CN113005160B - Method for preparing cellobiose by starch conversion - Google Patents
Method for preparing cellobiose by starch conversion Download PDFInfo
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- CN113005160B CN113005160B CN201911327630.9A CN201911327630A CN113005160B CN 113005160 B CN113005160 B CN 113005160B CN 201911327630 A CN201911327630 A CN 201911327630A CN 113005160 B CN113005160 B CN 113005160B
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- cellobiose
- starch
- phosphorylase
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- glucose
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- 229920002472 Starch Polymers 0.000 title claims abstract description 51
- 239000008107 starch Substances 0.000 title claims abstract description 51
- 235000019698 starch Nutrition 0.000 title claims abstract description 51
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 title claims description 55
- 108010048610 cellobiose phosphorylase Proteins 0.000 claims abstract description 34
- 108010056771 Glucosidases Proteins 0.000 claims abstract description 30
- 102000004366 Glucosidases Human genes 0.000 claims abstract description 30
- 108010028688 Isoamylase Proteins 0.000 claims abstract description 30
- 108010043943 Starch Phosphorylase Proteins 0.000 claims abstract description 29
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 239000000872 buffer Substances 0.000 claims description 13
- 229910021645 metal ion Inorganic materials 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 7
- 241000193448 Ruminiclostridium thermocellum Species 0.000 claims description 7
- 241001465752 Purpureocillium lilacinum Species 0.000 claims description 6
- 241000204666 Thermotoga maritima Species 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims 4
- 241000205088 Sulfolobus sp. Species 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 12
- 239000008103 glucose Substances 0.000 abstract description 12
- 238000000338 in vitro Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 abstract description 7
- 229950010772 glucose-1-phosphate Drugs 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 3
- 238000005457 optimization Methods 0.000 abstract description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 16
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 14
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 10
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 7
- 239000007995 HEPES buffer Substances 0.000 description 7
- 229910000160 potassium phosphate Inorganic materials 0.000 description 7
- 235000011009 potassium phosphates Nutrition 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 6
- 235000021317 phosphate Nutrition 0.000 description 5
- 102100033448 Lysosomal alpha-glucosidase Human genes 0.000 description 3
- 241000205101 Sulfolobus Species 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000007974 sodium acetate buffer Substances 0.000 description 3
- 229920000945 Amylopectin Polymers 0.000 description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- -1 IA) Proteins 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- 229920002774 Maltodextrin Polymers 0.000 description 2
- 239000005913 Maltodextrin Substances 0.000 description 2
- 241000160715 Sulfolobus tokodaii Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000589499 Thermus thermophilus Species 0.000 description 2
- GUBGYTABKSRVRQ-ASMJPISFSA-N alpha-maltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-ASMJPISFSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 229940035034 maltodextrin Drugs 0.000 description 2
- 229910001437 manganese ion Inorganic materials 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000203022 Acholeplasma laidlawii Species 0.000 description 1
- 108050004944 Alpha-glucan phosphorylases Proteins 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 235000021533 Beta vulgaris Nutrition 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000195620 Euglena Species 0.000 description 1
- 241000195619 Euglena gracilis Species 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 241000589564 Flavobacterium sp. Species 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000592795 Paenibacillus sp. Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010073135 Phosphorylases Proteins 0.000 description 1
- 102000009097 Phosphorylases Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- PBAYDYUZOSNJGU-UHFFFAOYSA-N chelidonic acid Natural products OC(=O)C1=CC(=O)C=C(C(O)=O)O1 PBAYDYUZOSNJGU-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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- 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
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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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)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01001—Phosphorylase (2.4.1.1)
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- 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
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/0102—Alpha-glucosidase (3.2.1.20)
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Abstract
The invention discloses a method for constructing an in-vitro multienzyme molecular machine and preparing cellobiose through multienzyme cascade catalysis, belonging to the field of enzyme catalysis preparation of cellobiose. The preparation method of cellobiose disclosed by the invention comprises the steps of hydrolyzing branched chains in starch by isoamylase, converting glucose units in debranched starch into glucose-1-phosphate and glucose by starch phosphorylase and glucosidase respectively, and synthesizing cellobiose by glucose-1-phosphate and glucose by cellobiose phosphorylase. By performing process optimization, the utilization rate of starch and the yield of cellobiose can be further improved. The technical method has the advantages of low cost and easy obtainment of substrates, low production cost, high product yield, simple separation and purification and the like, and can realize the large-scale production of cellobiose.
Description
Technical Field
The invention belongs to the field of biological manufacturing, and particularly relates to a method for preparing cellobiose by taking starch as a raw material and performing in-vitro multienzyme catalysis.
Background
Cellobiose is an oligosaccharide linked by beta-1, 4 glycosidic bonds, which has a sweetness of 30% of sucrose but almost zero calories, is mainly used in the food field as a functional sweetener, and is a soluble dietary fiber which can be used as a prebiotic to promote the proliferation of beneficial intestinal flora.
At present, the production of cellobiose mainly adopts dilute acid, enzyme or microorganism to hydrolyze high-purity cellulose. The acid hydrolysis cellulose has the advantages of non-single product, low cellobiose yield, high separation cost and easy environmental pressure. The difficulty of enzymolysis and microbial degradation processes is that cellulose has poor solubility, some pretreatment processes are needed, products are mixed, the yield is low, and the product cellobiose can inhibit the activity of cellulase. These deficiencies of the traditional approaches result in high prices of cellobiose, limiting the application prospects of such health food additives.
With the development of industrial enzyme biotechnology, scientists have begun to attempt enzymatic synthesis of cellobiose. Japanese scientists have tried to produce cellobiose from starch as a substrate using 3 enzymes (glucan phosphorylase, glucoamylase and cellobiose phosphorylase), however, the yield of cellobiose is only about 40% at maximum, resulting in a high cost for subsequent product separation.
Therefore, development of a cellobiase preparation method with low cost, low pollution and high yield is needed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for preparing cellobiose, which takes cheap starch as a substrate and catalytically produces cellobiose through an in-vitro multienzyme reaction system.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a method for preparing cellobiose by utilizing an enzyme catalytic reaction, which is characterized in that starch is taken as a substrate, and isoamylase (EC 3.2.1.68, IA), starch phosphorylase (alpha-glucan phosphorylase, EC 2.4.1.1, alpha GP), glucosidase (alpha-glucosidase, EC 3.2.1.20, alpha G) and cellobiose phosphorylase (cellobiose phosphorylase, EC 2.4.1.20, CBP) are added to construct a multienzyme catalytic reaction system for carrying out the enzyme catalytic reaction. In the invention, starch is taken as a substrate, starch is catalyzed by isoamylase to obtain debranched starch, the debranched starch is catalyzed by starch phosphorylase and glucosidase to be respectively converted into glucose-1-phosphate and glucose, and the glucose-1-phosphate and the glucose are catalyzed by cellobiose phosphorylase to generate cellobiose.
Preferably, the starch is any one or a mixture of more of soluble starch, soluble amylose, soluble amylopectin, amylodextrin, maltodextrin, maltopolysaccharide and maltose in any proportion.
Preferably, the concentration of starch in the enzyme-catalyzed reaction system is in the range of 1 to 200g/L, more preferably 5 to 50g/L, more preferably 8 to 20g/L, and most preferably 10g/L.
In a preferred embodiment, when the starch contains alpha-1, 6 glycosidic linkages (e.g., soluble starch, soluble amylopectin, amylodextrin, maltodextrin, maltopolysaccharide), an isoamylase (EC 3.2.1.68, ia) is added to the reaction system; more preferably, the isoamylase is added to the reaction system first, and after a period of time, the starch phosphorylase, the glucosidase and the cellobiose phosphorylase are added.
Preferably, the isoamylase is added to the reaction system first, and reacted at 10 to 99℃for 0.5 to 72 hours, more preferably at 30 to 95℃for 1 to 48 hours, still more preferably at 50 to 90℃for 1 to 12 hours, and most preferably at 85℃for 3 hours.
Preferably, when the starch is treated with the isoamylase, the concentration of the starch is 1 to 500g/L, more preferably 10 to 300g/L, still more preferably 50 to 250g/L, most preferably 200g/L; the amount of isoamylase to be used is 0.1 to 20U/mL, more preferably 0.5 to 10U/mL, still more preferably 1 to 8U/mL, and most preferably 5U/mL.
Preferably, the isoamylase is used to treat starch in a system containing buffer, metal ions.
Those skilled in the art will appreciate that a variety of buffers may be used in the present invention, such as sodium acetate buffer, HEPES buffer, citrate buffer such as sodium citrate buffer, and the like, preferably the buffer is sodium acetate buffer. Preferably, the pH of the buffer is 4.0-8.0, more preferably 4.5-6.5, most preferably 5.5. Preferably, the concentration of the buffer in the reaction system is 1 to 50mM, more preferably 2 to 20mM, still more preferably 3 to 10mM, and most preferably 5mM.
Those skilled in the art will appreciate that various metal ions are useful in the present invention, such as zinc ions, magnesium ions, manganese ions, etc., preferably the metal ion is a zinc example. Preferably, the concentration of zinc ions in the reaction system is 0.01 to 10mM, more preferably 0.1 to 5mM, still more preferably 0.2 to 1mM, and most preferably 0.5mM.
Preferably, the starch phosphorylase, glucosidase and cellobiose phosphorylase are added after the reaction system, and the reaction is continued at 10-95℃for 0.5-150 hours, more preferably at 20-80℃for 1-60 hours, still more preferably at 30-60℃for 6-48 hours, most preferably at 50℃for 12 hours
Preferably, buffer solution, phosphate and metal ions are also added into the reaction system.
Those skilled in the art will appreciate that various buffers may be used in the present invention, such as HEPES buffer, tris-HCl buffer, MOPS buffer, citrate buffer such as sodium citrate buffer, etc., preferably the buffer is HEPES buffer. Preferably, the pH of the buffer is between 5.0 and 8.0, more preferably between 6.0 and 7.5, most preferably 6.5. Preferably, the concentration of the buffer in the reaction system is 10 to 500mM, more preferably 20 to 150mM, still more preferably 50 to 120mM, and most preferably 100mM.
Those skilled in the art will appreciate that a variety of phosphates may be used in the present invention, such as potassium phosphate, sodium phosphate, etc., preferably the phosphate is potassium phosphate. Preferably, the concentration of phosphate in the reaction system is 1 to 50mM, more preferably 2 to 30mM, still more preferably 5 to 15mM, and most preferably 20mM.
Those skilled in the art will appreciate that a variety of metal ions may be used in the present invention, such as zinc ions, magnesium ions, manganese ions, etc., preferably the metal ions are zinc ions. Preferably, the concentration of zinc ions in the reaction system is 1 to 20mM, more preferably 2 to 15mM, still more preferably 3 to 10mM, and most preferably 5mM.
In the present invention, the starch phosphorylase, the glucosidase and the cellobiose phosphorylase added in the enzyme-catalyzed reaction system may be in any ratio.
Preferably, the ratio of starch phosphorylase, glucosidase and cellobiose phosphorylase is 1.5-3:0.5:1-3.
Further preferably, the ratio of starch phosphorylase, glucosidase and cellobiose phosphorylase is 2.5:0.5:1.2.
Preferably, the amount of starch phosphorylase used in the reaction system is 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, still more preferably 1 to 5U/mL, and most preferably 2.5U/mL.
Preferably, the amount of glucosidase in the reaction system is 0.1 to 50U/mL, more preferably 0.2 to 10U/mL, still more preferably 0.3 to 5U/mL, most preferably 0.5U/mL.
Preferably, the cellobiose phosphorylase is used in an amount of 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, still more preferably 1 to 5U/mL, and most preferably 1.2U/mL in the reaction system.
In the present invention, various sources of starch phosphorylase, cellobiose phosphorylase, isoamylase and glucosidase may be used. For example, the starch phosphorylase may be derived from Thermotoga maritima (Thermotoga maritima), clostridium thermocellum (Clostridium thermocellum), thermus thermophilus (Thermus thermophilus), etc., preferably the starch phosphorylase is derived from Thermotoga maritima; the cellobiose phosphorylase may be derived from clostridium thermocellum (Paenibacillus sp.), euglena (Euglena Gracilis), cholesteryl-free (Acholeplasma laidlawii), etc., and preferably the cellobiose phosphorylase is derived from clostridium thermocellum; the isoamylase may be derived from sulfolobus (Sulfolobus tokodaii), arabidopsis thaliana (Arabidopsis thaliana), flavobacterium (Flavobacterium sp.) and the like, and preferably the isoamylase is derived from sulfolobus; the glucosidase may be derived from Aspergillus niger (Aspergillus niger), beet (Beta vulgaris), paecilomyces lilacinus (Paecilomyces lilacinus), etc., preferably the glucosidase is derived from Paecilomyces lilacinus. The invention may also use starch phosphorylases, cellobiose phosphorylases, isoamylases and glucosidases having an amino acid sequence at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the various enzymes of the sources mentioned above.
The invention takes starch as a substrate, and adds isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase to prepare a four-enzyme reaction system, wherein the enzyme catalysis method comprises the following steps: hydrolyzing the branched chains in the starch by using isoamylase, and respectively converting glucose units in the debranched starch into glucose-1-phosphoric acid and glucose by using starch phosphorylase and glucosidase; glucose and glucose-1-phosphate are converted to cellobiose by cellobiose phosphorylase.
Since inorganic phosphorus is circulated during the reaction, the reaction can be started by adding a small amount of phosphate buffer solution, and the reaction can continuously occur, so that the use of phosphate does not cause environmental stress in actual production.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
in a multienzyme reaction system, starch is used as a raw material, the starch is converted into cellobiose through in-vitro multienzyme catalysis, and the conversion efficiency is obviously improved through process optimization, so that the separation cost of cellobiose is greatly reduced with high yield. The method has the advantages of simplicity, high raw material utilization rate, high cellobiose yield, low separation cost, environmental friendliness and the like, and can realize large-scale production of cellobiose.
Drawings
FIG. 1 is a schematic representation of an in vitro multi-enzyme catalytic pathway for the conversion of starch to cellobiose; wherein: IA is isoamylase, αgp is starch phosphorylase, αg is glucosidase, and CBP is cellobiose phosphorylase.
FIG. 2 shows SDS-PAGE for detection of 4 key enzymes; wherein: m is Marker, IA and alpha GP are purified by heat treatment, and alpha G and CBP are purified by Ni-NTA column.
FIG. 3 is a diagram showing the in vitro multienzyme catalysis of 10g/L IA treated starch cellobiose under initial conditions.
FIG. 4 is a plot of the reaction progress of 10g/L IA treated starch cellobiose catalyzed by multiple enzymes in vitro under optimal conditions.
FIG. 5 is a graph showing the reaction progress of in vitro multienzyme catalyzed high IA treated starch cellobiose under optimal conditions.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
The following materials were used in the examples of the present invention
Soluble starch, ACROS company product, product number: 424490020;
pET20b vector, novagen, madison, wis;
coli expressing strain BL21 (DE 3), invitrogen, carlsbad, calif.;
all enzymes of the invention are commercially available from Sigma, and all enzymes are also obtained by prokaryotic expression according to genetic engineering methods.
Example 1 in vitro Multi-enzyme catalyzed conversion of starch to cellobiose
The catalytic pathway for the conversion of starch to cellobiose by in vitro multi-enzyme catalytic systems is shown in figure 1. The key enzymes and key steps involved therein include: (1) Isoamylase (IA, EC 3.2.1.68) for hydrolyzing the branches of starch to give debranched starch; (2) Starch phosphorylase (αgp, EC 2.4.1.1) for liberating glucose-1-phosphate from debranched starch; (3) Glucosidase (αg, EC 3.2.1.20) for liberating glucose from debranched starch; (4) Cellobiose phosphorylase (CBP, EC 2.4.1.20) for catalyzing the production of glucose-1-phosphate and glucose into cellobiose.
In this example, the isoamylase was derived from sulfolobus (Sulfolobus tokodaii) and its gene was designated ST0928 on KEGG, and the genomic DNA of this strain was purchased from DSMZ, german collection of strains. Starch phosphorylase is derived from Thermotoga maritima (Thermotoga maritima), and its gene is numbered TM1168 on KEGG; the glucosidase is derived from paecilomyces lilacinus (Paecilomyces lilacinus), and the gene of the glucosidase is numbered QAQ81244 on KEGG; cellobiose phosphorylase is derived from clostridium thermocellum (Clostridium thermocellum), and the gene of cellobiose phosphorylase is numbered Cthe0275 on KEGG. These genomic DNAs are available from the official website of ATCC (www.atcc.org). These four genes were obtained by PCR from the corresponding genomic DNA using F1/R1, F2/R2, F3/R3 and F4/R4, respectively, wherein F1: GTTTAACTTTAAGAAGGAGATATAGTGCTGGAGAAACTTCCCGAG, R1: GTGGTGGTGGTGGTGCTCGAGTCAGAGAACCTTCTTCCAGAC, F2: CATCATCATCATCATCACAGCAGCGGCTTGAAAAAAACATGGTGGAAAGAAG, R2: GTGGTGGTGGTGGTGGTGCTCGAGTTCTTTCCAGATGTATACGCGCGCC, F3: GTTTAACTTTAAGAAGGAGATATACCATGGGTCAGAAAGGCTGGAAATTTC, R3: CAGTGGTGGTGGTGGTGGTGCTCGAGACTAATATTACGGCCCAGGGTCAC, F4: GTTTAACTTTAAGAAGGAGATATAATGGTTTTTTCACACAAGGATAGACC, R4: GTGGTGGTGGTGGTGGTGCTCGAGCTAATATTCAATCCTCCTATATACC and cloned into pET20b vectors (Novagen, madison, wis.) by Simple Cloning (You C, zhang XZ, zhangY-HP.2012.simple Cloning via direct transformation of PCR product (DNA Multi) to Escherichia coli and Bacillus subtilis.Appl.environmental.Microbiol.78 (5): 1593-5.) to obtain the corresponding expression vectors pET20b-StIA, pET20b-TmαGP, pET20b-PlαG and pET20b-PsCBP. Then, these four plasmids were transformed into E.coli expression bacterium BL21 (DE 3) (Invitrogen, carlsbad, calif.), respectively, and protein expression and purification were performed, and the results of protein purification are shown in FIG. 2.
The reaction system contains 5mM sodium acetate buffer (pH 5.5), 0.5mM divalent zinc ion, 5U/mL isoamylase and 200g/L starch, and the catalytic reaction is carried out at 85 ℃ for 3 hours to obtain the IA treated starch.
The reaction system then contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 20mM potassium phosphate (pH 6.5), 1U/mL starch phosphorylase, 1U/mL glucosidase, 2U/mL cellobiose phosphorylase, 10g/L IA treated starch, and the reaction was catalyzed at 50℃for 12 hours.
High performance liquid chromatography detects cellobiose concentration. 94.5. Mu.L of the reaction sample was taken and the reaction was stopped by adding 5.5. Mu.L of 10% sulfuric acid. The supernatant was centrifuged, and the cellobiose concentration was calculated by detecting the cellobiose peak area and peak height by HPLC.
After completion of the reaction, the final cellobiose concentration (FIG. 3) was 18mM and the conversion of 1 molecule cellobiose was 64.8% relative to starch (10 g/L, about 55.5mM glucose equivalent, 2 molecules glucose equivalent) calculated from the slope of the standard curve.
Example 2 increasing cellobiose yield by optimizing enzyme ratios
Preparation of isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase the preparation of the ia-treated starch was the same as in example 1.
The concentration of the optimal potassium phosphate is determined to be 20mM through gradual optimization, and the optimal enzyme adding amount is 2.5U/mL of starch phosphorylase, 0.5U/mL of glucosidase and 1.2U/mL of cellobiose phosphorylase. For cellobiose production, the reaction system contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 20mM potassium phosphate (pH 6.5), 2.5U/mL starch phosphorylase, 0.5U/mL glucosidase, 1.2U/mL cellobiose phosphorylase, 10g/L IA-treated starch, and the reaction was catalyzed at 50℃for 12 hours, and cellobiose was detected as in example 1.
After completion of the reaction, the final cellobiose concentration (FIG. 4) was 22.4mM, and the conversion to starch (10 g/L, about 55.5mM glucose equivalent) was 80.7%, which significantly increased the conversion and significantly decreased the glucose concentration compared to the initial conditions.
EXAMPLE 3 high concentration starch production cellobiose
Preparation of isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase the preparation of the ia-treated starch was the same as in example 1.
For cellobiose production, the reaction system contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 20mM potassium phosphate (pH 6.5), 12.5U/mL starch phosphorylase, 2.5U/mL glucosidase, 6U/mL cellobiose phosphorylase, 50g/L IA-treated starch, and the reaction was catalyzed at 50℃for 24 hours, and cellobiose was detected as in example 1.
After the end of the reaction, the final cellobiose concentration (FIG. 5) was 101mM (34.5 g/L) and the conversion was not significantly reduced compared to 10g/L starch.
The reaction system then contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 10mM potassium phosphate (pH 6.5), 25U/mL starch phosphorylase, 5U/mL glucosidase, 12U/mL cellobiose phosphorylase, 100g/L IA-treated starch, and the reaction was catalyzed at 50℃for 24 hours, and cellobiose was detected as in example 1.
After the reaction was completed, the final cellobiose concentration (FIG. 5) was 219mM (69 g/L), and the conversion was not significantly reduced compared to 10g/L starch.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (15)
1. A method for preparing cellobiose by enzyme catalysis is characterized in that starch treated by isoamylase is used as a substrate, a multienzyme catalytic reaction system is established by adding starch phosphorylase, glucosidase and cellobiose phosphorylase, and enzyme catalytic reaction is carried out, buffer solution, phosphate and metal ions are also added into the multienzyme catalytic reaction system, wherein the starch phosphorylase is derived from Thermotoga maritima, the glucosidase is derived from Paecilomyces lilacinus and the cellobiose phosphorylase is derived from Clostridium thermocellum, the adding ratio of the starch phosphorylase, the glucosidase and the cellobiose phosphorylase in the reaction system is 1.5-3:0.5:1-3, and the concentration of the phosphate is 1-50mM.
2. The method of claim 1, wherein the substrate is treated with an isoamylase in a system comprising a buffer, a metal ion.
3. The method of claim 1, wherein the isoamylase is derived from sulfolobus sp.
4. A method according to any one of claims 1 to 3, wherein the conditions of the isoamylase treatment are reaction at 30 to 95 ℃ for 1 to 48 hours.
5. The method of claim 4, wherein the conditions of the isoamylase treatment are reaction at 50-90 ℃ for 1-12 hours.
6. The method of claim 5, wherein the conditions of the isoamylase treatment are reaction at 85 ℃ for 3 hours.
7. The method of claim 1, wherein the reaction temperature of the multienzyme catalytic reaction system is between 10 ℃ and 95 ℃.
8. The method of claim 7, wherein the reaction temperature of the multienzyme catalytic reaction system is 20-80 ℃.
9. The method of claim 8, wherein the reaction temperature of the multienzyme catalytic reaction system is 30-60 ℃.
10. The method of claim 9, wherein the reaction temperature of the multienzyme catalytic reaction system is 50 ℃.
11. The method of claim 1, wherein the multienzyme catalytic reaction system has a reaction time of 0.5 to 150 hours.
12. The method of claim 11, wherein the reaction time of the multienzyme catalytic reaction system is 1 to 60 hours.
13. The method of claim 12, wherein the multienzyme catalytic reaction system has a reaction time of 6 to 48 hours.
14. The method of claim 13, wherein the multienzyme catalytic reaction system has a reaction time of 12 hours.
15. The method of claim 1, wherein the starch phosphorylase, glucosidase, and cellobiose phosphorylase are added in a ratio of 2.5:0.5:1.2 in the multi-enzyme catalytic reaction system.
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| JP2004222506A (en) * | 2003-01-17 | 2004-08-12 | Nikken Kasei Kk | METHOD FOR PRODUCING CELLOBIOSE FROM alpha-GLUCAN |
| JP2010148407A (en) * | 2008-12-24 | 2010-07-08 | Ezaki Glico Co Ltd | METHOD FOR CONVERTING GLUCOSE TO alpha-1,4-GLUCAN |
| CN109706200A (en) * | 2017-10-26 | 2019-05-03 | 中国科学院天津工业生物技术研究所 | A method of preparing laminaribiose |
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| JP2004222506A (en) * | 2003-01-17 | 2004-08-12 | Nikken Kasei Kk | METHOD FOR PRODUCING CELLOBIOSE FROM alpha-GLUCAN |
| JP2010148407A (en) * | 2008-12-24 | 2010-07-08 | Ezaki Glico Co Ltd | METHOD FOR CONVERTING GLUCOSE TO alpha-1,4-GLUCAN |
| CN109706200A (en) * | 2017-10-26 | 2019-05-03 | 中国科学院天津工业生物技术研究所 | A method of preparing laminaribiose |
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