 1.9 wt % to 3.0 wt %, 2.0 wt % to 3.0 wt %, 2.1 wt % to 3.0 wt %, 2.2 wt % to 3.0 wt %, 1.9 wt % to 2.7 wt %, 2.0 wt % to 2.7 wt %, 2.1 wt % to 2.7 wt %, 2.2 wt % to 2.7 wt %, 1.9 wt % to 2.5 wt %, 2.0 wt % to 2.5 wt %, 2.1 wt % to 2.5 wt %, or 2.2 wt % to 2.5 wt %, based on the total weight of the composition.

[0062] The content of sophorose in the composition may be 1.4 wt % or more, 1.5 wt % or more, 1.6 wt % or more, 1.7 wt % or more, 1.4 wt % to 3.0 wt %, 1.5 wt % to 3.0 wt %, 1.6 wt % to 3.0 wt %, 1.7 wt % to 3.0 wt %, 1.4 wt % to 2.5 wt %, 1.5 wt % to 2.5 wt %, 1.6 wt % to 2.5 wt %, 1.7 wt % to 2.5 wt %, 1.4 wt % to 2.0 wt %, 1.5 wt % to 2.0 wt %, 1.6 wt % to 2.0 wt %, or 1.7 wt % to 2.0 wt %, based on the total weight of the composition.

[0063] The content of maltose contained in the composition may be 2.6 wt % or more, 3.0 wt % or more, 3.4 wt % or more, 3.7 wt % or more, 2.6 wt % to 5.0 wt %, 3.0 wt % to 5.0 wt %, 3.4 wt % to 5.0 wt %, 3.7 wt % to 5.0 wt %, 2.6 wt % to 4.6 wt %, 3.0 wt % to 4.6 wt %, 3.4 wt % to 4.6 wt %, 3.7 wt % to 4.6 wt %, 2.6 wt % to 4.2 wt %, 3.0 wt % to 4.2 wt %, 3.4 wt % to 4.2 wt %, 3.7 wt % to 4.2 wt %, 2.6 wt % to 3.9 wt %, 3.0 wt % to 3.9 wt %, 3.4 wt % to 3.9 wt %, or 3.7 wt % to 3.9 wt %, based on the total weight of the composition.

[0064] The composition can further include an oligomer.

[0065] The content of the oligomer in the composition may be, but is not limited to, 29% to 35% by weight, 29% to 34% by weight, 29% to 33% by weight, 30% to 35% by weight, 30% to 34% by weight, 30% to 33% by weight, 31% to 35% by weight, 31% to 34% by weight, 32% to 35% by weight, or 32% to 34% by weight, based on the total composition content.

[0066] In one example of the present specification, it was confirmed that a composition having a low monosaccharide content and a high disaccharide content (SMB isolate) exhibited superior enzyme fermentation activity compared to a composition having a high monosaccharide content and a low disaccharide content (TGS). Therefore, compositions with high contents of trehalose, isomaltose, gentiobiose, cellobiose, sophorose, and maltose can be used to induce enzyme production in microorganisms. [Effects of the Invention]

[0067] The present invention relates to a method for producing disaccharides using beta-glucosidase and its cofactors, and a composition for inducing enzyme production in Trichoderma strains, including the produced disaccharides. Specifically, the method involves inducing beta-glucosidase and its cofactors, such as divalent metal ions such as manganese ions (Mn 2+ ), magnesium ions (Mg 2+ ), zinc ions (Zn 2+ ) or copper ions (Cu 2+ ), etc., can be added to increase the disaccharide production rate and obtain high concentrations of disaccharides. Furthermore, the disaccharide-rich composition produced by the production method of the present invention has an excellent effect of inducing enzyme production in Trichoderma strains, so that by applying a fermentation process that utilizes the produced disaccharides in high concentrations, it is possible to improve the enzyme productivity of Trichoderma. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention will be described in more detail below through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0069] Example 1. Preparation of a disaccharide composition Glucose powder (hydrated crystalline glucose, DAEJUNG CHEMICAL & METALS) with a purity of 95% or more was dissolved using steam to prepare 40 kg of glucose solution with a concentration of 71% w / w. 0.3 kg of β-glucosidase (SEQ ID NO: 1) at a concentration of 1,650 U / ml was added to the prepared glucose solution, and the solution was left to stand at 60°C for 7 to 21 days to prepare a composition containing disaccharides (Treated Glucose Syrup, TGS).

[0070] Example 2. Preparation of a disaccharide composition using the addition of a divalent metal cation To the glucose solution of Example 1, 0.3 kg of β-glucosidase (same as in Example 1) was added at a concentration of 1,650 U / ml, and divalent ions [manganese ions (Mn 2+ ), magnesium ions (Mg2+ ), zinc ions (Zn 2+ ) and copper ions (Cu 2+ ) each] was added at 1 mM, and the mixture was allowed to stand at 60°C for 7 to 21 days to produce a composition containing disaccharides.

[0071] Example 3. Confirmation of the increase in the disaccharide concentration contained in the disaccharide composition and the rate of increase or decrease of disaccharides by adding divalent metal cations The concentrations (g / L) and percentage changes (%) of the disaccharides trehalose, isomaltose, maltose, cellobiose, gentiobiose, and sophorose were measured for the compositions prepared in Examples 1 and 2, and are shown in Tables 1 and 2. The concentrations were analyzed using a Bio-LC system (Dionex ICS-3000, Sunnyvale, CA, United States) equipped with an electrochemical detector and a CarboPac PAI column.

[0072] [Table 1]

[0073] [Table 2]

[0074] Example 4. Separation of high-purity polysaccharides using SMB chromatography Simulated moving bed (SMB) chromatography was used to remove monosaccharides from the composition prepared in Example 2. SMB chromatography was performed using a Sequential Simulated Moving-Bed System (Novasep, France), which included eight serially connected columns (each 95 cm high and 2.5 cm in diameter), a feed pump, a recirculation pump, an eluent pump, a heat exchanger, and a valve for flow control. The operating conditions for SMB chromatography are shown in Table 3 below, and the results of the SMB chromatography separation experiment are shown in Table 4. The recovery rate was measured by comparing the amounts of disaccharides and trisaccharides injected into the mixture (TGS) with the amounts of disaccharides and trisaccharides in the raffinate using SMB chromatography, and the recovery rate was calculated using the following formula:

[0075] Recovery rate (%) = Raffinate (sugar content) / Feed (sugar content) x 100 *Sugar content = total amount of disaccharides and polysaccharides

[0076] [Table 3]

[0077] [Table 4]

[0078] As a result of the separation experiment using SMB chromatography, as shown in Table 4, it was confirmed that the purity of disaccharides and trisaccharides in the raffinate fraction using calcium ions was 81 to 89%, and the recovery rate was at the 89% level.

[0079] Example 5. NMR analysis of the produced disaccharide components Acetylation was performed for NMR analysis of the composition prepared in Example 2. The reagents used were acetic anhydride (extra pure, Junsei Chemical Co.) and zinc chloride (Kanto Chemical Co.), and the equipment conditions for separation and analysis of the acetylated sample are shown in Table 5 below.

[0080] [Table 5]

[0081] As a result, the presence of the conventional sugars trehalose, isomaltose, gentiobiose, cellobiose, maltose, sophorose, and maltose was confirmed in the analyzed sample, as well as the presence of a novel sugar, (1S)-1-hydroxyglucitol-D-glucopyranoside or 1-epi-1-hydroxyglucitol-D-glucopyranoside, in the analyzed sample.

[0082] Example 6. Analysis of sugar content in glucose solution and SMB separated solution enriched in disaccharides from glucose solution The following experiment was carried out to analyze the sugar content of the TGS prepared in Example 2 and its SMB fraction. The SMB fraction was obtained by subjecting the disaccharide composition prepared in Example 2 to SMB chromatography, resulting in the raffinate fraction of Example 4.

[0083] Specifically, the monosaccharide and disaccharide concentrations were analyzed using a Bio-LC system (Dionex ICS-3000, Sunnyvale, CA, United States) equipped with an electrochemical detector and a Carbo Pac PAI column to analyze the sugar content, as shown in Table 6. The column was eluted with 0.1 M NaOH (0-5 min) at 30°C, followed by a linear gradient of sodium acetate (0-0.2 M) at 1 mL / min for 5-35 min.

[0084] [Table 6]

[0085] As a result, as shown in Table 6, the monosaccharide content of TGS was 45.08%, and the monosaccharide content of SMB isolate was 28.04%, confirming that the monosaccharide content of SMB isolate was lower than that of TGS. In addition, the disaccharide content of TGS was 26.36%, and that of SMB isolate was 39.37%, confirming that the disaccharide content of SMB isolate was higher than that of TGS.

[0086] Example 7. Confirmation of the enzyme production induction effect depending on the disaccharide content of the composition To confirm whether the SMB isolate has a significant effect in inducing enzyme production in Trichoderma sp. strains, the enzyme fermentation activity was measured using TGS and SMB isolate as follows.

[0087] Specifically, TGS or SMB isolated solution was added to the medium, and the Trichoderma strain was diluted to a cell concentration of 190 or 200 g / L. After culturing at pH 4 and 28°C for 168 hours, the acidic cellulase enzyme activity was measured and shown in Table 7 below.

[0088] More specific measurements of the enzyme fermentation activity are shown in Examples 7-1 to 7-3 below.

[0089] Example 7-1. Preparation of enzyme-producing strains We prepared the enzyme-producing strains, Trichoderma reesei QM6a (ATCC13631), capable of producing acid cellulase (4-β-D-glucan 4-glucanohydrolase, EC 3.2.1.4), xylanase (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8), and phytase (3-phytases, EC 3.1.3.8, 6-phytases, EC 3.1.3.26, and 5-phytases, EC 3.1.3.72). Each strain was grown on potato dextrose agar plates at 30°C for 5 days to allow sporulation. After sporulation, the spores were resuspended in sterile NaCl solution (9 g / L) and 30% sterile glycerol was added. The mixture was stored in a 1.8 ml tube at −80°C.

[0090] Example 7-2. Fed-batch culture of enzyme-producing strains The Trichoderma reesei QM6a strain producing each enzyme in Example 7-1 was cultivated using fed-batch culture. The carbon source in the feed medium was supplied at a constant rate of 5.0 g / h when DO increased. The feed medium was supplied using a peristaltic pump. The fermentation medium consisted of 20.0 g / L carbohydrate, 5.0 g / L ammonium sulfate, 1.5 g / L magnesium sulfate, 0.5 g / L calcium chloride, 5.0 g / L dipotassium phosphate, 10.0 g / L yeast extract, 10.0 mg / L ferric sulfate, 4.0 mg / L cobalt chloride, 2.0 mg / L sodium molybdate, 0.4 mg / L boric acid, and 1.0 g / L Tween 80. The feed medium consisted of 600.0 g / L of carbohydrates, 6.0 g / L of yeast extract, and 1.0 g / L of sodium chloride, with a carbon source to yeast extract weight ratio of 1:0.01. For each enzyme activity experiment, TGS and SMB filtrate were added to the medium as carbohydrates. Fermentation conditions consisted of maintaining a culture temperature of 28°C and a pH of 4.0 using aqueous ammonia. The agitation speed was adjusted to 600 rpm to avoid DO (dissolved oxygen) limitation. The seed culture was prepared by inoculating 1 ml of frozen spore mixture into 150 ml of medium in a 500 ml Erlenmeyer flask and pre-incubating at 28°C and 200 rpm for 72 hours on a shaker, followed by inoculation with 10% (v / v) of the culture volume.

[0091] Example 7-3. Acid cellulase activity analysis The fermentation broth obtained in Example 7-2 was centrifuged to remove T. reesei cells and other solid matter. The culture supernatant was appropriately diluted for enzyme assays. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to liberate 1 μmol of D-glucose in 1 minute under standard assay conditions (5 mg / mL CMC, pH 4.8, 50°C).

[0092] Cellulase hydrolyzes cellulose to produce monosaccharides and oligosaccharides under specific temperature and pH conditions. Oligosaccharides with reducing ends and monosaccharides with reducing groups undergo a color reaction with DNS reagent at high temperatures. The color intensity of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis and the amount and reaction of reducing sugars. The cellulase activity in the solution is proportional, and can be calculated by measuring the absorbance of the reaction solution spectrophotometrically.

[0093] [Table 7]

[0094] As a result, as shown in Table 7, when TGS was added, the average activity of acidic cellulase was 2822.5 IU / mL, and when SMB isolation solution was added, the average activity of acidic cellulase was 3890 IU / mL, confirming that the addition of SMB isolation solution increased the average activity of acidic cellulase by a further 37.8%.

[0095] Example 8. Confirmation of enzyme production induction effect of each enzyme type in SMB separated solution To confirm whether the effect of SMB isolate on inducing enzyme production in Trichoderma strains differs depending on the type of enzyme, the fermentation activity of xylanase and phytase was measured using TGS and SMB isolate as follows.

[0096] Specifically, TGS or SMB isolated solution was added to the medium, and the Trichoderma strain was diluted to a cell concentration of 240 or 250 g / L. After culturing at pH 4 and 28°C for 168 hours, the enzyme activities of xylanase and phytase were measured and are shown in Table 8 below.

[0097] More specifically, the strain preparation and culture processes were measured in the same manner as described in Examples 7-1 and 7-2, and the analysis of each enzyme activity was shown in Examples 8-1 and 8-2 below.

[0098] Example 8-1. Analysis of neutral xylanase activity The fermentation broth obtained in Example 7-2 was centrifuged to remove T. reesei cells and other solid materials. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to liberate 1 μmol of D-xylose in 1 minute under standard assay conditions (1% xylan, pH 6.5, 50°C).

[0099] Xylanase decomposes xylan into oligosaccharides and monosaccharides under specific temperature and pH conditions. Terminally reduced oligosaccharides and monosaccharides with reducing groups react colorimetrically with DNS reagent. The color intensity of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis, and the amount of reducing sugars produced is proportional to the activity of xylanase in the reaction solution. Therefore, xylanase activity can be calculated spectrophotometrically.

[0100] Example 8-2. Analysis of phytase activity The fermentation broth obtained in Example 7-2 was centrifuged to remove T. reesei cells and other solid matter. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activity using international units (IU) per mL of supernatant. 1 IU is the phytase activity unit at 37°C and pH 5.5, and is defined as the amount of enzyme required to liberate 1 μmol of inorganic phosphorus from a 5.0 mmol / L sodium phytate solution for 1 minute.

[0101] Under certain temperature and pH conditions, phytase completely hydrolyzes the substrate sodium phytate to produce orthophosphate and inositol derivatives. In acidic solutions, it can form a yellow compound with ammonium vanadium molybdate, which can be measured colorimetrically at a wavelength of 415 nm.

[0102] [Table 8]

[0103] As a result, as shown in Table 8, the average neutral xylanase activity was 105,376 IU / mL when TGS was added, and 144,716 IU / mL when SMB isolate was added, confirming that the addition of SMB isolate increased the neutral xylanase fermentation activity by an additional 37.3%. In addition, the average phytase activity was 25,312 IU / mL when TGS was added, and 26,668 IU / mL when SMB isolate was added, confirming that the addition of SMB isolate increased the phytase fermentation activity by an additional 5.4%.

Claims

1. A composition comprising (1S)-1-hydroxyglucitol-D-glucopyranoside or 1-epi-1-hydroxyglucitol-D-glucopyranoside.

2. The composition of claim 1, further comprising any one or more selected from the group consisting of trehalose, isomaltose, gentiobiose, cellobiose, sophorose, and maltose.

3. The composition of claim 1 , wherein the composition induces enzyme production in a microorganism and / or increases the activity of an enzyme produced by a microorganism.

4. The composition of claim 3 , wherein the microorganism is a Trichoderma strain.

5. The composition of claim 3 , wherein the microorganism is Trichoderma reesei.

6. The composition of claim 3 , wherein the enzyme is an acid cellulase, a xylanase, or a phytase.

7. The composition of claim 1 , wherein the composition has a higher disaccharide content than monosaccharide content.

8. The composition of claim 1 , wherein the disaccharide content of the composition is 30% by weight or more based on the total weight of the composition.

9. The composition of claim 1 , wherein the monosaccharide content of the composition is 30% by weight or less based on the total weight of the composition.

10. 1) contacting glucose with beta-glucosidase; and 2) contacting the glucose or the reactant of step 1) with one or more ions selected from the group consisting of manganese ions (Mn 2+ ), magnesium ions (Mg 2+ ), zinc ions (Zn 2+ ), and copper ions (Cu 2+ ).

11. The method of claim 10, wherein the beta-glucosidase is from the genus Trichoderma.

12. 11. The method of claim 10, wherein the beta-glucosidase is derived from Trichoderma reesei.

13. The method of claim 10, wherein the composition further comprises any one or more selected from the group consisting of trehalose, isomaltose, cellobiose, maltose, gentiobiose, and sophorose.

14. 11. The method of claim 10, further comprising performing chromatography including divalent cations.

15. 15. The method of claim 14, wherein the chromatography is simulated moving bed chromatography.

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