CN103221370B - Preparation of isomerically pure substituted cyclohexanols - Google Patents
Preparation of isomerically pure substituted cyclohexanols Download PDFInfo
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- CN103221370B CN103221370B CN201180056293.XA CN201180056293A CN103221370B CN 103221370 B CN103221370 B CN 103221370B CN 201180056293 A CN201180056293 A CN 201180056293A CN 103221370 B CN103221370 B CN 103221370B
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- lipase
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- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical class OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title description 6
- 238000000034 method Methods 0.000 claims abstract description 58
- 108090001060 Lipase Proteins 0.000 claims abstract description 53
- 102000004882 Lipase Human genes 0.000 claims abstract description 51
- 239000004367 Lipase Substances 0.000 claims abstract description 51
- 235000019421 lipase Nutrition 0.000 claims abstract description 51
- 239000000203 mixture Substances 0.000 claims abstract description 20
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 22
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 22
- -1 dicarboxylic acids anhydride Chemical class 0.000 claims description 19
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Natural products CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 13
- 230000002255 enzymatic effect Effects 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 235000018102 proteins Nutrition 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 12
- 150000008065 acid anhydrides Chemical class 0.000 claims description 10
- CCOQPGVQAWPUPE-UHFFFAOYSA-N 4-tert-butylcyclohexan-1-ol Chemical compound CC(C)(C)C1CCC(O)CC1 CCOQPGVQAWPUPE-UHFFFAOYSA-N 0.000 claims description 9
- 241001453380 Burkholderia Species 0.000 claims description 9
- 150000002148 esters Chemical class 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
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- 150000002430 hydrocarbons Chemical class 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical compound O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000004215 Carbon black (E152) Substances 0.000 claims description 5
- 229930195733 hydrocarbon Natural products 0.000 claims description 5
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 241000134107 Burkholderia plantarii Species 0.000 claims description 2
- 125000003944 tolyl group Chemical group 0.000 claims 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 abstract description 9
- HPXRVTGHNJAIIH-PTQBSOBMSA-N cyclohexanol Chemical class O[13CH]1CCCCC1 HPXRVTGHNJAIIH-PTQBSOBMSA-N 0.000 abstract 2
- 239000002585 base Substances 0.000 description 82
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
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- DYLIWHYUXAJDOJ-OWOJBTEDSA-N (e)-4-(6-aminopurin-9-yl)but-2-en-1-ol Chemical compound NC1=NC=NC2=C1N=CN2C\C=C\CO DYLIWHYUXAJDOJ-OWOJBTEDSA-N 0.000 description 9
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- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 8
- 125000001072 heteroaryl group Chemical group 0.000 description 8
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- HVGZQCSMLUDISR-UHFFFAOYSA-N 2-Phenylethyl propanoate Chemical compound CCC(=O)OCCC1=CC=CC=C1 HVGZQCSMLUDISR-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
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- 229910052717 sulfur Inorganic materials 0.000 description 4
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 4
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- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- 125000005842 heteroatom Chemical group 0.000 description 3
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- 235000010755 mineral Nutrition 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
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- 150000003839 salts Chemical class 0.000 description 3
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- 125000004417 unsaturated alkyl group Chemical group 0.000 description 3
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- RCHLXMOXBJRGNX-UHFFFAOYSA-N 1-butylcyclohexan-1-ol Chemical compound CCCCC1(O)CCCCC1 RCHLXMOXBJRGNX-UHFFFAOYSA-N 0.000 description 2
- XNMQEEKYCVKGBD-UHFFFAOYSA-N 2-butyne Chemical compound CC#CC XNMQEEKYCVKGBD-UHFFFAOYSA-N 0.000 description 2
- MSXVEPNJUHWQHW-UHFFFAOYSA-N 2-methylbutan-2-ol Chemical compound CCC(C)(C)O MSXVEPNJUHWQHW-UHFFFAOYSA-N 0.000 description 2
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 2
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 description 2
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
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- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002140 imidazol-4-yl group Chemical group [H]N1C([H])=NC([*])=C1[H] 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- XBDUTCVQJHJTQZ-UHFFFAOYSA-L iron(2+) sulfate monohydrate Chemical compound O.[Fe+2].[O-]S([O-])(=O)=O XBDUTCVQJHJTQZ-UHFFFAOYSA-L 0.000 description 1
- VQTUBCCKSQIDNK-UHFFFAOYSA-N iso-butene Natural products CC(C)=C VQTUBCCKSQIDNK-UHFFFAOYSA-N 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- TVIDDXQYHWJXFK-UHFFFAOYSA-N n-Dodecanedioic acid Natural products OC(=O)CCCCCCCCCCC(O)=O TVIDDXQYHWJXFK-UHFFFAOYSA-N 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- BDJRBEYXGGNYIS-UHFFFAOYSA-N nonanedioic acid Chemical compound OC(=O)CCCCCCCC(O)=O BDJRBEYXGGNYIS-UHFFFAOYSA-N 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- RGSFGYAAUTVSQA-UHFFFAOYSA-N pentamethylene Natural products C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 1
- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000005561 phenanthryl group Chemical group 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 125000004574 piperidin-2-yl group Chemical group N1C(CCCC1)* 0.000 description 1
- 125000004482 piperidin-4-yl group Chemical group N1CCC(CC1)* 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Substances [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- LTUDISCZKZHRMJ-UHFFFAOYSA-N potassium;hydrate Chemical compound O.[K] LTUDISCZKZHRMJ-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 125000004353 pyrazol-1-yl group Chemical group [H]C1=NN(*)C([H])=C1[H] 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003509 tertiary alcohols Chemical class 0.000 description 1
- 125000000437 thiazol-2-yl group Chemical group [H]C1=C([H])N=C(*)S1 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
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- Analytical Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
Disclosed is a method for preparing isomerically pure substituted cyclohexanols starting from a mixture of cis/trans substituted cyclohexanols, which comprises reacting the cis/trans mixture of a substituted cyclohexanol with a dicarboxylic acid anhydride in the presence of a lipase, to give the trans semi-ester which is separated from the unreacted substituted cyclohexanol cis isomer.
Description
The present invention relates to the mixture of the hexalin be substituted from cis/trans, prepare the method for the pure hexalin be substituted of isometry substantially.
Can by using multiple technologies well known by persons skilled in the art, such as, distillation, chromatogram, crystallization, realize the mixture being separated cis/trans isomer.
WO 2005/073215 describes the method for the alkamine of production enantiomer-pure.It discloses under the condition that there is lipase with succinyl oxide to racemic alcohol carry out enantioselectivity acylations produce succinic acid half-ester, described succinic acid half-ester can with unreacted stage enantiomer separation.
EP 1069183 A2 teaches and carry out enantioselectivity acylations with succinyl oxide to racemic trans-2-methoxycyclohexanol under the immobilized existence from uncle's kirschner Pseudomonas alba (Pseudomonas burkholderia) lipase.
Usually, fractionation by distillation is for separating of cis/trans isomer.But when the hexalin be substituted, due to the distillation feature of hexalin be substituted, this method is not satisfied.Therefore, technical problem to be solved is the method for the mixture finding the hexalin be substituted for separating of cis/trans.
The present invention solves this problem by the method being provided for being separated the hexalin be substituted being in the pure form of basic isometry, and described method comprises:
(i) exist have as in SEQ ID No:2 the lipase of protein sequence shown, or there is the protein sequence identical with whole aminoacid sequence at least 75% shown in SEQ ID No:2 and under there is the condition of the lipase of the enzymatic activity of at least 50% of SEQ ID No:2, by the cis/trans mixture of hexalin that is substituted and dicarboxylic acids anhydride reactant, produce trans half esters
(ii) from the unreacted hexalin syn-isomerism body be substituted, trans half esters is separated,
(iii) the pure cis be substituted of isometry or trans cvclohexanol is separated.
Surprisingly, the lipase used in inventive method causes dicarboxylic acid anhydride to the acylations of the hexalin be substituted of trans forms with highly selective.The result of the inventive method obtains the cis or the trans isomer that are in the hexalin be substituted of the pure form of basic isometry.
" basic isometry is pure " means cis or trans product substantially not by another kind of isometry body pollution.Therefore, " basic isometry is pure " means to obtain at least 80%, preferably at least 90%, and more preferably at least 95%, the isomers of at least 96,97,98,99% especially.
The first step of inventive method:
Exist have as in SEQ ID No:2 the lipase of protein sequence shown, or there is the protein sequence identical with whole aminoacid sequence at least 75% shown in SEQ ID No:2 and under there is the condition of the lipase of the enzymatic activity of at least 50% of SEQ ID No:2, by the cis/trans mixture (following formula A1 to A3) of hexalin that is substituted and dicarboxylic acids anhydride reactant, produce half ester (following formula C1 to C3) and unreacted isomers (following formula B1 to B3).It should be noted that by using described lipase optionally acylations trans isomer.
In preferred embodiments, initial substance is the cis/trans mixture (above formula A3) of the hexalin that 4-is substituted, its exist have as in SEQ ID No:2 the lipase of protein sequence shown, or there is the protein sequence identical with whole aminoacid sequence at least 75% shown in SEQ ID No:2 and under there is the condition of the lipase of the enzymatic activity of at least 50% of SEQ ID No:2, with dicarboxylic acids anhydride reactant, produce half ester (above formula C3) and unreacted isomers (above formula B3).
What use in the inventive method is replaced by R1 according to the hexalin be substituted of formula A1 to A3.R1 can be any substituting group of inertia at reaction conditions.
Such as, R1 can be C that is that be substituted or that be unsubstituted
1-C
10alkyl, C that is that be substituted or that be unsubstituted
3-C
8-cycloalkyl, C that is that be substituted or that be unsubstituted
2-C
10-alkenyl or alkynyl, heterocycle that is that be substituted or that be unsubstituted, aryl that is that be substituted or that be unsubstituted.
" the C be unsubstituted
1-C
10" refer to the saturated alkyl of the straight or branched with 1 to 10 carbon atom, such as methyl, ethyl, propyl group, 1-methyl-ethyl, 1-methyl-propyl, 2-methyl-propyl, and 1-1 dimethyl ethyl etc.These are called the substituting group be unsubstituted in the context of the present invention.
Term " the C be unsubstituted
3-C
8-cycloalkyl " mean the monocyclic saturated hydrocarbon group base with 3 to 8 carbon ring member, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl or ring octyl group.
" the C be unsubstituted
2-C
10thiazolinyl " refer to have 2 to 10 carbon atoms and be in the unsaturated alkyl of straight or branched of double bond of any position, such as vinyl, 1-propenyl, 2-propenyl (allyl group), 1-methyl ethylene, 1-butylene base, crotyl, 3-butenyl, 1-methyl-1-propylene base, 2-methyl-1-propylene base, 1-methyl-2-propenyl, 2-methyl-2-propenyl etc.For purposes of the present invention, the ring unsaturated alkyl with 5 to 8 carbon ring member is also comprised, such as cyclopentadienyl, cyclohexadienyl, cycloheptadiene base, cyclooctadiene base in this definition.
Term " C
2-C
10-alkynyl " mean there are 2 to 10 carbon atoms and the unsaturated alkyl containing the straight or branched of at least one triple bond, such as ethynyl, 1-proyl, 2-propynyl (propargyl), ethyl acetylene base, 2-butyne base, 3-butynyl, 1-methyl-2-propynyl etc.
Term heterocycle means such as, " 5-; 6-, or 7 element heterocycles " wherein the ring members atom of heterocycle comprises 1,2,3 or 4 heteroatoms being selected from N, O and S except carbon atom, is interpreted as and means the saturated undersaturated and heteroaromatic (i.e. heteroaryl) with part.Example comprises:
◇ saturated with part undersaturated 5-, 6-or 7 element heterocycles, wherein the ring members atom of heterocycle comprise except carbon atom to be selected from N, O and S 1,2 or 3 heteroatomic, and its be saturated or part undersaturated, such as: pyrrolidin-2-yl, tetramethyleneimine-3 base, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, DOX-4-base, different
azoles alkane-3-base, different
azoles alkane-4-base, different
azoles alkane-5-base, isothiazolidine 3-base, isothiazolidine-4-base, isothiazolidine-5-base, pyrazolidine-3-base, pyrazolidine-4-base, pyrazolidine-5-base,
azoles alkane-2-base,
azoles alkane-4-base,
azoles alkane-5-base, thiazolidine-2-Ji, thiazolidine-4-base, thiazolidine-5-base, imidazolidine-2-base, imidazolidine-4-base, 2-pyrroline-2-base, 2-pyrroline-3-base, 3-pyrroline-2-base, 3-pyrroline-3-base, piperidin-2-yl, piperidines-3-base, piperidin-4-yl, 1,3-bis-
alkane-5-base, tetrahydropyrans-2-base, tetrahydropyran-4-base, tetramethylene sulfide-2-base, hexahydro-pyridazine-3-base, hexahydro-pyridazine-4-base, hexahydropyrimidine-2-base, hexahydropyrimidine-4-base, 5-hexahydropyrimidine base and piperazine-2-base;
◇ 5 Yuans heteroaryls (heteroaromatic base), wherein the ring members atom of heteroaryl comprises 1,2 or 3 heteroatoms being selected from N, O and S except carbon atom, such as, pyrroles-1-base, pyrroles-2-base, pyrroles-3-base, thiophene-2-base, thiene-3-yl-, furans-2-base, furans-3-base, pyrazol-1-yl, pyrazole-3-yl, pyrazoles-4-base, pyrazoles-5-base, imidazoles-1-base, imidazoles-2-base, imidazol-4 yl, imidazoles-5-base
azoles-2-base,
azoles-4-base,
azoles-5-base, different
azoles-3-base, different
azoles-4-base, different
azoles-5-base, thiazol-2-yl, thiazole-4-yl, thiazole-5-base, isothiazole-3-base, isothiazole-4-base, isothiazole-5-base, 1,2,4-triazolyl-1-base, 1,2,4-triazole-3-base 1,2,4-triazole-5-base, 1,2,4-
diazole-3-base, 1,2,4-
diazole-5-base and 1,2,4-thiadiazoles-3-base, 1,2,4-thiadiazoles-5-base;
◇ 6 Yuans heteroaryls (heteroaromatic base), wherein the ring members atom of heteroaryl comprises 1,2 or 3 heteroatoms being selected from N, O and S except carbon atom, such as pyridine-2-base, pyridin-3-yl, pyridin-4-yl, pyridazine-3-base, pyridazine-4-base, pyrimidine-2-base, pyrimidine-4-yl, pyrimidine-5-base, pyrazine-2-base and 1,3,5-triazine-2-base.
" aryl be unsubstituted " phenyl, naphthyl, anthryl or phenanthryl in particular.
In the context of the present invention, " being substituted " means, and by compared with the corresponding substituting group be unsubstituted, one or more H atom is by other atoms of inertia in process of the present invention or molecular radical (such as alkyl, N (alkyl)
2, O-alkyl, S-alkyl, CN, NO
2, I, Cl, Br, F, carbonyl, carboxyl, R3 is COOR3,5-of alkyl, 6-, or 7 element heterocycles, aryl-rear both as defined above) replaced.In the context of inert substituent, " alkyl " means the C of the straight or branched saturated hydrocarbyl for having 1 to 10 carbon atom
1-C
10-alkyl, such as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-etc.
Lipase-catalyzed acylation reaction of the present invention needs to use dicarboxylic acid anhydride.Each dicarboxylic acid anhydride in principle, is shown in that such as formula D can be used for this object.
Preferably, R2 is C
2-C
10alkyl.The example being ready to use in the dicarboxylic acid of method of the present invention is: propanedioic acid (i.e. daucic acid) acid anhydrides, succinic acid (i.e. succsinic acid) acid anhydrides, pentanedioic acid (i.e. glue acid) acid anhydrides, hexanodioic acid (i.e. adipic acid) acid anhydrides, pimelic acid (pimelic acid) acid anhydrides, suberic acid (i.e. cork acid) acid anhydrides, nonane diacid (i.e. lepargylic acid) acid anhydrides, sebacic acid (i.e. sebacic acid) acid anhydrides, undecane diacid acid anhydrides, dodecanedioic acid acid anhydrides.Particularly preferably use C
3-C
8dicarboxylic acid anhydride, such as succinyl oxide.
Preferably with equimolar amount, more preferably with the dicarboxylic acid anhydride used at least 10% excessive use the inventive method, to allow the complete acylations of trans isomer.The composition of initial substance, the per-cent namely in the mixture of hexalin that is substituted at cis/trans of trans isomer, measures decisive role for the dicarboxylic acid anhydride determining to use in the inventive method.Such as, for 30: 70 mixtures of cis/trans isomers, use the dicarboxylic acid anhydride of 0.7 equivalent or 0.8 excessive equivalent should play the object of the almost trans hexalin be substituted of acylations completely.
The lipase used in the inventive method is selected from such lipase, described lipase have according to the aminoacid sequence of SEQ ID No:2 or be derived from as SEQ ID No:2 show, show by disappearance, replace, to insert or its combination changes at the most 25%, preferably at the most 20%, more preferably at the most 15%, especially at the most 10,9,8,7,6,5,4,3,2,1% the sequence of amino-acid residue.This means, the lipase used in the inventive method has and the whole aminoacid sequence at least 75% shown in SEQ ID No:2, preferably at least 80%, more preferably at least 85%, especially at least 90,91,92,93,94,95,96,97,98,99% identical sequence.Percent sequence identity between two sequences is the function (i.e. Percent sequence identity=identical positional number/total number of positions x100) of the same position number that sequence has.
The lipase used in the inventive method can be expressed in the biology producing lipase.Producing that the biology of lipase means can be natural or by any biology of genetic modification (such as by being inserted into by lipase gene in the genome of biology) yielding lipase in next life, described lipase have according to the aminoacid sequence of SEQ ID No:2 or with the whole aminoacid sequence at least 75% shown in SEQ ID No:2, preferably at least 80%, more preferably at least 85%, especially at least 90,91,92,93,94,95,96,97,98,99% identical sequence.The example producing the biology of lipase is Aspergillus (Aspergillus), genus arthrobacter (Arthrobacter), Alkaligenes (Alcaligenes), bacillus (Bacillus), brevibacterium sp (Brevibacterium), Rhodopseudomonas (Pseudomonas), chromobacterium (Chromobacterium), Candida (Candida), Fusarium (Fusarium), geotrichum (Geotrichum), Humicola (Humicola), mucor (Mucor), Pichia (Pichia), Penicillium (Penicillium), Rhizomucor (Rhizomucor), the microorganism of Rhizopus (Rhizopus) or Thermus (Thermus).Preferably express in uncle's kirschner Pseudomonas alba (i.e. plant Burkholderia (Burkholderia plantarii)).
The preferred lipase gene be inserted in the biology producing lipase be a) as in SEQ ID No:1 the polynucleotide that define, b) in the total length in the sequence encoding district of SEQ ID No:1 with the sequence of SEQ ID No:1 at least about 50%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher identical polynucleotide.Percent sequence identity between two sequences is the function (i.e. Percent sequence identity=identical positional number/total number of positions x100) of the same position number that sequence has.
Can cultivate in a way known and produce the biology of lipase, such as, by fermenting in nutritional medium, described substratum except nutrition, trace element and suitable time microbiotic except, contain, such as, the buffer system of stabilizing protein and enzyme.Such as, at US 6596520 B1, particularly in embodiment 1 paragraph 1.1, describe the biology cultivated and produce lipase, wherein use plant Burkholderia as an example.
Compared with SEQ ID No:2, the derivative aminoacid sequence used in the inventive method should have at least 50%, preferably 65%, more preferably 80%, especially more than 90% the enzymatic activity of SEQ ID No:2.In this context, the enzymatic activity of SEQ ID No:2 means the ability causing the hexalin that trans selective acylations is substituted.Trans selective is at least 95%, more preferably at least 98%, even more preferably 99%.4-tert. butyl cyclohexanol can be used as reference substance.
Lipase activity (the people .Review:Lipase assays for conventional and molecular screening:an overview such as Gupta itself can be measured by currently known methods, Biotechnol.Appl.Biochem. (2003) 37,63-71).Catalytic activity is measured preferably by the test of use tributyrin in aqueous matrix.Phenylethyl alcohol test can be applied in organic system.
The 3 dimension shapes failing to be folded into expection produce protein or the enzyme of non-activity usually.Folding usually be called folding assistant's albumen specialization molecule supervision under carry out.In a preferred embodiment
◇ have according to the aminoacid sequence of SEQ ID No:2 or with the whole aminoacid sequence at least 75% shown in SEQ ID No:2, preferably at least 80%, more preferably at least 85%, especially at least 90,91,92,93,94,95,96,97,98, the lipase of 99% identical sequence by
◇ as in SEQ ID No:1 the polynucleotide that define, or in the total length in the sequence encoding district of SEQ ID No:1 with the sequence of SEQ ID No:1 at least about 50%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85% or 90%, even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher identical polynucleotide coded by, described polynucleotide exist
Express in ◇ plant Burkholderia.
Therefore, folding assistant's albumen and lipase coexpression in plant Burkholderia, described folding assistant's albumen have according to the aminoacid sequence of SEQ ID No:3 or be derived from as SEQ ID No:3 show, with in SEQ ID No:3 the whole aminoacid sequence at least 85% shown, especially at least 90,91,92,93,94,95,96,97,98,99% identical sequence.
The lipase used in method of the present invention can be used as the coarse extract of full cell with height to the preparation (such as not celliferous extract) of different purity of height purified form.Preferred use is in the lipase of the form of partial purification or highly purified protein soln.The plant Burkholderia cell used in preferred embodiments is little, has specific density comparable with the specific density of substratum.Preferably, by using suitable screen plate well known by persons skilled in the art to be separated described cell with the microfiltration of strong pump from supernatant liquor.Such as further concentrated by using ultrafiltration as is known to persons skilled in the art to reach.
Distinguishing in solution the lipase for conversion of substrate that uses with the stability usually with increase can be used for carrying out continuously or the immobilized lipase of batch reactions.Immobilized meaning uses method known to those skilled in the art to be usually combined in supporting agent on solid support, then in method according to the present invention (seeing below).Especially when implementation method continuously, immobilized lipase is used to be preferred embodiment.For this purpose, can such as advantageously use lipase to be retained in pillar or tubular reactor simultaneously.
There is the lipase in multiple fixing of possibility the inventive method.Can according to such as, as the people .Biotechnology Letters such as Persson 2000,22 (19): 1571-1575; The method that US6,596,520B1-particularly describe in embodiment 1, the coarse extract of immobilized whole-cell suspension or the supernatant liquor of cell culture, and purified protein soln.
The substratum (or fermented liquid) itself producing the biology of lipase also can at 50-150 DEG C, preferably 70-100 DEG C, more preferably 75-85 DEG C and even more preferably temperature (temperature out of the spray-drier) spraying dry of 80-85 DEG C.Also spraying dry can be carried out when there is carrier material.Must so that the mode of method of the present invention can be carried out to select supporting agent.Preferably, polysaccharide such as Star Dri 5 or mineral compound such as Na
2sO
4be used as supporting agent.Based on the solids content of fermented liquid, the weight of supporting agent is by weight 5 to 200%, preferably 10-200% by weight, more preferably 20-150% and particularly preferably 50-100% by weight by weight.Also the lipase solution that this class methods immobilization is purified can be used.Based on solid matter, residual moisture is less than 10%, and preferably it is less than 7%, is particularly preferably less than the residual moisture content of 5%.
In the method for the invention, use for initial substance (cis/trans mixture) 0.5-10%, preferably 0.5-5% by weight by weight, more preferably 0.5-1, especially by weight 1% (immobilized) lipase of amount.
Acylation reaction of the present invention can or under the condition of solvent not carry out.Preferably it is at organic solvent, such as hydrocarbon, ether, or carries out in alcohol.The solvent being particularly suitable for reacting is:
◇ fat hydrocarbon such as hexane, heptane or octane or its mixture, particularly sherwood oil, or
◇ is aromatic hydrocarbon based as benzene, toluene, dimethylbenzene, or
◇ ethers is methyl tert-butyl ether (MTBE), tetrahydrofuran (THF) (THF), Isosorbide-5-Nitrae-two such as
alkane, or
The ◇ alicyclic ring same clan such as pentamethylene, hexanaphthene, or
The ◇ tertiary alcohols such as trimethyl carbinol, tertiary amyl alcohol.
If use solvent, with organic solvent diluting initial substance (cis/trans mixture), make the 0.2-5 mole obtaining initial substance (cis/trans mixture), the solution of preferred 0.5-2 mole, preferred 0.6-1.2 mole.
Can react continuously or in batches.For the enforcement in technical scale, recommend continuous synthesis, particularly use the lipase having support.
The second step of inventive method:
Half ester needs to be separated half ester from unreacted isomers with the mixture of unreacted isomers.This can be completed easily by water extraction (such as half ester salt, the particularly water extraction of its basic metal or alkaline earth salt).Preferred embodiment is the water extraction under alkali (such as sodium carbonate or sodium hydroxide) exists.Preferably reason for this reason, pH should at 7.5-10, preferably 8-10, more preferably 8-9.5, in the scope of 9-9.5 especially.
3rd step of inventive method:
Which kind of isomers depending on alcohol is required, can check the organic phase containing syn-isomerism body, or containing being in the aqueous phase of trans isomer of half ester-formin.The trans isomer of alcohol that half ester is cut into corresponding acid and wants by usual method for hydrolysis can be used, such as, by with alkali (such as NaOH, KOH, Na
2cO
3) or acid (such as H
2sO
4, HCl) and process.
Hereinafter, the example by providing further describes the present invention.This explanation is not intended to limit the present invention.
Embodiment 1: preparation shaking flask pre-culture
The Erlenmeyer flask of two 1000ml is sealed, with aluminium foil covering and at 134 DEG C of sterilizing 30min with velveteen plug.Same at 134 DEG C of sterilizing 30min with the scaled glass cylinder of foil sealing 250ml.
Following component is used to prepare micro-salts solution: two liters are removed mineral water completely, 77.2g monohydrate potassium, 22.6g Zinc vitriol, 17.3g six ferrous sulfate hydrate ammonium, 5.7g Manganous sulfate monohydrate, 1.2g Salzburg vitriol, 0.5g Cobalt monosulfate heptahydrate and 3.0g CALCIUM CHLORIDE DIHYDRATE.
Make the 500ml substratum comprising following component: 3.8g dry yeast extract powder, 0.5g potassium primary phosphate, 1.5g Secondary ammonium phosphate, 0.5g bitter salt, 5g trace elements salts solution/500g water.Use phosphoric acid that pH is adjusted to 6.5.The substratum that filtration sterilization (0.22 μm) completes.By aseptic for 200ml substratum be transferred to two Erlenmeyer flasks each in, then use 1ml plant Burkholderia (LU8093) liquid storage flasks in each case.
Then in vibrator incubator (vibrator orbit radius=25mm), 30 DEG C and 200rpm hatch Erlenmeyer flask culture 12 hours.
Embodiment 2: prepare fermentor tank pre-culture
In stainless steel cask, make the substratum that indicates in 10 liters of embodiments 1 and use phosphoric acid that pH is adjusted to 6.5.Then by media transfer in the 21 liters of fermentor tanks being furnished with three traditional paddle stirrers.Then 30 DEG C are cooled to 121 DEG C of disinfection fermentation tanks 60 minutes.
Then be transferred in fermentor tank by aseptic for the Erlenmeyer flask pre-culture grown completely, and run the air that fermentor tank 8 hours: Ventilation Rate 0.5vvm compresses under the following conditions, steady temperature 30 DEG C, overburden pressure=0.1bar, rotating speed=1000rpm, regulates pH6.5 with the sodium hydroxide solution of 25% intensity and the phosphoric acid of 20% intensity.
Embodiment 3: produce lipase in fermentor tank
The fermentor tank that cubic capacity is 300 liters is loaded continuously with following initial substance:
150 liters are removed mineral water completely, 1207g dry yeast extract powder, 160g potassium primary phosphate, 480g Secondary ammonium phosphate, 432g bitter salt, 1600g trace elements salts solution (embodiment 1), 30ml
3062 (based on the foam reducing composition of polysiloxane).With the phosphoric acid of 20% intensity, the pH of substratum is adjusted to 4.5.
Be cooled to 30 DEG C 121 DEG C of disinfection fermentation tanks 60 minutes.Then the pre-culture aseptic inoculation fermentor tank of embodiment 2 is used, and run fermentor tank under the following conditions: the air that Ventilation Rate 0.4vvm compresses, steady temperature 30 DEG C, overburden pressure=0.3bar, rotating speed=550rpm, regulates pH6.5 with the sodium hydroxide solution of 25% intensity and the phosphoric acid of 20% intensity.
After the fermentation time of four hours, the filtration sterilization unit of passing hole size 0.2 μm pumps into rapeseed oil.In the first charging stage, carry out feed supplement according to following formula:
Feeding rate [g/l]=19.7*e
(0.11*t)
Wherein t represent in hour fermentation time
Stopped for the first charging stage after 17 hours.Thereafter and then, more rapeseed oils are pumped into according to following formula:
Feeding rate [g/l]=168.6*e
(0.0069*t)
After 96 hours, stop oil subsidy material, and continue to run fermentor tank until the oil consumption in substratum is most.After this, fermentor tank is cooled to 4 DEG C.And then, sample thief measure total dry matter (DM) and the enzymatic activity (unit/ml) of liquid.By infrared moisture analysis-e/or determining dry matter content.Use tributyrin as substrate titration determination enzymatic activity.Amount per minute being discharged the liquid of 1 μm of ol butyric acid from tributyrin is defined as an enzymatic unit.
In the fermentor tank of 300 liters, measured the dry matter content of 7.56%, enzymatic activity is 10256U/ml.Fermentor tank weight is 186.8kg.Total dry matter reaches 14.1kg.Total enzymatic activity reaches 1916MU.1MU=1000000 unit.
Embodiment 4: produce immobilized lipase
By spraying dry by the lipase immobilization that is present in fermented liquid over sodium sulfate.
For this purpose, 14.1kg sodium sulfate to be added in fermentor contents and to dissolve with the minimum speed of fermentor tank in 1 hour.After this, by whole fermentor contents spraying dry.Use 250m
3/ h nitrogen runs spray-drier.Temperature in is 180 DEG C.Temperature out is 75 DEG C.By two substance nozzle (two-substance nozzle) by fermentor contents nitrogen injection stream.Adjustment pump speed makes to reach desirable temperature out (about 15kg/h).Be separated dry powder by cyclonic separator and discharged from cyclonic separator by cell hub brake.
Obtain the dry powder amounting to 25.6kg.The residual moisture content of powder is 1.8%.By the sample dissolution of dry powder in water and use tributyrin measure enzymatic activity.The activity of powder is 67360 units/gDM.
The following transesterification of immobilized enzyme catalysis in organic medium:
Following test fixture is used for the suitability of the conversion in organic system: react in test reactor, described test reactor is made up of the 500ml jacketed vessel being furnished with the propeller type stirrer be made up of glass.Agitator is driven by magnetic coupling by the engine (RZR2051 type) from Heidolph.By thermostatted (Huber Ministat) heating unit.
At 50ml
weigh about 0.5g fixture in pipe and record accurate weight.
Thermostatted is set to 22 DEG C and agitator speed is 350rpm.
By glass funnel, load dry reactor with the 1-phenylethyl alcohol of 50.0g and the MTBE of 95.0g.Avoid any pollution of water because the only reaction of too much water resistance.
Added through the preweighted fixture containing lipase by glass funnel.
By adding the propionate initial action of 20.4g.
Add propionate after 60 minutes, sampling (about 1ml) and be filtered through immediately 0.2 μm of syringe filter (
30/02RC, Schleicher & Schuell).
The filtered sample of 100 μ l and 900 μ l HPLC elutriants (acetonitrile 20%, methyl alcohol 40%, trifluoroacetic acid 1% and water 39%) are placed in 2ml
in container.By 100 these solution of μ l then be placed in HPLC pipe and similarly supplement 900 μ l HPLC elutriants, wherein pipe be sealing.By HPLC analytic sample.
Unit of lipase activity in organic system is PEU (phenylethyl alcohol unit).1PEU is under the test conditions above, and per minute forms the amount of the lipase of 1 μm of ol phenylethyl propionate (PEP) from phenylethyl alcohol catalysis.
Parameter required for calculating:
Phenylethyl propionate concentration (PEP) [mmol/l]
Reaction volume [l]
Time (reaction times) [min]
Weight (amount of the enzyme of use) [g]
Calculate enzymatic activity
Calculate specific activity
The fixture obtained in embodiment has the specific activity of 842PEU/g fixture.
Embodiment 5: preparation cis-4-tert. butyl cyclohexanol
The cis/trans (30: 70) mixture (726mol, 1eq) of 113.4kg4-tert. butyl cyclohexanol is introduced and is equipped with in the reaction vessel of 453l MTBE (1.6mol).Add 58.1kg succinyl oxide (580mol; 0.8eq) and 1.1kg be fixed on Na
2sO
4on the lipase from plant Burkholderia (by weight 1%; See above example).At 20 DEG C of stirred reaction mixtures.Reaction method is checked by gas-chromatography.After the 23h reaction times, the trans isomer (table 1, No. 3) of 0.9GC area % still detected, then stirred reaction mixture 27h.
Table 1: isometry ratio cis/trans
Numbering | Detection | Cis (%) | Trans (%) |
1 | Start | 30.6 | 69.4 |
2 | After 17h | 94.6 | 5.4 |
3 | After 23h | 99.1 | 0.9 |
4 | After 50h | 99.9 | 0.1 |
By diatomite (Kieselgur, such as
) filter reaction mixture and with other MTBE rinsing vessel and MTBE.After MTBE phase distilled water being added into filtered reaction mixture and rinse step, add 25%NaOH solution until reach pH9.3 at 20 DEG C of substeps.Add other distilled water and be separated.Twice aqueous phase (pH9.3) is extracted again with MTBE.Combination organic phase, and remove solvent until obtain the suspension of white by distillation (50mbar, maximum 40 DEG C).This suspension (109kg) is concentrated further in rotary evaporator.
Table 2: isometry ratio cis/trans after extracting
Numbering | Detection | Cis (%) | Trans (%) |
1 | The organic phase of combination | 99.3 | 0.7 |
2 | After distillation | 99.6 | 0.4 |
For distillation, use the simple water distilling apparatus with the post (packed column, i.e. Raschigrings, 8x8mm) carrying packing material and the solid bridge (condenser of quenching) be heated.Transition temperature is 118 DEG C of 26mbar water spray vacuum.
Generally speaking, 24.8kg cis-4-(1,1-the dimethyl ethyl)-hexalin (73% output) being in white solid forms is obtained
Embodiment 6: preparation cis-4-tert. butyl cyclohexanol
Mixing 1g4-tert. butyl cyclohexanol (cis/trans: 30: 70; I.e. 1eq) and 10ml toluene (i.e. 0.6mol).Add 0.5g succinyl oxide (i.e. 0.8eq) and 0,1g and be fixed on Na
2sO
4on lipase (namely by weight 10% /). reaction conditions is in the same manner as in Example 1.
At 20 DEG C of stirred reaction mixture 24h also as embodiment 5 processes further.Analyze the detection after 1 and 24h churning time:
Numbering | Reaction times | Cis (%) | Trans (%) |
1 | 1h | 73.2 | 26.8 |
2 | 24h | 100 | - |
Embodiment 7: similar from embodiment 6 but different after 20 DEG C of 24h solvents (0.6mol)
Numbering | Solvent | Cis (%) | Trans (%) |
1 | MTBE | 99 | 0.1 |
2 | THF | 98 | - |
3 | Hexanaphthene | 79 | 19 |
4 | Sherwood oil | 81 | 17 |
Embodiment 8: but 20 DEG C 24hs after 1.2mol solvent similar to embodiment
Numbering | Solvent | Cis (%) | Trans (%) |
1 | MTBE | 100 | - |
2 | Toluene | 100 | - |
Claims (22)
1. prepare the method for the pure hexalin be substituted of basic isometry, described method comprises:
I) exist have as in SEQ ID No:2 the lipase of protein sequence shown, or there is the protein sequence identical with whole aminoacid sequence at least 75% shown in SEQ ID No:2 and under there is the condition of the lipase of the enzymatic activity of at least 50% of SEQ ID No:2, by the cis/trans mixture of hexalin that is substituted and dicarboxylic acids anhydride reactant, produce trans half esters
Ii) from the unreacted hexalin syn-isomerism body be substituted, trans half esters is separated,
Iii) cis be substituted or trans cvclohexanol is separated;
The mixture of the hexalin be substituted wherein used is being substituted relative to 4 of OH-group;
Wherein in step (i), use C
2-C
10dicarboxylic acid anhydride; And
Wherein said " basic isometry is pure " means the isomers obtaining at least 80%.
2. method according to claim 1, the lipase wherein used in step (i) be by a) as in SEQ ID No:1 the polynucleotide that define, coded by b) identical with the sequence at least 75% of SEQ ID No:1 in the total length in the sequence encoding district of SEQ ID No:1 polynucleotide.
3. method according to claim 2, wherein lipase is expressed in plant Burkholderia (Burkholderia plantarii).
4. method as claimed in one of claims 1-3, wherein uses succinyl oxide.
5. method as claimed in one of claims 1-3, the lipase wherein in step (i) is immobilized.
6. method according to claim 4, the lipase wherein in step (i) is immobilized.
7. the method any one of claim 1-3 and 6, wherein the reaction of step (i) is at hydrocarbon, the ether as solvent, or carries out in alcohol.
8. method according to claim 4, wherein the reaction of step (i) is at hydrocarbon, the ether as solvent, or carries out in alcohol.
9. method according to claim 5, wherein the reaction of step (i) is at hydrocarbon, the ether as solvent, or carries out in alcohol.
10. method according to claim 7, wherein solvent is selected from toluene, sherwood oil, MTBE, tetrahydrofuran (THF), or hexanaphthene.
The method of 11. according to Claim 8 or 9, wherein solvent is selected from toluene, sherwood oil, MTBE, tetrahydrofuran (THF), or hexanaphthene.
12. according to claim 1-3,6, method any one of 8-10, the separation wherein in step (ii) is undertaken by extracting at the pH of 8-10.
13. methods according to claim 4, the separation wherein in step (ii) is undertaken by extracting at the pH of 8-10.
14. methods according to claim 5, the separation wherein in step (ii) is undertaken by extracting at the pH of 8-10.
15. methods according to claim 7, the separation wherein in step (ii) is undertaken by extracting at the pH of 8-10.
16. methods according to claim 11, the separation wherein in step (ii) is undertaken by extracting at the pH of 8-10.
17. according to claim 1-3,6, method any one of 8-10,13-16, wherein use 4-tert. butyl cyclohexanol.
18. methods according to claim 4, wherein use 4-tert. butyl cyclohexanol.
19. methods according to claim 5, wherein use 4-tert. butyl cyclohexanol.
20. methods according to claim 7, wherein use 4-tert. butyl cyclohexanol.
21. methods according to claim 11, wherein use 4-tert. butyl cyclohexanol.
22. methods according to claim 12, wherein use 4-tert. butyl cyclohexanol.
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PCT/IB2011/055199 WO2012069974A1 (en) | 2010-11-26 | 2011-11-21 | Preparation of isomerically pure substituted cyclohexanols |
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Citations (2)
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---|---|---|---|---|
CN1280189A (en) * | 1999-07-09 | 2001-01-17 | Basf公司 | Immobilized lipase |
CN1914190A (en) * | 2004-01-29 | 2007-02-14 | 巴斯福股份公司 | Method for producing enantiomer-pure aminoalcohols |
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US4665028A (en) * | 1982-10-06 | 1987-05-12 | Novo Industri A/S | Method for production of an immobilized enzyme preparation by means of a crosslinking agent |
DE10151292A1 (en) * | 2001-10-22 | 2003-04-30 | Basf Ag | New bacterial lipase mutants, useful for enantioselective conversion, e.g. acylation of alcohols, have increased specific activity |
EP2248906A4 (en) * | 2008-01-23 | 2012-07-11 | Ajinomoto Kk | Method of producing l-amino acid |
KR20110087273A (en) * | 2008-09-29 | 2011-08-02 | 아커민 인코퍼레이티드 | Process for accelerated capture of carbon dioxide |
-
2011
- 2011-11-21 WO PCT/IB2011/055199 patent/WO2012069974A1/en active Application Filing
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CN1280189A (en) * | 1999-07-09 | 2001-01-17 | Basf公司 | Immobilized lipase |
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