CN107760743B - Method for preparing n-butyl-beta-D-glucoside by enzyme method - Google Patents
Method for preparing n-butyl-beta-D-glucoside by enzyme method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 14
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 39
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 38
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 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 claims abstract description 24
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000003054 catalyst Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 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 claims abstract description 12
- 239000008103 glucose Substances 0.000 claims abstract description 12
- 239000002608 ionic liquid Substances 0.000 claims abstract description 12
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 11
- JDTUPLBMGDDPJS-UHFFFAOYSA-N 2-methoxy-2-phenylethanol Chemical compound COC(CO)C1=CC=CC=C1 JDTUPLBMGDDPJS-UHFFFAOYSA-N 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 239000012295 chemical reaction liquid Substances 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- ZAQMJZVCEWHJNN-HOTMZDKISA-N (2R,3R,4S,5S,6R)-2-butyl-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Chemical compound CCCC[C@@]1(O)O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O ZAQMJZVCEWHJNN-HOTMZDKISA-N 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 241000589158 Agrobacterium Species 0.000 claims description 4
- 235000011437 Amygdalus communis Nutrition 0.000 claims description 4
- 241000220304 Prunus dulcis Species 0.000 claims description 4
- 235000020224 almond Nutrition 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 241000222393 Phanerochaete chrysosporium Species 0.000 claims description 3
- 241000204666 Thermotoga maritima Species 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 8
- 230000002194 synthesizing effect Effects 0.000 abstract description 6
- 238000006555 catalytic reaction Methods 0.000 abstract description 4
- -1 alkyl glycoside Chemical class 0.000 description 13
- 229930182470 glycoside Natural products 0.000 description 9
- 239000004094 surface-active agent Substances 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 230000002255 enzymatic effect Effects 0.000 description 6
- 229930182478 glucoside Natural products 0.000 description 6
- 102000004157 Hydrolases Human genes 0.000 description 4
- 108090000604 Hydrolases Proteins 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000009736 wetting Methods 0.000 description 2
- JVAZJLFFSJARQM-RMPHRYRLSA-N (2r,3r,4s,5s,6r)-2-hexoxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound CCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O JVAZJLFFSJARQM-RMPHRYRLSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- KAIPKTYOBMEXRR-UHFFFAOYSA-N 1-butyl-3-methyl-2h-imidazole Chemical compound CCCCN1CN(C)C=C1 KAIPKTYOBMEXRR-UHFFFAOYSA-N 0.000 description 1
- XREPTGNZZKNFQZ-UHFFFAOYSA-M 1-butyl-3-methylimidazolium iodide Chemical compound [I-].CCCCN1C=C[N+](C)=C1 XREPTGNZZKNFQZ-UHFFFAOYSA-M 0.000 description 1
- NUUDMTGMAZJCBY-UHFFFAOYSA-N 1-decyl-3-methyl-2h-imidazole Chemical compound CCCCCCCCCCN1CN(C)C=C1 NUUDMTGMAZJCBY-UHFFFAOYSA-N 0.000 description 1
- HOISBTKPPVRFDS-UHFFFAOYSA-M 1-decyl-3-methylimidazol-3-ium;bromide Chemical compound [Br-].CCCCCCCCCC[N+]=1C=CN(C)C=1 HOISBTKPPVRFDS-UHFFFAOYSA-M 0.000 description 1
- UXASSLBGWQQHRC-UHFFFAOYSA-N 1-heptyl-3-methyl-2h-imidazole Chemical compound CCCCCCCN1CN(C)C=C1 UXASSLBGWQQHRC-UHFFFAOYSA-N 0.000 description 1
- WGVGZVWOOMIJRK-UHFFFAOYSA-N 1-hexyl-3-methyl-2h-imidazole Chemical compound CCCCCCN1CN(C)C=C1 WGVGZVWOOMIJRK-UHFFFAOYSA-N 0.000 description 1
- DBAYTBHFIAGLLO-UHFFFAOYSA-N 1-methyl-3-nonyl-2h-imidazole Chemical compound CCCCCCCCCN1CN(C)C=C1 DBAYTBHFIAGLLO-UHFFFAOYSA-N 0.000 description 1
- KTUWFYALZIAAGE-UHFFFAOYSA-N 1-methyl-3-octyl-2h-imidazole Chemical compound CCCCCCCCN1CN(C)C=C1 KTUWFYALZIAAGE-UHFFFAOYSA-N 0.000 description 1
- HDXFBBSZRMZTFF-UHFFFAOYSA-N 1-methyl-3-pentyl-2h-imidazole Chemical compound CCCCCN1CN(C)C=C1 HDXFBBSZRMZTFF-UHFFFAOYSA-N 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
- 150000001241 acetals Chemical group 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003377 acid catalyst Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000005536 corrosion prevention Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000005461 lubrication Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical group 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for preparing n-butyl-beta-D-glucoside by an enzymatic method. The beta-glucosidase catalyst is prepared, and then applied to a 1-alkyl-3-methylimidazol ionic liquid micro-water system to catalyze the reaction of beta-D-glucose and n-butyl alcohol to synthesize n-butyl-beta-D-glucoside, wherein the yield (calculated by glucose) reaches 90-95%. The method is simple, high in selectivity, pure in product and mild in preparation condition, so that a large-scale application method for synthesizing the n-butyl-beta-D-glucoside through enzyme catalysis is provided.
Description
Technical Field
The invention relates to a synthesis method of n-butyl-beta-D-glucoside, in particular to a method for preparing n-butyl-beta-D-glucoside by an enzymatic method.
Background
The surfactant is an organic compound with a hydrophilic/lipophilic amphiphilic structure, which can obviously change the interface state of a solution system when a small amount of the surfactant is added. In the solution, the surfactant can produce a series of actions (or adverse actions) such as emulsification, dispersion, solubilization, washing, wetting, foaming, defoaming, moisturizing, lubrication, sterilization, softening, antistatic, corrosion prevention and the like, thereby meeting the requirements of various practical applications.
The surfactant has multiple types and wide application. At present, the variety of surfactants has reached thousands, and new surfactants are continuously being produced. With the continuous improvement of the living quality of people and the continuous improvement of the requirements on the ecological environment, the 'green chemistry' is generally regarded by people, which tends to lead the research and the production of the surfactant to develop towards the direction of nontoxic, nuisanceless, high-efficiency and all-natural green chemistry. The expression is as follows: the product is greened; the reaction process is green; the raw materials are green. The alkyl glycoside series which takes green natural materials as main synthetic raw materials completely accords with the development trend of green chemistry of the surfactant, and is a green surfactant with excellent performance.
Alkyl glycoside (APG) is a novel green nonionic surfactant developed in the early 80 th 20 th century. Alkyl glycoside synthesis techniques fall into two broad categories: chemical processes and enzymatic processes. The two-step glycoside conversion method in the chemical method is to use an acid catalyst to catalyze short carbon chain alcohol, such as n-butyl alcohol, to react with glucose to generate short carbon chain alkyl glycoside, such as n-butyl glycoside, and then to generate long carbon chain alkyl glycoside through acetal exchange reaction with long carbon chain fatty alcohol. Therefore, n-butyl glucoside is an intermediate product for synthesizing the long-carbon alkyl glucoside by a two-step chemical method, and is the key point of the two-step method. In addition, the n-butyl glucoside has excellent wetting and penetrating abilities, certain decontamination ability, acid resistance, alkali resistance, insensitivity to electrolyte and good compatibility with skin.
The reaction for synthesizing the alkyl glycoside by the enzyme catalysis method has the characteristics of high selectivity, pure product, high yield, mild preparation conditions and suitability for industrial scale production. Glucosidase (beta-Glucosidase, EC 3.2.1.21), a beta-D-glucoside hydrolase, is a class of hydrolases that catalyze the hydrolysis of beta-D-glucosidic bonds to form glucose and the corresponding aglycone. Studies have shown that beta-D-glucoside hydrolase can be used to catalyze the synthesis of alkyl glycosides. For example, Panitrarux et al catalyze C using beta-D-glucosidase hydrolase6-C12Glycosylation of alcohol; andersson et al synthesize hexyl glucoside by the transglycosidation reaction of p-nitrophenyl glucoside catalyzed by almond beta-glucosidase. However, the beta-D-glucoside hydrolase catalyzed glucoside yield is less than 60 percent because glucose has poor solubility in alcohol and the existence of water can promote hydrolysis side reaction. Organic solvents such as tert-butyl alcohol, 1, 4-dioxane, acetonitrile and the like are often added to a reaction substrate for synthesizing glucoside by beta-glucosidase to promote mutual dissolution of the substrates and maintain basic activity of the enzyme. However, β -glucosidase is easily inactivated in organic solvents, thereby affecting its catalytic efficiency. Therefore, the selection of a proper reaction medium to solve the problems is the key for realizing the enzymatic synthesis of the n-butyl-beta-D-glucoside.
The invention discloses a method for synthesizing n-butyl-beta-D-glucoside under catalysis of beta-glucosidase in ionic liquid, so that the yield of n-butyl-beta-D-glucoside (calculated by glucose) reaches 90-95%.
Disclosure of Invention
The invention aims to provide a method for preparing n-butyl-beta-D-glucose by an enzyme method, which is simple to operate and high in yield, aiming at the problems in the existing n-butyl-beta-D-glucose synthesis technology. The technical scheme adopted for solving the technical problem of the invention comprises the following steps: 1. preparing a beta-glucosidase catalyst; 2. preparing a substrate solution; 3. synthesizing n-butyl-beta-D-glucoside by enzyme catalysis; 4. and (5) separating a product.
In the scheme and the step 1, the beta-glucosidase catalyst is prepared by the following method: preparing the beta-glucosidase into a buffer solution with the protein mass percent of 5-20% by using a disodium hydrogen phosphate-citric acid buffer solution. Adding 0.5-2.5 mass percent of succinyl chloride into the buffer solution, and then carrying out oscillation reaction for 0.5-2 hours, wherein the mass ratio of the protein to the succinyl chloride in the buffer solution is 1: 0.5-3. And after the reaction is finished, centrifuging the reaction solution at 3000-5000 rpm for 5-10 minutes, collecting the precipitate, and freeze-drying to obtain the beta-glucosidase catalyst.
In the scheme, in the step 1, the beta-glucosidase is any one of almond beta-glucosidase, Agrobacterium (Agrobacterium) beta-glucosidase, white rot fungus (Phanerochaete chrysosporium) beta-glucosidase and Thermotoga maritima (Thermotoga maritima) beta-glucosidase.
In the scheme, in the step 1, the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 0.1-0.2M, and the pH value is 5.5-8.0.
In the scheme, step 2, the substrate solution is prepared by the following method: uniformly mixing beta-D-glucose and n-butyl alcohol according to a molar ratio of 1: 1-3, and adding 1-alkyl-3-methylimidazole (C)nMIm+N-2-10) BF4 -、Br-、I-And heating and stirring the ionic liquid of the salt at 80-100 ℃ to form a transparent liquid, wherein the mass percent of beta-D-glucose in the ionic liquid is 10-30%.
Said scheme, step 2, 1-alkyl-3-methylimidazole (C)nMIm+N-2-10) BF4 -、Br-、I-The ionic liquid of the salt is any one of 1-butyl-3-methylimidazole, 1-pentyl-3-methylimidazole, 1-hexyl-3-methylimidazole, 1-heptyl-3-methylimidazole, 1-octyl-3-methylimidazole, 1-nonyl-3-methylimidazole and 1-decyl-3-methylimidazole, and BF4 -、Br-、I-An ionic liquid of any one of the compositions.
In the scheme, in the step 3, 5U-10U of beta-glucosidase catalyst and deionized water are added into 2mL of substrate solution, the volume ratio of the deionized water is 0.2-0.5%, the reaction is carried out for 5-9 hours at 50-60 ℃, and reaction liquid is collected.
In the scheme, in the step 4, the reaction liquid obtained in the step 3 is extracted for 3 times by using diethyl ether, tert-butyl alcohol and ethyl acetate with the volume being 3 times that of the reaction liquid, the extract liquor is combined, and anhydrous MgSO is used4After drying, the product is obtained by reduced pressure distillation under 0.01 MPa. Product warp C18The column high performance liquid chromatography detection shows that the yield (calculated by glucose) of the n-butyl-beta-D-glucose reaches 90-95%.
Compared with the prior art, the invention mainly has the following advantages: enzymatic synthesis of n-butyl-beta-D-glucoside in 1-alkyl-3-methylimidazole (C)nMIm+N-2-10) BF of cation4 -、Br-、I-The method is carried out in the ionic liquid of the salt, only a small amount of water is needed for the reaction, and the yield reaches 90-95%. Simple process operation and mild conditions.
The specific implementation mode is as follows:
examples 1
1. Preparation of beta-glucosidase catalyst
Almond beta-glucosidase (manufactured by Beijing Baochidi technologies, Inc.) was prepared into an enzyme buffer solution with a protein mass percentage of 5% using 0.1M disodium hydrogen phosphate-citric acid buffer solution (manufactured by Nanchang rain dew laboratory instruments, Inc.) with a pH of 5.8. Succinyl chloride (produced by Shanghai purple reagent factory) with the mass percentage concentration of 0.5 percent is added into the enzyme buffer solution, then the oscillation reaction is carried out for 0.5 hour, and the mass ratio of the protein in the buffer solution to the succinyl chloride is 1: 0.5. After the reaction is finished, the reaction solution is centrifuged for 5 minutes at 3000rpm, and the precipitate is collected and freeze-dried to obtain the beta-glucosidase catalyst.
2. Preparation of substrate solution
beta-D-glucose (produced by Shiyao san Xue glucose Limited liability company) and n-butanol (Zibohai Zhenghua chemical with limited shares)Manufactured by Inc.) was mixed uniformly in a molar ratio of 1:1, and 1-butyl-3-methylimidazolium tetrafluoroborate (C) was added4MImBF4) (produced by Jiangxi Jinkai chemical Co., Ltd.), and is heated and stirred at 80 ℃ until a uniform transparent liquid is formed, wherein the mass percent of the beta-D-glucose in the ionic liquid is 10%.
3. Enzymatic synthesis of n-butyl-beta-D-glucoside
Adding 5U of beta-glucosidase catalyst and deionized water into 2mL of substrate solution, wherein the volume ratio of the deionized water is 0.2%, reacting for 5 hours at 50 ℃, and collecting reaction liquid.
4. Product separation
Extracting the reaction solution obtained in step 3 with 3 times volume of diethyl ether (produced by refining factory in Laiyang economic technology development area) for 3 times, mixing extractive solutions, and adding anhydrous MgSO4(produced by lianyun gang fengtai biotechnology limited) and then distilled under reduced pressure of 0.01MPa to obtain the product. Product warp C18The column high performance liquid phase method detects that the yield of the butyl-beta-D-glucose (calculated by glucose) reaches 90 percent.
EXAMPLES example 2
1. Preparation of beta-glucosidase catalyst
Agrobacterium beta-glucosidase (Megazyme, USA) was prepared into an enzyme solution with a protein content of 10% by mass using 0.15M disodium hydrogen phosphate-citric acid buffer (manufactured by Nanchang rain dew laboratory instruments, Inc.) with a pH of 6.5. Adding succinyl chloride (produced by Shanghai purple reagent factory) with the mass percentage concentration of 1.5% into the enzyme solution, and then oscillating for reaction for 1 hour, wherein the mass ratio of the protein to the succinyl chloride in the buffer solution is 1: 2. And after the reaction is finished, centrifuging the reaction solution at 4000rpm for 7 minutes, collecting the precipitate, and freeze-drying to obtain the beta-glucosidase catalyst.
2. Preparation of substrate solution
Mixing beta-D-glucose (produced by SANXUEglucose Limited liability company of ShiYAO group) and n-butanol (produced by Zibohai Zhengji chemical Co., Ltd.) at a molar ratio of 1:2, and adding 1-decyl-3-methylimidazolium bromide (C)10MIm Br) (manufactured by Kokat Industrial Co., Ltd., Lanzhou), heated and stirred at 100 deg.CUntil a clear liquid is formed. The mass percentage of the beta-D-glucose in the ionic liquid is 20%.
3. Enzymatic synthesis of n-butyl-beta-D-glucoside
Adding 8U beta-glucosidase catalyst and deionized water into 2mL substrate solution, wherein the volume ratio of the deionized water is 0.3%, reacting for 7 hours at 60 ℃, and collecting reaction liquid.
4. Product separation
Extracting the reaction solution obtained in step 3 with 3 times volume of tert-butanol (Zibo Jinlin chemical Co., Ltd.) for 3 times, mixing the extractive solutions, and adding anhydrous MgSO4(produced by lianyun gang fengtai biotechnology limited) and then distilled under reduced pressure of 0.01MPa to obtain the product. Product warp C18The column high performance liquid chromatography detection shows that the yield of the butyl-beta-D-glucose (calculated by glucose) reaches 92 percent.
EXAMPLE 3
1. Preparation of beta-glucosidase catalyst
White rot fungus (Phanerochaete chrysosporium) beta-glucosidase (Megazyme, USA) was prepared into an enzyme solution with a protein mass percentage of 20% using 0.2M disodium hydrogen phosphate-citric acid buffer solution (Nanchang rain dew laboratory instruments, Inc.) with a pH of 7.5. Succinyl chloride (produced by Shanghai purple reagent factory) with the mass percentage concentration of 2.5 percent is added into the buffer solution, and then oscillation reaction is carried out for 2 hours, wherein the mass ratio of the protein to the succinyl chloride in the buffer solution is 1: 3. And after the reaction is finished, centrifuging the reaction solution at 5000rpm for 10 minutes, collecting the precipitate, and freeze-drying to obtain the beta-glucosidase catalyst.
2. Preparation of substrate solution
Mixing beta-D-glucose (produced by SANXUEglucose Limited liability company of ShiYAO group) and n-butanol (produced by Zibohai chemical Co., Ltd.) at a molar ratio of 1:3, and adding 1-butyl-3-methylimidazolium iodide (C)4Mm I) (manufactured by kokter industrial & trade limited, lanzhou), and heated and stirred at 90 ℃ until a transparent liquid is formed, and the mass percentage of beta-D-glucose in the ionic liquid is 30%.
3. Enzymatic synthesis of n-butyl-beta-D-glucoside
Adding 10U beta-glucosidase catalyst and deionized water into 2mL substrate solution, wherein the volume ratio of the deionized water is 0.5%, reacting for 9 hours at 55 ℃, and collecting reaction liquid.
4. Product separation
Extracting the reaction solution obtained in step 3 with 3 times volume of ethyl acetate (produced by Jiangsu Sopp Co., Ltd.) for 3 times, mixing the extractive solutions, and adding anhydrous MgSO4(produced by lianyun gang fengtai biotechnology limited) and then distilled under reduced pressure of 0.01MPa to obtain the product. Product warp C18The column high performance liquid chromatography detection shows that the yield of the butyl-beta-D-glucose (calculated by glucose) reaches 95 percent.
Claims (4)
1. A method for preparing n-butyl-beta-D-glucoside by an enzyme method is characterized in that a beta-glucosidase catalyst is adopted to catalyze beta-D-glucose and n-butanol in a substrate solution to react; collecting reaction liquid after reacting for 5-9 hours, extracting for 3 times by using diethyl ether, tertiary butanol and ethyl acetate with the volume of 3 times, combining extract liquid, and using anhydrous MgSO4After drying, carrying out reduced pressure distillation under 0.01MPa to obtain a product; the product is detected by a C18 column high performance liquid chromatography, and the yield of n-butyl-beta-D-glucose is 90-95% in terms of glucose;
the beta-glucosidase catalyst is prepared by the following method: preparing a buffer solution with the weight percentage of protein of 5-20% by using 0.1-0.2M, pH disodium hydrogen phosphate-citric acid buffer solution with the value of 5.5-8.0 for beta-glucosidase; adding succinyl chloride with the mass percentage concentration of 0.5-2.5% into a buffer solution, carrying out oscillation reaction for 0.5-2 hours, wherein the mass ratio of protein to succinyl chloride in the solution is 1: 0.5-3, centrifuging reaction liquid at 3000-5000 rpm for 5-10 minutes after the reaction is finished, collecting precipitates, and carrying out freeze drying to obtain a beta-glucosidase catalyst;
the substrate solution was prepared as follows: uniformly mixing beta-D-glucose and n-butyl alcohol according to a molar ratio of 1: 1-3, and adding BF of 1-alkyl-3-methylimidazole4 -、Br-、I-The ionic liquid formed by salt, wherein the 1-alkyl-3-methylimidazole is CnMIm +, n is 2-10; in thatHeating and stirring at 80-100 ℃ to form transparent liquid, wherein the mass percentage of the beta-D-glucose in the ionic liquid is 10-30%.
2. The method for preparing n-butyl- β -D-glucoside by the enzymatic method of claim 1, wherein the β -glucosidase is any one of almond β -glucosidase, Agrobacterium β -glucosidase, white rot fungus Phanerochaete chrysosporium β -glucosidase, Thermotoga maritima β -glucosidase.
3. The method for preparing n-butyl-beta-D-glucoside by using the enzymatic method according to claim 1, wherein 5U-10U of beta-glucosidase catalyst and deionized water are added into 2mL of substrate solution, the volume ratio of the deionized water is 0.2-0.5%, the reaction is carried out for 5-9 hours at 50-60 ℃, and reaction liquid is collected.
4. The method for preparing n-butyl-beta-D-glucoside by enzyme method according to claim 1, characterized in that the product separation adopts the following method: extracting the reaction solution with 3 times volume of diethyl ether, tert-butanol and ethyl acetate for 3 times, mixing the extractive solutions, and extracting with anhydrous MgSO4After drying, carrying out reduced pressure distillation under 0.01MPa to obtain a product; the product is detected by a C18 column high performance liquid chromatography, and the yield of n-butyl-beta-D-glucose is 90-95% in terms of glucose.
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| Alkyl glucoside synthesis using Thai rosewood β-glucosidase;Kriengsak Lirdprapamongkol;《Biotechnology Letters》;20001231;第1889–1894页 * |
| Enzymatic-catalyzed synthesis of alkylglycosides in monophasic;Ali Ismail;《Journal of Biotechnology》;19990415;第145–149页 * |
| Factors affecting the specificity of β-glucosidase from Fusarium;M. Makropoulou;《International Journal of Biological Macromolecules》;19981231;第97–101页 * |
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