CN106701859A - Process for double-enzyme coupling-chemical synthesis of epsilon-caprolactone - Google Patents
Process for double-enzyme coupling-chemical synthesis of epsilon-caprolactone Download PDFInfo
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- CN106701859A CN106701859A CN201710088911.8A CN201710088911A CN106701859A CN 106701859 A CN106701859 A CN 106701859A CN 201710088911 A CN201710088911 A CN 201710088911A CN 106701859 A CN106701859 A CN 106701859A
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- caprolactone
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- 238000000034 method Methods 0.000 title claims abstract description 91
- PAPBSGBWRJIAAV-UHFFFAOYSA-N ε-Caprolactone Chemical compound O=C1CCCCCO1 PAPBSGBWRJIAAV-UHFFFAOYSA-N 0.000 title abstract description 12
- 238000003786 synthesis reaction Methods 0.000 title abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 298
- 108090000790 Enzymes Proteins 0.000 claims abstract description 298
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims abstract description 92
- 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 31
- 239000008103 glucose Substances 0.000 claims abstract description 31
- 230000000694 effects Effects 0.000 claims abstract description 22
- 102100021851 Calbindin Human genes 0.000 claims abstract 8
- 101000898082 Homo sapiens Calbindin Proteins 0.000 claims abstract 8
- 101001021643 Pseudozyma antarctica Lipase B Proteins 0.000 claims abstract 8
- 229940088598 enzyme Drugs 0.000 claims description 319
- 108010015776 Glucose oxidase Proteins 0.000 claims description 129
- 238000004132 cross linking Methods 0.000 claims description 120
- 239000004366 Glucose oxidase Substances 0.000 claims description 117
- 229940116332 glucose oxidase Drugs 0.000 claims description 117
- 235000019420 glucose oxidase Nutrition 0.000 claims description 117
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 108
- 108010031797 Candida antarctica lipase B Proteins 0.000 claims description 93
- 238000006243 chemical reaction Methods 0.000 claims description 69
- JBFHTYHTHYHCDJ-UHFFFAOYSA-N gamma-caprolactone Chemical compound CCC1CCC(=O)O1 JBFHTYHTHYHCDJ-UHFFFAOYSA-N 0.000 claims description 62
- 101000966369 Rhizopus oryzae Lipase Proteins 0.000 claims description 57
- 239000000843 powder Substances 0.000 claims description 46
- 239000000047 product Substances 0.000 claims description 46
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 45
- 239000000126 substance Substances 0.000 claims description 32
- 239000002608 ionic liquid Substances 0.000 claims description 28
- 150000003254 radicals Chemical class 0.000 claims description 28
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 21
- 239000003054 catalyst Substances 0.000 claims description 21
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 19
- 239000003431 cross linking reagent Substances 0.000 claims description 19
- 239000011259 mixed solution Substances 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 19
- 238000001556 precipitation Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 19
- 238000004108 freeze drying Methods 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 17
- 238000005119 centrifugation Methods 0.000 claims description 17
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 16
- -1 1- ethyl-3-methylimidazole tetrafluoroborates Chemical class 0.000 claims description 15
- NJMWOUFKYKNWDW-UHFFFAOYSA-N 1-ethyl-3-methylimidazolium Chemical compound CCN1C=C[N+](C)=C1 NJMWOUFKYKNWDW-UHFFFAOYSA-N 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 15
- 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 description 15
- 238000000605 extraction Methods 0.000 claims description 15
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 15
- 239000011736 potassium bicarbonate Substances 0.000 claims description 14
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 14
- 239000011541 reaction mixture Substances 0.000 claims description 14
- 238000004220 aggregation Methods 0.000 claims description 12
- 230000002776 aggregation Effects 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- IBZJNLWLRUHZIX-UHFFFAOYSA-N 1-ethyl-3-methyl-2h-imidazole Chemical class CCN1CN(C)C=C1 IBZJNLWLRUHZIX-UHFFFAOYSA-N 0.000 claims description 8
- 239000012429 reaction media Substances 0.000 claims description 8
- LZBCVRCTAYKYHR-UHFFFAOYSA-N acetic acid;chloroethene Chemical class ClC=C.CC(O)=O LZBCVRCTAYKYHR-UHFFFAOYSA-N 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000012805 post-processing Methods 0.000 claims description 4
- 239000006227 byproduct Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims 9
- 238000010168 coupling process Methods 0.000 claims 9
- 238000005859 coupling reaction Methods 0.000 claims 9
- QQZOPKMRPOGIEB-UHFFFAOYSA-N 2-Oxohexane Chemical compound CCCCC(C)=O QQZOPKMRPOGIEB-UHFFFAOYSA-N 0.000 claims 1
- SEACXNRNJAXIBM-UHFFFAOYSA-N triethyl(methyl)azanium Chemical compound CC[N+](C)(CC)CC SEACXNRNJAXIBM-UHFFFAOYSA-N 0.000 claims 1
- 150000004965 peroxy acids Chemical class 0.000 abstract description 20
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 12
- 238000011065 in-situ storage Methods 0.000 abstract description 12
- 230000001590 oxidative effect Effects 0.000 abstract description 10
- 230000003647 oxidation Effects 0.000 abstract description 7
- 238000007254 oxidation reaction Methods 0.000 abstract description 7
- 239000007800 oxidant agent Substances 0.000 abstract description 6
- 125000002252 acyl group Chemical group 0.000 abstract description 5
- 230000007423 decrease Effects 0.000 abstract description 3
- 239000000758 substrate Substances 0.000 abstract description 2
- 230000002779 inactivation Effects 0.000 abstract 1
- 230000005764 inhibitory process Effects 0.000 abstract 1
- 239000008363 phosphate buffer Substances 0.000 description 16
- 239000012467 final product Substances 0.000 description 15
- 238000002156 mixing Methods 0.000 description 15
- 239000000460 chlorine Substances 0.000 description 7
- 238000006220 Baeyer-Villiger oxidation reaction Methods 0.000 description 5
- 241000219095 Vitis Species 0.000 description 5
- 235000009754 Vitis X bourquina Nutrition 0.000 description 5
- 235000012333 Vitis X labruscana Nutrition 0.000 description 5
- 235000014787 Vitis vinifera Nutrition 0.000 description 5
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 4
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 4
- 239000005515 coenzyme Substances 0.000 description 4
- 108010058646 cyclohexanone oxygenase Proteins 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 101710197852 Baeyer-Villiger monooxygenase Proteins 0.000 description 3
- 101710137307 FAD-containing monooxygenase EthA Proteins 0.000 description 3
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 3
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 3
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 3
- JFDZBHWFFUWGJE-UHFFFAOYSA-N benzonitrile Chemical compound N#CC1=CC=CC=C1 JFDZBHWFFUWGJE-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 229920005862 polyol Polymers 0.000 description 3
- 150000003077 polyols Chemical class 0.000 description 3
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 2
- 239000004342 Benzoyl peroxide Substances 0.000 description 2
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- GZCGUPFRVQAUEE-VANKVMQKSA-N aldehydo-L-glucose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-VANKVMQKSA-N 0.000 description 2
- 235000019400 benzoyl peroxide Nutrition 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- CGZZMOTZOONQIA-UHFFFAOYSA-N cycloheptanone Chemical compound O=C1CCCCCC1 CGZZMOTZOONQIA-UHFFFAOYSA-N 0.000 description 2
- 229910001882 dioxygen Inorganic materials 0.000 description 2
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000003999 initiator Substances 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 150000002596 lactones Chemical class 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 229920001610 polycaprolactone Polymers 0.000 description 2
- 239000004632 polycaprolactone Substances 0.000 description 2
- 230000003244 pro-oxidative effect Effects 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 241000588624 Acinetobacter calcoaceticus Species 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 206010068052 Mosaicism Diseases 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 108090000417 Oxygenases Proteins 0.000 description 1
- 102000004020 Oxygenases Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229910052593 corundum Inorganic materials 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- CVIFVNPRIBETLU-BTVCFUMJSA-N hydrogen peroxide;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OO.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O CVIFVNPRIBETLU-BTVCFUMJSA-N 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000003765 sex chromosome Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 239000012209 synthetic fiber Substances 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 229910001845 yogo sapphire Inorganic materials 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention relates to a process for double-enzyme coupling-chemical synthesis of epsilon-caprolactone; hydrogen peroxide generated in situ from GOD oxidation of glucose by a GOD/CALB or GOD/ROL double-enzyme cross-linked enzyme aggregate is used as an oxidant, at the same time, peroxy acid is generated in situ through catalyzing oxidation of an acyl donor by CALB or ROL in the cross-linked enzyme aggregate, and then epsilon-caprolactone (epsilon-CL) is generated through oxidation of cyclohexanone by peroxy acid. The yield of the epsilon-caprolactone reaches 97% or more, and the selectivity of the epsilon-caprolactone is 100%. The process reduces enzyme inactivation caused by too high concentration of hydrogen peroxide, avoids an easily explosiveness risk possibly caused by direct addition of peroxy acid, reduces the influence of decrease of enzyme micro-environment pH on the enzyme activity, and can reduce the inhibition of a substrate on an enzyme.
Description
Technical field
The invention belongs to chemistry-enzyme technology field, and in particular to a kind of dual-enzyme coupling-chemical method synthesis ε-oneself in
The technique of ester, i.e., urged using glucose oxidase (GOD)/double enzyme crosslinking enzyme aggregates of candida antarctica lipase B (CALB)
Change grape is glycoxidative and acry radical donor crosses hydrolysis lotus root connection reaction generation peroxy acid in situ, and then using the life of peroxy acid peroxyester
Into the technique of caprolactone (ε-CL).
Background technology
6-caprolactone is a kind of quite varied organic synthesis intermediate of purposes, can be used to producing synthetic fibers, plastics, thin
Film, coating and plasticizer etc., the polycaprolactone (PCL) obtained by being particularly polymerized by 6-caprolactone or with other monomers is a class
The macromolecular material of Wholly-degradable, with good biocompatibility, it is non-toxic and good ooze the property of medicine, in biomedical work
Good application is obtained in journey.
The main method of current industrial production caprolactone is chemical Baeyer-Villiger (Baeyer-Villiger) oxidizing process,
I.e. under 50 DEG C of normal pressures using peroxy acid be oxidizing cyclohexanone come prepare 6-caprolactone (H.A.Wittcoff,
B.G.Reubeu, J.S.Plotkin (Eds.), Industrial Organic Chemicals, John Wiley, NJ, 2004,
292pp).The subject matter of this method is the risk that peroxy acid has blast in storage and transportation, usual Baeyer-
It is anhydrous peroxyacetic acid that Villiger oxidizing process prepares the most frequently used peroxide of ε-CL, but in the market only has the water of Peracetic acid
Solution can be supplied.Therefore, economic, safe and efficient caprolactone synthetic method is found significant.
The B of Chinese patent CN 102408404 disclose a kind of method that 6-caprolactone is prepared by molecular oxygen oxidation cyclohexanone,
The method is oxidant with excessive oxygen, and adds pro-oxidant benzaldehyde and initiator azodiisobutyronitrile or benzoyl peroxide first
Acyl or its mixture, in 30-55 DEG C of heating stirring reaction 7-28h, rotated evaporation, column chromatography for separation, finally give ε-oneself in
Ester products, yield is up to 85.1%.The problem of this method be need add pro-oxidant and initiator, complex operation and
Benzaldehyde, azodiisobutyronitrile are poisonous, and benzoyl peroxide is extremely unstable, and three is inflammable, there is safe operation sex chromosome mosaicism；
CN102351836A discloses one kind with cyclohexanone as raw material, H2O2It is oxidant, benzonitrile is solvent, CaO or Al2O3To urge
Agent, 1-7h, the technique for preparing caprolactone are reacted at 60-80 DEG C.But this technique used catalyst needs high temperature to activate for a long time
Or impregnation process, it is unfavorable for extensive preparation.
The Baeyer-Villiger oxidation method of enzymatic is another approach for preparing caprolactone.The method utilizes Baeyer-Villiger list
Oxygenase (BVMOs) is catalyst, and molecular oxygen is oxidant, catalysis ring-type oxidation of ketones generation lactone (K.Balke, M.Kadow,
H.Mallin,S.Sass,U.T.Bornscheuer,Discovery,application and protein engineering
Baeyer-Villiger monooxygenases organic synthesis.Org.Biomol.Chem.2012,10,
6249–6265).However, the monooxygenase of most of catalysis Baeyers-Villiger reaction is required for NADPH as coenzyme, FAD is
Prothetic group.And the recycling of NADPH is difficult.And, the less stable of this fermentoid, reaction yield is relatively low, lacks commercialization enzyme
Preparation these problems all strongly limit the commercial Application of Baeyer-Villiger monooxygenase.Mallin etc. (H.Mallin,
H.Wulf,U.T.Bornscheuer,A self-sufficient Baeyer–Villiger biocatalysis system
for the synthesis ofε-caprolactone from cyclohexanol,Enzyme and Microbial
The 283-287 of Technology 53 (2013)) by a kind of heat stable polyols dehydrogenase (PDH-loopN) of recombination expression and
Cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus uses RelizymeTMHA403 carriers carry out co-immobilization,
Polyol dehydrogenase-cyclohexanone monooxygenase co-immobilization enzyme is prepared for, and is catalyzed cyclohexanol synthesis under the conditions of pH9.0 with it
6-caprolactone, conversion ratio is 34%.Meanwhile, they are also catalyzed with the free enzyme system of polyol dehydrogenase-cyclohexanone monooxygenase
Cyclohexanol is reacted, and 6-caprolactone yield is 55%.The system constructs coenzyme circulation certainly by starting material of cyclohexanol
Meet system, coenzyme NADP 11 circulation is capable of achieving without adding extra cosubstrate.But deficiency is, in immobilization process
The loss of CHMO vigor is larger, causes gained immobilized enzyme catalysis ability to significantly reduce, and conversion ratio is only 34%.And free enzymatic
When, although effect makes moderate progress, but 6-caprolactone yield is not also high, and resolvase stablizes poor, is not easily recycled, it is difficult to profit repeatedly
With, cause reaction cost high, be not suitable for industrial applications.Chinese patent CN104195194A disclose a kind of two enzymes method production ε-
Caprolactone technique, the technology utilization alcohol dehydrogenase and cyclohexanone monooxygenase co-immobilization enzyme or resolvase are catalyst, with sky
Gas is oxidant, with phosphate buffer-butyl acetate two-phase system as reaction medium, by cyclohexanol direct oxidation synthesize ε-oneself
Lactone.The system there is also above-mentioned similar problem, i.e., carry out two kinds of co-immobilizations of enzyme, its process using carrier immobilized method
Cumbersome, enzyme activity loss is big, and reaction cost is high, and although free enzymatic avoids immobilization process, but there is also stabilization
The problems such as differing from, be not easily recycled, so as to limit its plant-scale application.
In sum, the subject matter of chemical method is that peroxy acid uses the blast wind brought with storage and transportation process
Danger.The Baeyer of enzymatic-Villiger reaction can avoid the use of peroxy acid, but subject matter is a lack of commercialization enzyme and coenzyme
Circulatory problems, the use cost of enzyme is higher.Due to the risk that directly addition peroxy acid is present, in-situ preparation peroxy acid will be solved effectively
Certainly this problem.Some hydrolases can be catalyzed the hydrolysis excessively of carboxylic acid or carboxylate and hydrogen peroxide, so that in-situ preparation
Peroxy acid.Therefore, using enzymatic in-situ preparation peroxy acid, while the peroxy acid peroxyester generation 6-caprolactone of generation will
Effectively solve the above problems.In order to solve the above problems, the present invention investigated one and be prepared in oneself with dual-enzyme coupling-chemical method
The new method of ester, will glucose oxidase (GOD) and candida antarctica lipase B (CALB) or Rhizopus oryzae lipase
(ROL) co-precipitation crosslinking, is made the double enzyme crosslinking enzyme aggregates of GOD/CALB or the double enzyme crosslinking enzyme aggregates of GOD/ROL, using this
The cross-linked enzyme aggregate oxidizing glucose hydrogen peroxide for producing in situ is oxidant, while the cross-linked enzyme aggregate and catalyzing acyl
Donor such as ethyl acetate oxidation is in situ to produce peroxy acid, and then using peroxy acid peroxyester generation 6-caprolactone.
The content of the invention
It is an object of the invention to provide the method that a kind of dual-enzyme coupling-chemical method synthesizes 6-caprolactone, to solve above-mentioned skill
Art problem.
Technical scheme is as follows：
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：To adding ion in reactor
Liquid is added thereto to glucose, cyclohexanone and acry radical donor as reaction medium, makes concentration of glucose be 1.0-3.0mol/
L, acry radical donor concentration is 0.5-2.0mol/L, adds double enzyme crosslinking enzyme aggregates for catalyst carries out reacting prepared product, its
In the usage amount of double enzyme crosslinking enzyme aggregates be 25-50g/L, the ionic liquid is 1- ethyl-3-methylimidazole tetrafluoro boric acids
Salt ([EMIM] [BF4]), 1- ethyl-3-methylimidazoles villaumite ([EMIM] [Cl]) or triethyl methyl ammonium tetrafluoroborate
([Et3MeN][BF4])。
Described pair of enzyme crosslinking enzyme aggregate is the double enzyme crosslinking enzyme aggregates of GOD/CALB or the double enzyme crosslinking enzyme aggregations of GOD/ROL
Body.
The cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.02-0.1mol/hL.
The present invention also further comprise to reaction system add inorganic weak bases in and by-product acetic acid step, its is inorganic weak
Base amount is 0.2-1.0mol/L.Described inorganic weak bases are KHCO3。
The acry radical donor is ethyl acetate, acetic anhydride or acetic acid 2- chloroethene esters.
Described to react on 25-40 DEG C, 150-250rpm reacts 8-15h under the conditions of throughput 1.0-5.0vvm.
Unless otherwise specified, ventilated in the present invention as filtrated air.
The present invention still further comprises post-processing step, and the post processing is dissolved with ethyl acetate after terminating for reaction and reacted
Mixed liquor, leaches double enzyme crosslinking enzyme aggregates, and filtrate uses toluene extraction product through vacuum-concentrcted.
The extract 10%NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.
The preparation method of the double enzyme crosslinking enzyme aggregates of described GOD/CALB is as follows：GOD enzyme powders and CALB enzyme powders are mixed
Thing, with buffer solution, the buffer solution is phosphate buffer, and the pH value of the phosphate buffer is 6.0-7.0, in 4
DEG C stirring is lower adds ammonium sulfate, makes its concentration for 0.4-0.6kg/L, then to crosslinking agent glutaraldehyde is added in this solution, makes its dense
It is 0.1-0.5% to spend, and is crosslinked 2-3h, the mixed solution centrifugation that will be obtained, and removes supernatant, and precipitation is washed with deionized, cold
It is lyophilized dry, both obtained the double enzyme crosslinking enzyme aggregates of GOD/CALB.
The double enzyme crosslinking enzyme aggregate more specifically preparation methods of described GOD/CALB are as follows：
By 0.75-3.0KgGOD enzyme powders (>=500U/g) and CALB enzyme powders (>=200U/g) mixture, with the pH of 5-20L
6.0-7.0 phosphate buffers dissolve, and make its concentration be 0.4- in ammonium sulfate is added under 120rpm low rate mixings under the conditions of 4 DEG C
0.6kg/L, continues to stir 1h, adds crosslinking agent glutaraldehyde to make its concentration for 0.1-0.5%, is crosslinked 2-3h, and mixed solution is in 4
DEG C, 5000 × g centrifugation 5min remove supernatant, and precipitation is washed with deionized, freeze-drying, obtain final product the double enzymes of GOD/CALB and hand over
Connection enzyme aggregate.
GOD enzyme powders and the enzyme activity of CALB enzyme powders that the preparation of the double enzyme crosslinking enzyme aggregates of described GOD/CALB is added
The ratio between be 5:1-10:1.
The preparation method of the double enzyme crosslinking enzyme aggregates of described GOD/ROL is as follows：By GOD enzyme powders and ROL enzyme powder mixtures,
With pH 6.5-8.0 buffer solutions, lower addition acetone is stirred under the conditions of 4 DEG C, its addition is 2-5L/L, then to this solution
Middle addition crosslinking agent glutaraldehyde, makes its concentration for 0.1-0.5%, is crosslinked 2-3h, the mixed solution centrifugation for obtaining, and removes supernatant
Liquid, precipitation is washed with deionized, freeze-drying, has both obtained the double enzyme crosslinking enzyme aggregates of GOD/ROL.
The double enzyme crosslinking enzyme aggregate more specifically preparation methods of described GOD/ROL are as follows：
By 0.75-3.0KgGOD enzyme powders (>=500U/g) and ROL enzyme powders (>=500U/g) mixture, with the pH of 5-20L
6.5-8.0 phosphate buffers dissolve, and in acetone is added under 120rpm low rate mixings under the conditions of 4 DEG C, its addition is 2-5L/L,
Continue to stir 1.5h, add crosslinking agent glutaraldehyde to make its concentration for 0.1-0.5%, be crosslinked 2-3h, mixed solution is in 4 DEG C, 5000
× g is centrifuged 5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregations of GOD/ROL
Body.
GOD enzyme powders that the preparation of the double enzyme crosslinking enzyme aggregates of described GOD/ROL is added and the enzyme activity of ROL enzyme powders it
Than being 8:1-12:1.
The more specifically technical scheme that the present invention is used is as follows：
(1) glucose oxidase (GOD)/double enzyme crosslinking enzyme aggregates of candida antarctica lipase B (CALB) are prepared.
By 0.75-3.0KgGOD enzyme powders (>=500U/g) and CALB enzyme powders (>=200U/g) mixture, (the ratio between enzyme activity is
5:1-10:1), dissolved with the pH 6.0-7.0 phosphate buffers of 5-20L, in addition under 120rpm low rate mixings under the conditions of 4 DEG C
Ammonium sulfate makes its concentration for 0.4-0.6kg/L, continues to stir 1h, adds crosslinking agent glutaraldehyde to make its concentration for 0.1-0.5%, hands over
Connection 2-3h, in 4 DEG C, 5000 × g centrifugation 5min remove supernatant to mixed solution, and precipitation is washed with deionized, freeze-drying,
Obtain final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
(2) with the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of cross-linked enzyme aggregate is 25-
50g/L.To reaction medium is added in jacketed stirred reactor, reaction medium is ionic liquid 1- ethyl-3-methylimidazoles four
Borofluoride ([EMIM] [BF4]), 1- ethyl-3-methylimidazoles villaumite ([EMIM] [Cl]) or triethyl methyl ammonium tetrafluoro boric acid
Salt [Et3MeN][BF4]), then it is added thereto to glucose, cyclohexanone and acry radical donor ethyl acetate, acetic anhydride or acetic acid 2- chlorine
Ethyl ester, makes concentration of glucose for 1.0-3.0mol/L, and acry radical donor concentration is 0.5-2mol/L, and cyclohexanone is using continuous dropwise addition
Mode is added, and addition speed is 0.02-0.1mol/hL, and adds KHCO to reaction system3Its consumption is 0.2-1.0mol/
L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.React on 25-40 DEG C, 150-250rpm, throughput 1.0-
8-15h is reacted under the conditions of 5.0vvm.Reaction terminates rear reaction mixture and is dissolved with isometric ethyl acetate, leaches GOD/CALB double
Enzyme crosslinking enzyme aggregate, filter vacuum is concentrated under reduced pressure, with isometric toluene extraction product, extract 10%NaHCO3Washing,
Dried with anhydrous magnesium sulfate again, vacuum-concentrcted obtains product.Constituted with chromatographic product.As a result, cyclohexanone turns
Rate is 96-99%, and 6-caprolactone yield is 97-99%, and 6-caprolactone is selectively 100%.Reaction leaches GOD/ after terminating
The double enzyme crosslinking enzyme aggregates of CALB, after being washed through ethyl acetate, can reuse 10-15 times.Raffinate ionic liquid also may be used
Recycle.
More specifically another technical scheme that the present invention is used is as follows：
(1) glucose oxidase (GOD)/double enzyme crosslinking enzyme aggregates of Rhizopus oryzae lipase (ROL) are prepared.
By 0.75-3.0KgGOD enzyme powders (>=500U/g) and ROL enzyme powders (>=500U/g) mixture, (the ratio between enzyme activity is 8:
1-12:1), dissolved with the pH 6.5-8.0 phosphate buffers of 5-20L, in adding third under 120rpm low rate mixings under the conditions of 4 DEG C
Ketone, its addition is 2-5L/L, continues to stir 1.5h, adds crosslinking agent glutaraldehyde to make its concentration for 0.1-0.5%, is crosslinked 2-
3h, in 4 DEG C, 5000 × g centrifugation 5min remove supernatant to mixed solution, and precipitation is washed with deionized, and freeze-drying is obtained final product
The double enzyme crosslinking enzyme aggregates of GOD/ROL.
(2) with the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/ROL as catalyst, the usage amount of cross-linked enzyme aggregate is 25-
50g/L.To reaction medium is added in jacketed stirred reactor, reaction medium is ionic liquid 1- ethyl-3-methylimidazoles four
Borofluoride ([EMIM] [BF4]), 1- ethyl-3-methylimidazoles villaumite ([EMIM] [Cl]) or triethyl methyl ammonium tetrafluoro boric acid
Salt [Et3MeN][BF4]), then it is added thereto to glucose, cyclohexanone and acry radical donor ethyl acetate, acetic anhydride or acetic acid 2- chlorine
Ethyl ester, makes concentration of glucose for 1.0-3.0mol/L, and acry radical donor concentration is 0.5-2mol/L, and cyclohexanone is using continuous dropwise addition
Mode is added, and addition speed is 0.02-0.1mol/hL, and adds KHCO to reaction system3Its consumption is 0.2-1.0mol/
L, adds the double enzyme crosslinking enzyme aggregates of GOD/ROL to start reaction.React on 25-40 DEG C, 150-250rpm, throughput 1.0-
8-10h is reacted under the conditions of 5.0vvm.Reaction terminates rear reaction mixture and is dissolved with isometric ethyl acetate, leaches GOD/ROL double
Enzyme crosslinking enzyme aggregate, filter vacuum is concentrated under reduced pressure, with isometric toluene extraction product, extract 10%NaHCO3Washing,
Dried with anhydrous magnesium sulfate again, vacuum-concentrcted obtains product.Constituted with chromatographic product.As a result, cyclohexanone turns
Rate is 98-99%, and 6-caprolactone yield is 98-99%, and 6-caprolactone is selectively 100%.Reaction leaches GOD/ after terminating
The double enzyme crosslinking enzyme aggregates of ROL, after being washed through ethyl acetate, can reuse 15-20 times.Raffinate ionic liquid can also be followed
Ring is used.
The method that the present invention is provided can utilize GOD/CALB or GOD/ROL systems to continue, the in situ of stabilization produces peroxide
Change hydrogen and peroxy acid, being directly added into for peroxy acid uses brought risk of explosion in effectively prevent conventional chemical methods, it is in situ
Hydrogen peroxide supply can reduce H2O2Loss of the excessive concentration to enzyme activity.Reaction is additionally added KHCO3With accessory substance in neutralization reaction
Acetic acid, to reduce the decline of enzyme microenvironment pH caused by acetic acid, so that enzyme keeps activity and selectivity higher.
The enzyme that the present invention prepares used by the double enzyme crosslinking enzyme aggregates of GOD/CALB or GOD/ROL is respectively glucose oxidase
(GOD), candida antarctica lipase B (CALB) and Rhizopus oryzae lipase (ROL), have commercialization enzyme to supply, can be convenient
For the preparation of the double enzyme crosslinking enzyme aggregates of GOD/CALB of the invention or GOD/ROL.
Advantages of the present invention is as follows：
(1) in-situ hydrogen peroxide, reduces the too high enzyme for causing of concentration of hydrogen peroxide and inactivates；
(2) in-situ preparation peroxy acid, it is to avoid direct addition peroxy acid may cause explosive risk；
(3) KHCO is added3Neutralizing by-product acetic acid reduces influence of the decline of enzyme microenvironment pH to enzymatic activity.
(4) cyclohexanone is added by the way of continuous dropwise addition, reduces suppression of the substrate to enzyme.
(5) cyclohexanone conversion ratio of the present invention is 96-99%, and 6-caprolactone yield is 97-99%, and 6-caprolactone is selectively
100%.The conversion ratio and yield of remote ultra-traditional preparation method.
(6) the safer simultaneous reactions condition easy to operate of the more traditional chemical method of the method for the present invention is gentleer, with GOD/
The double enzyme crosslinking enzyme aggregates of CALB or GOD/ROL are catalyst, without adding the catalyst such as strong acid, enzyme catalyst and reaction medium
Ionic liquid can be repeatedly used, and be a kind of friendly 6-caprolactone synthetic method of green.
Specific embodiment
Clear, complete description will be carried out to the technical scheme in the embodiment of the present invention below.Obviously, described implementation
Example is only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
Embodiment 1
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：It is glycoxidative that grape is prepared first
Enzyme (the GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB), its process is as follows：By 0.75Kg GOD enzyme powders
(the ratio between enzyme activity is 5 to (>=500U/g) and CALB enzyme powders (>=200U/g) mixture:1), with the phosphate-buffereds of pH 6.0 of 5L
Liquid dissolves, and in 3kg ammonium sulfate is added under 120rpm low rate mixings under the conditions of 4 DEG C, continues to stir 1h, adds crosslinking agent glutaraldehyde to make
Its concentration is 0.1%, is crosslinked 2h, and in 4 DEG C, 5000 × g centrifugation 5min remove supernatant, precipitation deionized water to mixed solution
Washing, freeze-drying obtains final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 50g/
L.To addition ionic liquid 1- ethyl-3-methylimidazoles tetrafluoroborate ([EMIM] in 100L jacketed stirred reactors
[BF4]) 60L, then glucose, cyclohexanone and acry radical donor ethyl acetate are added thereto to, make concentration of glucose for 1mol/L, second
Acetoacetic ester concentration is 0.5mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.02mol/hL, and is added
Enter KHCO3Its consumption is 0.2mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.25 DEG C are reacted on,
150rpm, 8h is reacted under the conditions of throughput 5.0vvm.Reaction mixture is dissolved with isometric ethyl acetate and leached after should terminating
The double enzyme crosslinking enzyme aggregates of GOD/CALB, filtrate uses isometric toluene extraction product through vacuum-concentrcted, extract use
10%NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.With chromatographic product group
Into.As a result, cyclohexanone conversion ratio is 96.6%, and 6-caprolactone yield is 98.1%, and 6-caprolactone is selectively 100%.Reaction
The double enzyme crosslinking enzyme aggregates of GOD/CALB are leached after end, after being washed through ethyl acetate, can be reused 10 times.Raffinate from
Sub- liquid also can be recycled.
Embodiment 2
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：It is glycoxidative that grape is prepared first
Enzyme (the GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB), its process is as follows：By 1.0Kg GOD enzyme powders (>=
500U/g) (the ratio between enzyme activity is 6 with CALB enzyme powders (>=200U/g) mixture:1), with the phosphate buffers of pH 7.0 of 10L
Dissolving, in 5kg ammonium sulfate is added under 120rpm low rate mixings under the conditions of 4 DEG C, continues to stir 1h, adds crosslinking agent glutaraldehyde to make it
Concentration is 0.2%, is crosslinked 2.5h, and in 4 DEG C, 5000 × g centrifugation 5min remove supernatant, precipitation deionized water to mixed solution
Washing, freeze-drying obtains final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 25g/
L.To addition ionic liquid 1- ethyl-3-methylimidazoles tetrafluoroborate ([EMIM] in 500L jacketed stirred reactors
[BF4]) 300L, then glucose, cyclohexanone and acry radical donor acetic anhydride are added thereto to, make concentration of glucose for 1.5mol/L,
Acetic anhydride concentration is 0.75mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.03mol/hL, and
Add KHCO3Its consumption is 0.6mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.40 DEG C are reacted on,
200rpm, 10h is reacted under the conditions of throughput 4.0vvm.Reaction terminates the dissolving of rear reaction mixture isometric ethyl acetate and leaches
The double enzyme crosslinking enzyme aggregates of GOD/CALB, filter vacuum is concentrated under reduced pressure, and uses toluene extraction product, extract 10%NaHCO3Wash
Wash, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.As a result, cyclohexanone
Conversion ratio is 98.0%, and 6-caprolactone yield is 98.7%, and 6-caprolactone is selectively 100%.Reaction leaches GOD/ after terminating
The double enzyme crosslinking enzyme aggregates of CALB, after being washed through ethyl acetate, can reuse 12 times.Raffinate ionic liquid is also recycled
Use.
Embodiment 3
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：It is glycoxidative that grape is prepared first
Enzyme (the GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB), its process is as follows：By 1.5Kg GOD enzyme powders (>=
500U/g) (the ratio between enzyme activity is 8 with CALB enzyme powders (>=200U/g) mixture:1), with the phosphate buffers of pH 6.5 of 15L
Dissolving, in 6kg ammonium sulfate is added under 120rpm low rate mixings under the conditions of 4 DEG C, continues to stir 1h, adds crosslinking agent glutaraldehyde to make it
Concentration is 0.3%, is crosslinked 3h, and in 4 DEG C, 5000 × g centrifugation 5min remove supernatant to mixed solution, and precipitation is washed with deionized water
Wash, freeze-drying, obtain final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 30g/
L.To addition ionic liquid 1- ethyl-3-methylimidazoles villaumite ([EMIM] [Cl]) 600L in 1000L jacketed stirred reactors,
Glucose, cyclohexanone and acry radical donor acetic acid 2- chloroethene esters are added thereto to again, make concentration of glucose be 2.5mol/L, acetic acid 2-
Chloroethene ester concentration is 1mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.05mol/hL, and is added
KHCO3Its consumption is 0.5mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.React on 40 DEG C, 200rpm,
12h is reacted under the conditions of throughput 1.0vvm.Reaction terminates the dissolving of rear reaction mixture isometric ethyl acetate and leaches GOD/
The double enzyme crosslinking enzyme aggregates of CALB, filter vacuum is concentrated under reduced pressure, with isometric toluene extraction product, extract 10%NaHCO3
Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.As a result, hexamethylene
Ketone conversion ratio is 98.3%, and 6-caprolactone yield is 98.5%, and 6-caprolactone is selectively 100%.Reaction is leached after terminating
The double enzyme crosslinking enzyme aggregates of GOD/CALB, after being washed through ethyl acetate, can reuse 11 times.Raffinate ionic liquid also may be used
Recycle.
Embodiment 4
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：It is glycoxidative that grape is prepared first
Enzyme (the GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB), its process is as follows：By 2.0Kg GOD enzyme powders (>=
500U/g) (the ratio between enzyme activity is 7 with CALB enzyme powders (>=200U/g) mixture:1), with the phosphate buffers of pH 7.0 of 20L
Dissolving, in 8kg ammonium sulfate is added under 120rpm low rate mixings under the conditions of 4 DEG C, continues to stir 1h, adds crosslinking agent glutaraldehyde to make it
Concentration is 0.5%, is crosslinked 3h, and in 4 DEG C, 5000 × g centrifugation 5min remove supernatant to mixed solution, and precipitation is washed with deionized water
Wash, freeze-drying, obtain final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 35g/
L.To addition ionic liquid triethyl methyl ammonium tetrafluoroborate [Et in 500L jacketed stirred reactors3MeN][BF4])
350L, then glucose, cyclohexanone and acry radical donor ethyl acetate are added thereto to, make concentration of glucose for 2.5mol/L, acyl group
Donor concentrations are 1mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.05mol/hL, and is added
KHCO3Its consumption is 0.5mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.React on 40 DEG C, 200rpm,
10h is reacted under the conditions of throughput 2.0vvm.Reaction terminates the dissolving of rear reaction mixture isometric ethyl acetate and leaches GOD/
The double enzyme crosslinking enzyme aggregates of CALB, filter vacuum is concentrated under reduced pressure, with isometric toluene extraction product, extract 10%NaHCO3
Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.As a result, hexamethylene
Ketone conversion ratio is 98.7%, and 6-caprolactone yield is 97.5%, and 6-caprolactone is selectively 100%.Reaction is leached after terminating
The double enzyme crosslinking enzyme aggregates of GOD/CALB, after being washed through ethyl acetate, can reuse 13 times.Raffinate ionic liquid also may be used
Recycle.
Embodiment 5
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB) are prepared first, its
Process is as follows：By 2.5Kg GOD enzyme powders (>=500U/g) and CALB enzyme powders (>=200U/g) mixture, (the ratio between enzyme activity is 9:
1), dissolved with the phosphate buffers of pH 6.8 of 20L, in adding 12kg ammonium sulfate under 120rpm low rate mixings under the conditions of 4 DEG C, after
Continuous stirring 1h, adds crosslinking agent glutaraldehyde to make its concentration be 0.4%, is crosslinked 3h, and mixed solution is in 4 DEG C, 5000 × g centrifugations
5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 25g/
L.To addition ionic liquid 1- ethyl-3-methylimidazoles tetrafluoroborate ([EMIM] in 1000L jacketed stirred reactors
[BF4]) 700L, then glucose, cyclohexanone and acry radical donor acetic acid 2- chloroethene esters are added thereto to, make the concentration of glucose be
2.0mol/L, acry radical donor concentration is 1.5mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is
0.08mol/hL, and add KHCO3Its consumption is 0.8mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start instead
Should.30 DEG C are reacted on, 150rpm reacts 15h under the conditions of throughput 3.0vvm.Reaction terminates the isometric second of rear reaction mixture
Acetoacetic ester dissolving leaches the double enzyme crosslinking enzyme aggregates of GOD/CALB, and filter vacuum is concentrated under reduced pressure, with isometric toluene extraction product,
Extract 10%NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Use chromatographic
Product is constituted.As a result, cyclohexanone conversion ratio is 97.7%, and 6-caprolactone yield is 98.4%, and 6-caprolactone is selectively
100%.Reaction leaches the double enzyme crosslinking enzyme aggregates of GOD/CALB after terminating, after being washed through ethyl acetate, can reuse 10
It is secondary.Raffinate ionic liquid also can be recycled.
Embodiment 6
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB) are prepared first, its
Process is as follows：By 3.0Kg GOD enzyme powders (>=500U/g) and CALB enzyme powders (>=200U/g) mixture, (the ratio between enzyme activity is 10:
1), dissolved with the phosphate buffers of pH 7.0 of 20L, in adding 12kg ammonium sulfate under 120rpm low rate mixings under the conditions of 4 DEG C, after
Continuous stirring 1h, adds crosslinking agent glutaraldehyde to make its concentration be 0.2%, is crosslinked 3h, and mixed solution is in 4 DEG C, 5000 × g centrifugations
5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 30g/
L.To addition ionic liquid triethyl methyl ammonium tetrafluoroborate [Et in 1000L jacketed stirred reactors3MeN][BF4]
700L, then glucose, cyclohexanone and acry radical donor acetic acid 2- chloroethene esters are added thereto to, make concentration of glucose for 2.5mol/L,
Acry radical donor concentration is 1.0mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.08mol/hL, and
Add KHCO3Its consumption is 1.0mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.40 DEG C are reacted on,
150rpm, 10h is reacted under the conditions of throughput 4.0vvm.Reaction terminates the dissolving of rear reaction mixture isometric ethyl acetate and leaches
The double enzyme crosslinking enzyme aggregates of GOD/CALB, filter vacuum is concentrated under reduced pressure, and with isometric toluene extraction product, extract uses 10%
NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.Knot
Really, cyclohexanone conversion ratio is 98.1%, and 6-caprolactone yield is 98.5%, and 6-caprolactone is selectively 100%.After reaction terminates
The double enzyme crosslinking enzyme aggregates of GOD/CALB are leached, after being washed through ethyl acetate, can be reused 15 times.Raffinate ionic liquid
Also can be recycled.
Embodiment 7
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB) are prepared first, its
Process is as follows：By 1.75Kg GOD enzyme powders (>=500U/g) and CALB enzyme powders (>=200U/g) mixture, (the ratio between enzyme activity is 8:
1), dissolved with the phosphate buffers of pH 7.0 of 20L, in adding 10kg ammonium sulfate under 120rpm low rate mixings under the conditions of 4 DEG C, after
Continuous stirring 1h, adds crosslinking agent glutaraldehyde to make its concentration be 0.3%, is crosslinked 3h, and mixed solution is in 4 DEG C, 5000 × g centrifugations
5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 30g/
L.To addition ionic liquid 1- ethyl-3-methylimidazoles tetrafluoroborate ([EMIM] in 500L jacketed stirred reactors
[BF4]) 350L, then glucose, cyclohexanone and acry radical donor ethyl acetate are added thereto to, make concentration of glucose be 2.5mol/
L, acry radical donor concentration is 1.0mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.1mol/hL,
And add KHCO3Its consumption is 1.0mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.40 DEG C are reacted on,
180rpm, 12h is reacted under the conditions of throughput 2.0vvm.Reaction terminates the dissolving of rear reaction mixture isometric ethyl acetate and leaches
The double enzyme crosslinking enzyme aggregates of GOD/CALB, filter vacuum is concentrated under reduced pressure, and with isometric toluene extraction product, extract uses 10%
NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.Knot
Really, cyclohexanone conversion ratio is 98.0%, and 6-caprolactone yield is 98.6%, and 6-caprolactone is selectively 100%.After reaction terminates
The double enzyme crosslinking enzyme aggregates of GOD/CALB are leached, after being washed through ethyl acetate, can be reused 10 times.Raffinate ionic liquid
Also can be recycled.
Embodiment 8
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking aggregations of candida antarctica lipase B (CALB) are prepared first, its
Process is as follows：By 2.0Kg GOD enzyme powders (>=500U/g) and CALB enzyme powders (>=200U/g) mixture, (the ratio between enzyme activity is 10:
1), dissolved with the phosphate buffers of pH 7.0 of 20L, in adding 10kg ammonium sulfate under 120rpm low rate mixings under the conditions of 4 DEG C, after
Continuous stirring 1h, adds crosslinking agent glutaraldehyde to make its concentration be 0.2%, is crosslinked 2.5h, and mixed solution is in 4 DEG C, 5000 × g centrifugations
5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/CALB.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/CALB as catalyst, the usage amount of double enzyme crosslinking enzyme aggregates is 30g/
L.To addition ionic liquid triethyl methyl ammonium tetrafluoroborate ([Et in 1000L jacketed stirred reactors3MeN][BF4])
600L, then glucose, cyclohexanone and acry radical donor acetic acid 2- chloroethene esters are added thereto to, make concentration of glucose for 2.5mol/L,
Acry radical donor concentration is 1mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.05mol/hL, and is added
Enter KHCO3Its consumption is 0.5mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/CALB to start reaction.40 DEG C are reacted on,
180rpm, 10h is reacted under the conditions of throughput 3.0vvm.Reaction terminates the dissolving of rear reaction mixture isometric ethyl acetate and leaches
The double enzyme crosslinking enzyme aggregates of GOD/CALB, filter vacuum is concentrated under reduced pressure, and with isometric toluene extraction product, extract uses 10%
NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.Knot
Really, cyclohexanone conversion ratio is 98.5%, and 6-caprolactone yield is 98.5%, and 6-caprolactone is selectively 100%.After reaction terminates
The double enzyme crosslinking enzyme aggregates of GOD/CALB are leached, after being washed through ethyl acetate, can be reused 10 times.Raffinate ionic liquid
Also can be recycled.
Embodiment 9
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking enzyme aggregates of Rhizopus oryzae lipase (ROL) are prepared first, and its process is such as
Under：By 0.75KgGOD enzyme powders (>=500U/g) and ROL enzyme powders (>=500U/g) mixture, (the ratio between enzyme activity is 12:1) 5L, is used
The dissolving of the phosphate buffers of pH 6.5, in adding acetone under 120rpm low rate mixings under the conditions of 4 DEG C, its addition is 2L/L,
Continue to stir 1.5h, add crosslinking agent glutaraldehyde to make its concentration be 0.2%, be crosslinked 2h, mixed solution is in 4 DEG C, 5000 × g centrifugations
5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/ROL.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/ROL as catalyst, the usage amount of cross-linked enzyme aggregate is 25g/L.To
Ionic liquid 1- ethyl-3-methylimidazoles tetrafluoroborate ([EMIM] [BF is added in 500L jacketed stirred reactors4])
350L, then glucose, cyclohexanone and acry radical donor ethyl acetate are added thereto to, make concentration of glucose for 1.0mol/L, acyl group
Donor concentrations are 0.5mol/L, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.02mol/hL, and to anti-
System is answered to add KHCO3Its consumption is 0.2mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/ROL to start reaction.React on 40
DEG C, 150rpm reacts 8h under the conditions of throughput 1.0vvm.Reaction terminates rear reaction mixture and is dissolved with isometric ethyl acetate,
The double enzyme crosslinking enzyme aggregates of GOD/ROL are leached, filter vacuum is concentrated under reduced pressure, and with isometric toluene extraction product, extract is used
10%NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.With chromatographic product group
Into.As a result, cyclohexanone conversion ratio is 98.5%, and 6-caprolactone yield is 99.3%, and 6-caprolactone is selectively 100%.Reaction
The double enzyme crosslinking enzyme aggregates of GOD/ROL are leached after end, after being washed through ethyl acetate, can be reused 18 times.Raffinate from
Sub- liquid also can be recycled.
Embodiment 10
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking enzyme aggregates of Rhizopus oryzae lipase (ROL) are prepared first, and its process is such as
Under：By 1.5KgGOD enzyme powders (>=500U/g) and ROL enzyme powders (>=500U/g) mixture, (the ratio between enzyme activity is 10:1) 10L, is used
The dissolving of the phosphate buffers of pH 7.0, in adding acetone under 120rpm low rate mixings under the conditions of 4 DEG C, its addition is 3L/L,
Continue to stir 1.5h, add crosslinking agent glutaraldehyde to make its concentration for 0.25%, be crosslinked 2h, mixed solution in 4 DEG C, 5000 × g from
Heart 5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/ROL.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/ROL as catalyst, the usage amount of cross-linked enzyme aggregate is 30g/L.To
Ionic liquid triethyl methyl ammonium tetrafluoroborate ([Et is added in 1000L jacketed stirred reactors3MeN][BF4]) 600L,
Glucose, cyclohexanone and acry radical donor acetic anhydride are added thereto to again, make concentration of glucose for 2.0mol/L, acry radical donor concentration
It is 1.0mol/L, cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.05mol/hL, and is added to reaction system
Enter KHCO3Its consumption is 0.5mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/ROL to start reaction.40 DEG C are reacted on,
200rpm, 9h is reacted under the conditions of throughput 2.0vvm.Reaction terminates rear reaction mixture and is dissolved with isometric ethyl acetate, leaches
The double enzyme crosslinking enzyme aggregates of GOD/ROL, filter vacuum is concentrated under reduced pressure, and with isometric toluene extraction product, extract uses 10%
NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.Knot
Really, cyclohexanone conversion ratio is 99.2%, and 6-caprolactone yield is 98.9%, and 6-caprolactone is selectively 100%.After reaction terminates
The double enzyme crosslinking enzyme aggregates of GOD/ROL are leached, after being washed through ethyl acetate, can be reused 20 times.Raffinate ionic liquid
Also can be recycled.
Embodiment 11
A kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：
Glucose oxidase (GOD)/double enzyme crosslinking enzyme aggregates of Rhizopus oryzae lipase (ROL) are prepared first, and its process is such as
Under：By 3.0KgGOD enzyme powders (>=500U/g) and ROL enzyme powders (>=500U/g) mixture, (the ratio between enzyme activity is 12:1) 20L, is used
The dissolving of the phosphate buffers of pH 7.5, in adding acetone under 120rpm low rate mixings under the conditions of 4 DEG C, its addition is 5L/L,
Continue to stir 1.5h, add crosslinking agent glutaraldehyde to make its concentration be 0.5%, be crosslinked 3h, mixed solution is in 4 DEG C, 5000 × g centrifugations
5min, removes supernatant, and precipitation is washed with deionized, freeze-drying, obtains final product the double enzyme crosslinking enzyme aggregates of GOD/ROL.
With the double enzyme crosslinking enzyme aggregates of above-mentioned GOD/ROL as catalyst, the usage amount of cross-linked enzyme aggregate is 50g/L.To
Addition ionic liquid 1- ethyl-3-methylimidazoles villaumite ([EMIM] [Cl]) 300L in 500L jacketed stirred reactors, then to
Glucose, cyclohexanone and acry radical donor acetic acid 2- chloroethene esters are wherein added, makes concentration of glucose for 3.0mol/L, acry radical donor is dense
It is 2mol/L to spend, and cyclohexanone is added by the way of continuous dropwise addition, and addition speed is 0.1mol/hL, and is added to reaction system
Enter KHCO3Its consumption is 1.0mol/L, adds the double enzyme crosslinking enzyme aggregates of GOD/ROL to start reaction.35 DEG C are reacted on,
250rpm, 10h is reacted under the conditions of throughput 5.0vvm.Reaction terminates rear reaction mixture and is dissolved with isometric ethyl acetate, filters
Go out the double enzyme crosslinking enzyme aggregates of GOD/ROL, filter vacuum is concentrated under reduced pressure, and with isometric toluene extraction product, extract uses 10%
NaHCO3Washing, then dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.Constituted with chromatographic product.Knot
Really, cyclohexanone conversion ratio is 99.4%, and 6-caprolactone yield is 99.2%, and 6-caprolactone is selectively 100%.After reaction terminates
The double enzyme crosslinking enzyme aggregates of GOD/ROL are leached, after being washed through ethyl acetate, can be reused 17 times.Raffinate ionic liquid
Also can be recycled.
Claims (10)
1. a kind of dual-enzyme coupling-chemical method synthesizes the technique of 6-caprolactone, comprises the following steps that：To adding ionic liquid in reactor
Body is added thereto to glucose, cyclohexanone and acry radical donor as reaction medium, makes concentration of glucose for 1.0-3.0mol/L,
Acry radical donor concentration is 0.5-2.0mol/L, adds double enzyme crosslinking enzyme aggregates for catalyst carries out reacting prepared product, wherein
The usage amount of double enzyme crosslinking enzyme aggregates is 25-50g/L, and the ionic liquid is 1- ethyl-3-methylimidazole tetrafluoroborates
([EMIM][BF4]), 1- ethyl-3-methylimidazoles villaumite ([EMIM] [Cl]) or triethyl methyl ammonium borofluoride ([Et3MeN]
[BF4])。
2. the technique that a kind of dual-enzyme coupling according to claim 1-chemical method synthesizes 6-caprolactone, it is characterised in that：Ring
Hexanone is added by the way of continuous dropwise addition, and addition speed is 0.02-0.1mol/hL.
3. the technique that a kind of dual-enzyme coupling according to claim 1-chemical method synthesizes 6-caprolactone, it is characterised in that：To
Reaction system add inorganic weak bases in and by-product acetic acid, its consumption be 0.2-1.0mol/L.
4. the technique that a kind of dual-enzyme coupling according to claim 1-chemical method synthesizes 6-caprolactone, it is characterised in that：Institute
Acry radical donor is stated for ethyl acetate, acetic anhydride or acetic acid 2- chloroethene esters.
5. the technique that a kind of dual-enzyme coupling according to claim 1-chemical method synthesizes 6-caprolactone, it is characterised in that：Institute
State and react on 25-40 DEG C, 150-250rpm reacts 8-15h under the conditions of throughput 1.0-5.0vvm.
6. the technique that a kind of dual-enzyme coupling according to claim 1-chemical method synthesizes 6-caprolactone, it is characterised in that：Also
Including post-processing step, the post processing dissolves reaction mixture after terminating for reaction with ethyl acetate, leaches double enzyme crosslinking enzymes
Aggregation, filtrate uses toluene extraction product through vacuum-concentrcted；Preferably, the extract 10%NaHCO3Washing, then
Dried with anhydrous magnesium sulfate, vacuum-concentrcted obtains product.
7. the technique that a kind of dual-enzyme coupling according to claim 1-chemical method synthesizes 6-caprolactone, it is characterised in that：Institute
It is the double enzyme crosslinking enzyme aggregations of glucose oxidase/candida antarctica lipase B (GOD/CALB) to state double enzyme crosslinking enzyme aggregates
Body or the double enzyme crosslinking enzyme aggregates of glucose oxidase/Rhizopus oryzae lipase (GOD/ROL).
8. a kind of dual-enzyme coupling according to claim any one of 1-7-chemical method synthesizes the technique of 6-caprolactone, its feature
It is：The preparation method of the double enzyme crosslinking enzyme aggregates of described GOD/CALB is as follows：By GOD enzyme powders and CALB enzyme powder mixtures,
With pH 6.0-7.0 buffer solutions, lower addition ammonium sulfate is stirred under the conditions of 4 DEG C, make its concentration for 0.4-0.6kg/L, then to
Crosslinking agent glutaraldehyde is added in this solution, makes its concentration for 0.1-0.5%, be crosslinked 2-3h, the mixed solution centrifugation for obtaining is removed
Supernatant, precipitation is washed with deionized, freeze-drying, has both obtained the double enzyme crosslinking enzyme aggregates of GOD/CALB；Described GOD/ROL
The preparation method of double enzyme crosslinking enzyme aggregates is as follows：It is molten with pH 6.5-8.0 buffer solutions by GOD enzyme powders and ROL enzyme powder mixtures
Solution, stirs lower addition acetone under the conditions of 4 DEG C, and its addition is 2-5L/L, then to crosslinking agent glutaraldehyde is added in this solution, is made
Its concentration is 0.1-0.5%, is crosslinked 2-3h, the mixed solution centrifugation for obtaining, and removes supernatant, and precipitation is washed with deionized,
Freeze-drying, had both obtained the double enzyme crosslinking enzyme aggregates of GOD/ROL.
9. the technique that a kind of dual-enzyme coupling according to claim 8-chemical method synthesizes 6-caprolactone, it is characterised in that institute
The ratio between GOD enzyme powders that the preparation of the double enzyme crosslinking enzyme aggregates of the GOD/CALB that states is added and the enzyme activity of CALB enzyme powders are 5:1-
10:1；The ratio between GOD enzyme powders that the preparation of the double enzyme crosslinking enzyme aggregates of described GOD/ROL is added and the enzyme activity of ROL enzyme powders
It is 8:1-12:1.
10. the technique that a kind of dual-enzyme coupling according to claim 3-chemical method synthesizes 6-caprolactone, it is characterised in that：Institute
The inorganic weak bases stated are KHCO3。
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| CN114058650A (en) * | 2021-11-16 | 2022-02-18 | 北京化工大学 | Method for preparing epoxy vegetable oil by double-enzyme coupling reaction system |
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| CN110938663B (en) * | 2019-11-30 | 2022-12-16 | 华南理工大学 | Method for improving yield of epsilon-caprolactone based on modification of ribosome binding site |
| CN114058650A (en) * | 2021-11-16 | 2022-02-18 | 北京化工大学 | Method for preparing epoxy vegetable oil by double-enzyme coupling reaction system |
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