CN110283799B - Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof - Google Patents
Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof Download PDFInfo
- Publication number
- CN110283799B CN110283799B CN201910375432.3A CN201910375432A CN110283799B CN 110283799 B CN110283799 B CN 110283799B CN 201910375432 A CN201910375432 A CN 201910375432A CN 110283799 B CN110283799 B CN 110283799B
- Authority
- CN
- China
- Prior art keywords
- leu
- mutant
- glu
- lys
- ile
- 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
- 150000001299 aldehydes Chemical class 0.000 title claims abstract description 38
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 title claims abstract description 37
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 title claims abstract description 36
- 102000005602 Aldo-Keto Reductases Human genes 0.000 claims abstract description 23
- 108010084469 Aldo-Keto Reductases Proteins 0.000 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 23
- 230000009467 reduction Effects 0.000 claims abstract description 21
- -1 secondary alcohol compound Chemical class 0.000 claims abstract description 20
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims description 12
- 102000004316 Oxidoreductases Human genes 0.000 claims description 11
- 108090000854 Oxidoreductases Proteins 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
- 229910052736 halogen Inorganic materials 0.000 claims description 7
- 150000002367 halogens Chemical class 0.000 claims description 7
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims description 6
- 125000001424 substituent group Chemical group 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 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 5
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 3
- 150000003333 secondary alcohols Chemical class 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 238000011946 reduction process Methods 0.000 claims 1
- 244000063299 Bacillus subtilis Species 0.000 abstract description 17
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 13
- 230000035772 mutation Effects 0.000 abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 9
- 230000003287 optical effect Effects 0.000 abstract description 7
- 101001110310 Lentilactobacillus kefiri NADP-dependent (R)-specific alcohol dehydrogenase Proteins 0.000 abstract description 5
- 150000001298 alcohols Chemical class 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 5
- ZMPGBVQQIQSQED-MRVPVSSYSA-N methyl (2r)-2-(2-chlorophenyl)-2-hydroxyacetate Chemical compound COC(=O)[C@H](O)C1=CC=CC=C1Cl ZMPGBVQQIQSQED-MRVPVSSYSA-N 0.000 abstract description 2
- 238000006722 reduction reaction Methods 0.000 description 19
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 16
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 229940088598 enzyme Drugs 0.000 description 12
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 11
- 150000001413 amino acids Chemical group 0.000 description 11
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000012071 phase Substances 0.000 description 8
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000000543 intermediate Substances 0.000 description 5
- 125000004343 1-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])(*)C([H])([H])[H] 0.000 description 4
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 4
- RZZMZYZXNJRPOJ-BJDJZHNGSA-N Ala-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](C)N RZZMZYZXNJRPOJ-BJDJZHNGSA-N 0.000 description 4
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 4
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 4
- CNQAFFMNJIQYGX-DRZSPHRISA-N Ala-Phe-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 CNQAFFMNJIQYGX-DRZSPHRISA-N 0.000 description 4
- LSMDIAAALJJLRO-XQXXSGGOSA-N Ala-Thr-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LSMDIAAALJJLRO-XQXXSGGOSA-N 0.000 description 4
- PNQWAUXQDBIJDY-GUBZILKMSA-N Arg-Glu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNQWAUXQDBIJDY-GUBZILKMSA-N 0.000 description 4
- URAUIUGLHBRPMF-NAKRPEOUSA-N Arg-Ser-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O URAUIUGLHBRPMF-NAKRPEOUSA-N 0.000 description 4
- VYZBPPBKFCHCIS-WPRPVWTQSA-N Arg-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N VYZBPPBKFCHCIS-WPRPVWTQSA-N 0.000 description 4
- BZMWJLLUAKSIMH-FXQIFTODSA-N Asn-Glu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BZMWJLLUAKSIMH-FXQIFTODSA-N 0.000 description 4
- FTCGGKNCJZOPNB-WHFBIAKZSA-N Asn-Gly-Ser Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FTCGGKNCJZOPNB-WHFBIAKZSA-N 0.000 description 4
- UDSVWSUXKYXSTR-QWRGUYRKSA-N Asn-Gly-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O UDSVWSUXKYXSTR-QWRGUYRKSA-N 0.000 description 4
- OOWSBIOUKIUWLO-RCOVLWMOSA-N Asn-Gly-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O OOWSBIOUKIUWLO-RCOVLWMOSA-N 0.000 description 4
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 4
- VHQOCWWKXIOAQI-WDSKDSINSA-N Asp-Gln-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O VHQOCWWKXIOAQI-WDSKDSINSA-N 0.000 description 4
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 4
- PYXXJFRXIYAESU-PCBIJLKTSA-N Asp-Ile-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PYXXJFRXIYAESU-PCBIJLKTSA-N 0.000 description 4
- XLILXFRAKOYEJX-GUBZILKMSA-N Asp-Leu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLILXFRAKOYEJX-GUBZILKMSA-N 0.000 description 4
- GWIJZUVQVDJHDI-AVGNSLFASA-N Asp-Phe-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O GWIJZUVQVDJHDI-AVGNSLFASA-N 0.000 description 4
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 4
- ITGFVUYOLWBPQW-KKHAAJSZSA-N Asp-Thr-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O ITGFVUYOLWBPQW-KKHAAJSZSA-N 0.000 description 4
- UXIPUCUHQBIQOS-SRVKXCTJSA-N Asp-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UXIPUCUHQBIQOS-SRVKXCTJSA-N 0.000 description 4
- AKJRHDMTEJXTPV-ACZMJKKPSA-N Glu-Asn-Ala Chemical compound C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AKJRHDMTEJXTPV-ACZMJKKPSA-N 0.000 description 4
- GZWOBWMOMPFPCD-CIUDSAMLSA-N Glu-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N GZWOBWMOMPFPCD-CIUDSAMLSA-N 0.000 description 4
- VIIBEIQMLJEUJG-LAEOZQHASA-N Gly-Ile-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O VIIBEIQMLJEUJG-LAEOZQHASA-N 0.000 description 4
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 4
- 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 4
- JBJNKUOMNZGQIM-PYJNHQTQSA-N His-Arg-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JBJNKUOMNZGQIM-PYJNHQTQSA-N 0.000 description 4
- STWGDDDFLUFCCA-GVXVVHGQSA-N His-Glu-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O STWGDDDFLUFCCA-GVXVVHGQSA-N 0.000 description 4
- SOYCWSKCUVDLMC-AVGNSLFASA-N His-Pro-Arg Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCCNC(=N)N)C(=O)O SOYCWSKCUVDLMC-AVGNSLFASA-N 0.000 description 4
- TZCGZYWNIDZZMR-NAKRPEOUSA-N Ile-Arg-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](C)C(=O)O)N TZCGZYWNIDZZMR-NAKRPEOUSA-N 0.000 description 4
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 4
- MQFGXJNSUJTXDT-QSFUFRPTSA-N Ile-Gly-Ile Chemical compound N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)O MQFGXJNSUJTXDT-QSFUFRPTSA-N 0.000 description 4
- VOBYAKCXGQQFLR-LSJOCFKGSA-N Ile-Gly-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O VOBYAKCXGQQFLR-LSJOCFKGSA-N 0.000 description 4
- ADDYYRVQQZFIMW-MNXVOIDGSA-N Ile-Lys-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ADDYYRVQQZFIMW-MNXVOIDGSA-N 0.000 description 4
- GLYJPWIRLBAIJH-FQUUOJAGSA-N Ile-Lys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N GLYJPWIRLBAIJH-FQUUOJAGSA-N 0.000 description 4
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 description 4
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 4
- XYUBOFCTGPZFSA-WDSOQIARSA-N Leu-Arg-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 XYUBOFCTGPZFSA-WDSOQIARSA-N 0.000 description 4
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 4
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 4
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 4
- SYRTUBLKWNDSDK-DKIMLUQUSA-N Leu-Phe-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYRTUBLKWNDSDK-DKIMLUQUSA-N 0.000 description 4
- AKVBOOKXVAMKSS-GUBZILKMSA-N Leu-Ser-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O AKVBOOKXVAMKSS-GUBZILKMSA-N 0.000 description 4
- LCNASHSOFMRYFO-WDCWCFNPSA-N Leu-Thr-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O LCNASHSOFMRYFO-WDCWCFNPSA-N 0.000 description 4
- BTEMNFBEAAOGBR-BZSNNMDCSA-N Leu-Tyr-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BTEMNFBEAAOGBR-BZSNNMDCSA-N 0.000 description 4
- JCFYLFOCALSNLQ-GUBZILKMSA-N Lys-Ala-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JCFYLFOCALSNLQ-GUBZILKMSA-N 0.000 description 4
- OVIVOCSURJYCTM-GUBZILKMSA-N Lys-Asp-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O OVIVOCSURJYCTM-GUBZILKMSA-N 0.000 description 4
- 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 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
- VEGLGAOVLFODGC-GUBZILKMSA-N Lys-Glu-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O VEGLGAOVLFODGC-GUBZILKMSA-N 0.000 description 4
- SQJSXOQXJYAVRV-SRVKXCTJSA-N Lys-His-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N SQJSXOQXJYAVRV-SRVKXCTJSA-N 0.000 description 4
- IUWMQCZOTYRXPL-ZPFDUUQYSA-N Lys-Ile-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O IUWMQCZOTYRXPL-ZPFDUUQYSA-N 0.000 description 4
- QKXZCUCBFPEXNK-KKUMJFAQSA-N Lys-Leu-His Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 QKXZCUCBFPEXNK-KKUMJFAQSA-N 0.000 description 4
- MEQLGHAMAUPOSJ-DCAQKATOSA-N Lys-Ser-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O MEQLGHAMAUPOSJ-DCAQKATOSA-N 0.000 description 4
- UGCIQUYEJIEHKX-GVXVVHGQSA-N Lys-Val-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O UGCIQUYEJIEHKX-GVXVVHGQSA-N 0.000 description 4
- LUYURUYVNYGKGM-RCWTZXSCSA-N Met-Pro-Thr Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O LUYURUYVNYGKGM-RCWTZXSCSA-N 0.000 description 4
- KVNOBVKRBOYSIV-SZMVWBNQSA-N Met-Pro-Trp Chemical compound CSCC[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N KVNOBVKRBOYSIV-SZMVWBNQSA-N 0.000 description 4
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 4
- 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 4
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 4
- APJPXSFJBMMOLW-KBPBESRZSA-N Phe-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 APJPXSFJBMMOLW-KBPBESRZSA-N 0.000 description 4
- TXPUNZXZDVJUJQ-LPEHRKFASA-N Pro-Asn-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N2CCC[C@@H]2C(=O)O TXPUNZXZDVJUJQ-LPEHRKFASA-N 0.000 description 4
- MHHQQZIFLWFZGR-DCAQKATOSA-N Pro-Lys-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O MHHQQZIFLWFZGR-DCAQKATOSA-N 0.000 description 4
- KAAPNMOKUUPKOE-SRVKXCTJSA-N Ser-Asn-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KAAPNMOKUUPKOE-SRVKXCTJSA-N 0.000 description 4
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 4
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 4
- BVOVIGCHYNFJBZ-JXUBOQSCSA-N Thr-Leu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O BVOVIGCHYNFJBZ-JXUBOQSCSA-N 0.000 description 4
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 4
- ZOCJFNXUVSGBQI-HSHDSVGOSA-N Thr-Trp-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O ZOCJFNXUVSGBQI-HSHDSVGOSA-N 0.000 description 4
- IXEGQBJZDIRRIV-QEJZJMRPSA-N Trp-Asn-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O IXEGQBJZDIRRIV-QEJZJMRPSA-N 0.000 description 4
- GEGYPBOPIGNZIF-CWRNSKLLSA-N Trp-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O GEGYPBOPIGNZIF-CWRNSKLLSA-N 0.000 description 4
- KOVXHANYYYMBRF-IRIUXVKKSA-N Tyr-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O KOVXHANYYYMBRF-IRIUXVKKSA-N 0.000 description 4
- HSBZWINKRYZCSQ-KKUMJFAQSA-N Tyr-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O HSBZWINKRYZCSQ-KKUMJFAQSA-N 0.000 description 4
- GXAZTLJYINLMJL-LAEOZQHASA-N Val-Asn-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N GXAZTLJYINLMJL-LAEOZQHASA-N 0.000 description 4
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 description 4
- DHINLYMWMXQGMQ-IHRRRGAJSA-N Val-His-His Chemical compound C([C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 DHINLYMWMXQGMQ-IHRRRGAJSA-N 0.000 description 4
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 4
- RLVTVHSDKHBFQP-ULQDDVLXSA-N Val-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 RLVTVHSDKHBFQP-ULQDDVLXSA-N 0.000 description 4
- 108010041407 alanylaspartic acid Proteins 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 4
- FDPIMTJIUBPUKL-UHFFFAOYSA-N dimethylacetone Natural products CCC(=O)CC FDPIMTJIUBPUKL-UHFFFAOYSA-N 0.000 description 4
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 4
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 4
- 108010077515 glycylproline Proteins 0.000 description 4
- 108010092114 histidylphenylalanine Proteins 0.000 description 4
- 108010077158 leucinyl-arginyl-tryptophan Proteins 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 4
- 108010073969 valyllysine Proteins 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 2
- WAPNOHKVXSQRPX-SPBYTNOZSA-N 1-phenylethanol Chemical class [13CH3][13CH](O)C1=CC=CC=C1 WAPNOHKVXSQRPX-SPBYTNOZSA-N 0.000 description 2
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 2
- 239000005541 ACE inhibitor Substances 0.000 description 2
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 2
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- BNMRSWQOHIQTFL-JSGCOSHPSA-N Gly-Val-Phe Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 BNMRSWQOHIQTFL-JSGCOSHPSA-N 0.000 description 2
- SBVMXEZQJVUARN-XPUUQOCRSA-N Gly-Val-Ser Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O SBVMXEZQJVUARN-XPUUQOCRSA-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
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 2
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 2
- MMJJFXWMCMJMQA-STQMWFEESA-N Phe-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CC=CC=C1 MMJJFXWMCMJMQA-STQMWFEESA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- WMIUTJPFHMMUGY-ZFWWWQNUSA-N Trp-Pro-Gly Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)NCC(=O)O WMIUTJPFHMMUGY-ZFWWWQNUSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 2
- 238000011914 asymmetric synthesis Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000010531 catalytic reduction reaction Methods 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 description 2
- 229960003009 clopidogrel Drugs 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- ZJYKSSGYDPNKQS-LLVKDONJSA-N ethyl (2r)-2-hydroxy-4-phenylbutanoate Chemical compound CCOC(=O)[C@H](O)CCC1=CC=CC=C1 ZJYKSSGYDPNKQS-LLVKDONJSA-N 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012847 fine chemical Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 108010015792 glycyllysine Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 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 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 108010045397 lysyl-tyrosyl-lysine Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 101150075118 sub1 gene Proteins 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 description 1
- OJRHUICOVVSGSY-RXMQYKEDSA-N (2s)-2-chloro-3-methylbutan-1-ol Chemical compound CC(C)[C@H](Cl)CO OJRHUICOVVSGSY-RXMQYKEDSA-N 0.000 description 1
- WRGWHECSFKIQJE-PHDIDXHHSA-N (3r,5r)-6-cyano-3,5-dihydroxyhexanoic acid Chemical compound N#CC[C@@H](O)C[C@@H](O)CC(O)=O WRGWHECSFKIQJE-PHDIDXHHSA-N 0.000 description 1
- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 1
- ZBFFNPODXBJBPW-VIFPVBQESA-N (R)-phenylacetylcarbinol Chemical compound CC(=O)[C@H](O)C1=CC=CC=C1 ZBFFNPODXBJBPW-VIFPVBQESA-N 0.000 description 1
- WDFGIPRSKLXATJ-UHFFFAOYSA-N 2-ethyl-2-hydroxy-4-phenylbutanoic acid Chemical compound CCC(O)(C(O)=O)CCC1=CC=CC=C1 WDFGIPRSKLXATJ-UHFFFAOYSA-N 0.000 description 1
- KWOLFJPFCHCOCG-UHFFFAOYSA-N Acetophenone Natural products CC(=O)C1=CC=CC=C1 KWOLFJPFCHCOCG-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000005862 Angiotensin II Human genes 0.000 description 1
- 101800000734 Angiotensin-1 Proteins 0.000 description 1
- 102400000344 Angiotensin-1 Human genes 0.000 description 1
- 101800000733 Angiotensin-2 Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- XPCFTKFZXHTYIP-PMACEKPBSA-N Benazepril Chemical compound C([C@@H](C(=O)OCC)N[C@@H]1C(N(CC(O)=O)C2=CC=CC=C2CC1)=O)CC1=CC=CC=C1 XPCFTKFZXHTYIP-PMACEKPBSA-N 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101000783577 Dendroaspis angusticeps Thrombostatin Proteins 0.000 description 1
- 101000783578 Dendroaspis jamesoni kaimosae Dendroaspin Proteins 0.000 description 1
- 108010061435 Enalapril Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 101001110286 Homo sapiens Ras-related C3 botulinum toxin substrate 1 Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 101710149643 Integrin alpha-IIb Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 206010024264 Lethargy Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102000007466 Purinergic P2 Receptors Human genes 0.000 description 1
- 108010085249 Purinergic P2 Receptors Proteins 0.000 description 1
- 102100022122 Ras-related C3 botulinum toxin substrate 1 Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- XPNGNIFUDRPBFJ-UHFFFAOYSA-N alpha-methylbenzylalcohol Natural products CC1=CC=CC=C1CO XPNGNIFUDRPBFJ-UHFFFAOYSA-N 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 1
- 229950006323 angiotensin ii Drugs 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 239000003524 antilipemic agent Substances 0.000 description 1
- 229940127218 antiplatelet drug Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 229960001770 atorvastatin calcium Drugs 0.000 description 1
- FQCKMBLVYCEXJB-MNSAWQCASA-L atorvastatin calcium Chemical compound [Ca+2].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.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 FQCKMBLVYCEXJB-MNSAWQCASA-L 0.000 description 1
- UGUUDTWORXNLAK-UHFFFAOYSA-N azidoalcohol Chemical class ON=[N+]=[N-] UGUUDTWORXNLAK-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229960004530 benazepril Drugs 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- GKIRPKYJQBWNGO-OCEACIFDSA-N clomifene Chemical compound C1=CC(OCCN(CC)CC)=CC=C1C(\C=1C=CC=CC=1)=C(\Cl)C1=CC=CC=C1 GKIRPKYJQBWNGO-OCEACIFDSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 150000003997 cyclic ketones Chemical class 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 229960000873 enalapril Drugs 0.000 description 1
- GBXSMTUPTTWBMN-XIRDDKMYSA-N enalapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)CC1=CC=CC=C1 GBXSMTUPTTWBMN-XIRDDKMYSA-N 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000003944 halohydrins Chemical class 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 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 1
- BRHPBVXVOVMTIQ-ZLELNMGESA-N l-leucine l-leucine Chemical compound CC(C)C[C@H](N)C(O)=O.CC(C)C[C@H](N)C(O)=O BRHPBVXVOVMTIQ-ZLELNMGESA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 229940002661 lipitor Drugs 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- QCCDLTOVEPVEJK-UHFFFAOYSA-N phenylacetone Chemical class CC(=O)CC1=CC=CC=C1 QCCDLTOVEPVEJK-UHFFFAOYSA-N 0.000 description 1
- 239000000106 platelet aggregation inhibitor Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000036316 preload Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 229960003401 ramipril Drugs 0.000 description 1
- HDACQVRGBOVJII-JBDAPHQKSA-N ramipril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](C[C@@H]2CCC[C@@H]21)C(O)=O)CC1=CC=CC=C1 HDACQVRGBOVJII-JBDAPHQKSA-N 0.000 description 1
- 239000003087 receptor blocking agent Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002693 spinal anesthesia Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of biology, and relates to a plurality of mutants of ketoreductase and application thereof, wherein the mutants are obtained by carrying out mutation on wild Bacillus subtilis aldo-ketoreductase (BsAKR (YvgN)), and in particular relates to a plurality of mutants of ketoreductase and a preparation method thereof. Also relates to a method for obtaining an S or R configuration secondary alcohol compound with optical activity by catalyzing the reduction of alpha-keto acid ester compounds and 1-acetophenone compounds by using the aldehyde ketone reductase BsAKR (YvgN) and the mutant thereof. Compared with a wild type, the stereoselectivity of the mutant is improved or reversed, the substrate is catalyzed to obtain chiral alcohols with two configurations (R or S type), such as optically pure (R) -o-chloromandelic acid methyl ester, (S) -2-hydroxy-4-phenyl ethyl butyrate and (R) -2-hydroxy-4-phenyl ethyl butyrate, and the mutant has good application value in the field of chiral alcohol preparation.
Description
Technical Field
The invention relates to the technical field of biology, and relates to a plurality of mutants of ketoreductase and application thereof, wherein the mutants are obtained by carrying out mutation on wild Bacillus subtilis aldo-ketoreductase (BsAKR (YvgN)), and in particular relates to a plurality of mutants of ketoreductase and a preparation method thereof. Also relates to a method for obtaining an S or R configuration secondary alcohol compound with optical activity by catalyzing the reduction of alpha-keto acid ester compounds and 1-acetophenone compounds by using the aldehyde ketone reductase BsAKR (YvgN) and the mutant thereof.
Background
The chiral carbon atom of the chiral alcohol is connected with an active hydroxyl functional group, so that the chiral alcohol becomes a key synthetic intermediate for fine chemical engineering, medicine and agricultural application. For example, ethyl chiral alcohol (3R, 5R) -6-cyano-3,5-dihydroxyhexanoate (ethyl (3R, 5R) -6-cyanoo-3, 5-dihydrohexanoate) is a hypolipidemic agent developed by the company Peucedani, atorvastatin calcium (Lipitor)TM) Key chiral intermediates in the synthesis process. In addition, chiral alcohols (such as chiral halohydrins) containing other active functional groups can also be converted into other chiral modules with wider application values, such as chiral epoxy compounds, chiral diols, chiral hydroxy nitriles, chiral hydroxy azides, chiral amino alcohols and the like.
The alpha-keto ester compound is widely applied in the fields of chemical industry and medicine as an important synthetic intermediate, and the R-and S-forms of the alcohol have important application values. (R) -O-chloromandelic acid methyl ester (molecular formula is o-Cl-C)6H4CH(OH)COOCH3Molecular weight 200.62, cas number: 32345-59-8) is an important chiral intermediate for synthesizing clopidogrel, a platelet aggregation inhibitor. Clopidogrel is an Adenosine Diphosphate (ADP) receptor blocker, can be combined with an ADP receptor on the surface of a platelet membrane to prevent fibrinogen from being treatedBinds with glycoprotein GPIIb/IIIa receptor, thereby inhibiting platelet aggregation, is mainly used for treating acute myocardial infarction, and has wide market at home. (R) -2-hydroxy-4-phenylbutyric acid Ethyl ester [ Ethyl (R) -2-hydroxy-4-phenylbutyrate, (R) -HPBE]Are important precursors for the manufacture of various Angiotensin Converting Enzyme (ACE) inhibitors, such as benazepril, enalapril and ramipril, which are widely used in the treatment of hypertension and congestive heart failure. ACE inhibitors interfere with the conversion of angiotensin I to angiotensin II, and are beneficial in reducing cardiac preload. And meanwhile, the ACE inhibitor can prevent or reverse cardiovascular remodeling and inhibit hypertrophy and hyperplasia of cardiac muscle and blood vessels. Occupies a large share in the domestic antihypertensive drug market. In addition, (R) -phenylacetylcarbinol is a precursor of (-) -ephedrine, a drug used to prevent hypotension during spinal anesthesia, and is also used to treat asthma, lethargy.
Optically pure 1-phenyl ethanol compounds, in particular alpha halogen substituted 1-phenyl ethanol compounds are important chiral intermediates in the synthesis process of medicaments and other fine chemicals. At present, various methods for obtaining optically pure chiral purity are developed, including two major classes of kinetic resolution and asymmetric synthesis. The theoretical yield of the asymmetric synthesis method of the prochiral carbonyl compound can reach 100 percent, and the method is an important way for obtaining the optically pure 1-phenyl ethanol compound. Researchers have developed chemical synthesis methods for synthesizing optically pure chiral alcohols by using chiral metal ligands as catalysts, and some of them are applied to industrial production, however, their application in synthesis of drugs and the like is limited due to their harsh conditions and heavy metal residue problems. Biocatalysis has received increasing attention in the synthesis of chiral alcohols due to its advantages of being environmentally friendly, mild in reaction conditions, high in regio-and stereoselectivity, and the like.
Aldo Ketoreductases (AKRs) are a type that depends on NAD (P)+The oxidoreductase of (1), comprising more than 190 members. The structure is composed of (alpha/beta)8The structure comprises a barrel-shaped structure and a loop region, an active center consists of loop4, loop7 and loop C, and catalytic active sites comprise Tyr, asp, lys and His. Aldehyde ketonesThe substrate of the proenzyme is prochiral ketone, and the type is wide, such as alpha-ketoester, beta-ketoester, aliphatic chain ketone, cyclic ketone and the like. In order to improve the practicability of the aldehyde ketone reductase in the process of asymmetrically catalyzing and reducing alpha-keto ester and 1-phenyl ethyl ketone compounds, a method for changing the catalytic property of the enzyme by rational protein design and directed evolution is used for modifying the stereoselectivity of the aldehyde ketone reductase by researchers. It has been proposed in the prior art that the substrate profile of an aldehyde-ketone reductase can be influenced by Protein modifications of the loop region of the aldehyde-ketone reductase (Protein Eng, des Sel 2014 27. Furthermore, zhang et al semi-rational design of a thermostable aldoketoreductase from thermatopaa maritima for the synthesis of enantiomerically pure ethyl-2-hydroxy-4-phenylbutyric acid showed that W21 and W86 of this AKR play an important role in determining enantioselectivity (Sci Rep 2017.
The aldehyde ketone reductase BsAKR (YvgN) which is obtained from the bacillus subtilis by gene mining in the laboratory shows poor activity and stereoselectivity on alpha-keto ester and 1-acetophenone in a wild state. The stereoselectivity of the enzyme is improved and reversed through rational design and protein directed evolution, the biocatalytic preparation of the optically pure chiral alcohol is realized, and the application value is very important.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the aldehyde ketone reductase BsAKR (YvgN) and also provides a reductase mutant obtained by mutating the aldehyde ketone reductase, and the aldehyde ketone reductase BsAKR (YvgN) and the mutant can improve the enantioselectivity and the activity of the aldehyde ketone reductase BsAKR (YvgN) and the mutant to alpha-keto acid ester compounds or 1-acetophenone compounds, so that the corresponding chiral alcohol route obtained by reducing the aldehyde ketone reductase is efficient and feasible.
The invention provides aldehyde ketone reductase BsAKR (YvgN), which is determined to be wild type bacillus subtilis aldehyde ketone reductase by sequencing, and the sequence of the aldehyde ketone reductase BsAKR is shown in SEQ ID NO. 1.
The invention also provides a mutant of the aldehyde ketone reductase with improved property, which is obtained by mutation of a wild type bacillus subtilis aldehyde ketone reductase BsAKR (YvgN) gene (SEQ ID NO. 1), the aldehyde ketone reductase is expressed in escherichia coli engineering bacteria, and the aldehyde ketone reductase and the mutant thereof can reduce alpha-keto acid ester compounds to obtain S or R configuration secondary alcohols with optical activity.
Further, the aldehyde ketone reductase gene is connected to an expression plasmid to obtain the recombinant aldehyde ketone reductase. The expression plasmid is:
the recombinant aldone reductase comprises an amino acid sequence with at least 75 percent of identity with SEQ ID NO. 02.
Further, the present invention provides mutants of aldehyde ketone reductase BsaKR (YvgN) obtained by mutation at position 25 or 113, or mutation at both positions 25 and 113 of recombinant aldehyde ketone reductase (i.e., SEQ ID NO. 2).
The mutant of the aldehyde ketone reductase BsAKR (YvgN) corresponds to the 25 th residue of SEQ ID NO.2 as a smaller amino acid residue, and the 113 th residue as a larger amino acid residue, and the amino acids are important for improving the activity and the enantioselectivity of the aldehyde ketone reductase.
The smaller amino acids involved in the present invention are Ala, ser, thr, val, ile and Leu, preferably serine (Ser); the larger amino acid residues involved are Pro, phe, try, leu, preferably phenylalanine (Phe).
In some embodiments of the invention, the aldone reductase mutants containing mutations exhibit higher activity and stereoselectivity for alpha-keto acid ester compounds and 1-acetophenone compounds, and comprise one or two mutations, namely, a mutation at position 25 or position 113, or a mutation at positions 25 and 113 simultaneously, and a mutation at position 25 of an amino acid sequence to Ser; 113 th amino acid sequence is mutated to Phe.
The amino acid sequence of the aldehyde ketone reductase mutant is preferably shown in SEQ ID NO.4,6 and 8.
Embodiments of the invention include nucleic acids encoding the aldoketoreductase mutants having at least 75% sequence identity to the nucleic acid sequence encoding the BsaKR (YvgN) mutant of the invention.
The sequence of the nucleic acid capable of coding the mutant is shown in SEQ ID NO.3,5 and 7.
Related embodiments of the invention also include vectors comprising these nucleic acids and host cells comprising such vectors.
The invention also provides application of the aldone reductase and the mutant thereof in reducing alpha-keto acid esters and 1-acetophenone compounds. The wild-type aldo-keto reductase BsAKR (YvgN) and the mutant thereof, or the cell containing the wild-type or mutant enzyme can be used as a catalyst to catalyze and asymmetrically reduce alpha-keto ester or 1-acetophenone compounds to obtain corresponding optical pure chiral products.
The wild-type aldone reductase BsAKR (YvgN) and the mutant thereof, or cells containing the wild-type or mutant enzyme can be used as a catalyst to catalyze and asymmetrically reduce alpha-keto acid esters and 1-acetophenone compounds to obtain corresponding optical homochiral products with different configurations (S and R configurations).
The alpha-ketonic acid ester compound is shown as a formula I, and the 1-phenyl ethyl ketone compound is shown as a formula II:
wherein,
R1is C1-C4 alkyl, phenyl or phenyl with substituent, and the substituent of the phenyl is halogen or C1-C4 alkyl;
R2is C1-C4 alkyl;
R3is a halogenated C1-C4 alkyl group.
R4Is halogen, halogenated C1-C4 alkyl;
preferably, the first and second air flow paths are arranged in parallel,
R1is phenyl or phenyl with substituent, and the substituent is halogen;
R2is C1-C2 alkyl;
R3is halogenated C1-C4 alkyl, and the halogen of the halogenated alkyl is F or Cl.
R4Is chlorine, halogenated C1-C4 alkyl;
some typical structures are described below:
one embodiment of the invention provides a method for asymmetrically reducing alpha-keto acid esters and 1-acetophenone compounds, which comprises the following steps:
in a phosphate buffer solution of pH5-7, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of aldehyde ketone reductase or its mutant, alpha-keto acid ester or 1-phenyl ethyl ketone compound is reduced to produce optically active chiral secondary alcohol.
The dosage of aldone reductase mutant is 0.02-40g/L, the dosage of glucose dehydrogenase is 0.01-5g/L, the dosage of glucose is 6-200g/L, and NADP+The dosage is 0.1-0.5mmol, the concentration of the alpha-ketonic acid ester or the 1-phenyl ethyl ketone compound is 3-100g/L, the buffer solution is a phosphate buffer solution with the pH value of 5-7, and the reaction temperature is 30-40 ℃.
The aldehyde ketone reductase or the mutant thereof can exist in various forms such as cells, crude enzyme powder, enzyme solution, immobilized enzyme and the like in various feasible ways.
The invention has the beneficial technical effects that: compared with a naturally-occurring ketoreductase wild type, the activity and enantioselectivity of the aldehyde ketone reductase mutant on alpha-ketonic acid esters and 1-acetophenone compounds are reversed and improved by mutating bacillus subtilis aldehyde ketone reductase BsAKR (YvgN).
Drawings
FIG. 1 shows the construction of genes of wild-type aldo-keto reductase BsAKR (YvgN) and its mutant enzyme, and recombinant expression vector containing the genes.
FIG. 2 shows the HPLC detection results of the reduction of substrate Sub1 catalyzed by wild-type aldo-keto reductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8);
sub1: sub1 substrate control; rac-1: product racemate control
FIG. 3 shows the HPLC results of the reduction of substrate Sub2 catalyzed by wild-type aldoketoreductase BsAKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 4 shows the HPLC detection results of the reduction of substrate Sub3 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 5 shows the HPLC detection results of the wild-type aldoketoreductase BsAKR (YvgN) mutant (SEQ ID NO.4, 8) enzyme catalyzing the reduction of the substrate Sub 4.
FIG. 6 shows the HPLC detection results of the wild-type aldoketoreductase BsAKR (YvgN) mutant (SEQ ID NO.4, 8) enzyme catalyzing the reduction of the substrate Sub 5.
FIG. 7 shows the HPLC results of the reduction of substrate Sub6 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 8 shows the HPLC results of the reduction of the substrate Sub7 catalyzed by wild-type aldoketoreductase BsAKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 9 shows the HPLC results of the reduction of substrate Sub8 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 10 shows the HPLC results of the reduction of substrate Sub9 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 11 shows the HPLC results of the reduction of the substrate Sub10 catalyzed by wild-type aldone reductase BsAKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO. 4).
FIG. 12 shows the HPLC results of the reduction of substrate Sub11 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 13 shows the HPLC detection results of the wild-type aldoketoreductase BsAKR (YvgN) mutant (SEQ ID NO.4, 6) enzyme catalyzing the reduction of the substrate Sub 12.
FIG. 14 shows the HPLC results of the reduction of substrate Sub13 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
Detailed Description
The aldehyde ketone reductase mutant of the present invention, and the reduction of α -keto acid esters and 1-phenylacetones using the enzyme are described below by way of specific embodiments. Unless otherwise specified, the protocols used in the present invention are well known to those skilled in the art. Furthermore, the examples are to be construed as illustrative, and not restrictive.
Definitions of certain terms.
Asymmetric reduction is a method for obtaining an optically pure corresponding reduction product by reducing a prochiral compound, and in the invention, the aldehyde ketone reductase catalyzes an alpha-keto acid ester compound or a 1-phenyl acetophenone compound to selectively obtain corresponding alcohol with S or R configuration.
Enantiomeric excess is defined as the amount of one isomer a in an enantiomeric mixture which is more abundant than the other isomer B in the total amount, abbreviated ee, and is expressed by the formula (a-B)/(a + B) × 100%, and the enantiomeric excess is used to indicate the optical purity of a chiral compound. The higher the ee value, the higher the optical purity.
The 20 amino acids are abbreviated as follows
ASP | D | Aspartic acid | Ile | I | Isoleucine |
Thr | T | Threonine | Leu | L | Leucine |
Ser | S | Serine | Thr | T | Tyrosine |
Glu | E | Glutamic acid | Phe | F | Phenylalanine |
Pro | P | Proline | His | H | Histidine |
Gly | G | Glycine | Lys | L | Lysine |
Ala | A | Alanine | Arg | R | Arginine |
Cys | C | Cysteine | Trp | W | Tryptophan |
Val | V | Valine | Gln | Q | Glutamine |
Met | M | Methionine | Asn | N | Asparagine |
The amino acids are classified into larger and smaller amino acids according to the steric hindrance of amino acid side chain groups, the smaller amino acids involved in the invention are Ala, ser, thr, val, ile and Leu, and the larger amino acid residues are Pro, phe, try and Leu.
The examples relate to the formulation of the culture medium.
LB liquid medium: 0.5% yeast extract, 1% peptone and 1% sodium chloride (for example, preparing solid medium, adding 1.5% agar before sterilization), and autoclaving at 115 deg.C for 30min.
Example 1 extraction of Bacillus subtilis genomic DNA
a) After bacillus subtilis is cultured by LB liquid overnight, the fermentation liquor is centrifuged for 5min by 3000r/min in a 1mL centrifuge tube, the supernatant is discarded to collect the bacteria, and the steps can be repeated for several times to obtain enough cells; b) Extracting the genome of the bacillus cereus according to the operation instruction of the bacterial genome DNA extraction kit.
Example 2 cloning of the wild-type BsAKR (YvgN) Gene
The Bacillus subtilis genomic DNA obtained in example 1 was used as a template for PCR reaction in the following reaction system:
and (3) amplification procedure: 94 ℃ below zero: 10min (30s at 94 ℃, 30s at 45 ℃,30 s at 72 ℃) and 10min at 72 ℃.
Primer 1: bsaKR (YvgN) -EcoR I-F: CCGGAATTCGATGCCAACAAGTTTAAAA;
Primer 2: bsaKR (YvgN) -Xho I-R: CCGCTCGAGAAACAGAAGCTCATCAGG;
Restriction sites are underlined;
the DNA fragment obtained by PCR amplification is purified by a gel recovery kit. E.coli DH 5. Alpha. Containing pET28b MBP Plasmid was cultured overnight in LB broth at 37 ℃ and 220r/min, and Plasmid extraction was performed using the reference TIAnprep Mini Plasmid Kit.
The target fragment and the plasmid pET28b MBP plasmid are subjected to double restriction enzyme digestion, and the restriction enzyme digestion system is as follows:
and recovering fragments of the enzyme digestion product by using a gel recovery kit, and connecting the fragments by using T4 ligase.
Example 3 E.coli Rosetta (DE 3) preparation and transformation of competent cells
a) 0.4mL of the seed culture medium is inoculated into 20mL of LB liquid culture medium for culture for 3h; b) 2mL of thalli are enriched in a 1.5mL EP tube twice at 3000r/min for 5min, and the supernatant is discarded; c) Adding 100 μ l ice-cold TSS solution, re-suspending thallus, ice-cooling for 30min; d) Adding 20 μ L of the connecting solution, gently rotating and mixing, and performing ice bath for 30min; e) Heat shock was performed at 42 ℃ for 60s, ice bath was performed for 2min, and 600. Mu.L of LB liquid medium was added. Culturing at 37 ℃, and performing shaking culture at 150r/min for 1h; f) mu.L of each was plated on LB-resistant plates.
EXAMPLE 4 construction of mutants
The construction of the mutant pools was performed by overlap extension PCR using the mutant and flanking primers. (designed mutation primers are as follows:.)
PCR amplification conditions: 94 ℃ below zero: 10Min (94 ℃ 30s,45 ℃ 30s,72 ℃ 30 s) 35 cycles, 72 ℃: and (5) 10min. The resulting gene fragment was purified and amplified by PCR as follows
The desired fragment was obtained by PCR as described in example 2 and ligated into the pET28b MBP vector and transformed into E.coli Rosetta (DE 3) as described in example 3. Respectively obtaining wild aldehyde ketone reductase BsAKR (YvgN) and mutants thereof.
Example 5 mutant expression
a) Inoculating a single colony into a 4ml LB liquid culture medium with kanamycin resistance, and culturing at 37 ℃ and 200rpm for 6 hours to obtain a seed solution; b) Inoculating 20 μ l seed solution into 20ml LB liquid culture medium with kanamycin resistance, culturing at 37 deg.C and 200rpm until OD600 of the culture solution reaches 0.8-1.0, adding 0.5mM IPTG, and cooling to 20 deg.C for inducing expression for 20 hr; c) Collecting thallus by centrifuging culture solution at 4000rpm × 15min, washing twice with normal saline, reselecting thallus with 0.1M pH6.0 sodium phosphate buffer solution, adding lysozyme with final concentration of 1mg/ml, crushing at 30 deg.C and 200rpm for 1h, centrifuging at 4 deg.C and 10000rpm, collecting supernatant, and screening.
Example 6 expression of glucose dehydrogenase
Coli Rosetta (DE 3) containing the pET22b-GDH plasmid was expressed as described in example 5 to obtain glucose dehydrogenase.
Example 7 catalytic reduction of crude enzyme solution
Typical substrates referred to in this example are shown below:
reaction system: the supernatants were mixed together in 450. Mu.l portions each as described in examples 5 and 6 to a final concentration of 0.3mM NADP+8mg of glucose and 4mg of substrate (100 mu.l of methanol for dissolution) and reacting for 6 hours at 30 ℃; extracted three times with equal volumes of ethyl acetate.
Example 8 high Performance liquid phase analysis of substrates and products
The substrate of the high performance liquid phase analysis comprises Sub1-13, and the structure of Sub1-13 is as follows:
the conditions for detecting Sub1, 2 and the product are as follows: a chromatographic column: AD-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub3, 4, 5 and the product are as follows: a chromatographic column: OD-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub6, 8 and the product were: a chromatographic column: OD-H, mobile phase: n-hexane: isopropanol =98, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub7, 9 and the product are as follows: a chromatographic column: OB-H, mobile phase: n-hexane: isopropanol =95, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub10 and the product were: a chromatographic column: OJ-H, mobile phase: n-hexane: isopropanol =95, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub11 and the product were: a chromatographic column: OJ-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub12 and the product were: a chromatographic column: OD-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub13 and the product were: a chromatographic column: AD-H, mobile phase: n-hexane: isopropanol =95, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The detection results of the wild-type aldo-keto reductase (SEQ ID NO. 2) and its mutant (SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO. 8) catalyzing the above-mentioned substrate are shown in Table 1:
TABLE 1 detection results of aldehyde ketone reductase BsAKR (YvgN) wild type and its mutant for catalytic reduction of alpha-keto acid esters and 1-acetophenone compounds
a) Conversion [% ]; b) Reduction product alcohol enantiomer excess (ee%); c) Absolute configuration of the reduction product alcohol; NA No activity was detected.
Sequence listing
<110> Shenyang university of pharmacy
<120> aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 1
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg tttttaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactggc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 2
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 2
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Phe Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Trp Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
<210> 3
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 3
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg ttagcaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactggc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 4
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 4
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Ser Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Trp Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
<210> 5
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 5
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg tttttaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactttc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 6
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 6
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Phe Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Phe Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
<210> 7
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 7
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg ttagcaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactttc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 8
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 8
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Ser Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Phe Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
Claims (10)
1. The mutant of aldehyde ketone reductase is characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO.4,6 and 8.
2. A nucleic acid encoding the aldoketoreductase mutant of claim 1.
3. The nucleic acid of claim 2, having the nucleic acid sequence set forth in SEQ ID No.3,5,7.
4. An expression vector comprising the nucleic acid of claim 2 or 3 and capable of expression in a host cell.
5. A host cell comprising the nucleic acid of claim 2 or 3 or the expression vector of claim 4.
6. The host cell of claim 5, wherein the host cell is E.coli.
7. The use of the aldo-keto reductase mutant of claim 1 in the reduction of alpha-keto acid esters or 1-acetophenone compounds.
8. The use of claim 7, wherein the reduction process comprises: in a phosphate buffer solution of pH5-7, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of the aldehyde ketone reductase mutant as described in claim 1, reducing the alpha-keto acid ester or 1-acetophenone compound to produce an optically active chiral secondary alcohol.
9. The use according to claim 8, wherein the aldone reductase is present in an amount of 0.02-40g/L, the glucose dehydrogenase is present in an amount of 0.01-5g/L, the glucose is present in an amount of 6-200g/L, and the NADP is present+The dosage is 0.1-0.5mmol, the substrate concentration is 3-100g/L, the buffer solution is phosphate buffer solution with pH of 5-7, and the reaction temperature is 30-40 ℃.
10. The use of claim 7, wherein the α -ketonate compound is of formula I, and the 1-acetophenone compound is of formula II:
wherein:
R1is C1-C4 alkyl, phenyl or phenyl with substituent, and the substituent of the phenyl is halogen or C1-C4 alkyl;
R2is C1-C4 alkyl;
R3is a halogenated C1-C4 alkyl group;
R4is halogen, halogenated C1-C4 alkyl.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910375432.3A CN110283799B (en) | 2019-05-07 | 2019-05-07 | Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910375432.3A CN110283799B (en) | 2019-05-07 | 2019-05-07 | Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110283799A CN110283799A (en) | 2019-09-27 |
CN110283799B true CN110283799B (en) | 2022-11-01 |
Family
ID=68002053
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910375432.3A Active CN110283799B (en) | 2019-05-07 | 2019-05-07 | Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110283799B (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114540321B (en) * | 2022-01-27 | 2024-04-02 | 沈阳药科大学 | Preparation method of R-2-sulfonyl-1-phenylethanol derivative |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105624125A (en) * | 2014-11-26 | 2016-06-01 | 南京博优康远生物医药科技有限公司 | Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate |
CN107058248A (en) * | 2017-04-26 | 2017-08-18 | 浙江工业大学 | One kind restructuring aldehyde Ketoreductase mutant, gene, carrier, engineering bacteria and its application |
CN109055327A (en) * | 2018-07-23 | 2018-12-21 | 浙江工业大学 | Aldehyde Ketoreductase mutant and its application |
-
2019
- 2019-05-07 CN CN201910375432.3A patent/CN110283799B/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105624125A (en) * | 2014-11-26 | 2016-06-01 | 南京博优康远生物医药科技有限公司 | Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate |
CN107058248A (en) * | 2017-04-26 | 2017-08-18 | 浙江工业大学 | One kind restructuring aldehyde Ketoreductase mutant, gene, carrier, engineering bacteria and its application |
CN109055327A (en) * | 2018-07-23 | 2018-12-21 | 浙江工业大学 | Aldehyde Ketoreductase mutant and its application |
Non-Patent Citations (5)
Title |
---|
Key sites insight on the stereoselectivity of four mined aldo-keto reductases toward α-keto esters and halogen-substituted acetophenones;Wenhe Zhang等;《Appl Microbiol Biotechnol》;20190604;第103卷(第15期);第6119-6128页 * |
MULTISPECIES: aldo/keto reductase [Bacillus],NCBI Reference Sequence: WP_003221597.1;无;《NCBI GenBank》;20180823;第1页 * |
The Aldo-Keto Reductases(AKRs):Overview;Trevor M.Penning;《Chem Biol Interact》;20150605;第236-246页 * |
无.MULTISPECIES: aldo/keto reductase [Bacillus],NCBI Reference Sequence: WP_003221597.1.《NCBI GenBank》.2018, * |
醛酮还原酶的挖掘与立体选择性改造研究;李衡宇等;《第十二届中国酶工程学术研讨会》;20190808;第1页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110283799A (en) | 2019-09-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102618513B (en) | Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound | |
CN111094557B (en) | Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol | |
CN104099305A (en) | Carbonyl reductase mutant as well as gene and application thereof | |
CN108546690B (en) | Short-chain dehydrogenase and mutant thereof, and preparation and application of gene | |
CN112359036B (en) | Nitrilase mutant with improved catalytic activity and reaction specificity and application thereof | |
CN106701723B (en) | D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and its application and reaction product | |
CN110283799B (en) | Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof | |
CN111454918B (en) | Enol reductase mutant and application thereof in preparation of (R) -citronellal | |
CN110592035B (en) | Carbonyl reductase mutant, recombinant expression vector and application of carbonyl reductase mutant in production of chiral alcohol | |
CN117264935A (en) | Phenylalanine ammonia lyase mutant and application thereof | |
CN113583988A (en) | Amino acid dehydrogenase mutant and application thereof | |
CN113322291A (en) | Synthesis method of chiral amino alcohol compound | |
CN113444702B (en) | Enone reductase mutant and application thereof | |
CN109593739A (en) | Recombinate ketone acid reduction enzyme mutant, gene, engineering bacteria and its application | |
CN110272879B (en) | Aldehyde ketone reductase BcAKR and mutant and application thereof | |
CN110257350B (en) | Aldehyde ketone reductase BmAKR1, mutant and application thereof | |
CN115896081A (en) | Aspartase mutant and application thereof | |
CN110257349B (en) | Aldehyde ketone reductase BmAKR2, mutant and application thereof | |
CN110669743A (en) | P450 monooxygenase mutant from deinococcus radiodurans and application thereof | |
CN114752574B (en) | Enzyme catalysis system, catalase, preparation method and application | |
CN106047826B (en) | Aldehyde dehydrogenase, its recombinant expression transformants and the application in the synthesis of statin precursor | |
CN113755539B (en) | Dihydropyrimidine amino hydrolase and application thereof | |
CN114774491B (en) | Method for preparing (2S, 3R) -2- (phthalimidomethyl) -3-hydroxybutyrate | |
CN114214261B (en) | Escherichia coli genetically engineered bacterium for expressing esterase EstS and application thereof | |
CN114015665A (en) | Engineered NADPH-dependent phenylglycine dehydrogenase and application thereof |
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 |