CN103160547A - 一种醇脱氢酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 - Google Patents
一种醇脱氢酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 Download PDFInfo
- Publication number
- CN103160547A CN103160547A CN201310132479XA CN201310132479A CN103160547A CN 103160547 A CN103160547 A CN 103160547A CN 201310132479X A CN201310132479X A CN 201310132479XA CN 201310132479 A CN201310132479 A CN 201310132479A CN 103160547 A CN103160547 A CN 103160547A
- Authority
- CN
- China
- Prior art keywords
- ethyl
- chloro
- gly
- seq
- alcohol dehydrogenase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010021809 Alcohol dehydrogenase Proteins 0.000 title claims abstract description 24
- 102000007698 Alcohol dehydrogenase Human genes 0.000 title claims abstract description 22
- ZAJNMXDBJKCCAT-RXMQYKEDSA-N ethyl (3r)-4-chloro-3-hydroxybutanoate Chemical compound CCOC(=O)C[C@@H](O)CCl ZAJNMXDBJKCCAT-RXMQYKEDSA-N 0.000 title abstract 4
- 230000003197 catalytic effect Effects 0.000 title description 2
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 5
- 241000894006 Bacteria Species 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 9
- 150000002148 esters Chemical class 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 239000003054 catalyst Substances 0.000 abstract description 2
- OHLRLMWUFVDREV-UHFFFAOYSA-N ethyl 4-chloro-3-oxobutanoate Chemical compound CCOC(=O)CC(=O)CCl OHLRLMWUFVDREV-UHFFFAOYSA-N 0.000 abstract 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion 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](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 abstract 2
- 229950006238 nadide Drugs 0.000 abstract 2
- 230000002349 favourable effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 25
- 238000006555 catalytic reaction Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 230000003287 optical effect Effects 0.000 description 11
- 238000006722 reduction reaction Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 230000000968 intestinal effect Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 239000012064 sodium phosphate buffer Substances 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- DKPFZGUDAPQIHT-UHFFFAOYSA-N butyl acetate Chemical compound CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 6
- 241000222122 Candida albicans Species 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 230000003139 buffering effect Effects 0.000 description 4
- 229940095731 candida albicans Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- 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 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000012044 organic layer Substances 0.000 description 3
- 229940049547 paraxin Drugs 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 235000011009 potassium phosphates Nutrition 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- RDRMWJBLOSRRAW-BYULHYEWSA-N Asp-Asn-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O RDRMWJBLOSRRAW-BYULHYEWSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 241000228393 Sporidiobolus salmonicolor Species 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 108010016616 cysteinylglycine Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010073969 valyllysine Proteins 0.000 description 2
- 238000009834 vaporization Methods 0.000 description 2
- 230000008016 vaporization Effects 0.000 description 2
- XXDIJWSZFWZBRM-QCEWEWFLSA-N (3z,5e,8r,9e,11z,14s,16s,17e,19e,24r)-8,14,16-trihydroxy-24-methyl-1-oxacyclotetracosa-3,5,9,11,17,19-hexaen-2-one Chemical compound C[C@@H]1CCC\C=C\C=C\[C@@H](O)C[C@@H](O)C\C=C/C=C/[C@H](O)C\C=C\C=C/C(=O)O1 XXDIJWSZFWZBRM-QCEWEWFLSA-N 0.000 description 1
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 1
- MCKSLROAGSDNFC-ACZMJKKPSA-N Ala-Asp-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MCKSLROAGSDNFC-ACZMJKKPSA-N 0.000 description 1
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 1
- GSHKMNKPMLXSQW-KBIXCLLPSA-N Ala-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C)N GSHKMNKPMLXSQW-KBIXCLLPSA-N 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- 108010053754 Aldehyde reductase Proteins 0.000 description 1
- WKPXXXUSUHAXDE-SRVKXCTJSA-N Arg-Pro-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O WKPXXXUSUHAXDE-SRVKXCTJSA-N 0.000 description 1
- PLVAAIPKSGUXDV-WHFBIAKZSA-N Asn-Gly-Cys Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)C(=O)N PLVAAIPKSGUXDV-WHFBIAKZSA-N 0.000 description 1
- GQRDIVQPSMPQME-ZPFDUUQYSA-N Asn-Ile-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O GQRDIVQPSMPQME-ZPFDUUQYSA-N 0.000 description 1
- NCFJQJRLQJEECD-NHCYSSNCSA-N Asn-Leu-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O NCFJQJRLQJEECD-NHCYSSNCSA-N 0.000 description 1
- RCFGLXMZDYNRSC-CIUDSAMLSA-N Asn-Lys-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O RCFGLXMZDYNRSC-CIUDSAMLSA-N 0.000 description 1
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 1
- PAYPSKIBMDHZPI-CIUDSAMLSA-N Asp-Leu-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O PAYPSKIBMDHZPI-CIUDSAMLSA-N 0.000 description 1
- IVPNEDNYYYFAGI-GARJFASQSA-N Asp-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N IVPNEDNYYYFAGI-GARJFASQSA-N 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 241000222128 Candida maltosa Species 0.000 description 1
- VKAWJBQTFCBHQY-GUBZILKMSA-N Cys-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CS)N VKAWJBQTFCBHQY-GUBZILKMSA-N 0.000 description 1
- LHJDLVVQRJIURS-SRVKXCTJSA-N Cys-Phe-Asp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CS)N LHJDLVVQRJIURS-SRVKXCTJSA-N 0.000 description 1
- IRKLTAKLAFUTLA-KATARQTJSA-N Cys-Thr-Lys Chemical compound C[C@@H](O)[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CCCCN)C(O)=O IRKLTAKLAFUTLA-KATARQTJSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- MLZRSFQRBDNJON-GUBZILKMSA-N Gln-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MLZRSFQRBDNJON-GUBZILKMSA-N 0.000 description 1
- XXLBHPPXDUWYAG-XQXXSGGOSA-N Gln-Ala-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XXLBHPPXDUWYAG-XQXXSGGOSA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- SGVGIVDZLSHSEN-RYUDHWBXSA-N Gln-Tyr-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O SGVGIVDZLSHSEN-RYUDHWBXSA-N 0.000 description 1
- RFDHKPSHTXZKLL-IHRRRGAJSA-N Glu-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N RFDHKPSHTXZKLL-IHRRRGAJSA-N 0.000 description 1
- UGSVSNXPJJDJKL-SDDRHHMPSA-N Glu-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N UGSVSNXPJJDJKL-SDDRHHMPSA-N 0.000 description 1
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 1
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 1
- XQHSBNVACKQWAV-WHFBIAKZSA-N Gly-Asp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XQHSBNVACKQWAV-WHFBIAKZSA-N 0.000 description 1
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 1
- XPJBQTCXPJNIFE-ZETCQYMHSA-N Gly-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 1
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- YIFUFYZELCMPJP-YUMQZZPRSA-N Gly-Leu-Cys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O YIFUFYZELCMPJP-YUMQZZPRSA-N 0.000 description 1
- RYAOJUMWLWUGNW-QMMMGPOBSA-N Gly-Val-Gly Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O RYAOJUMWLWUGNW-QMMMGPOBSA-N 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- XINDHUAGVGCNSF-QSFUFRPTSA-N His-Ala-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XINDHUAGVGCNSF-QSFUFRPTSA-N 0.000 description 1
- VLPMGIJPAWENQB-SRVKXCTJSA-N His-Cys-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O VLPMGIJPAWENQB-SRVKXCTJSA-N 0.000 description 1
- HQKADFMLECZIQJ-HVTMNAMFSA-N His-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N HQKADFMLECZIQJ-HVTMNAMFSA-N 0.000 description 1
- STGQSBKUYSPPIG-CIUDSAMLSA-N His-Ser-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 STGQSBKUYSPPIG-CIUDSAMLSA-N 0.000 description 1
- FFYYUUWROYYKFY-IHRRRGAJSA-N His-Val-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O FFYYUUWROYYKFY-IHRRRGAJSA-N 0.000 description 1
- XENGULNPUDGALZ-ZPFDUUQYSA-N Ile-Asn-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N XENGULNPUDGALZ-ZPFDUUQYSA-N 0.000 description 1
- NZOCIWKZUVUNDW-ZKWXMUAHSA-N Ile-Gly-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O NZOCIWKZUVUNDW-ZKWXMUAHSA-N 0.000 description 1
- QHUREMVLLMNUAX-OSUNSFLBSA-N Ile-Thr-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)O)N QHUREMVLLMNUAX-OSUNSFLBSA-N 0.000 description 1
- RQZFWBLDTBDEOF-RNJOBUHISA-N Ile-Val-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N RQZFWBLDTBDEOF-RNJOBUHISA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 description 1
- FGNQZXKVAZIMCI-CIUDSAMLSA-N Leu-Asp-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N FGNQZXKVAZIMCI-CIUDSAMLSA-N 0.000 description 1
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 1
- NHHKSOGJYNQENP-SRVKXCTJSA-N Leu-Cys-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N NHHKSOGJYNQENP-SRVKXCTJSA-N 0.000 description 1
- KGCLIYGPQXUNLO-IUCAKERBSA-N Leu-Gly-Glu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O KGCLIYGPQXUNLO-IUCAKERBSA-N 0.000 description 1
- UCDHVOALNXENLC-KBPBESRZSA-N Leu-Gly-Tyr Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 UCDHVOALNXENLC-KBPBESRZSA-N 0.000 description 1
- KPYAOIVPJKPIOU-KKUMJFAQSA-N Leu-Lys-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O KPYAOIVPJKPIOU-KKUMJFAQSA-N 0.000 description 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- LMVOVCYVZBBWQB-SRVKXCTJSA-N Lys-Asp-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LMVOVCYVZBBWQB-SRVKXCTJSA-N 0.000 description 1
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 1
- IZJGPPIGYTVXLB-FQUUOJAGSA-N Lys-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N IZJGPPIGYTVXLB-FQUUOJAGSA-N 0.000 description 1
- OBZHNHBAAVEWKI-DCAQKATOSA-N Lys-Pro-Asn Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O OBZHNHBAAVEWKI-DCAQKATOSA-N 0.000 description 1
- HKXSZKJMDBHOTG-CIUDSAMLSA-N Lys-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN HKXSZKJMDBHOTG-CIUDSAMLSA-N 0.000 description 1
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 1
- XXDIJWSZFWZBRM-CAOGAUQTSA-N Macrolactin A Natural products CC1CCCC=CC=CC(O)CC(O)CC=C/C=C/C(O)CC=CC=C/C(=O)O1 XXDIJWSZFWZBRM-CAOGAUQTSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- SMVTWPOATVIXTN-NAKRPEOUSA-N Met-Ser-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SMVTWPOATVIXTN-NAKRPEOUSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- SEPNOAFMZLLCEW-UBHSHLNASA-N Phe-Ala-Val Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O SEPNOAFMZLLCEW-UBHSHLNASA-N 0.000 description 1
- JJHVFCUWLSKADD-ONGXEEELSA-N Phe-Gly-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](C)C(O)=O JJHVFCUWLSKADD-ONGXEEELSA-N 0.000 description 1
- YKUGPVXSDOOANW-KKUMJFAQSA-N Phe-Leu-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YKUGPVXSDOOANW-KKUMJFAQSA-N 0.000 description 1
- DSXPMZMSJHOKKK-HJOGWXRNSA-N Phe-Phe-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O DSXPMZMSJHOKKK-HJOGWXRNSA-N 0.000 description 1
- AFNJAQVMTIQTCB-DLOVCJGASA-N Phe-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=CC=C1 AFNJAQVMTIQTCB-DLOVCJGASA-N 0.000 description 1
- ZVJGAXNBBKPYOE-HKUYNNGSSA-N Phe-Trp-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(O)=O)C1=CC=CC=C1 ZVJGAXNBBKPYOE-HKUYNNGSSA-N 0.000 description 1
- IEIFEYBAYFSRBQ-IHRRRGAJSA-N Phe-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N IEIFEYBAYFSRBQ-IHRRRGAJSA-N 0.000 description 1
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 1
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 1
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 1
- ABSSTGUCBCDKMU-UWVGGRQHSA-N Pro-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 ABSSTGUCBCDKMU-UWVGGRQHSA-N 0.000 description 1
- WCNVGGZRTNHOOS-ULQDDVLXSA-N Pro-Lys-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O WCNVGGZRTNHOOS-ULQDDVLXSA-N 0.000 description 1
- KWMZPPWYBVZIER-XGEHTFHBSA-N Pro-Ser-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWMZPPWYBVZIER-XGEHTFHBSA-N 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 241001123231 Schwanniomyces capriottii Species 0.000 description 1
- KCNSGAMPBPYUAI-CIUDSAMLSA-N Ser-Leu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KCNSGAMPBPYUAI-CIUDSAMLSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- XFTYVCHLARBHBQ-FOHZUACHSA-N Thr-Gly-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O XFTYVCHLARBHBQ-FOHZUACHSA-N 0.000 description 1
- 241000006364 Torula Species 0.000 description 1
- 241000235006 Torulaspora Species 0.000 description 1
- ACGIVBXINJFALS-HKUYNNGSSA-N Trp-Phe-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N ACGIVBXINJFALS-HKUYNNGSSA-N 0.000 description 1
- RCLOWEZASFJFEX-KKUMJFAQSA-N Tyr-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RCLOWEZASFJFEX-KKUMJFAQSA-N 0.000 description 1
- MOCXXGZHHSPNEJ-AVGNSLFASA-N Tyr-Cys-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O MOCXXGZHHSPNEJ-AVGNSLFASA-N 0.000 description 1
- HVHJYXDXRIWELT-RYUDHWBXSA-N Tyr-Glu-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O HVHJYXDXRIWELT-RYUDHWBXSA-N 0.000 description 1
- SMUWZUSWMWVOSL-JYJNAYRXSA-N Tyr-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N SMUWZUSWMWVOSL-JYJNAYRXSA-N 0.000 description 1
- WOCYUGQDXPTQPY-FXQIFTODSA-N Val-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N WOCYUGQDXPTQPY-FXQIFTODSA-N 0.000 description 1
- YFOCMOVJBQDBCE-NRPADANISA-N Val-Ala-Glu Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N YFOCMOVJBQDBCE-NRPADANISA-N 0.000 description 1
- REJBPZVUHYNMEN-LSJOCFKGSA-N Val-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N REJBPZVUHYNMEN-LSJOCFKGSA-N 0.000 description 1
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 1
- OGNMURQZFMHFFD-NHCYSSNCSA-N Val-Asn-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N OGNMURQZFMHFFD-NHCYSSNCSA-N 0.000 description 1
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 1
- YTPLVNUZZOBFFC-SCZZXKLOSA-N Val-Gly-Pro Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N1CCC[C@@H]1C(O)=O YTPLVNUZZOBFFC-SCZZXKLOSA-N 0.000 description 1
- IECQJCJNPJVUSB-IHRRRGAJSA-N Val-Tyr-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CO)C(O)=O IECQJCJNPJVUSB-IHRRRGAJSA-N 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229960002298 aminohydroxybutyric acid Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- OBNCKNCVKJNDBV-UHFFFAOYSA-N butanoic acid ethyl ester Natural products CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 description 1
- OAIYNRAQCIOEBD-UHFFFAOYSA-N butyl acetate;hydrate Chemical compound O.CCCCOC(C)=O OAIYNRAQCIOEBD-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000010531 catalytic reduction reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- ZAJNMXDBJKCCAT-UHFFFAOYSA-N ethyl 4-chloro-3-hydroxybutanoate Chemical compound CCOC(=O)CC(O)CCl ZAJNMXDBJKCCAT-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- YQGDEPYYFWUPGO-UHFFFAOYSA-N gamma-amino-beta-hydroxybutyric acid Chemical compound [NH3+]CC(O)CC([O-])=O YQGDEPYYFWUPGO-UHFFFAOYSA-N 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 1
- 108010010096 glycyl-glycyl-tyrosine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 108010087810 leucyl-seryl-glutamyl-leucine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- 239000010948 rhodium Substances 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种氨基酸序列如SEQIDNO:2所示的醇脱氢酶在4-氯乙酰乙酸乙酯不对称还原制备(R)-4-氯-3羟基丁酸乙酯中的应用。即以氨基酸序列如SEQIDNO:2所示的醇脱氢酶为催化剂,以4-氯乙酰乙酸乙酯为底物,以NADH为辅因子,不对称还原制备(R)-4-氯-3羟基丁酸乙酯。本发明首次将氨基酸序列如SEQIDNO:2所示的醇脱氢酶应用于4-氯乙酰乙酸乙酯不对称还原制备(R)-4-氯-3羟基丁酸乙酯中,取得很好的效果,其酶活高达5.6U/mg,其对底物的得率高达94%、产物的对映体过量值为100%,且产量高,大大降低了生产成本。
Description
技术领域
本发明属于生物技术领域,涉及一种醇脱氢酶的应用,尤其涉及一种醇脱氢酶(alcohol dehydrogenase)在以4-氯乙酰乙酸乙酯为底物不对称还原反应生产(R)-4-氯-3羟基丁酸乙酯中的应用。
背景技术
(R)-4-氯-3羟基丁酸乙酯(Ethyl 4-chloro-3-hydroxybutanoate, (R)-CHBE)是一种重要的有机中间体,可用于很多活性药物的合成,如(-)-大内酰亚胺A((-)-macrolactin A)、L-肉碱(L-carnitine)和R-Y-氨基-β-羟基丁酸(GABOB)的关键手性中间体[1,2]。
以4-氯乙酰乙酸乙酯(4-chloroacetoacetate Ethyl COBE)作为还原反应的潜手性底物,易于合成且价格低廉,以其为底物进行不对称还原反应获取(R)-CHBE是非常经济有效的制备途径。
迄今为止关于COBE不对称还原制备手性CHBE已进行了很多的研究报道。概括起来主要有化学法和生物法。
化学催化不对称还原法,所用催化剂包括价格昂贵的铑、釕等金属,采用化学法合成手性CHBE的缺点是产物的光学纯度不够高,且催化还原反应需要很高的氢气压,耗能高,污染大[3]。
微生物法分为酶催化和全细胞催化法,全细胞法则分为采用野生酵母和基因工程菌催化COBE为(R)-CHBE两种。许多微生物都能还原COBE,但绝大多数微生物的还原产物是S构型的,只有少数微生物能获得R型还原产物,如赭色掷孢酵母(Sporobolomyces salmonicolor) 、麦芽糖假丝酵母(Candida maltosa) 、卡斯太拉德巴利酵母(Debaryomyces castellii)及掷孢圆酵母(Torulaspora nagoyaensis)等,但产物的手性选择性即对映体过量(ee) 值只有8%~63%[4,5]。采用野生酵母进行催化所获得的产物的光学活性往往很低,需要筛选到高立体选择性的优良微生物菌株非常困难,所以近来的研究着重集中于运用重组大肠杆菌不对称合成具有高立体选择性的(R)-CHBE。Hiroaki Yamamoto等[6]将来自于Candida parapsilosis醇脱氢酶基因在大肠杆菌E.coli W3110中表达,重组菌在水相体系中催化COBE不对称还原(辅底物为2-propanol),得到产物(R)-CHBE,光学纯度99% e.e。 Kataoka. M等[7]醛基还原酶基因 (来自Sporobolomyces salmonicolor) 和葡萄糖脱氢酶基因 (来自Bacillus megaterium) 在大肠杆菌中共表达将此转化菌株的细胞用于在水/乙酸正丁酯两相体系中催化COBE的不对称还原。在定期添加辅酶NADP、葡萄糖和底物以及控制 pH 值的条件下,得到在有机相中的产物(R)-CHBE 浓度为268 g/L,摩尔转化率和产物的光学纯度分别为94.1%和91.7% e.e. 。
综上所述,现有催化COBE为(R)-CHBE的技术存在底物得率低、产物光学活性低、成本高等问题。
本专利中涉及到的还原酶是醇脱氢酶,其包含336个氨基酸,其在Genbank中的收录号为EEQ43320.1 (http://www.ncbi.nlm.nih.gov/protein/EEQ43320.1 ),其氨基酸序列如SEQ ID NO:2所示。编码该蛋白的基因含有1011 bp碱基 ,其在Genbank中的收录号为KC236900,(http://www.ncbi.nlm.nih.gov/nuccore/KC236900 )其基因序列如SEQ ID NO:1所示。至今未发现该醇脱氢酶用于COBE不对称还原制备(R)-CHBE的报道。
【参考文献】:
[1] Yan Q Ni, Jian-He Xu . Biocatalytic ketone reduction: A green and efficient access to enantiopure alcohols . Biotechnol Adv (2011)
[2] Ying Liu, Zhinan Xu . Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate with two co-existing, recombinant Escherichia
coli strains . Biotechnology Letters (2005) 27: 119–125
[3] Zhou Y -G, Tang W, Wang W-B , Li W, Zhang X. J A m Chem Soc , 2002 , 124 (18) : 4952
[4] ShimizuS, Kataoka M, Kita K. J Mol Catal B, 1998, 5 (124) : 321
[5] Kita K, Kataoka M, ShimizuS. J Biosci Bioeng, 1999, 88(6) : 591
[6] Hiroaki Yamamoto , Akinobu Matsuyama , Yoshinori Kobayashi . Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with Recombinant Escherichia coli Cell, 2002 ,66(2),481-483
发明内容
本发明所要解决的技术问题是提供一种醇脱氢酶在COBE不对称还原制备(R)-CHBE中的应用。
为解决上述技术问题,本发明所采用的技术方案如下:
一种氨基酸序列如SEQ ID NO:2所示的醇脱氢酶在4-氯乙酰乙酸乙酯(COBE)不对称还原制备(R)-4-氯-3羟基丁酸乙酯((R)-CHBE)中的应用。
即以氨基酸序列如SEQ ID NO:2所示的醇脱氢酶为催化剂,以4-氯乙酰乙酸乙酯为底物,不对称还原制备(R)-4-氯-3羟基丁酸乙酯。
具体反应是将表达基因序列如SEQ ID NO:1所示的重组菌,25~50g/L的4-氯乙酰乙酸乙酯,在pH6.0~7.5、20~30℃、180~280rpm条件下,与200mmol/L~2mol/L异丙醇,反应16~20h,得到(R)-4-氯-3羟基丁酸乙酯。其中,加入异丙醇可生成NADH,且使反应循环进行,降低了加入量以减少了生产成本。
发明人运用现代生物信息学工具,结合分子生物学技术,采用基因工程的手段从白色念珠菌 Candida albicans克隆醇脱氢酶的基因,在大肠杆菌中表达后发现其在水相中能够高效的催化COBE为(R)-CHBE,e.e值为100%。同时,通过在水相和水/有机相中反应、分批添加底物COBE等方式,解除了底物和产物对细胞和酶的抑制作用,显著的提高了转化效果。通过对醇脱氢酶的基因进行重组表达,获得了具有新型催化功能的酶蛋白,开发了该条基因的新功能——催化非天然底物COBE为高立体选择性的(R)-CHBE。
有益效果:本发明首次将氨基酸序列如SEQ ID NO:2所示的醇脱氢酶应用于COBE不对称还原制备(R)-CHBE中,取得很好的效果,其酶活高达5.6U/mg。氨基酸序列如SEQ ID NO:2所示的醇脱氢酶对底物COBE的得率高(大于90%)、产物CHBE的光学活性高(e.e%为100%),且产量高,大大降低了生产成本。
附图说明:
图1 为醇脱氢酶基因的构建图。
具体实施方式:
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:重组大肠杆菌E.coli Rosetta(pET-22b-CA)
1、醇脱氢酶基因的获取
白色念珠菌 Candida albicans(购于 Centraalbureauvoor Schimmelcultures (CBS) Fungal Biodiversiry Centre),培养基YPD(g·L-1):酵母提取物10g,蛋白胨20g,葡萄糖20g,补蒸馏水至1L。
将白色念珠菌 Candida albicans接种于5mL YPD液体培养基中30℃培养至对数生长期,使用基因组DNA提取试剂盒(北京天为生物工程有限公司酵母基因组提取试剂盒,GD2415 Yeast gDNA Kit)提取基因组。
构建表达载体所用的引物加设酶切位点,引物序列如下:
上游引物(CA-sense含NdeI)为:5'- GGAATTC CATATGTCAATTCCATCTACTCAGTACG -3'
下游引物(CA-anti含BamHI)为:5'- CGC GGATCCTTATGGATTAAACACGACTCTTCCT -3'
所有引物均由美吉生物公司合成。
基因的PCR条件:
94 ℃变性7 min,按如下参数循环30次:94 ℃变性1 min,60 ℃退火50 s,72 ℃延伸1.5 min。最后72 ℃延伸10 min。
2、表达载体的构建
用Nde I 及BamH I分别酶切pET-22b (pET-22b购于Novagen(默克中国))及所扩增含有两个酶切位点的目的基因,分别胶回收已双酶切的目的片段和表达载体,将已双酶切的表达载体pET-22b与目的基因用T4连接酶进行连接过夜, 将10 uL的连接产物pET-22b-CA加入100uL的Rosetta(DE3)感受态细胞中,冰上放置30 min,42 ℃热激90 sec。冰上放置2min。加入预热的0.45mL LB。220 rpm 37℃ 1h。将200 uL菌液加入分别含有100 μg/mL的氨苄青霉素和34μg/mL氯霉素的LB平板上,37℃过夜培养12-16,得到重组菌E.coli Rosseta(含pET-22b-CADH)。构建图谱见图1。
3、酶活的测定
挑取重组菌E.coli Rosseta(pET-22b-CA)及出发大肠杆菌Rosseta(DE3)至含100μg/mL氨苄青霉素和34μg/mL氯霉素的LB液体培养基中,37℃振荡培养过夜。然后按2 %接种量分别接种到含100μg/mL氨苄青霉素和34μg/mL氯霉素的LB新鲜培养基中,37 ℃培养至OD600约为0.6时,加入IPTG至终浓度0.8 mmol·L-1,25℃,220rpm,诱导表达10 h后,离心(4℃,5000rpm,15min),菌泥用100mM磷酸纳缓冲(pH7.0)重悬,超声破碎细胞(功率300W,超声5s,间歇5s,共5min),离心(4℃,12000rpm,15min),测定上清中的酶活。
酶反应体系包括100mM 磷酸钠缓冲液(pH7.0),5mM NADH,20mM COBE,30℃,340nm处测定吸光值的下降。酶活定义为每分钟内氧化1μmol NADH所需要的酶量为一个酶活单位U。蛋白采用Brandford法进行测定。
结果显示,出发大肠杆菌Rosseta(DE3)的比酶活为0.12U/mg protein,而重组菌E.coli Rosseta(pET22b-CA)的比酶活为5.6U/mg protein。
实施例2:重组大肠杆菌E.coli Rosseta(pET22b-CA)的发酵
挑取重组菌E.coli Rosseta(pET-22b-CA)至含抗生素的LB培养液,37℃振荡培养过夜。然后按2 %接种量分别接种到新鲜培养液中,37 ℃培养至OD600约为0.6时,加入IPTG至终浓度0.8 mmol·L-1,25℃,220rpm,诱导表达10 h后,8000 rpm,4 ℃离心10 min,弃上清,沉淀备用。
实施例3:
取实施例2的沉淀用磷酸钠缓冲(100 mmol·L-1, pH 6.5)洗涤两次,称取2g(湿重)的大肠杆菌菌泥,悬浮于总体系25 mL的反应液中(pH 6.5磷酸钾缓冲与醋酸丁酯体积比为1:1)。加入异丙醇150mmol/L,COBE 25g/L,30℃,180rpm,12h。产物(R)-CHBE的产量为20.5 g/L,产物的得率为:82%,光学纯度e.e%为100%。
产物的检测方法如下(以下实施例中产物的检测方法和实施例3相同):
对于水相反应:反应结束后, 8000 rpm离心10 min分离有机层和水层。小心吸取上层乙酸乙酯过有机膜,加入内标,保存测样。
对于水/有机两相反应:反应结束后8000 rpm离心10 min分离有机层和水层。小心吸取上层乙酸乙酯过有机膜,保存测样。
检测产物旋光性的样品处理(乙酰化):取5-10 ul 样品,加入乙酸酐0.2 mL,无水吡啶0.1mL,密闭条件下沸水浴中保持l h,冷却至室温,加入l mL乙酸乙酯,用水洗涤两次(每次l mL),然后用同样体积的饱和食盐水洗涤两次,分出有机层用无水硫酸钠干燥过夜。
测定COBE和CHB浓度以及产物的旋光性都使用气相7820A(Agilent)进行检测。测定COBE和CHB浓度,色谱柱为PEG-20M 毛细管柱(HP-FFAP;30m×0.32mm×0.25mm;Agilent),检测器FID温度220℃,汽化室温度220℃,柱温130℃。对产物的旋光性进行分析,色谱柱CP-Chirasil-Dex CB (25m*25mm*25um;Aglilent),检测器FID温度250℃,汽化室温度250℃,柱温120℃,R型和S型CHBE的出峰时间分别为:16.813min和17.413min。
实施例4:
取实施例2的沉淀用磷酸钠缓冲(100 mmol·L-1, pH 6.5)洗涤两次,称取5g(湿重)的大肠杆菌菌泥,悬浮于总体系25 mL的反应液中(pH 6.5磷酸钾缓冲)。加入异丙醇150mmol/L,COBE 25g/L,30℃,180rpm,12h。产物(R)-CHBE的产量为23.5 g/L,产物的得率为:94%,光学纯度e.e%为100%。
实施例5:
取实施例2的沉淀用磷酸钠缓冲(100 mmol·L-1, pH 6.5)洗涤两次,称取5g(湿重)的大肠杆菌菌泥,悬浮于总体系25 mL的反应液中(pH 6.5磷酸钾缓冲与醋酸丁酯体积比为1:1)。加入异丙醇300mmol/L,COBE 50g/L,30℃,180rpm,12h。产物(R)-CHBE的产量为37.2g/L,产物的得率为:74.3%,光学纯度e.e%为100%。
实施例6:
取实施例4的沉淀用磷酸钠缓冲(100 mmol·L-1, pH 7.0)洗涤两次,称取2g(湿重)的大肠杆菌菌泥,悬浮于15 mL的pH 7.0磷酸钠缓冲中。超声处理细胞(功率300W,超声5s,间歇5s,共5min),加入葡萄糖500mmol/L,COBE 50g/L(0h、2h、4h、6h、10h各10g/L ),GDH 200U,NAD 0.1mmol/L,25℃,220rpm,16h。产物(R)-CHBE的产量为46.5g/L,产物的得率为:93%,光学纯度e.e%为100%。
实施例7:
取实施例4的沉淀用磷酸钠缓冲(100 mmol·L-1, pH 7.0)洗涤两次,称取2g(湿重)的大肠杆菌菌泥,悬浮于15 mL的pH 7.0磷酸钠缓冲中。超声处理细胞(功率300W,超声5s,间歇5s,共5min),加入甲酸钠 500mmol/L,COBE 50g/L(0、2、4、6、10h各10g/L ),FDH 200U,NAD 0.1mmol/L,25℃,220rpm,16h。产物(R)-CHBE的产量为43.8g/L,产物的得率为:87.6%,光学纯度e.e%为100%。
<110> 南京工业大学
<120> 一种醇脱氢酶在催化生成(R)-4-氯-3-羟基丁酸乙酯中的应用
<130> njut080822
<140> 2012-10-29
<141> 2012-10-29
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1011
<212> DNA
<213> Candida albicans
<313> (1)..(3)
<400> 1
atgtcaattc catctactca gtacggattt ttttataata aagctagtgg tcttaacttg 60
aaaaaagact tgccggttaa caagccaggt gctggtcaat tgcttttaaa ggttgatgca 120
gttggccttt gtcattcaga tttacatgtt ctctatgaag gtttggattg tggtgataat 180
tatgtgatgg gccacgaaat tgctgggact gttgctgaac taggtgaaga ggtgagtgag 240
tttgcagttg gagatcgtgt cgcttgtgtc ggccccaatg gatgtggtct ttgtaaacac 300
tgtcttactg gtaacgataa tgtttgtacc aagtcgtttt tggattggtt tggattgggt 360
tacaatggag gttacgagca attcttgtta gtcaagagac caagaaactt ggtcaagatc 420
cctgacaatg ttacttccga ggaagctgca gctattacgg atgccgtatt gactccttac 480
catgctatca agtctgcagg tgttggtcca gcaagtaata tattaattat cggagctggt 540
ggattaggag gtaacgctat tcaagttgca aaagcatttg gtgcgaaggt tactgttttg 600
gataaaaagg ataaggcaag agaccaagct aaggcctttg gagctgacca ggtttacagt 660
gaattaccag acagcgtttt acctgggtca ttcagtgctt gttttgattt tgtttcggtt 720
caggcaacat acgatttgtg tcaaaagtat tgtgagccaa agggtactat tgttcccgta 780
ggtctaggtg caacttcgct taacataaat cttgctgatt tagatcttcg tgaaattacc 840
gtcaagggct cattctgggg taccctgatg gacttaagag aagcatttga attggctgca 900
cagggaaagg tcaaaccaaa tgttgctcat gctccattgt cagaattgcc taagtatatg 960
gagaagttga gagccggtgg ttatgaagga agagtcgtgt ttaatccata a 1011
<210> 2
<211> 336
<212> PRT
<213> Candida albicans
<400> 2
Met Ser Ile Pro Ser Thr Gln Tyr Gly Phe Phe Tyr Asn Lys Ala Ser
1 5 10 15
Gly Leu Asn Leu Lys Lys Asp Leu Pro Val Asn Lys Pro Gly Ala Gly
20 25 30
Gln Leu Leu Leu Lys Val Asp Ala Val Gly Leu Cys His Ser Asp Leu
35 40 45
His Val Leu Tyr Glu Gly Leu Asp Cys Gly Asp Asn Tyr Val Met Gly
50 55 60
His Glu Ile Ala Gly Thr Val Ala Glu Leu Gly Glu Glu Val Ser Glu
65 70 75 80
Phe Ala Val Gly Asp Arg Val Ala Cys Val Gly Pro Asn Gly Cys Gly
85 90 95
Leu Cys Lys His Cys Leu Thr Gly Asn Asp Asn Val Cys Thr Lys Ser
100 105 110
Phe Leu Asp Trp Phe Gly Leu Gly Tyr Asn Gly Gly Tyr Glu Gln Phe
115 120 125
Leu Leu Val Lys Arg Pro Arg Asn Leu Val Lys Ile Pro Asp Asn Val
130 135 140
Thr Ser Glu Glu Ala Ala Ala Ile Thr Asp Ala Val Leu Thr Pro Tyr
145 150 155 160
His Ala Ile Lys Ser Ala Gly Val Gly Pro Ala Ser Asn Ile Leu Ile
165 170 175
Ile Gly Ala Gly Gly Leu Gly Gly Asn Ala Ile Gln Val Ala Lys Ala
180 185 190
Phe Gly Ala Lys Val Thr Val Leu Asp Lys Lys Asp Lys Ala Arg Asp
195 200 205
Gln Ala Lys Ala Phe Gly Ala Asp Gln Val Tyr Ser Glu Leu Pro Asp
210 215 220
Ser Val Leu Pro Gly Ser Phe Ser Ala Cys Phe Asp Phe Val Ser Val
225 230 235 240
Gln Ala Thr Tyr Asp Leu Cys Gln Lys Tyr Cys Glu Pro Lys Gly Thr
245 250 255
Ile Val Pro Val Gly Leu Gly Ala Thr Ser Leu Asn Ile Asn Leu Ala
260 265 270
Asp Leu Asp Leu Arg Glu Ile Thr Val Lys Gly Ser Phe Trp Gly Thr
275 280 285
Ser Met Asp Leu Arg Glu Ala Phe Glu Leu Ala Ala Gln Gly Lys Val
290 295 300
Lys Pro Asn Val Ala His Ala Pro Leu Ser Glu Leu Pro Lys Tyr Met
305 310 315 320
Glu Lys Leu Arg Ala Gly Gly Tyr Glu Gly Arg Val Val Phe Asn Pro
325 330 335
Claims (3)
1.一种醇脱氢酶在4-氯乙酰乙酸乙酯不对称还原制备(R)-4-氯-3羟基丁酸乙酯中的应用,其特征在于以氨基酸序列如SEQ ID NO:2所示的醇脱氢酶为催化剂,以4-氯乙酰乙酸乙酯为底物,以NADH为辅因子,不对称还原制备(R)-4-氯-3羟基丁酸乙酯。
2.根据权利要求1所述的应用,其特征在于以氨基酸序列如SEQ ID NO:2所示的醇脱氢酶为催化剂,以4-氯乙酰乙酸乙酯为底物,以NADH为辅因子,醇脱氢酶的酶活为5.6U/mg protein。
3.根据权利要求2所述的应用,其特征在于表达基因序列如SEQ ID NO:1所示的重组菌,与200mmol/L~300mmol/L 异丙醇、1.5~50g/L的4-氯乙酰乙酸乙酯,在pH6.0~7.5、20~30℃、180~280rpm条件下反应16~20h,得到(R)-4-氯-3羟基丁酸乙酯。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310132479XA CN103160547A (zh) | 2013-04-17 | 2013-04-17 | 一种醇脱氢酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310132479XA CN103160547A (zh) | 2013-04-17 | 2013-04-17 | 一种醇脱氢酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN103160547A true CN103160547A (zh) | 2013-06-19 |
Family
ID=48584081
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310132479XA Pending CN103160547A (zh) | 2013-04-17 | 2013-04-17 | 一种醇脱氢酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103160547A (zh) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103709058A (zh) * | 2013-12-10 | 2014-04-09 | 苏州汉酶生物技术有限公司 | 一种左旋肉碱的合成方法 |
CN104988085A (zh) * | 2014-12-19 | 2015-10-21 | 常州大学 | (r)-4-氯-3-羟基丁酸乙酯及其衍生物的生物合成方法 |
CN105274160A (zh) * | 2015-11-11 | 2016-01-27 | 南京工业大学 | 一种酶法不对称还原制备(S)-N-boc-3-羟基哌啶的方法 |
CN106316873A (zh) * | 2015-07-08 | 2017-01-11 | 黄冈华阳药业有限公司 | 一种左旋肉碱的新型制备方法 |
CN109053479A (zh) * | 2018-10-15 | 2018-12-21 | 兆科药业(合肥)有限公司 | 一种季胺内盐的合成方法 |
CN109943482A (zh) * | 2019-03-06 | 2019-06-28 | 泰州市惠利生物科技有限公司 | 一种利用酶膜反应器耦合萃取制备r-4-氯-3-羟基丁酸乙酯的方法 |
CN114908129A (zh) * | 2021-09-30 | 2022-08-16 | 上海康鑫化工有限公司 | 用于制备(r)-4-氯-3-羟基丁酸乙酯的脱氢酶 |
-
2013
- 2013-04-17 CN CN201310132479XA patent/CN103160547A/zh active Pending
Non-Patent Citations (4)
Title |
---|
FARGO CD ET AL: "Genbank:KC236900", 《GENBANK》 * |
KITA K ET AL: "S.Diversity of 4-chloroacetoatate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis", 《BIOSCIENCE AND BIOENGINEERING》 * |
朱清禾等: "Rhodococcus erythropolis 手性醇脱氢酶的克隆表达及其性质", 《微生物学报》 * |
金永琴等: "乙醛还原酶工程菌的构建以及与葡萄糖脱氢酶工程菌偶联还原制备R-CHBE", 《化工进展》 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103709058A (zh) * | 2013-12-10 | 2014-04-09 | 苏州汉酶生物技术有限公司 | 一种左旋肉碱的合成方法 |
CN104988085A (zh) * | 2014-12-19 | 2015-10-21 | 常州大学 | (r)-4-氯-3-羟基丁酸乙酯及其衍生物的生物合成方法 |
CN106316873A (zh) * | 2015-07-08 | 2017-01-11 | 黄冈华阳药业有限公司 | 一种左旋肉碱的新型制备方法 |
CN105274160A (zh) * | 2015-11-11 | 2016-01-27 | 南京工业大学 | 一种酶法不对称还原制备(S)-N-boc-3-羟基哌啶的方法 |
CN105274160B (zh) * | 2015-11-11 | 2020-06-12 | 南京工业大学 | 一种酶法不对称还原制备(S)-N-boc-3-羟基哌啶的方法 |
CN109053479A (zh) * | 2018-10-15 | 2018-12-21 | 兆科药业(合肥)有限公司 | 一种季胺内盐的合成方法 |
CN109053479B (zh) * | 2018-10-15 | 2021-09-07 | 兆科药业(合肥)有限公司 | 一种季胺内盐的合成方法 |
CN109943482A (zh) * | 2019-03-06 | 2019-06-28 | 泰州市惠利生物科技有限公司 | 一种利用酶膜反应器耦合萃取制备r-4-氯-3-羟基丁酸乙酯的方法 |
CN109943482B (zh) * | 2019-03-06 | 2022-03-29 | 江苏惠利生物科技有限公司 | 一种利用酶膜反应器耦合萃取制备r-4-氯-3-羟基丁酸乙酯的方法 |
CN114908129A (zh) * | 2021-09-30 | 2022-08-16 | 上海康鑫化工有限公司 | 用于制备(r)-4-氯-3-羟基丁酸乙酯的脱氢酶 |
CN114908129B (zh) * | 2021-09-30 | 2023-05-30 | 上海康鑫化工有限公司 | 用于制备(r)-4-氯-3-羟基丁酸乙酯的脱氢酶 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103160547A (zh) | 一种醇脱氢酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 | |
CN104726507A (zh) | 一种醛酮还原酶在催化生成(r)-4-氯-3羟基丁酸乙酯中的应用 | |
CN101372699B (zh) | 一种羰酰还原酶在生产(s)-4-氯-3羟基丁酸乙酯中的应用 | |
CN102212567B (zh) | 一种l-2-氨基丁酸的生产方法 | |
CN103173503A (zh) | 重组大肠杆菌表达酮还原酶生物制备 (s)-4-氯-3-羟基丁酸乙酯的方法 | |
CN103122355B (zh) | 重组耐热醛酮还原酶基因、编码酶、载体、工程菌及应用 | |
CN103642765A (zh) | 醇脱氢酶突变体及其应用 | |
CN101613672A (zh) | 一种不对称转化制备(s)-4-氯-3-羟基丁酸乙酯的重组大肠杆菌及其构建方法 | |
CN109055327A (zh) | 醛酮还原酶突变体及其应用 | |
CN110423717A (zh) | 多酶重组细胞及多酶级联催化合成d-泛解酸内酯的方法 | |
CN102952814A (zh) | 重组耐热短链脱氢酶基因、编码酶、载体、工程菌及应用 | |
CN102808002B (zh) | 一种生物法合成甲基乙偶姻及其衍生物的重组细胞和方法 | |
CN105543186A (zh) | 一种醇脱氢酶lc3及其基因和应用 | |
CN105274160A (zh) | 一种酶法不对称还原制备(S)-N-boc-3-羟基哌啶的方法 | |
CN101962661B (zh) | 一种羰酰还原酶在生产(s)-4-氯-3羟基丁酸乙酯中的应用 | |
CN103289970A (zh) | 酮还原酶lek、编码基因、突变体及应用 | |
CN102978249A (zh) | 6-氰基-(3r,5r)-二羟基己酸叔丁酯的生物制备方法 | |
CN103320403A (zh) | 酮还原酶lek突变体及应用 | |
CN109402154A (zh) | 一种利用协同调控策略提高重组菌产异荭草苷产量的方法 | |
CN101555491B (zh) | 提高酿酒酵母发酵生产酒精产量的方法 | |
CN103695443A (zh) | 一种新型羰基还原酶、其基因及应用 | |
CN103255183B (zh) | 一种利用羰基还原酶不对称还原制备(s)-4-氯-3羟基丁酸乙酯的方法及应用 | |
CN102286556B (zh) | 一种山梨糖还原酶在生物法制备(s)-4-氯-3羟基丁酸乙酯中的应用 | |
CN104372038A (zh) | 两步催化制备(r)-4-氰基-3-羟基丁酸乙酯的方法 | |
CN103290072B (zh) | 一种酶法不对称还原制备(s)-4-氯-3羟基丁酸乙酯的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130619 |