CN108048438B - 一种卤代醇脱卤酶突变体及其应用 - Google Patents
一种卤代醇脱卤酶突变体及其应用 Download PDFInfo
- Publication number
- CN108048438B CN108048438B CN201810133923.2A CN201810133923A CN108048438B CN 108048438 B CN108048438 B CN 108048438B CN 201810133923 A CN201810133923 A CN 201810133923A CN 108048438 B CN108048438 B CN 108048438B
- Authority
- CN
- China
- Prior art keywords
- mutant
- hhec
- halohydrin dehalogenase
- fermentation
- reaction
- 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.)
- Active
Links
- 108010013164 halohydrin dehalogenase Proteins 0.000 title claims abstract description 31
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- XUKUURHRXDUEBC-KAYWLYCHSA-N Atorvastatin Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-KAYWLYCHSA-N 0.000 claims abstract description 9
- XUKUURHRXDUEBC-UHFFFAOYSA-N Atorvastatin Natural products C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CCC(O)CC(O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229960005370 atorvastatin Drugs 0.000 claims abstract description 9
- -1 -tert-butyl dihydroxyhexanoate Chemical compound 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 238000000855 fermentation Methods 0.000 claims description 28
- 230000004151 fermentation Effects 0.000 claims description 28
- 239000000758 substrate Substances 0.000 claims description 22
- 239000002609 medium Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 9
- 229930027917 kanamycin Natural products 0.000 claims description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 9
- 229960000318 kanamycin Drugs 0.000 claims description 9
- 229930182823 kanamycin A Natural products 0.000 claims description 9
- NQTYMGFSEOSJKM-RKDXNWHRSA-N tert-butyl (3r,5r)-6-cyano-3,5-dihydroxyhexanoate Chemical compound CC(C)(C)OC(=O)C[C@H](O)C[C@H](O)CC#N NQTYMGFSEOSJKM-RKDXNWHRSA-N 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 229930195727 α-lactose Natural products 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- 230000003698 anagen phase Effects 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 claims description 2
- 239000012429 reaction media Substances 0.000 claims description 2
- FIKPWJZUGTVXCO-SFYZADRCSA-N tert-butyl (3r,5s)-6-chloro-3,5-dihydroxyhexanoate Chemical compound CC(C)(C)OC(=O)C[C@H](O)C[C@H](O)CCl FIKPWJZUGTVXCO-SFYZADRCSA-N 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 34
- 102000004190 Enzymes Human genes 0.000 abstract description 34
- 230000000694 effects Effects 0.000 abstract description 13
- 230000003197 catalytic effect Effects 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 150000001298 alcohols Chemical class 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 27
- 238000000034 method Methods 0.000 description 18
- 239000000047 product Substances 0.000 description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 241000588724 Escherichia coli Species 0.000 description 13
- 238000006555 catalytic reaction Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 5
- 239000004593 Epoxy Substances 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- DCRWPTBMWMGADO-AVGNSLFASA-N Lys-Glu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DCRWPTBMWMGADO-AVGNSLFASA-N 0.000 description 4
- 108010005233 alanylglutamic acid Proteins 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- DANUJARGWMPVQX-UHFFFAOYSA-N tert-butyl hexanoate Chemical compound CCCCCC(=O)OC(C)(C)C DANUJARGWMPVQX-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- QPDUVFSVVAOUHE-XVKPBYJWSA-N Gly-Gln-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)CN)C(O)=O QPDUVFSVVAOUHE-XVKPBYJWSA-N 0.000 description 3
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- FIKPWJZUGTVXCO-UHFFFAOYSA-N tert-butyl 6-chloro-3,5-dihydroxyhexanoate Chemical compound CC(C)(C)OC(=O)CC(O)CC(O)CCl FIKPWJZUGTVXCO-UHFFFAOYSA-N 0.000 description 3
- HWPXGQCMZITGFN-XVYDVKMFSA-N Ala-Cys-His Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HWPXGQCMZITGFN-XVYDVKMFSA-N 0.000 description 2
- UQJUGHFKNKGHFQ-VZFHVOOUSA-N Ala-Cys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UQJUGHFKNKGHFQ-VZFHVOOUSA-N 0.000 description 2
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 2
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 2
- XRUJOVRWNMBAAA-NHCYSSNCSA-N Ala-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 XRUJOVRWNMBAAA-NHCYSSNCSA-N 0.000 description 2
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 2
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 2
- YJHKTAMKPGFJCT-NRPADANISA-N Ala-Val-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O YJHKTAMKPGFJCT-NRPADANISA-N 0.000 description 2
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 2
- KWKQGHSSNHPGOW-BQBZGAKWSA-N Arg-Ala-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)NCC(O)=O KWKQGHSSNHPGOW-BQBZGAKWSA-N 0.000 description 2
- COXMUHNBYCVVRG-DCAQKATOSA-N Arg-Leu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O COXMUHNBYCVVRG-DCAQKATOSA-N 0.000 description 2
- NPAVRDPEFVKELR-DCAQKATOSA-N Arg-Lys-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O NPAVRDPEFVKELR-DCAQKATOSA-N 0.000 description 2
- QEYJFBMTSMLPKZ-ZKWXMUAHSA-N Asn-Ala-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O QEYJFBMTSMLPKZ-ZKWXMUAHSA-N 0.000 description 2
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 2
- RXBGWGRSWXOBGK-KKUMJFAQSA-N Asp-Lys-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RXBGWGRSWXOBGK-KKUMJFAQSA-N 0.000 description 2
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- PRBLYKYHAJEABA-SRVKXCTJSA-N Gln-Arg-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O PRBLYKYHAJEABA-SRVKXCTJSA-N 0.000 description 2
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 2
- SYDJILXOZNEEDK-XIRDDKMYSA-N Glu-Arg-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O SYDJILXOZNEEDK-XIRDDKMYSA-N 0.000 description 2
- IRXNJYPKBVERCW-DCAQKATOSA-N Glu-Leu-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IRXNJYPKBVERCW-DCAQKATOSA-N 0.000 description 2
- VGBSZQSKQRMLHD-MNXVOIDGSA-N Glu-Leu-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VGBSZQSKQRMLHD-MNXVOIDGSA-N 0.000 description 2
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 2
- QJVZSVUYZFYLFQ-CIUDSAMLSA-N Glu-Pro-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O QJVZSVUYZFYLFQ-CIUDSAMLSA-N 0.000 description 2
- BXSZPACYCMNKLS-AVGNSLFASA-N Glu-Ser-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BXSZPACYCMNKLS-AVGNSLFASA-N 0.000 description 2
- VHPVBPCCWVDGJL-IRIUXVKKSA-N Glu-Thr-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VHPVBPCCWVDGJL-IRIUXVKKSA-N 0.000 description 2
- YYPFZVIXAVDHIK-IUCAKERBSA-N Gly-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN YYPFZVIXAVDHIK-IUCAKERBSA-N 0.000 description 2
- CUYLIWAAAYJKJH-RYUDHWBXSA-N Gly-Glu-Tyr Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CUYLIWAAAYJKJH-RYUDHWBXSA-N 0.000 description 2
- QPCVIQJVRGXUSA-LURJTMIESA-N Gly-Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QPCVIQJVRGXUSA-LURJTMIESA-N 0.000 description 2
- KAJAOGBVWCYGHZ-JTQLQIEISA-N Gly-Gly-Phe Chemical compound [NH3+]CC(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KAJAOGBVWCYGHZ-JTQLQIEISA-N 0.000 description 2
- HHSOPSCKAZKQHQ-PEXQALLHSA-N Gly-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)CN HHSOPSCKAZKQHQ-PEXQALLHSA-N 0.000 description 2
- CQMFNTVQVLQRLT-JHEQGTHGSA-N Gly-Thr-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O CQMFNTVQVLQRLT-JHEQGTHGSA-N 0.000 description 2
- VXZZUXWAOMWWJH-QTKMDUPCSA-N His-Thr-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O VXZZUXWAOMWWJH-QTKMDUPCSA-N 0.000 description 2
- GQKSJYINYYWPMR-NGZCFLSTSA-N Ile-Gly-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N GQKSJYINYYWPMR-NGZCFLSTSA-N 0.000 description 2
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 2
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 2
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 description 2
- QQUJSUFWEDZQQY-AVGNSLFASA-N Lys-Gln-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCCN QQUJSUFWEDZQQY-AVGNSLFASA-N 0.000 description 2
- ZMMDPRTXLAEMOD-BZSNNMDCSA-N Lys-His-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZMMDPRTXLAEMOD-BZSNNMDCSA-N 0.000 description 2
- CRIODIGWCUPXKU-AVGNSLFASA-N Lys-Pro-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(O)=O CRIODIGWCUPXKU-AVGNSLFASA-N 0.000 description 2
- CUHGAUZONORRIC-HJGDQZAQSA-N Lys-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N)O CUHGAUZONORRIC-HJGDQZAQSA-N 0.000 description 2
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 2
- DBMLDOWSVHMQQN-XGEHTFHBSA-N Met-Ser-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DBMLDOWSVHMQQN-XGEHTFHBSA-N 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- BYAIIACBWBOJCU-URLPEUOOSA-N Phe-Ile-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BYAIIACBWBOJCU-URLPEUOOSA-N 0.000 description 2
- SJRQWEDYTKYHHL-SLFFLAALSA-N Phe-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O SJRQWEDYTKYHHL-SLFFLAALSA-N 0.000 description 2
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 description 2
- VPFGPKIWSDVTOY-SRVKXCTJSA-N Pro-Glu-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O VPFGPKIWSDVTOY-SRVKXCTJSA-N 0.000 description 2
- FEVDNIBDCRKMER-IUCAKERBSA-N Pro-Gly-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)CNC(=O)[C@@H]1CCCN1 FEVDNIBDCRKMER-IUCAKERBSA-N 0.000 description 2
- ZJXXCGZFYQQETF-CYDGBPFRSA-N Pro-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@@H]1CCCN1 ZJXXCGZFYQQETF-CYDGBPFRSA-N 0.000 description 2
- FYXCBXDAMPEHIQ-FHWLQOOXSA-N Pro-Trp-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CCCCN)C(=O)O FYXCBXDAMPEHIQ-FHWLQOOXSA-N 0.000 description 2
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 2
- PZZJMBYSYAKYPK-UWJYBYFXSA-N Ser-Ala-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O PZZJMBYSYAKYPK-UWJYBYFXSA-N 0.000 description 2
- KNCJWSPMTFFJII-ZLUOBGJFSA-N Ser-Cys-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KNCJWSPMTFFJII-ZLUOBGJFSA-N 0.000 description 2
- HJEBZBMOTCQYDN-ACZMJKKPSA-N Ser-Glu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HJEBZBMOTCQYDN-ACZMJKKPSA-N 0.000 description 2
- YQQKYAZABFEYAF-FXQIFTODSA-N Ser-Glu-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQQKYAZABFEYAF-FXQIFTODSA-N 0.000 description 2
- XGQKSRGHEZNWIS-IHRRRGAJSA-N Ser-Pro-Tyr Chemical compound N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O XGQKSRGHEZNWIS-IHRRRGAJSA-N 0.000 description 2
- KBLYJPQSNGTDIU-LOKLDPHHSA-N Thr-Glu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O KBLYJPQSNGTDIU-LOKLDPHHSA-N 0.000 description 2
- DMWNPLOERDAHSY-MEYUZBJRSA-N Tyr-Leu-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DMWNPLOERDAHSY-MEYUZBJRSA-N 0.000 description 2
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 2
- 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 2
- JFAWZADYPRMRCO-UBHSHLNASA-N Val-Ala-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JFAWZADYPRMRCO-UBHSHLNASA-N 0.000 description 2
- LJSZPMSUYKKKCP-UBHSHLNASA-N Val-Phe-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 LJSZPMSUYKKKCP-UBHSHLNASA-N 0.000 description 2
- KSFXWENSJABBFI-ZKWXMUAHSA-N Val-Ser-Asn Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KSFXWENSJABBFI-ZKWXMUAHSA-N 0.000 description 2
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 2
- 108010077245 asparaginyl-proline Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000002526 effect on cardiovascular system Effects 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 108010020688 glycylhistidine Proteins 0.000 description 2
- 108010077515 glycylproline Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 108010018006 histidylserine Proteins 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 2
- 108010000761 leucylarginine Proteins 0.000 description 2
- 108010089256 lysyl-aspartyl-glutamyl-leucine Proteins 0.000 description 2
- 108010009298 lysylglutamic acid Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 108010005942 methionylglycine Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- 108010053725 prolylvaline Proteins 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- MNWBNISUBARLIT-UHFFFAOYSA-N sodium cyanide Chemical compound [Na+].N#[C-] MNWBNISUBARLIT-UHFFFAOYSA-N 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 108010084932 tryptophyl-proline Proteins 0.000 description 2
- QLWOUBCORTYSPP-UHFFFAOYSA-N 1h-imidazol-1-ium;hydroxide Chemical compound O.C1=CNC=N1 QLWOUBCORTYSPP-UHFFFAOYSA-N 0.000 description 1
- LJBWEZVYRBKOCI-UHFFFAOYSA-N 2,4,6-triaminoquinazoline Chemical class N1=C(N)N=C(N)C2=CC(N)=CC=C21 LJBWEZVYRBKOCI-UHFFFAOYSA-N 0.000 description 1
- WQKHERPPDYPMNX-UHFFFAOYSA-N 6-chloro-3,4-dihydro-2h-naphthalen-1-one Chemical compound O=C1CCCC2=CC(Cl)=CC=C21 WQKHERPPDYPMNX-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- UULLJGQFCDXVTQ-CYDGBPFRSA-N Arg-Pro-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UULLJGQFCDXVTQ-CYDGBPFRSA-N 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- OREPWMPAUWIIAM-ZPFDUUQYSA-N Gln-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N OREPWMPAUWIIAM-ZPFDUUQYSA-N 0.000 description 1
- LHRXAHLCRMQBGJ-RYUDHWBXSA-N Gly-Glu-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)CN LHRXAHLCRMQBGJ-RYUDHWBXSA-N 0.000 description 1
- ALOBJFDJTMQQPW-ONGXEEELSA-N Gly-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)CN ALOBJFDJTMQQPW-ONGXEEELSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- HQEPKOFULQTSFV-JURCDPSOSA-N Ile-Phe-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)O)N HQEPKOFULQTSFV-JURCDPSOSA-N 0.000 description 1
- FHPZJWJWTWZKNA-LLLHUVSDSA-N Ile-Phe-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N FHPZJWJWTWZKNA-LLLHUVSDSA-N 0.000 description 1
- 238000008214 LDL Cholesterol Methods 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- AOKZOUGUMLBPSS-PMVMPFDFSA-N Phe-Trp-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(O)=O AOKZOUGUMLBPSS-PMVMPFDFSA-N 0.000 description 1
- WFHYFCWBLSKEMS-KKUMJFAQSA-N Pro-Glu-Phe Chemical compound N([C@@H](CCC(=O)O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C(=O)[C@@H]1CCCN1 WFHYFCWBLSKEMS-KKUMJFAQSA-N 0.000 description 1
- AJBQTGZIZQXBLT-STQMWFEESA-N Pro-Phe-Gly Chemical compound C([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 AJBQTGZIZQXBLT-STQMWFEESA-N 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- JPIDMRXXNMIVKY-VZFHVOOUSA-N Ser-Ala-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPIDMRXXNMIVKY-VZFHVOOUSA-N 0.000 description 1
- XKFJENWJGHMDLI-QWRGUYRKSA-N Ser-Phe-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O XKFJENWJGHMDLI-QWRGUYRKSA-N 0.000 description 1
- 108010045306 T134 peptide Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- SKHPKKYKDYULDH-HJGDQZAQSA-N Thr-Asn-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SKHPKKYKDYULDH-HJGDQZAQSA-N 0.000 description 1
- PZVGOVRNGKEFCB-KKHAAJSZSA-N Thr-Asn-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N)O PZVGOVRNGKEFCB-KKHAAJSZSA-N 0.000 description 1
- XIULAFZYEKSGAJ-IXOXFDKPSA-N Thr-Leu-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 XIULAFZYEKSGAJ-IXOXFDKPSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- YKCXQOBTISTQJD-BZSNNMDCSA-N Tyr-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N YKCXQOBTISTQJD-BZSNNMDCSA-N 0.000 description 1
- XUIOBCQESNDTDE-FQPOAREZSA-N Tyr-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O XUIOBCQESNDTDE-FQPOAREZSA-N 0.000 description 1
- PAPWZOJOLKZEFR-AVGNSLFASA-N Val-Arg-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PAPWZOJOLKZEFR-AVGNSLFASA-N 0.000 description 1
- YMTOEGGOCHVGEH-IHRRRGAJSA-N Val-Lys-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O YMTOEGGOCHVGEH-IHRRRGAJSA-N 0.000 description 1
- CEKSLIVSNNGOKH-KZVJFYERSA-N Val-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](C(C)C)N)O CEKSLIVSNNGOKH-KZVJFYERSA-N 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003741 agents affecting lipid metabolism Substances 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000012152 bradford reagent Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000004868 gas analysis Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 108010055015 haloalcohol dehalogenase Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/002—Nitriles (-CN)
- C12P13/004—Cyanohydrins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y405/00—Carbon-halide lyases (4.5)
- C12Y405/01—Carbon-halide lyases (4.5.1)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种卤代醇脱卤酶突变体及其在合成阿托伐他汀关键中间体(3R,5R)6‑腈基‑3,5‑二羟基己酸叔丁酯中的应用,所述卤代醇脱卤酶突变体HheC‑A氨基酸序列SEQ ID NO.2所示。本发明提供了的1株卤代醇脱卤酶突变体,与野生酶相比,其催化活性提高了22倍,比活力提高了约20倍。将突变体HheC‑A用于全细胞催化D3转化A7的反应过程,当D3的投料量为50g/L时,9小时内D3转化率为92%,A7得率72%。突变体HheC‑A催化D3转化为A7的产物得率是野生酶HheC的4.8倍。
Description
(一)技术领域
本发明涉及一种卤代醇脱卤酶的突变体及其应用。
(二)背景技术
心脑血管疾病是一种严重威胁人类,具有高患病率、高致残率和高死亡率的特点,全世界每年死于心脑血管疾病的人数高达1500万人,居各种死因首位。2015年国内调血脂类药物总体市场已超过200亿元人民币,他汀类药物独占鳌头,国内他汀药物终端零售价市场达到了151.43亿元的规模,占整个降血脂用药市场近80%。其中,阿托伐他汀上市近20年的临床证明,药物安全性高、不良反应小,在降低LDL胆固醇治疗方面优于其它他汀类药物。2015年,中国阿托伐他汀总体市场达到了83.19亿元,同比上一年增长了7.97%,并仍呈现逐年上涨的趋势。
随着市场竞争的日益加剧以及企业社会责任的落实,阿托伐他丁的生产技术亟需改进和创新,一方面需减少工业生产对环境资源的破坏及能源的浪费,另一方面可降低企业成本和患者用药成本,以提升整个行业的经济效益和社会效益。采用生物催化技术升级阿托伐他汀药物关鍵中间体合成技术,能够建立节能、绿色、高效的新生产工艺。卤代醇脱卤酶具有一步转化法替代高能耗和高溶剂用量的化学合成步骤,具备高效催化的特性,适用于建立低能耗、低成本和环保的阿托伐他汀侧链合成新工艺。
(三)发明内容
本发明目的是提供一种高效催化合成阿托伐他汀关键中间体(3R,5R)6-腈基-3,5-二羟基己酸叔丁酯(A7)合成的卤代醇脱卤酶突变体及其应用。
本发明采用的技术方案是:
本发明提供一种卤代醇脱卤酶突变体(HheC-A),所述卤代醇脱卤酶突变体HheC-A氨基酸序列SEQ ID NO.2所示。本发明所述卤代醇脱卤酶突变体HheC-A基因是将源于根癌农杆菌Agrobacterium tumefaciens卤代醇脱卤酶HheC(氨基酸序列SEQ ID NO.4)的人工合成基因,进行体外定向进化后,最终筛选得到的突变体。
本发明还提供一种所述卤代醇脱卤酶突变体的编码基因,所述编码基因的核苷酸序列如SEQ ID NO.1所示。
本发明还涉及一种所述编码基因构建的重组载体及重组载体转化制备的重组基因工程菌。优选以pET28a(+)质粒为表达载体,以大肠杆菌为表达宿主(大肠杆菌BL21(DE3))。
本发明还提供一种所述卤代醇脱卤酶突变体在合成阿托伐他汀关键中间体(3R,5R)6-腈基-3,5-二羟基己酸叔丁酯中的应用,所述的应用以含卤代醇脱卤酶突变体编码基因的重组基因工程菌经发酵培养获得的含湿菌体的发酵液为反应介质,以(3R,5S)6-氯-3,5-二羟基己酸叔丁酯(D3)为底物构成pH 8.0-9.0的反应体系,在35℃进行转化反应,反应完全后,将反应液分离纯化,获得(3R,5R)6-腈基-3,5-二羟基己酸叔丁酯(A7)。
进一步,所述反应体系中,湿菌体用量为10~30g/L发酵液,所述底物终浓度为50g/L发酵液。
进一步,所述发酵液按如下方法制备:
(1)菌种活化:将含卤代醇脱卤酶突变体编码基因的重组基因工程菌(优选(Escherichia coli)BL21(DE3)(pET28a-hheC-A))接种到含有50μg/ml卡那霉素的种子培养基中,37℃培养至对数生长中期,获得种子液;种子培养基组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO40.71g/L、MgSO4·7H2O0.49g/L、卡那霉素50μg/L,溶剂为去离子水,pH7.0;
(2)发酵培养:将种子液按体积浓度5%的接种量接种到发酵培养基中(装液量70%),30℃培养6h;加入终浓度为5.0-7.0mM的α-乳糖,控制发酵温度22℃,继续发酵20h,得到所述的发酵液;所述发酵培养基质量组成:1%的蛋白胨、0.5%的酵母提取粉、54mM甘油、2.8mM葡萄糖、25mM Na2HPO4、25mM KH2PO4、50mM NH4Cl、5mM Na2SO4、2mM MgSO4、卡那霉素50μg/L,溶剂为去离子水,pH值自然。
与现有技术相比,本发明的有益效果主要体现在:本发明提供了的1株卤代醇脱卤酶突变体,与野生酶相比,其催化活性提高了22倍,比活力提高了约20倍。将突变体HheC-A用于全细胞催化D3转化A7的反应过程,当D3的投料量为50g/L时,9小时内D3转化率为92%(部分水解),A7得率72%。突变体HheC-A催化D3转化为A7的产物得率是野生酶HheC的4.8倍。
(四)附图说明
图1为pET28a-hheC载体的结构示意图。
图2为突变体酶活与野生型酶HheC的酶活比较。
图3为催化过程中底物D3、中间环氧产物和产物A7的变化。(A):野生型卤代醇脱卤酶HheC全细胞催化D3转化A7的反应过程;(B):突变体HheC-A全细胞催化D3转化A7的反应过程。
图4底物D3、中间环氧产物和产物A7气相分析色谱图。(A):0.5g/L D3(D3标准品溶解在乙酸乙酯中)的气相分析色谱图;(B):0.5g/L A7(A7标准品溶解在乙酸乙酯中)的气相分析色谱图;(C):催化反应过程取样进行气相分析的典型色图谱。其中标记的色谱峰a、色谱峰b和色谱峰c分别是底物D3、中间环氧产物和产物A7。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
LB培养基组成:酵母粉5.0g/L、蛋白胨10.0g/L、NaCl 10.0g/L,溶剂为去离子水,pH值自然。
种子培养基组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO43.4g/L、NH4Cl 2.67g/L、Na2SO40.71g/L、MgSO4·7H2O 0.49g/L、卡那霉素50μg/L,溶剂为去离子水,pH7.0。
发酵培养基组成:1%的蛋白胨、0.5%的酵母提取粉、54mM甘油、2.8mM葡萄糖、25mM Na2HPO4、25mM KH2PO4、50mM NH4Cl、5mM Na2SO4、2mM MgSO4、卡那霉素50μg/L,溶剂为去离子水,pH值自然。
实施例1、根癌农杆菌A.tumefaciens卤代醇脱卤酶HheC基因的人工合成及其突变体HheC-A的筛选
一、构建高效A.tumefaciens卤代醇脱卤酶HheC基因的大肠杆菌
根据GenBank数据库中根癌农杆菌A.tumefaciens卤代醇脱卤酶HheC基因的氨基酸序列(ID:AF397296),由基因合成公司合成密码子优化的HheC基因(序列如SEQ ID NO.3,氨基酸序列为SEQ ID NO.4所示),该基因合成时直接***到pET28a质粒的NdeI/Hind III酶切位点之间,如图1所示,得到重组表达质粒pET28a-hheC。
将重组质粒pET28a-hheC转化到E.coli BL21(DE3)的高效感受态细胞,在含卡那霉素的LB琼脂平板上筛选,得到重组大肠杆菌E.coli BL21(DE3)(pET28a-hheC)。
二、卤代醇脱卤酶HheC基因的体外定向进化
首先采用易错PCR方法进行定向进化和筛选。根据合成的hheC序列(SEQ IDNO.3),设计PCR扩增引物(hheC-F:5′-GCAGCCATATGAGCACAGCGATTGTGAC-3′,
hheC-R:5′-CAGCAAGCTTTCATTCAGGCATTCCAGGCCAG-3′),下划线处为Nde I和HindIII的酶切位点。以质粒pET28a-hheC为模板,采用如表1所示的PCR体系进行易错PCR反应。
表1 易错PCR体系的组成
10×易错PCR缓冲液 | 5.0μL |
MnCl<sub>2</sub>(5mM) | 4.0μL |
10mM dNTP | 1.0μL |
10μM hheC-F | 2.0μL |
10μM hheC-R | 2.0μL |
Taq DNA聚合酶(5U/μL) | 1.5μL |
ddH<sub>2</sub>O | 34.5μL |
总体积 | 50μL |
PCR扩增程序为:95℃预变性3min;95℃变性30s,56℃退火40s,72℃延伸50s,35个循环;72℃,8min,4℃保温。
PCR结束后,将PCR产物进行胶回收试剂盒纯化,并通过一步克隆法(南京诺唯赞生物科技有限公司)法,克隆到pET28a质粒的Nde I/Hind III酶切位点之间。连接产物转化到E.coli BL21(DE3)菌株中,构建易错PCR文库。
采用基于检测氯离子显色浓度的高通量方法筛选易错PCR文库(Kurtovic S,Jansson R,Mannervik B.2007.Arch Biochem Biophys 464:284-287;Bergmann JG,SanikJ.1957.Anal.Chem.29:241-243.),可以快速检测对底物(3R,5S)6-氯-3,5-二羟基己酸叔丁酯(D3)催化活性显著提高的突变菌落。挑选的阳性克隆,用于进一步复筛,并测序验证,而后用于下一轮的易错PCR。通过五轮的易错PCR筛选得到催化活性提高5.6倍的突变体HheC-05,氨基酸突变位点如下:V9I、Q87R、Y177T、K203R、V205Y、W237T。
为进一步提高卤代醇脱卤酶对底物D3的催化活性,对HheC-05进行同源建模,通过分子对接,筛选最优构型,分析底物结合位点的关键氨基酸残基,包括Q72、A83、P84、T134和P135,进行定点饱和突变,通过Quickchange的方法引入NNK简并密码子(N:A/C/G/T;K:G/T)来替代目的氨基酸。
在72位引入NNK简并密码子的定点突变引物为:
72F:
5’-GCGGTGACCTCAGCTTATGGCNNKGTGGATGTACTGGTGAGCAAC-3’
72R:
5’-GTTGCTCACCAGTACATCCACMNNGCCATAAGCTGAGGTCACCGC-3’
在83和84位引入NNK简并密码子的定点突变引物为:
8384-F:
5’-GTGAGCAACGATATTTTCNNKNNKGAATTCAGACCGATTGATAAG-3’
8384-R:
5’-CTTATCAATCGGTCTGAATTCMNNMNNGAAAATATCGTTGCTCAC-3’
在134和135位引入NNK简并密码子的定点突变引物为:
1345-F:
5’-ATCATCTTTATTACGAGCGCGNNKNNKTTTGGACCGTGGAAAGAACTG-3’
1345-R:
5’-CAGTTCTTTCCACGGTCCAAAMNNMNNCGCGCTCGTAATAAAGATGAT-3’
Quickchange法重叠延伸PCR具体实施条件如下:
PCR反应体系均为:2×Phanta Max Buffer 25μL,dNTP Mix 1μL,10μM正向引物1μL,10μM反向引物1μL,模板DNA1μL,Max聚合酶1μL,双蒸水补齐至50μL;
PCR反应扩增条件:94℃预变性5min;随后进行94℃ 1min,56℃ 50s,72℃ 50s30个循环;最后保存在4℃。
在上述通过PCR扩增得到的片段中加入限制性内切酶DpnI,置于37℃金属浴中温浴30分钟进行酶切消化,将体系置于80℃金属浴中温浴30分钟灭酶。最后将酶切后的PCR产物转化至大肠杆菌BL21(DE3)感受态细胞中。
采用上述的基于氯离子浓度显色的高通量筛选方法,对突变文库进行筛选,最终筛选得到突变体HheC-A(核苷酸序列如SEQ ID NO.1,氨基酸序列为SEQ ID NO.2所示)。
实施例2卤代醇脱卤酶突变体HheC-A与亲本的催化特性比较
一、卤代醇脱卤酶的诱导表达
将E.coli BL21(DE3)(pET28a-hheC-A)、E.coli BL21(DE3)(pET28a-hheC-05)和野生型E.coli BL21(DE3)(pET28a-hheC)分别接种在含有50μg/ml卡那霉素的种子培养基中,37℃摇床培养至对数生长中期,获得种子液。
将新鲜培养的种子液按体积浓度5%的接种量接种到发酵培养基中,30℃,培养6h;加入终浓度为5.6-7.0mM的α-乳糖,控制发酵温度22℃,继续发酵12h,得到表达卤代醇脱卤酶的菌液。
二、卤代醇脱卤酶的纯化
将发酵的菌液在4℃、9000×g离心10min,收集细胞;采用20mM的磷酸缓冲液(pH7.0)重新悬浮细胞,采用高压匀浆机进行细胞破碎;细胞破碎液进行离心,12000×g离心10min,收集上清液;将获得的上清液加入Ni-NTA亲和层析柱,上样后使用1×BindingBuffer洗脱直至吸光值平稳,分别加入终浓度50mM、100mM、150mM、200mM、250mM咪唑水溶液洗脱目的蛋白。通过酶活测定和SDS-PAGE分析,发现突变酶主要出现在150mM咪唑水溶液中,且条带单一。将含有目的蛋白的洗脱液通过GE PD-10脱盐柱,使用20mM磷酸缓冲液(pH7.0)洗下目的蛋白。之后使用蛋白超滤离心管浓缩,从而得到酶液。
三、卤代醇脱卤酶的酶学性质的测定
(1)样品酶活测定:吸取上述制备的酶液0.05mL,加入0.44mL的50mM Tris/SO4(pH7.0)缓冲液,置于30℃水浴预热5min;加入0.01ml的质量浓度2%D3的甲醇溶液,开始酶的反应;反应20min后,加入0.2mL的饱和Hg(SCN)2乙醇溶液和1.8mL的60g/L FeNH4(SO4)2·12H2O(溶解在1mol/L HNO3中)溶液,在460nm检测反应液的吸光度,与标准氯离子浓度标准曲线的吸光值对照,测定反应液中的氯离子浓度。
氯离子浓度标准曲线绘制:①配置浓度为0mM、6mM、12mM、18mM、24mM和30mM的NaCl水溶液;②采用96孔板,各孔中分别加入各个NaCl水溶液40μL、饱和硫氰酸汞乙醇溶液60μL和150μL的60g/L FeNH4(SO4)2·12H2O(溶解在1mol/L HNO3中)溶液,在460nm检测反应液的吸光值,绘制氯离子浓度标准曲线。以氯离子浓度(mM)为横坐标x,A460的吸光值为纵坐标y,线性拟合,可得到标准曲线方程:y=0.0745x-0.023,R2=0.995。
酶活单位定义:1U酶活单位定义为每分钟酶催化反应产生1.0μmol氯离子所需的酶量。
突变体HheC-05、突变酶HheC-A的酶活与野生型酶HheC的酶活比较如图2所示。其中,由易错PCR筛选的突变体HheC-05,其催化的相对活性是野生酶的5倍;经过关键氨基酸残基饱和突变,作进一步突变和筛选的突变体HheC-A,其相对酶活是野生酶的22倍。
(2)酶液样品的蛋白质浓度测定:Bradford法测定溶液中蛋白浓度,取200μL待测样品(酶液)加入2mL Bradford试剂(Bio-Rad),混匀后迅速在595nm下测定吸光值,空白为pH6.5磷酸盐缓冲液,每组样品三个平行。以牛血清蛋白(BSA)作为标样蛋白样品绘制标准曲线:首先配制1.0mg/mL的标准BSA水溶液;在各支试管中分别加入0、0.01、0.02、0.04、0.06、0.08、0.1mL的标准BSA水溶液,然后用去离子水补充到0.1mL;在各支试管中分别加入5.0mL考马斯亮兰G-250试剂,漩涡震荡混匀;放置约5min后,进行分光光度计分析。以蛋白质浓度(mg/mL)为横坐标x,以吸光值为纵坐标y,其标准曲线方程为y=0.0042x+0.0082,R2=0.958。将酶液样品的吸光值对照标准曲线可得酶液的蛋白浓度,不同样品蛋白浓度不同。
(3)动力学参数测定:改变步骤(1)底物D3的添加量,终浓度分别为0.05mM、0.1mM、0.2mM、0.5mM、1.0mM、2.0mM和5.0mM,测定其初始反应速率,并根据米氏方程的双倒数法作图,进行线性拟合,计算得到酶的动力学参数,包括Km和kcat值。
突变酶HheC-A的酶活与野生型酶HheC的酶催化动力学参数比较如表1所示。
表1 野生型酶HheC与突变体HheC-A的催化性质比较
卤代醇脱卤酶 | 比活力(U/mg) | K<sub>m</sub>(mM) | k<sub>cat</sub>(s<sup>-1</sup>) | k<sub>cat</sub>/K<sub>m</sub>(s<sup>-1</sup>·mM<sup>-1</sup>) |
HheC | 0.35±0.021 | 12.8±0.26 | 98 | 7.6 |
突变体HheC-A | 7.2±0.15 | 4.6±0.17 | 223 | 48.5 |
实施例3突变体HheC-A在催化(3R,5S)6-氯-3,5-二羟基己酸叔丁酯(D3)转化为(3R,5R)6-腈基-3,5-二羟基己酸叔丁酯(A7)中的应用
(1)菌种活化
将表达突变体HheC-A的大肠杆菌(Escherichia coli)BL21(DE3)(pET28a-hheC-A)命名为E.coliIEF-hheCA。将该菌株的单菌落接种到含有50μg/ml卡那霉素的种子培养基中,37℃培养至对数生长中期,获得种子液。将表达亲本的菌株(Escherichia coli)BL21(DE3)(pET28a-hheC)单菌落接种到含有50μg/ml卡那霉素的种子培养基中,37℃培养至对数生长中期,获得种子液。
(2)2L发酵罐中的菌剂制备
将新鲜培养的种子液按照体积浓度5%的接种量,接种到1.5L的发酵培养基(装液量70%)中,30℃培养6h;加入终浓度为7.0mM的α-乳糖,控制发酵温度22℃,继续发酵20h,得到脱卤酶突变体HheC-A或脱卤酶亲本HheC的发酵液,菌体细胞含量为10-30g/L。
(3)50mL全细胞催化转化
采用50mL的全细胞酶液(即脱卤酶突变体HheC-A或脱卤酶亲本HheC的发酵液),加入2.5g底物D3,取1.7mL的30%的NaCN(过量),溶解于3.4mL的水中,制成约质量浓度10%NaCN水溶液,用于自动滴定控制反应液pH在8.0-9.0之间;将反应液置于35℃水浴进行催化反应9h。
催化反应为非均相反应,过程取样检测底物D3、中间环氧产物和产物A7的动态变化,其中脱卤酶亲本HheC和脱卤酶突变体HheC-A的催化过程分别如图3中A和图3中B所示。取样方法为:连续搅拌过程中,取出100μL的反应液到1mL的乙酸乙酯中;12000×g离心5min;取上清用于气相色谱分析。同时,分别取D3标准品和A7标准品,用乙酸乙酯配制0.5g/L标准品溶液进行气相分析色谱检测。
气相色谱分析方法:毛细管色谱柱:DB1701 30m×0.53mm×1.5μm;柱温:100℃以20℃/min升温至200℃,保温20min;进样口温度:140℃;检测器温度:260℃;载气(N2):5ml/min;分流比:20:1;进样量:1μL;空白溶液:乙酸乙酯。
底物D3的出峰时间一般为4.8-5.0min,中间产物环氧物的出峰时间为3.4-3.5min,产物A7的出峰时间为6.5-7.0min。其气相色谱图如图4所示。
脱卤酶亲本HheC的催化反应结果:底物D3投料量2.5g,在50mL反应体系中,其投料浓度为50g/L经过9h的全细胞催化,测得底物D3残留为1.35g,即底物转化率为46%(部分水解);而产物A7生成为0.36g,产物得率为15%(部分水解)。
脱卤酶突变体HheC的催化反应结果:底物D3投料量2.5g,在50mL反应体系中,其投料浓度为50g/L经过9h的全细胞催化,测得底物D3残留为0.2g,即底物转化率为92%(部分水解);而产物A7生成为1.8g,产物生成率约为72%(部分水解)。所以,脱卤酶突变体HheC-A催化底物D3转化生成A7,其产物得率是野生型脱卤酶HheC的4.8倍。
序列表
<110> 浙江宏元药业股份有限公司、浙江工业大学
<120> 一种卤代醇脱卤酶突变体及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 765
<212> DNA
<213> Agrobacterium tumefaciens
<400> 1
atgagcacag cgattgtgac gaaccttaaa cattttggcg gcatgggctc agctctgaga 60
cttagcgaag cgggacatac ggtggcatgc catgatgaat ctttcaagca aaaggatgaa 120
ctggaagcgt ttgcggaaac atatccgcag ctgaaaccga tgagcgaaca ggaaccggcg 180
gaactgattg aagcggtgac ctcagcttat ggccatgtgg atgtactggt gagcaacgat 240
attttcccag gagaattcag accgattgat aagtatgcgg tggaagatta tcgcggtgcg 300
gtggaagcgc tgcagattag accgtttgcg ctggtcaatg ctgtagcgag ccaaatgaaa 360
aagcgcaaat caggccatat catctttatt acgagcgcgg cgtcctttgg accgtggaaa 420
gaactgagca cgtatacgtc tgctcgtgcg ggcgcttgca ccttagcgaa tgcgctgtct 480
aaagaactgg gcgaatacaa cattccggtg tttgcgattg gaccgaacac tcttcactct 540
gaagatagcc cgtatttcta tccgacagaa ccgtggaaaa caaatccgga acatgtagct 600
catgtgcgaa aatacacggc gcttcaacgc ctgggcacac aaaaagaact gggcgaactg 660
gttgcgtttc tggctagcgg cagctgcgat tatcttacgg gccaggtgtt tacgcttgct 720
ggcggctttc ctatgatcga acgctggcct ggaatgcctg aatga 765
<210> 2
<211> 254
<212> PRT
<213> Agrobacterium tumefaciens
<400> 2
Met Ser Thr Ala Ile Val Thr Asn Leu Lys His Phe Gly Gly Met Gly
1 5 10 15
Ser Ala Leu Arg Leu Ser Glu Ala Gly His Thr Val Ala Cys His Asp
20 25 30
Glu Ser Phe Lys Gln Lys Asp Glu Leu Glu Ala Phe Ala Glu Thr Tyr
35 40 45
Pro Gln Leu Lys Pro Met Ser Glu Gln Glu Pro Ala Glu Leu Ile Glu
50 55 60
Ala Val Thr Ser Ala Tyr Gly His Val Asp Val Leu Val Ser Asn Asp
65 70 75 80
Ile Phe Pro Gly Glu Phe Arg Pro Ile Asp Lys Tyr Ala Val Glu Asp
85 90 95
Tyr Arg Gly Ala Val Glu Ala Leu Gln Ile Arg Pro Phe Ala Leu Val
100 105 110
Asn Ala Val Ala Ser Gln Met Lys Lys Arg Lys Ser Gly His Ile Ile
115 120 125
Phe Ile Thr Ser Ala Ala Ser Phe Gly Pro Trp Lys Glu Leu Ser Thr
130 135 140
Tyr Thr Ser Ala Arg Ala Gly Ala Cys Thr Leu Ala Asn Ala Leu Ser
145 150 155 160
Lys Glu Leu Gly Glu Tyr Asn Ile Pro Val Phe Ala Ile Gly Pro Asn
165 170 175
Thr Leu His Ser Glu Asp Ser Pro Tyr Phe Tyr Pro Thr Glu Pro Trp
180 185 190
Lys Thr Asn Pro Glu His Val Ala His Val Arg Lys Tyr Thr Ala Leu
195 200 205
Gln Arg Leu Gly Thr Gln Lys Glu Leu Gly Glu Leu Val Ala Phe Leu
210 215 220
Ala Ser Gly Ser Cys Asp Tyr Leu Thr Gly Gln Val Phe Thr Leu Ala
225 230 235 240
Gly Gly Phe Pro Met Ile Glu Arg Trp Pro Gly Met Pro Glu
245 250
<210> 3
<211> 765
<212> DNA
<213> Agrobacterium tumefaciens
<400> 3
atgagcacag cgattgtgac gaacgttaaa cattttggcg gcatgggctc agctctgaga 60
cttagcgaag cgggacatac ggtggcatgc catgatgaat ctttcaagca aaaggatgaa 120
ctggaagcgt ttgcggaaac atatccgcag ctgaaaccga tgagcgaaca ggaaccggcg 180
gaactgattg aagcggtgac ctcagcttat ggccaagtgg atgtactggt gagcaacgat 240
attttcgcac cagaattcca accgattgat aagtatgcgg tggaagatta tcgcggtgcg 300
gtggaagcgc tgcagattag accgtttgcg ctggtcaatg ctgtagcgag ccaaatgaaa 360
aagcgcaaat caggccatat catctttatt acgagcgcga cgccctttgg accgtggaaa 420
gaactgagca cgtatacgtc tgctcgtgcg ggcgcttgca ccttagcgaa tgcgctgtct 480
aaagaactgg gcgaatacaa cattccggtg tttgcgattg gaccgaacta tcttcactct 540
gaagatagcc cgtatttcta tccgacagaa ccgtggaaaa caaatccgga acatgtagct 600
catgtgaaaa aagtcacggc gcttcaacgc ctgggcacac aaaaagaact gggcgaactg 660
gttgcgtttc tggctagcgg cagctgcgat tatcttacgg gccaggtgtt ttggcttgct 720
ggcggctttc ctatgatcga acgctggcct ggaatgcctg aatga 765
<210> 4
<211> 254
<212> PRT
<213> Agrobacterium tumefaciens
<400> 4
Met Ser Thr Ala Ile Val Thr Asn Val Lys His Phe Gly Gly Met Gly
1 5 10 15
Ser Ala Leu Arg Leu Ser Glu Ala Gly His Thr Val Ala Cys His Asp
20 25 30
Glu Ser Phe Lys Gln Lys Asp Glu Leu Glu Ala Phe Ala Glu Thr Tyr
35 40 45
Pro Gln Leu Lys Pro Met Ser Glu Gln Glu Pro Ala Glu Leu Ile Glu
50 55 60
Ala Val Thr Ser Ala Tyr Gly Gln Val Asp Val Leu Val Ser Asn Asp
65 70 75 80
Ile Phe Ala Pro Glu Phe Gln Pro Ile Asp Lys Tyr Ala Val Glu Asp
85 90 95
Tyr Arg Gly Ala Val Glu Ala Leu Gln Ile Arg Pro Phe Ala Leu Val
100 105 110
Asn Ala Val Ala Ser Gln Met Lys Lys Arg Lys Ser Gly His Ile Ile
115 120 125
Phe Ile Thr Ser Ala Thr Pro Phe Gly Pro Trp Lys Glu Leu Ser Thr
130 135 140
Tyr Thr Ser Ala Arg Ala Gly Ala Cys Thr Leu Ala Asn Ala Leu Ser
145 150 155 160
Lys Glu Leu Gly Glu Tyr Asn Ile Pro Val Phe Ala Ile Gly Pro Asn
165 170 175
Tyr Leu His Ser Glu Asp Ser Pro Tyr Phe Tyr Pro Thr Glu Pro Trp
180 185 190
Lys Thr Asn Pro Glu His Val Ala His Val Lys Lys Val Thr Ala Leu
195 200 205
Gln Arg Leu Gly Thr Gln Lys Glu Leu Gly Glu Leu Val Ala Phe Leu
210 215 220
Ala Ser Gly Ser Cys Asp Tyr Leu Thr Gly Gln Val Phe Trp Leu Ala
225 230 235 240
Gly Gly Phe Pro Met Ile Glu Arg Trp Pro Gly Met Pro Glu
245 250
Claims (9)
1.一种卤代醇脱卤酶突变体,其特征在于所述卤代醇脱卤酶突变体氨基酸序列为SEQID NO.2所示。
2.一种权利要求1所述卤代醇脱卤酶突变体的编码基因。
3.如权利要求2所述的编码基因,其特征在于所述编码基因的核苷酸序列如SEQ IDNO.1所示。
4.一种权利要求2所述编码基因构建的重组载体。
5.一种权利要求4所述重组载体转化制备的重组基因工程菌。
6.一种权利要求1所述卤代醇脱卤酶突变体在合成阿托伐他汀关键中间体(3R,5R)6-腈基-3,5-二羟基己酸叔丁酯中的应用。
7.如权利要求6所述的应用,其特征在于所述的应用以含卤代醇脱卤酶突变体编码基因的重组基因工程菌经发酵培养获得的含湿菌体的发酵液为反应介质,以(3R,5S)6-氯-3,5-二羟基己酸叔丁酯为底物构成pH 8.0-9.0的反应体系,在35℃进行转化反应,反应完全后,将反应液分离纯化,获得(3R,5R)6-腈基-3,5-二羟基己酸叔丁酯。
8.如权利要求7所述的应用,其特征在于所述湿菌体用量为10~30g/L发酵液,所述底物终浓度为50g/L发酵液。
9.如权利要求7所述的应用,其特征在于所述发酵液按如下方法制备:
(1)菌种活化:将含卤代醇脱卤酶突变体编码基因的重组基因工程菌接种到含有50μg/ml卡那霉素的种子培养基中,37℃培养至对数生长中期,获得种子液;种子培养基组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO40.71g/L、MgSO4·7H2O 0.49g/L、卡那霉素50μg/L,溶剂为去离子水,pH7.0;
(2)发酵培养:将种子液按体积浓度5%的接种量接种到发酵培养基中,30℃培养6h;加入终浓度为5.0-7.0mM的α-乳糖,控制发酵温度22℃,继续发酵20h,得到所述的发酵液;所述发酵培养基质量组成:1%的蛋白胨、0.5%的酵母提取粉、54mM甘油、2.8mM葡萄糖、25mMNa2HPO4、25mM KH2PO4、50mM NH4Cl、5mM Na2SO4、2mM MgSO4、卡那霉素50μg/L,溶剂为去离子水,pH值自然。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810133923.2A CN108048438B (zh) | 2018-02-09 | 2018-02-09 | 一种卤代醇脱卤酶突变体及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810133923.2A CN108048438B (zh) | 2018-02-09 | 2018-02-09 | 一种卤代醇脱卤酶突变体及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108048438A CN108048438A (zh) | 2018-05-18 |
CN108048438B true CN108048438B (zh) | 2020-08-18 |
Family
ID=62125864
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810133923.2A Active CN108048438B (zh) | 2018-02-09 | 2018-02-09 | 一种卤代醇脱卤酶突变体及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108048438B (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109295119B (zh) * | 2018-10-18 | 2021-08-06 | 浙江宏元药业股份有限公司 | 一种生产他汀类药物中间体的生物催化方法 |
CN109295044A (zh) * | 2018-10-26 | 2019-02-01 | 浙江大学 | 一种卤醇脱卤酶突变体及其应用 |
CN109439700A (zh) * | 2018-11-09 | 2019-03-08 | 浙江宏元药业股份有限公司 | 一种脱卤酶高效催化合成他汀类药物中间体的方法 |
CN113481179A (zh) * | 2021-06-24 | 2021-10-08 | 江苏阿尔法药业股份有限公司 | 一种脱卤酶和制备方法及在制备阿托伐他汀中间体中的应用 |
CN115820598B (zh) * | 2022-08-26 | 2024-07-09 | 天津大学 | 有机溶剂耐受性提高的卤代烷脱卤酶DhaA突变体及其应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007128469A1 (en) * | 2006-05-03 | 2007-11-15 | Dsm Ip Assets B.V. | Process for the preparation of enantiomerically enriched nitriles |
WO2011036072A1 (de) * | 2009-09-25 | 2011-03-31 | Ernst-Moritz-Arndt-Universität | Verfahren zur umwandlung von 3-halogen-1,2-propandiol und seinen mono- und diesterderivaten zu 1,2,3-propantriol oder monoacylglyceriden durch enzymatische umsetzung |
CN102827853A (zh) * | 2012-09-06 | 2012-12-19 | 江苏阿尔法药业有限公司 | 一种卤醇脱卤酶基因突变体及其应用 |
CN104651290A (zh) * | 2015-02-10 | 2015-05-27 | 浙江大学 | 工程菌及其在制备阿托伐他汀药物中间体中的应用 |
CN104673733A (zh) * | 2015-02-10 | 2015-06-03 | 浙江大学 | 工程菌及其在制备(r)-6-氰基-5-羟基-3-羰基己酸叔丁酯中的应用 |
CN105567655A (zh) * | 2014-10-14 | 2016-05-11 | 南京博优康远生物医药科技有限公司 | 一种卤醇脱卤酶及其在合成他汀类药物中间体中的应用 |
-
2018
- 2018-02-09 CN CN201810133923.2A patent/CN108048438B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007128469A1 (en) * | 2006-05-03 | 2007-11-15 | Dsm Ip Assets B.V. | Process for the preparation of enantiomerically enriched nitriles |
WO2011036072A1 (de) * | 2009-09-25 | 2011-03-31 | Ernst-Moritz-Arndt-Universität | Verfahren zur umwandlung von 3-halogen-1,2-propandiol und seinen mono- und diesterderivaten zu 1,2,3-propantriol oder monoacylglyceriden durch enzymatische umsetzung |
CN102827853A (zh) * | 2012-09-06 | 2012-12-19 | 江苏阿尔法药业有限公司 | 一种卤醇脱卤酶基因突变体及其应用 |
CN105567655A (zh) * | 2014-10-14 | 2016-05-11 | 南京博优康远生物医药科技有限公司 | 一种卤醇脱卤酶及其在合成他汀类药物中间体中的应用 |
CN104651290A (zh) * | 2015-02-10 | 2015-05-27 | 浙江大学 | 工程菌及其在制备阿托伐他汀药物中间体中的应用 |
CN104673733A (zh) * | 2015-02-10 | 2015-06-03 | 浙江大学 | 工程菌及其在制备(r)-6-氰基-5-羟基-3-羰基己酸叔丁酯中的应用 |
Non-Patent Citations (1)
Title |
---|
定向进化提高卤代醇脱卤酶HHDH的催化活性;王金玉;《中国优秀硕士学位论文全文数据库 基础科学辑》;20170215(第2期);第A006-560页 * |
Also Published As
Publication number | Publication date |
---|---|
CN108048438A (zh) | 2018-05-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108048438B (zh) | 一种卤代醇脱卤酶突变体及其应用 | |
US11492606B2 (en) | Nitrilase mutant, construction method therefor, and application thereof | |
US11162124B2 (en) | Alcohol dehydrogenase mutant and application thereof in cofactor regeneration | |
CN108467860B (zh) | 一种高产γ-氨基丁酸的方法 | |
US10829755B2 (en) | Genetically engineered arginine deiminase modified by site-directed mutagenesis | |
CN111518783B (zh) | 重组(R)-ω-转氨酶、突变体及其在制备西他列汀中的应用 | |
CN112626056A (zh) | 一种腈水合活性专一性提高的腈水解酶突变体及其应用 | |
CN114480512B (zh) | 氧化还原酶及其突变体在生物合成圆柚酮中的应用 | |
Zhang et al. | Efficient production of ethyl (R)-4-chloro-3-hydroxybutanoate by a novel alcohol dehydrogenase from Lactobacillus curieae S1L19 | |
CN113201516B (zh) | 一种对硝基苄基酯酶突变体及应用 | |
CN111454918B (zh) | 一种烯醇还原酶突变体及其在制备(r)-香茅醛中的应用 | |
CN107142287A (zh) | 青蒿醛双键还原酶DBR1及其重组菌在制备二氢‑β‑紫罗兰酮中的应用 | |
CN113444702B (zh) | 烯酮还原酶突变体及其应用 | |
CN113736803B (zh) | 一种表达乙醛脱氢酶的重组枯草芽孢杆菌及其应用 | |
CN112553185B (zh) | 一种腈水解活性专一性提高的腈水解酶突变体及其应用 | |
CN107779459B (zh) | 葡萄糖脱氢酶dna分子、载体和菌株及应用 | |
CN108004225A (zh) | 一种成团泛菌来源的苯丙氨酸氨基变位酶的突变体 | |
CN113025594B (zh) | 多肽、核酸及其在合成香叶醇上的应用 | |
CN110846288B (zh) | 一种谷胱甘肽双功能酶突变体及其应用 | |
CN109370997B (zh) | 一种苯丙氨酸氨基变位酶突变体 | |
CN113481175B (zh) | 一种活性和立体选择性提高的烯键还原酶突变体及其编码基因以及应用 | |
CN109337891B (zh) | 一种热稳定性提高的苯丙氨酸氨基变位酶突变体 | |
CN114317631B (zh) | 单胺氧化酶在制备托品酮中的应用 | |
CN110452899B (zh) | 一种葡萄糖异构酶、突变体及其在制备d-果糖中的应用 | |
CN112481231B (zh) | 一种兼具酰基转移酶和谷丙转氨酶活性的双功能酶 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |