WO2016165520A1 - 肌醇的制备方法 - Google Patents

肌醇的制备方法 Download PDF

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WO2016165520A1
WO2016165520A1 PCT/CN2016/076526 CN2016076526W WO2016165520A1 WO 2016165520 A1 WO2016165520 A1 WO 2016165520A1 CN 2016076526 W CN2016076526 W CN 2016076526W WO 2016165520 A1 WO2016165520 A1 WO 2016165520A1
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inositol
cellulose
enzyme
phosphorylase
phosphate
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French (fr)
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张以恒
游淳
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张以恒
游淳
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Priority to KR1020177032707A priority Critical patent/KR102017024B1/ko
Priority to JP2017554491A priority patent/JP6685322B2/ja
Priority to EP16779495.7A priority patent/EP3305905B1/en
Publication of WO2016165520A1 publication Critical patent/WO2016165520A1/zh
Priority to US15/783,996 priority patent/US10597682B2/en
Priority to US16/291,377 priority patent/US11028414B2/en

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    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/0102—Cellobiose phosphorylase (2.4.1.20)
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    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01049—Cellodextrin phosphorylase (2.4.1.49)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03025—Inositol-phosphate phosphatase (3.1.3.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/02—Phosphotransferases (phosphomutases) (5.4.2)
    • C12Y504/02002—Phosphoglucomutase (5.4.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01001—Phosphorylase (2.4.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y505/00—Intramolecular lyases (5.5)
    • C12Y505/01—Intramolecular lyases (5.5.1)
    • C12Y505/01004—Inositol-3-phosphate synthase (5.5.1.4)

Definitions

  • the invention relates to a method for preparing inositol, in particular to a method for converting starch or cellulose and their derivatives into inositol by multi-enzyme catalysis in vitro, and belongs to the field of enzyme catalytic production of inositol.
  • Inositol also known as cyclohexanol
  • Inositol is one of the water-soluble vitamin B families. Inositol is an essential substance for the growth of humans, animals and microorganisms, and is widely used in medicine, food, feed and other industries.
  • the global demand is about 5,000 tons per year. Due to the current high price of inositol, the market prospect of inositol has not been fully developed. For example, the global feed production in 2013 was 960 million tons. With 0.5% inositol, the feed industry's required inositol production should reach 190-4.8 million tons per year. Under this circumstance, the current domestic and even global production is far from meeting the demand.
  • inositol is mainly due to the traditional high temperature pressure hydrolysis of phytic acid (inositol hexaphosphate).
  • the process equipment has strict material requirements, large one-time investment, and the operating pressure can only be controlled within a certain range, which limits the improvement of raw material utilization rate, and the crude product refining process is complicated, the loss is large, the production cost is high, and the process is high.
  • a large amount of phosphoric acid pollutants will be produced, which is serious to the water source and environmental pollution.
  • atmospheric pressure hydrolysis has been developed to reduce energy consumption and pollution.
  • the hotspots of inositol production are studied in the chemical synthesis method and the microbial enzymatic method.
  • problems such as high cost and low yield in different degrees.
  • the technical problem to be solved by the present invention is to provide an enzyme catalytic conversion method of inositol, which can produce inositol by multi-enzyme catalyzing starch or cellulose and their derivatives and glucose in vitro, which has inositol yield and transformation High rate, low production cost and no pollution.
  • the technical solution adopted by the present invention is:
  • the invention first discloses a preparation method of inositol, comprising the following steps:
  • glucan phosphorylase EC 2.4.1.1
  • glucose phosphatase EC 5.4.2.2
  • inositol-3 - Inositol-3-phophate synthase EC 5.5.1.4
  • inositol monophosphatase EC 3.1.3.25
  • the concentration of the substrate in the step (1) is 10 g/L; the amount of the glucan phosphorylase is 0.05 U/mL, and the amount of the glucose phosphate mutase is 1 U/mL, the muscle
  • the amount of the alcohol-3-phosphate synthase is 0.05 U/mL, and the amount of the inositol monophosphatase is 2 U/mL; the enzyme
  • the conditions for the catalytic reaction are a reaction at 40-80 ° C for 10-100 hours; preferably, a reaction at 60 ° C for 40 hours.
  • starch debranching enzyme and maltose phosphorylase (EC 2.4.1.8) or starch debranching enzyme and glucanotransferase (EC 2.4.1.25) to the multi-enzyme reaction system can effectively increase inositol Yield and conversion rate; preferably, the amount of the starch debranching enzyme is 1 U / mL, the amount of the maltose phosphorylase (EC 2.4.1.8) or glucanotransferase is 1 U / mL; Wherein the debranching enzyme is any one or two of isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41);
  • the concentration of the substrate is 10 g/L; the amount of the glucan phosphorylase is 5 U/mL, and the amount of the glucose phosphate mutase is 1 U/mL, the inositol-3 - the amount of phosphate synthase is 5 U / mL, the amount of the inositol monophosphatase is 2 U / mL, the amount of the starch debranching enzyme is 1 U / mL, the maltose phosphorylase or glucanotransferase The dosage is 1U/mL;
  • the enzyme catalyzes the reaction at 40-100 ° C for 10-100 hours; preferably, the enzyme catalyzes the reaction at 40-80 ° C for 40 hours; most preferably, the enzyme catalyzes the reaction at 80 ° C for 40 hours.
  • the redundant glucose is converted into inositol, and polyphosphate glucokinase (EC 2.7.1.63) and polyphosphate are added to the multi-enzyme reaction system; preferably, The polyphosphate glucokinase is used in an amount of 1 U/mL, and the polyphosphate is used in an amount of 10 mM; wherein the polyphosphate is preferably sodium polyphosphate.
  • the multi-enzyme reaction system further comprises the following components: a buffer solution, an inorganic phosphate, a divalent magnesium ion, and a zinc ion or a manganese ion; preferably, the amount of each component is: buffer 100 mM, inorganic phosphate 10 mM, bivalent Magnesium ions 5 mM, zinc ions or manganese ions 0.5 mM; wherein the buffer is preferably HEPES buffer, more preferably, the HEPES buffer has a pH of 7.2.
  • the residual starch residue will be pure amylose.
  • a small amount of alpha amylase (EC 3.2.1.1) can be added to promote the hydrolysis of the starch residue, further increasing the production of inositol.
  • the alpha amylase is used in an amount of 0.1 U/ml.
  • the invention adopts starch or starch derivative as a substrate, and adds glucan phosphorylase, glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase to prepare a multi-enzyme reaction system, and multi-enzyme catalysis
  • the pathway comprises: converting one glucose unit of the starch or starch derivative into glucose-1-phosphate by dextran phosphorylase; converting glucose-1-phosphate to glucose-6-phosphate by glucose phosphate mutase; Inositol-3-phosphate synthase converts glucose-6-phosphate to inositol-3-phosphate, which converts inositol-3-phosphate to inositol.
  • the two enzymes catalyzed by the conversion of glucose-6-phosphate to inositol-3-phosphate by inositol-3-phosphate synthase and inositol-3-phosphate by inositol monophosphatase are irreversible The reaction, so the enzyme catalytic system can obtain a high conversion rate. The high yield and high conversion rate can greatly reduce the separation cost of the final inositol.
  • amylopectin Since starch is a mixture of amylose (20-30%) and amylopectin (70-80%). Branches in amylopectin are linked to the backbone by alpha-1,6 glycosidic linkages, whereas dextran phosphorylase does not decompose alpha-1,6 glycosidic linkages.
  • the present invention is in a multi-enzyme reaction system A debranching enzyme, isoamylase or pullulanase, which is capable of decomposing the ⁇ -1,6 glycosidic linkage in starch, is added.
  • the present invention also adds maltose phosphorylase to the reaction system to decompose maltose into glucose-1-phosphate and glucose; more preferably, the present invention Adding polyphosphoric acid and polyphosphate glucokinase to the multi-enzyme reaction system, converting glucose to glucose-6-phosphate, converting into inositol by inositol-3-phosphate synthase and inositol monophosphatase, and finally starch and All of the glucose units in its derivatives are converted to inositol, thereby increasing the yield and conversion of inositol.
  • the maltose phosphorylase can be replaced by a 4-a-glucanotransferase (EC. 2.4.1.25), which can polymerize short-chain oligosaccharides into long-chain oligosaccharides, and Long-chain oligosaccharides can be reused by dextran phosphorylase to increase starch utilization.
  • a 4-a-glucanotransferase EC. 2.4.1.25
  • Long-chain oligosaccharides can be reused by dextran phosphorylase to increase starch utilization.
  • the starch of the present invention is preferably a soluble starch; the starch derivative comprises any one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, malt polysaccharide or maltose.
  • the invention also discloses another preparation method of inositol, comprising the following steps:
  • the invention uses cellulose as a substrate, and adds cellulase, fibrin phosphophosphorylase, cellobiose phosphorylase, glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase, and prepares more Enzyme reaction system
  • the multi-enzyme catalytic pathway comprises: hydrolyzing cellulose by cellulase to produce fibrin and cellobiose; and dextrose unit of fibrin or cellobiose by fibrin phosphophosphophosphorylase and cellobiose phosphorylase Conversion to glucose-1-phosphate; conversion of glucose-1-phosphate to glucose-6-phosphate by glucose phosphate mutase; conversion of glucose-6-phosphate to inositol-3- by inositol-3-phosphate synthase Phosphoric acid, which converts inositol-3-phosphate to inositol by inositol monophosphatas
  • step (1) preferably comprises first mixing 10 g/L of cellulose or cellulose derivative and 5 U/ml cellulase in an ice water bath, placing in an ice water bath for 5 minutes, and then centrifuging at 4 ° C to remove the supernatant. a mixture of cellulase and cellulose; this treatment is capable of removing almost all of the glucosidases in the commercial cellulase, thus preventing the glucosidase from hydrolyzing cellobiose to produce a large amount of glucose, so that the main hydrolyzate is Cellobiose and fiber polysaccharides.
  • the concentration of the mixture of cellulase and cellulose is 10 g/L; the amount of the fibrin-phosphorylphosphatase is 5 U/mL, and the amount of the cellobiose phosphorylase is 5 U.
  • the enzyme-catalyzed reaction is carried out at 20 to 100 ° C for 10 to 100 hours; preferably, at 40 to 80 ° C for 72 hours; most preferably, at 50 ° C for 72 hours.
  • the multi-enzyme reaction system in step (1) is added to polyphosphoglucose (EC 2.7.1.63) and polyphosphate; preferably, the polyphosphate glucokinase The amount is 5 U/mL, and the polyphosphate is used in an amount of 10 mM; wherein the polyphosphate is preferably sodium polyphosphate.
  • the multi-enzyme reaction system further contains the following components: a buffer, an inorganic phosphate, a divalent magnesium ion, and a zinc ion or a manganese ion; preferably, the amount of each component is used.
  • a buffer 100 mM, inorganic phosphate 10 mM, divalent magnesium ion 5 mM, zinc ion or manganese ion 0.5 mM; wherein the buffer is preferably 100 mM HEPES buffer; further preferably, the pH of the HEPES buffer It is 7.2.
  • the invention further adds polyphosphoric acid glucokinase and polyphosphoric acid in the multi-enzyme reaction system, and converts the final product glucose hydrolyzed into glucose-6-phosphate, and then inositol-3-phosphate synthase and inositol monophosphate.
  • the enzyme catalyzes the conversion to inositol, which ultimately converts all of the glucose units in the cellulose and its derivatives to inositol.
  • the cellulose derivative of the present invention comprises any one of a pretreated product of cellulose, a fibrous polysaccharide or a cellobiose;
  • the pretreated product of the cellulose of the present invention is preferably a product obtained by subjecting cellulose to a concentrated phosphoric acid treatment.
  • Any of the enzymes in the multi-enzyme reaction system of the present invention may be replaced by any enzyme having an equivalent function, preferably an equally functional mutant enzyme obtained by protein engineering.
  • isoamylase, maltose phosphorylase, polyphosphate glucokinase and sodium polyphosphate are additionally added, and the amount of glucan phosphorylase and inositol-3-phosphate synthase is increased, and finally the inositol is converted.
  • the in vitro multi-enzyme catalyzed by the invention converts cellulose into an inositol experiment.
  • a microcrystalline cellulose (Avicel) is used as a substrate, and cellulase, fibrin phosphate phosphorylase, cellobiose are added.
  • Phosphorylase glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase, catalyzed reaction, the final inositol conversion rate is 14%; regenerated amorphous cellulose as substrate , adding cellulase, cellulase phosphorylase, cellobiose phosphorylase, glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase, catalytic reaction, final inositol conversion rate It is 48%.
  • polyphosphoglucose kinase and sodium polyphosphate were additionally added to the above reaction system, and the final inositol conversion rate reached 65%, and the conversion rate was remarkably improved.
  • the invention adopts starch or cellulose and their derivatives as raw materials in a multi-enzyme reaction system, and is converted into inositol by multi-enzyme catalysis in vitro, and an enzyme which can promote hydrolysis of starch or cellulose is added and optimized by process optimization.
  • the by-product (glucose) enzyme significantly increases the conversion efficiency, and the high yield and high conversion rate greatly reduce the separation cost of inositol.
  • the method of the invention has the advantages of simplicity, high utilization rate of raw materials, high yield of inositol, low production cost and low pollution, and can achieve inositol Large-scale production.
  • enzyme reaction means a chemical reaction carried out under the action of a biocatalyst-enzyme.
  • glucose polymer means starch or cellulose obtained by polymerizing glucose molecules.
  • glucose oligomer means partially hydrolyzed starch, starch dextrin, malt polysaccharide, maltose, fibrin or cellobiose, and the like.
  • IA isoamylase
  • PA pullulanase
  • ⁇ GP glucan phosphorylase
  • PGM glucose phosphate mutase
  • IMP inositol monophosphatase
  • MP maltose phosphorylase (the enzyme can be replaced by glucanotransferase)
  • PPGK polyphosphate glucokinase
  • Figure 2 shows four key enzymes by SDS-PAGE; among them, column 1, dextran phosphorylase; column 2, glucose phosphate mutase; column 3, inositol-3-phosphate synthase; Column, inositol monophosphatase;
  • Figure 3 is an analysis of inositol by HPLC; wherein, Figure 3a is a distinction between inositol, glucose, glucose-1-phosphate, and glucose-6-phosphate by HPLC; Figure 3b is a quantitative analysis of inositol by HPLC, by inositol The intensity of the peak can be quantitatively determined as the concentration of inositol;
  • Figure 4 is a product obtained by enzymatic reaction using soluble starch as a substrate by HPLC, and the characteristic peak of inositol indicated by an arrow;
  • Figure 5 is a product obtained by enzymatic reaction using maltodextrin as a substrate by HPLC, and the characteristic peak of inositol indicated by an arrow;
  • Figure 6 is a schematic diagram of an in vitro multi-enzyme catalytic pathway for converting cellulose to inositol; among them, Cellulase, cellulase; CDP, fibrin phosphorylase; CBP, cellobiose phosphorylase; PGM, glucose phosphate mutase ;IPS, inositol-3-phosphate synthase; IMP, inositol monophosphatase; PPGK, polyphosphate glucokinase;
  • Figure 7 shows two key enzymes for the conversion of cellulose to inositol by SDS-PAGE; wherein, column 1, cellobiose phosphorylase, column 2, fibrin phosphorylase;
  • Fig. 8 is a product obtained by enzymatic reaction using microcrystalline cellulose (Avicel) as a substrate by HPLC, and the characteristic peak of inositol indicated by an arrow.
  • pET20b vector Novagen, Madison, WI;
  • E. coli expression strain BL21 (DE3), Invitrogen, Carlsbad, CA;
  • enzymes in the present invention are commercially available from Sigma; however, they can all be obtained by prokaryotic expression according to genetic engineering methods;
  • Cellulase was purchased from Sigma, product number C2730;
  • Maltose phosphorylase was purchased from Sigma under the product number M8284;
  • Alpha amylase was purchased from Sigma, product number 10065;
  • Starch is converted to inositol by an in vitro multi-enzyme catalytic system ( Figure 1).
  • These key enzymes include: (1) glucan phosphorylase ( ⁇ GP, EC 2.4.1.1), release of glucose-1-phosphate from starch; (2) glucose phosphate mutase (PGM, EC 5.4.2.2) , catalyzing glucose-1-phosphate to glucose-6-phosphate; (3) inositol-3-phosphate synthase (IPS, EC 5.5.1.4), which catalyzes glucose-6-phosphate as inositol-3-phosphate; 4) Inositol monophosphatase (IMP, EC 3.1.3.25), which dephosphorylates inositol-3-phosphate to inositol. Since the last two enzyme reactions are irreversible, the enzyme catalyst system can obtain a high conversion rate.
  • glucan phosphorylase ⁇ GP, EC 2.4.1.1
  • PGM glucose phosphate mutase
  • the glucan phosphorylase is derived from Thermotoga maritima
  • the gene is numbered TM1168 on KEGG
  • the glucose phosphate mutase is also derived from Thermotoga maritima
  • the gene is numbered TM0769 on KEGG
  • the phosphate synthase is derived from Archaeoglobus fulgidus
  • the gene is numbered AF1794 on KEGG
  • the inositol monophosphatase is also derived from Thermotoga maritima
  • the gene is numbered TM1415 on KEGG.
  • HPLC can be used to distinguish inositol, glucose, glucose-1-phosphate or glucose-6-phosphate from the reaction solution (Fig. 3a); and inositol can be quantified, as shown in Figure 3b.
  • concentration of inositol is directly proportional to the intensity of the inositol peak in HPLC; the mobile phase of HPLC is 5 mM dilute sulfuric acid.
  • the final concentration of the final inositol (Fig. 4) was 1.6 g/L, and the conversion was 16%.
  • the isoamylase is derived from Sulfolobus tokodaii, the gene is numbered ST0928 on KEGG, and the genomic DNA of the strain is Germany. Professor Georg Fuchs from Freiburg is kindly provided.
  • the phosphatidylinositol polyphosphate is derived from Thermobifida fusca, and the gene is numbered Tfu1811 on KEGG.
  • the genomic DNA of this strain is provided by Professor David Wilson of Cornell University.
  • the glucanotransferase is derived from Thermococcus litoralis, the gene numbered OCC_10078 on KEGG, and the genomic DNA of this strain is available on the ATCC official website (www.atcc.org).
  • Glucan phosphorylase, glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase were prepared in the same manner as in Experimental Example 1; maltose phosphorylase was purchased from Sigma, product number M8284.
  • glucan phosphorylase glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase were the same as in Experimental Example 1; preparation of polyphosphate glucokinase was the same as in Experimental Example 4, pullulanase ( Pullulanase, EC 3.2.1.41) was purchased from Sigma under the product number P1067; maltose phosphorylase was purchased from Sigma under the product number M8284.
  • the soluble starch is first treated with pullulan at 37 ° C, followed by the addition of other enzymes, and then reacted at 80 ° C.
  • glucan phosphorylase glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase were the same as in Experimental Example 1; preparation of isoamylase and polyphosphate glucokinase was the same as in Experimental Example 4, maltose Phosphorylase was purchased from Sigma, product number M8284;
  • FIG. 6 A schematic diagram of the conversion of cellulose to inositol by an in vitro multi-enzyme catalytic system is shown in Figure 6.
  • Cellulase was obtained from Sigma, product number C2730; preparation of glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase was the same as in Experimental Example 1.
  • fibrous polysaccharide phosphorylase (Cthe_2989) and cellobiose phosphorylase (Cthe_0275) are derived from Clostridium thermocellum. These two genes were obtained by PCR using different primers from the corresponding genomic DNA (genomic DNA available from the ATCC official website ( www.atcc.org/ )), and by Simple Cloning (You, C., et The method of al. (2012)) was cloned into the pET20b vector to obtain the corresponding expression vectors pET20b-CthCDP and pET20b-CthCBP. Both plasmids were transformed into E. coli expressing strain BL21 (DE3), and protein expression and purification were carried out. The results of protein purification are shown in Fig. 7.
  • microcrystalline cellulose (Avicel) was used as a substrate.
  • Commercially available cellulase (5 U/ml) and cellulose (10 g/L) were first mixed on an ice water bath, placed in an ice water bath for 5 minutes, centrifuged at 4 ° C, and the supernatant was removed. Precipitated as a mixture of cellulose and cellulase capable of binding to cellulose. This treatment removes almost all of the glucosidases in the commercial cellulase, thus preventing the glucosidase from hydrolyzing cellobiose to produce large amounts of glucose, so that the main hydrolysates are cellobiose and fibrin.
  • Cellulase is a product from Sigma, product number C2730; preparation of glucose phosphate mutase, inositol-3-phosphate synthase, inositol monophosphatase is the same as experimental example 1; fibrin phosphorylase and fiber The preparation of disaccharide phosphorylase was the same as in Experimental Example 7.
  • This experiment uses Regenerated Amorphous cellulose (RAC), which is the product of Avicel after concentrated phosphoric acid treatment (Zhang, YHP, et al. (2006).” A Transition from Cellulose Swelling to Cellulose Dissolution By o-Phosphoric Acid: Evidence from Enzymatic Hydrolysis and Supramolecular Structure. "Biomacromolecules 7(2): 644-648.) is a substrate.
  • Commercially available cellulase (5 U/ml) and the cellulose (10 g/L) were first mixed on an ice water bath, placed in an ice water bath for 5 minutes, centrifuged at 4 ° C, and the supernatant was removed. Precipitated as a mixture of cellulose and cellulase capable of binding to cellulose.
  • Cellulase is a product from Sigma, product number C2730; preparation of glucose phosphate mutase, inositol-3-phosphate synthase, inositol monophosphatase is the same as experimental example 1; fibrin phosphorylase and fiber The preparation of disaccharide phosphorylase was the same as in Experimental Example 7.
  • Cellulase is a product from Sigma, product number C2730; preparation of glucose phosphate mutase, inositol-3-phosphate synthase, inositol monophosphatase is the same as experimental example 1; fibrin phosphorylase and fiber The preparation of disaccharide phosphorylase was the same as in Experimental Example 7.
  • Cellulase is a product from Sigma, product number C2730; preparation of glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphatase is the same as in experimental example 1; preparation of polyphosphate glucokinase Experimental Example 4; Preparation of Fibrous Polysaccharide Phosphorylase and Cellobiose Phosphorylase Same as Experimental Example 7.
  • Example 8 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL fibrosis phosphorylase, 5 U/mL in a 0.75 ml reaction system Cellobiose phosphorylase, 1 U/mL glucose phosphate mutase, 5 U/mL inositol-3-phosphate synthase and 2 U/mL inositol monophosphatase, 10 g/L of cellulose as in Example 8.

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Abstract

提供了一种肌醇的制备方法，以淀粉、纤维素或它们的衍生物为底物，添加能够促进淀粉或纤维素水解的酶以及利用副产物葡萄糖的酶，建立多酶反应体系，将底物转化为肌醇，提升了原料的转化效率和肌醇的产率。

Description

肌醇的制备方法 技术领域
本发明涉及肌醇的制备方法，尤其涉及体外多酶催化将淀粉或纤维素以及它们的衍生物转化为肌醇的方法，属于肌醇的酶催化生产领域。
背景技术
肌醇又名环已六醇，是水溶性维生素B族中的一种。肌醇是人、动物与微生物生长的必需物质，广泛应用于医药、食品、饲料等行业。目前全球的需求量大概在每年5,000吨左右，由于目前肌醇的高售价，使得肌醇的市场前景还未得到充分的开发，例如2013年全球饲料产量为9.6亿吨，如果都加入0.2-0.5％的肌醇，那么饲料工业所需肌醇的产量应该达到每年190-480万吨。在这种情况下，目前中国国内，甚至全世界的产量都远远不能满足需求。
目前肌醇的生产主要还是传统的高温加压水解植酸(六磷酸肌醇)。该工艺设备材质要求严格，一次性投资大，操作压力只能控制在一定的范围内，限制了原材料利用率的提高，且粗产品精制工艺复杂，损失较多，生产成本较高，而且该工艺会产生大量的磷酸污染物，对水源，环境污染严重。近些年，为了减少能耗和污染开发了常压水解法。目前研究肌醇生产的热点在化学合成法和微生物酶解法，然而均不同程度的存在成本高、产率低等问题。
因此，亟待开发一种低成本，低污染，高产率的生产肌醇的新方法。
发明内容
本发明所要解决的技术问题是提供一种肌醇的酶催化转化方法，通过体外多酶催化淀粉或纤维素以及它们的衍生物和葡萄糖生产肌醇的方法，该方法具有肌醇产率和转化率高，生产成本低，无污染等优点。
为解决上述技术问题，本发明所采取的技术方案是：
本发明首先公开了一种肌醇的制备方法，包括以下步骤：
(1)以淀粉或淀粉衍生物为底物，加入葡聚糖磷酸化酶(α-Glucan phosphorylase，EC 2.4.1.1)，葡萄糖磷酸变位酶(Phosphoglucomutase，EC 5.4.2.2)，肌醇-3-磷酸合成酶(Inositol-3-phophate synthase，EC 5.5.1.4)和肌醇单磷酸酶(Inositol monophosphatase，EC 3.1.3.25)建立多酶反应体系，进行酶催化反应；(2)将反应产物进行分离、纯化，即得。
其中，步骤(1)所述底物的浓度为10g/L；所述葡聚糖磷酸化酶的用量为0.05U/mL，所述葡萄糖磷酸变位酶的用量为1U/mL，所述肌醇-3-磷酸合成酶的用量为0.05U/mL，所述肌醇单磷酸酶的用量为2U/mL；所述酶 催化反应的条件是在40-80℃反应10-100小时；优选为，在60℃反应40小时。
在所述多酶反应体系中加入淀粉去分支酶和麦芽糖磷酸化酶(EC 2.4.1.8)，或者，淀粉去分支酶和葡聚糖转移酶(EC 2.4.1.25)，能够有效提高肌醇的得率和转化率；优选的，所述淀粉去分支酶的用量为1U/mL，所述麦芽糖磷酸化酶(maltose phosphorylase，EC 2.4.1.8)或葡聚糖转移酶的用量为1U/mL；其中，所述去分支酶为异淀粉酶(isoamylase，EC 3.2.1.68)或普鲁兰酶(pullulanase，EC 3.2.1.41)中的任意一种或两种；
更优选的，所述底物的浓度为10g/L；所述葡聚糖磷酸化酶的用量为5U/mL，所述葡萄糖磷酸变位酶的用量为1U/mL，所述肌醇-3-磷酸合成酶的用量为5U/mL，所述肌醇单磷酸酶的用量为2U/mL，所述淀粉去分支酶的用量为1U/mL，所述麦芽糖磷酸化酶或葡聚糖转移酶的用量为1U/mL；
所述酶催化反应在40-100℃反应10-100小时；优选的，所述酶催化反应在40-80℃反应40小时；最优选的，所述酶催化反应在80℃反应40小时。
为了进一步提高肌醇的得率，将冗余的葡萄糖转化为肌醇，在所述多酶反应体系中加入聚磷酸葡萄糖激酶(polyphosphate glucokinase，EC 2.7.1.63)和聚磷酸盐；优选的，所述聚磷酸葡萄糖激酶的用量为1U/mL，所述聚磷酸盐的用量为10mM；其中，所述聚磷酸盐优选为聚磷酸钠。
所述多酶反应体系还含有以下各成分：缓冲液、无机磷酸根、二价镁离子和锌离子或锰离子；优选的，各成分的用量为：缓冲液100mM，无机磷酸根10mM，二价镁离子5mM，锌离子或锰离子0.5mM；其中，所述缓冲液优选为HEPES缓冲液，更优选的，HEPES缓冲液的pH值7.2。
反应结束后，残余的淀粉残渣将是纯的直链淀粉，此时可加入少量的α淀粉酶(EC 3.2.1.1)促进淀粉残渣的水解，进一步提高肌醇的产量。优选的，α淀粉酶的用量为0.1U/ml。
本发明以淀粉或淀粉衍生物为底物，加入葡聚糖磷酸化酶，葡萄糖磷酸变位酶，肌醇-3-磷酸合成酶和肌醇单磷酸酶，配制多酶反应体系，多酶催化途径包括：由葡聚糖磷酸化酶将淀粉或淀粉衍生物中的一个葡萄糖单元转化为葡萄糖-1-磷酸；由葡萄糖磷酸变位酶将葡萄糖-1-磷酸转化为葡萄糖-6-磷酸；由肌醇-3-磷酸合成酶将葡萄糖-6-磷酸转化为肌醇-3-磷酸，由肌醇单磷酸酶将肌醇-3-磷酸转化为肌醇。由于由肌醇-3-磷酸合成酶将葡萄糖-6-磷酸转化为肌醇-3-磷酸和由肌醇单磷酸酶将肌醇-3-磷酸转化为肌醇的两个酶催化反应是不可逆反应，所以该酶催化体系能够得到很高的转化率。而高得率和高转化率可以大大降低最终肌醇的分离成本。
由于淀粉是直链淀粉(20-30％)和支链淀粉(70-80％)的混合物。支链淀粉中的支链是以α-1,6糖苷键与主链相连的，而葡聚糖磷酸化酶并不能分解α-1,6糖苷键。为了提高肌醇的转化率，本发明在多酶反应体系中 加入了能够分解淀粉中α-1,6糖苷键的去分支酶—异淀粉酶或普鲁兰酶。由于葡聚糖磷酸化酶水解淀粉的最终产物是麦芽糖，为了利用麦芽糖，本发明还在反应体系中加入麦芽糖磷酸化酶，将麦芽糖分解为葡萄糖-1-磷酸和葡萄糖；更优选的，本发明在多酶反应体系中再加入聚磷酸和聚磷酸葡萄糖激酶，将葡萄糖转化为葡萄糖-6-磷酸，由肌醇-3-磷酸合成酶和肌醇单磷酸酶转化为肌醇，最终将淀粉及其衍生物中所有的葡萄糖单元转化为肌醇，从而提高肌醇的得率和转化率。在这里，麦芽糖磷酸化酶可被葡聚糖转移酶(4-a-glucanotransferase，EC.2.4.1.25)替换，该酶可以将短链的寡聚糖聚合成为长链的寡聚糖，而该长链的寡聚糖又可被葡聚糖磷酸化酶重新利用，从而能够提高淀粉的利用率。
本发明所述淀粉优选为可溶性淀粉；所述淀粉衍生物包括部分水解淀粉、淀粉糊精、麦芽糊精、麦芽多糖或麦芽糖中的任意一种或多种。
本发明还公开了另一种肌醇的制备方法，包括以下步骤：
(1)以纤维素或纤维素衍生物为底物，加入纤维素酶，纤维多糖磷酸化酶(cellodextrin phosphorylase，EC 2.4.1.49)、纤维二糖磷酸化酶(cellobiose phosphorylase，EC 2.4.1.20)、葡萄糖磷酸变位酶(EC 5.4.2.2)、肌醇-3-磷酸合成酶(EC 5.5.1.4)和肌醇单磷酸酶(EC 3.1.3.25)建立多酶反应体系，进行酶催化反应；(2)将反应产物分离、纯化，即得。
本发明以纤维素为底物，加入纤维素酶，纤维多糖磷酸化酶、纤维二糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶，配制多酶反应体系，该多酶催化途径包括：由纤维素酶水解纤维素生成纤维多糖和纤维二糖；由纤维多糖磷酸化酶、纤维二糖磷酸化酶将纤维多糖或纤维二糖的一个葡萄糖单元转化为葡萄糖-1-磷酸；由葡萄糖磷酸变位酶将葡萄糖-1-磷酸转化为葡萄糖-6-磷酸；由肌醇-3-磷酸合成酶将葡萄糖-6-磷酸转化为肌醇-3-磷酸，由肌醇单磷酸酶将肌醇-3-磷酸转化为肌醇。
其中，步骤(1)优选为，先将10g/L纤维素或纤维素衍生物和5U/ml纤维素酶在冰水浴上混合，放置于冰水浴中5分钟，然后4℃离心，去上清，得到纤维素酶和纤维素的混合物；该处理能够去除商业化纤维素酶中几乎所有的葡萄糖苷酶，这样可以避免葡萄糖苷酶水解纤维二糖生成大量的葡萄糖，从而使主要的水解产物是纤维二糖和纤维多糖。
在多酶反应体系中，所述纤维素酶和纤维素的混合物的浓度为10g/L；所述纤维多糖磷酸化酶的用量为5U/mL，所述纤维二糖磷酸化酶的用量为5U/mL，所述葡萄糖磷酸变位酶的用量为1U/mL，所述肌醇-3-磷酸合成酶的用量为5U/mL，所述肌醇单磷酸酶的用量为2U/mL；所述酶催化反应是在20-100℃反应10-100小时；优选为，在40-80℃反应72小时；最优选为，在50℃反应72小时。
为了进一步提高肌醇的得率，步骤(1)中的多酶反应体系加入聚磷酸葡萄糖激酶(EC 2.7.1.63)和聚磷酸盐；优选的，所述聚磷酸葡萄糖激酶的 用量为5U/mL，所述聚磷酸盐的用量为10mM；其中，所述聚磷酸盐优选为聚磷酸钠。
为了提高酶催化反应的效率和产品的收率，所述多酶反应体系还含有以下各成分：缓冲液、无机磷酸根、二价镁离子和锌离子或锰离子；优选的，各成分的用量为：缓冲液100mM，无机磷酸根10mM，二价镁离子5mM，锌离子或锰离子0.5mM；其中，所述缓冲液优选为100mM HEPES缓冲液；进一步优选的，所述HEPES缓冲液的pH值是7.2。
本发明在多酶反应体系中再加入聚磷酸葡萄糖激酶和聚磷酸，将纤维素水解后的最终产物葡萄糖转化为葡萄糖-6-磷酸，再由肌醇-3-磷酸合成酶和肌醇单磷酸酶催化转化为肌醇，最终将纤维素及其衍生物中所有的葡萄糖单元转化为肌醇。
本发明所述纤维素衍生物包括纤维素经过预处理后的产物，纤维多糖或纤维二糖中的任意一种；
其中，纤维素预处理方法有多种，例如酸水解法、酶水解法、物理法等；本发明所述纤维素经过预处理后的产物，优选为纤维素经过浓磷酸处理后的产物。
本发明多酶反应体系中的任何一种酶还可以为任何一种具有同等功能的酶所替换，优选为通过蛋白质工程改造所获得的具有同等功能的突变酶。
本发明体外多酶催化将淀粉转化为肌醇试验中，在一个反应体系中，以可溶性淀粉为原料，加入葡聚糖磷酸化酶，葡萄糖磷酸变位酶，肌醇-3-磷酸合成酶和肌醇单磷酸酶，进行催化反应，最终肌醇的转化率为16％。在上述反应体系中另外加入异淀粉酶，麦芽糖磷酸化酶，聚磷酸葡萄糖激酶和聚磷酸钠，同时提高葡聚糖磷酸化酶和肌醇-3-磷酸合成酶的用量，最终肌醇的转化率达到72％，转化率显著提高。
本发明体外多酶催化将纤维素转化为肌醇实验中，在一个反应体系中，以微晶形的纤维素(Avicel)为底物，加入纤维素酶、纤维多糖磷酸化酶，纤维二糖磷酸化酶，葡萄糖磷酸变位酶，肌醇-3-磷酸合成酶和肌醇单磷酸酶，进行催化反应，最终肌醇的转化率为14％；以重生的非晶形的纤维素为底物，加入纤维素酶、纤维多糖磷酸化酶，纤维二糖磷酸化酶，葡萄糖磷酸变位酶，肌醇-3-磷酸合成酶和肌醇单磷酸酶，进行催化反应，最终肌醇的转化率为48％。在上述反应体系中另外加入聚磷酸葡萄糖激酶和聚磷酸钠，最终肌醇的转化率达到65％，转化率显著提高。
本发明技术方案与现有技术相比，具有以下有益效果：
本发明在一个多酶反应体系中，以淀粉或纤维素以及它们的衍生物为原料，通过体外多酶催化转化为肌醇，并通过过程优化，添加能促进淀粉或纤维素水解的酶以及利用副产物(葡萄糖)的酶，使转化效率显著提高，高得率和高转化率又大大降低肌醇的分离成本。本发明方法具有简便，原料利用率高、肌醇产率高，生产成本低，污染低等优点，可以实现肌醇的 规模化生产。
本发明所涉及到的术语及定义
除非另外定义，否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。
术语“酶催化反应”意指在生物催化剂-酶作用下进行的化学反应。
术语“葡萄糖多聚物”意指葡萄糖分子聚合而成的淀粉或纤维素。
术语“葡萄糖寡聚物”意指部分水解的淀粉、淀粉糊精、麦芽多糖、麦芽糖、纤维多糖或纤维二糖等。
附图说明
图1为转化淀粉生成肌醇的体外多酶催化途径的示意图；其中，IA，异淀粉酶；PA，普鲁兰酶；αGP，葡聚糖磷酸化酶；PGM，葡萄糖磷酸变位酶；IPS，肌醇-3-磷酸合成酶；IMP，肌醇单磷酸酶；MP，麦芽糖磷酸化酶(该酶可被葡聚糖转移酶替换)；PPGK，聚磷酸葡萄糖激酶；
图2为SDS-PAGE检测4个关键酶；其中，第1列，葡聚糖磷酸化酶；第2列，葡萄糖磷酸变位酶；第3列，肌醇-3-磷酸合成酶；第4列，肌醇单磷酸酶；
图3为利用HPLC分析肌醇；其中，图3a为用HPLC方式区分肌醇、葡萄糖、葡萄糖-1-磷酸和葡萄糖-6-磷酸；图3b为用HPLC定量分析肌醇的浓度，通过肌醇峰的强度可以定量得到肌醇的浓度；
图4为利用HPLC检测以可溶性淀粉为底物，经过酶促反应后的产物，箭头所指表示肌醇的特征峰；
图5为利用HPLC检测以麦芽糊精为底物，经过酶促反应后的产物，箭头所指表示肌醇的特征峰；
图6为转化纤维素生成肌醇的体外多酶催化途径的示意图；其中，Cellulase，纤维素酶；CDP，纤维多糖磷酸化酶；CBP，纤维二糖磷酸化酶；PGM，葡萄糖磷酸变位酶；IPS，肌醇-3-磷酸合成酶；IMP，肌醇单磷酸酶；PPGK，聚磷酸葡萄糖激酶；
图7为SDS-PAGE检测转化纤维素生成肌醇的2个关键酶；其中，第1列，纤维二糖磷酸化酶，第2列，纤维多糖磷酸化酶；
图8为利用HPLC检测以微晶形纤维素(Avicel)为底物，经过酶促反应后的产物，箭头所指表示肌醇的特征峰。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的，不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这 些修改或替换均落入本发明的保护范围。
实验材料
可溶性淀粉，soluble starch，ACROS公司产品，产品编号：424490020；
麦芽糊精，ALDRICH公司产品，产品编号419672；
pET20b载体，Novagen，Madison，WI；
大肠杆菌表达菌BL21(DE3)，Invitrogen，Carlsbad，CA；
本发明中的大部分酶(除了肌醇单磷酸酶，聚磷酸葡萄糖激酶和葡聚糖转移酶)能在Sigma公司购买得到；但是都可以按照基因工程方法通过原核表达获得；
纤维素酶从Sigma公司购买，产品编号为C2730；
麦芽糖磷酸化酶从Sigma公司购买，产品编号为M8284；
α淀粉酶从Sigma公司购买，产品编号为10065；
Avicel，微晶形纤维素，从Sigma公司购买，产品编号为11365。
实验例1体外多酶催化将淀粉转化为肌醇
通过一个体外多酶催化体系将淀粉转化为肌醇(图1)。这些关键酶包括：(1)葡聚糖磷酸化酶(αGP，EC 2.4.1.1)，从淀粉上释放出葡萄糖-1-磷酸；(2)葡萄糖磷酸变位酶(PGM，EC 5.4.2.2)，催化葡萄糖-1-磷酸到葡萄糖-6-磷酸；(3)肌醇-3-磷酸合成酶(IPS，EC 5.5.1.4)，其催化葡萄糖-6-磷酸为肌醇-3-磷酸；(4)肌醇单磷酸酶(IMP，EC 3.1.3.25)，其将肌醇-3-磷酸脱磷成为肌醇。由于最后两个酶反应是不可逆反应，所以该酶催化体系能够得到很高的转化率。
在本发明中，葡聚糖磷酸化酶来源于Thermotoga maritima，基因在KEGG上的编号为TM1168，葡萄糖磷酸变位酶也来源于Thermotoga maritima，基因在KEGG上的编号为TM0769，肌醇-3-磷酸合成酶来源于Archaeoglobus fulgidus，基因在KEGG上的编号为AF1794，肌醇单磷酸酶也来源于Thermotoga maritima，基因在KEGG上的编号为TM1415，这些基因组DNA都可从ATCC的官方网站(www.atcc.org)上获得。这四个基因分别用不同的引物从相应的基因组DNA中通过PCR获取，并通过Simple Cloning(You,C.,et al.(2012)."Simple Cloning via Direct Transformation of PCR Product(DNA Multimer)to Escherichia coli and Bacillus subtilis."Appl.Environ.Microbiol.78(5):1593-1595.)的方法克隆至pET20b载体((Novagen,Madison,WI)中，获得相应的表达载体pET20b-TmαGP，pET20b-AfIPS，pET20b-TmPGM和pET20b-TmIMP。这四个质粒都转化至大肠杆菌表达菌BL21(DE3)(Invitrogen,Carlsbad,CA)中，并进行蛋白质表达与纯化，蛋白质纯化的结果如图2所示。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，0.05U/mL的 葡聚糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，0.05U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的可溶性淀粉，在60℃进行催化反应，反应40个小时。
根据保持时间的不同，HPLC可以用来区分反应液中的肌醇、葡萄糖、葡萄糖-1-磷酸或葡萄糖-6-磷酸(图3a)；并且可以对肌醇进行定量，如图3b所示，肌醇的浓度与HPLC中肌醇特征峰的强度是成正比的；HPLC的流动相为5mM的稀硫酸。
反应结束后，最终肌醇(图4)的终浓度是1.6g/L，转化率为16％。
实验例2体外多酶催化将淀粉转化为肌醇
葡聚糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，0.05U/mL的葡聚糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，0.05U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的可溶性淀粉，在40℃进行催化反应，反应40个小时。
反应结束后，最终肌醇的终浓度是0.9g/L，转化率为9％。
实验例3体外多酶催化将淀粉转化为肌醇
葡聚糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，0.05U/mL的葡聚糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，0.05U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的可溶性淀粉，在80℃进行催化反应，反应40个小时。
反应结束后，最终肌醇的终浓度是3.6g/L，转化率为36％。
实验例4通过过程优化和添加促进淀粉水解的酶，利用体外多酶催化将淀粉转化为肌醇
由于淀粉是有分支链，单纯采用葡聚糖磷酸化酶并不能完全将淀粉水解，因为葡聚糖磷酸化酶只会作用于α-1，4糖苷键，而分支链是以α-1，6糖苷键与主链连接的。这需要加入异淀粉酶(isoamylase，EC 3.2.1.68)水解α-1，6糖苷键。最后，淀粉被这两种酶水解的最终产物是麦芽糖和葡萄糖，为了将这些最终产物转化为肌醇，还需要加入麦芽糖磷酸化酶(maltose phosphorylase，EC 2.4.1.8)和聚磷酸葡萄糖激酶(polyphosphate glucokinase，EC 2.7.1.63)。
在本发明中，异淀粉酶来源于Sulfolobus tokodaii，基因在KEGG上的 编号为ST0928，该菌株的基因组DNA是德国
Figure PCTCN2016076526-appb-000001
Freiburg的Georg Fuchs教授友情提供。聚磷酸葡萄糖激酶来源于Thermobifida fusca，基因在KEGG上的编号为Tfu1811，该菌株的基因组DNA是美国康奈尔大学的David Wilson教授友情提供。葡聚糖转移酶来源于Thermococcus litoralis，基因在KEGG上的编号为OCC_10078，该菌株的基因组DNA可从ATCC的官方网站(www.atcc.org)上获得。这三个基因分别用不同的引物从相应的基因组DNA中通过PCR获取，并通过Simple Cloning(You,C.,et al.(2012)."Simple Cloning via Direct Transformation of PCR Product(DNA Multimer)to Escherichia coli and Bacillus subtilis."Appl.Environ.Microbiol.78(5):1593-1595.)的方法克隆至pET20b载体中，获得相应的表达载体pET20b-StIA，pET20b-TfuPPGK和pET20b-Ti4GT，这三个质粒都转化至大肠杆菌表达菌BL21(DE3)中，并进行蛋白质表达与纯化。
葡聚糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1；麦芽糖磷酸化酶从Sigma公司购买，产品编号为M8284。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的葡聚糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，1U/mL的异淀粉酶，1U/mL的麦芽糖磷酸化酶，1U/mL的聚磷酸葡萄糖激酶，10mM聚磷酸钠，10g/L的可溶性淀粉，在80℃进行催化反应，反应40个小时。最终肌醇(图4)的终浓度为7.2g/L，转化率达到了72％。
实验例5通过过程优化和添加促进淀粉水解的酶，利用体外多酶催化将淀粉转化为肌醇
葡聚糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1；聚磷酸葡萄糖激酶的制备同实验例4，普鲁兰酶(pullulanase，EC 3.2.1.41)从Sigma公司购买，产品编号为P1067；麦芽糖磷酸化酶从Sigma公司购买，产品编号为M8284。
由于从sigma公司购买的普鲁兰酶不能在高温(80℃)下反应，因此先在37℃用普鲁兰酶处理可溶性淀粉，随后再加入其他的酶，再在80℃反应。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，1U/mL的普鲁兰酶，10mM聚磷酸钠，10g/L的可溶性淀粉，在37℃进行催化反应，反应10个小时后加入5U/mL的葡聚糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，1U/mL的麦芽糖磷酸化酶，1U/mL的聚磷酸葡萄糖激酶，在80℃进行催化反应，反应40个小时。最终肌醇(图4)的终浓度为7.3g/L，转化率达到了73％。
随后在反应体系中加入少量的α淀粉酶促进残渣淀粉的水解，提高肌醇的产量，α淀粉酶的用量为0.1U/ml，反应先在37℃反应6小时，随后继续在80℃反应24小时，最终肌醇(图4)的终浓度为8.8g/L，转化率达到了88％。
实验例6体外多酶催化将麦芽糊精转化为肌醇
葡聚糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1；异淀粉酶、聚磷酸葡萄糖激酶的制备同实验例4，麦芽糖磷酸化酶从Sigma公司购买，产品编号为M8284；
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的葡聚糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，1U/mL的异淀粉酶，1U/mL的麦芽糖磷酸化酶，1U/mL的聚磷酸葡萄糖激酶，10mM聚磷酸钠，10g/L的麦芽糊精(ALDRICH公司产品，产品编号419672)，在80℃进行催化反应，反应40个小时。最终肌醇(图5)的终浓度为7.8g/L，转化率达到了78％。
实验例7体外多酶催化将纤维素转化为肌醇
通过一个体外多酶催化体系将纤维素转化为肌醇的示意图见图6。
纤维素酶是来自于Sigma公司的产品，产品编号为C2730；葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1。
纤维多糖磷酸化酶(Cthe_2989)和纤维二糖磷酸化酶(Cthe_0275)都是来源于Clostridium thermocellum。这两个基因分别用不同的引物从相应的基因组DNA(基因组DNA可从ATCC的官方网站(www.atcc.org/)上获得)中通过PCR获取，并通过Simple Cloning(You,C.,et al.(2012))的方法克隆至pET20b载体中，获得相应的表达载体pET20b-CthCDP和pET20b-CthCBP。这两个质粒都转化至大肠杆菌表达菌BL21(DE3)中，并进行蛋白质表达与纯化，蛋白质纯化的结果如图7所示。
本实验采用微晶形纤维素(Avicel)为底物。首先将商业化的纤维素酶(5U/ml)和纤维素(10g/L)在冰水浴上混合，放置于冰水浴中5分钟，在4℃离心，去上清。沉淀为纤维素和能与纤维素结合的纤维素酶的混合物。该处理能够去除商业化纤维素酶中几乎所有的葡萄糖苷酶，这样可以避免葡萄糖苷酶水解纤维二糖生成大量的葡萄糖，从而使主要的水解产物是纤维二糖和纤维多糖。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的纤维多糖磷酸化酶，5U/mL纤维二糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的如上所述的纤维素和纤维素酶的混合物，在50℃进行催化反应，反应72个小时。最 终肌醇的终浓度是1.4g/L，转化率为14％，如图8所示。
实验例8体外多酶催化将纤维素转化为肌醇
纤维素酶是来自于Sigma公司的产品，产品编号为C2730；葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶、肌醇单磷酸酶的制备同实验例1；纤维多糖磷酸化酶和纤维二糖磷酸化酶的制备同实验例7。
本实验采用重生的非晶形的纤维素(Regenerated Amorphous cellulose(RAC)，这是Avicel经过浓磷酸处理后的产物)(Zhang,Y.H.P.,et al.(2006)."A Transition from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric Acid: Evidence from Enzymatic Hydrolysis and Supramolecular Structure."Biomacromolecules 7(2):644-648.)为底物。首先将商业化的纤维素酶(5U/ml)和该纤维素(10g/L)在冰水浴上混合，放置于冰水浴中5分钟，在4℃离心，去上清。沉淀为纤维素和能与纤维素结合的纤维素酶的混合物。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的纤维多糖磷酸化酶，5U/mL纤维二糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的如上所述的纤维素和纤维素酶的混合物，在50℃进行催化反应，反应72个小时。最终肌醇的终浓度是4.8g/L，转化率为48％。
实验例9体外多酶催化将纤维素转化为肌醇
纤维素酶是来自于Sigma公司的产品，产品编号为C2730；葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶、肌醇单磷酸酶的制备同实验例1；纤维多糖磷酸化酶和纤维二糖磷酸化酶的制备同实验例7。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的纤维多糖磷酸化酶，5U/mL纤维二糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的如实验例8的纤维素和纤维素酶的混合物，在40℃进行催化反应，反应72个小时。最终肌醇的终浓度是2.3g/L，转化率为23％。
实验例10体外多酶催化将纤维素转化为肌醇
纤维素酶是来自于Sigma公司的产品，产品编号为C2730；葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶、肌醇单磷酸酶的制备同实验例1；纤维多糖磷酸化酶和纤维二糖磷酸化酶的制备同实验例7。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的纤维多糖磷酸化酶，5U/mL纤维二糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶， 5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的如实验例8的纤维素和纤维素酶的混合物，在80℃进行催化反应，反应72个小时。最终肌醇的终浓度是1.9g/L，转化率为19％。
实验例11体外多酶催化将纤维素转化为肌醇
由于纤维素水解后的最终产物为葡萄糖，为了将其转化为肌醇，还需要加入聚磷酸葡萄糖激酶和聚磷酸。
纤维素酶是来自于Sigma公司的产品，产品编号为C2730；葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶的制备同实验例1；聚磷酸葡萄糖激酶的制备同实验例4；纤维多糖磷酸化酶、纤维二糖磷酸化酶的制备同实验例7。
在一个0.75毫升的反应体系中含有100mM的HEPES缓冲液(pH 7.2)，10mM的无机磷酸根，5mM的二价镁离子，0.5mM锌离子，5U/mL的纤维多糖磷酸化酶，5U/mL纤维二糖磷酸化酶，1U/mL的葡萄糖磷酸变位酶，5U/mL肌醇-3-磷酸合成酶和2U/mL的肌醇单磷酸酶，10g/L的如实验例8的纤维素和纤维素酶的混合物，5U/mL的聚磷酸葡萄糖激酶，10mM聚磷酸钠，在50℃进行催化反应，反应72个小时。最终肌醇(图8)的终浓度为6.5g/L，转化率达到了65％。

Claims (9)

  1. 一种肌醇的制备方法，其特征在于，包括以下步骤：
    (1)以淀粉或淀粉衍生物为底物，加入葡聚糖磷酸化酶，葡萄糖磷酸变位酶，肌醇-3-磷酸合成酶和肌醇单磷酸酶建立多酶反应体系，进行酶催化反应；(2)将反应产物进行分离、纯化，即得。
  2. 按照权利要求1所述的制备方法，其特征在于：所述多酶反应体系中还含有淀粉去分支酶，麦芽糖磷酸化酶和α淀粉酶，或者，淀粉去分支酶，葡聚糖转移酶和α淀粉酶；所述淀粉去分支酶为异淀粉酶或普鲁兰酶中的任意一种或两种。
  3. 按照权利要求2所述的制备方法，其特征在于：所述多酶反应体系中还含有聚磷酸葡萄糖激酶和聚磷酸盐；其中，所述聚磷酸盐优选为聚磷酸钠。
  4. 按照权利要求1所述的制备方法，其特征在于：步骤(1)所述淀粉衍生物包括部分水解淀粉、淀粉糊精、麦芽糊精、麦芽多糖或麦芽糖中的任意一种或多种。
  5. 一种肌醇的制备方法，其特征在于，包括以下步骤：
    (1)以纤维素或纤维素衍生物为底物，加入纤维素酶，纤维多糖磷酸化酶、纤维二糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶建立多酶反应体系，进行酶催化反应；(2)将反应产物分离、纯化，即得。
  6. 按照权利要求5所述的制备方法，其特征在于：步骤(1)优选为，先将纤维素或纤维素衍生物和纤维素酶混合，然后离心，去上清，得到纤维素酶和纤维素的混合物；所述纤维素酶和纤维素的混合物中的纤维素酶不含葡萄糖苷酶；
    在纤维素酶和纤维素的混合物中，加入纤维多糖磷酸化酶、纤维二糖磷酸化酶、葡萄糖磷酸变位酶、肌醇-3-磷酸合成酶和肌醇单磷酸酶，建立多酶反应体系。
  7. 按照权利要求6所述的制备方法，其特征在于：所述多酶反应体系中还含有聚磷酸葡萄糖激酶和聚磷酸盐；其中，所述聚磷酸盐优选为聚磷酸钠。
  8. 按照权利要求5所述的制备方法，其特征在于：步骤(1)所述纤维素衍生物包括纤维素经过预处理后的产物，纤维多糖或纤维二糖中的任意一种。
  9. 按照权利要求1或5所述的制备方法，其特征在于，所述多酶反应体系还含有以下成分：缓冲液、无机磷酸根、二价镁离子，锌离子或锰离子。
PCT/CN2016/076526 2015-04-17 2016-03-16 肌醇的制备方法 WO2016165520A1 (zh)

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