CN114958801B - 产物耐受型腈水解酶突变体及其应用 - Google Patents
产物耐受型腈水解酶突变体及其应用 Download PDFInfo
- Publication number
- CN114958801B CN114958801B CN202210260482.9A CN202210260482A CN114958801B CN 114958801 B CN114958801 B CN 114958801B CN 202210260482 A CN202210260482 A CN 202210260482A CN 114958801 B CN114958801 B CN 114958801B
- Authority
- CN
- China
- Prior art keywords
- ala
- leu
- glu
- gly
- mutant
- 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
- 108010033272 Nitrilase Proteins 0.000 title claims abstract description 82
- 102000004190 Enzymes Human genes 0.000 claims abstract description 75
- 108090000790 Enzymes Proteins 0.000 claims abstract description 75
- 238000006243 chemical reaction Methods 0.000 claims abstract description 61
- 239000000758 substrate Substances 0.000 claims abstract description 21
- 150000001413 amino acids Chemical class 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000003054 catalyst Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 35
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 239000000047 product Substances 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 19
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- YGFXLCAKCKPSQQ-UHFFFAOYSA-N 2-(1-cyanocyclohexyl)acetic acid Chemical compound OC(=O)CC1(C#N)CCCCC1 YGFXLCAKCKPSQQ-UHFFFAOYSA-N 0.000 claims description 17
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 14
- CYMZDPHCBAWUHZ-UHFFFAOYSA-N 1-(cyanomethyl)cyclohexane-1-carbonitrile Chemical compound N#CCC1(C#N)CCCCC1 CYMZDPHCBAWUHZ-UHFFFAOYSA-N 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 235000018102 proteins Nutrition 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 238000011068 loading method Methods 0.000 claims description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 6
- 235000004279 alanine Nutrition 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- 235000003704 aspartic acid Nutrition 0.000 claims description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 229930027917 kanamycin Natural products 0.000 claims description 6
- 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 6
- 229960000318 kanamycin Drugs 0.000 claims description 6
- 229930182823 kanamycin A Natural products 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000012880 LB liquid culture medium Substances 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims description 4
- 230000014509 gene expression Effects 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 239000000385 dialysis solution Substances 0.000 claims description 3
- 239000012149 elution buffer Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 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 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 1
- 238000010353 genetic engineering Methods 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 40
- 230000003197 catalytic effect Effects 0.000 abstract description 17
- 239000012429 reaction media Substances 0.000 abstract description 14
- 239000002253 acid Substances 0.000 abstract description 9
- 238000006555 catalytic reaction Methods 0.000 abstract description 6
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 241000726118 Acidovorax facilis Species 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 22
- 241000588724 Escherichia coli Species 0.000 description 20
- UGJMXCAKCUNAIE-UHFFFAOYSA-N Gabapentin Chemical compound OC(=O)CC1(CN)CCCCC1 UGJMXCAKCUNAIE-UHFFFAOYSA-N 0.000 description 16
- 239000011734 sodium Substances 0.000 description 12
- 230000000284 resting effect Effects 0.000 description 11
- 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 8
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 8
- FMQGYTMERWBMSI-HJWJTTGWSA-N Val-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N FMQGYTMERWBMSI-HJWJTTGWSA-N 0.000 description 8
- 108010005233 alanylglutamic acid Proteins 0.000 description 8
- 229960002870 gabapentin Drugs 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 6
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical class NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 5
- 238000009776 industrial production Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N methyl cyanide Natural products CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 238000002741 site-directed mutagenesis Methods 0.000 description 5
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 4
- BUANFPRKJKJSRR-ACZMJKKPSA-N Ala-Ala-Gln Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CCC(N)=O BUANFPRKJKJSRR-ACZMJKKPSA-N 0.000 description 4
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 4
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 4
- YAXNATKKPOWVCP-ZLUOBGJFSA-N Ala-Asn-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O YAXNATKKPOWVCP-ZLUOBGJFSA-N 0.000 description 4
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 4
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 4
- OSRZOHXQCUFIQG-FPMFFAJLSA-N Ala-Phe-Pro Chemical compound C([C@H](NC(=O)[C@@H]([NH3+])C)C(=O)N1[C@H](CCC1)C([O-])=O)C1=CC=CC=C1 OSRZOHXQCUFIQG-FPMFFAJLSA-N 0.000 description 4
- UCDOXFBTMLKASE-HERUPUMHSA-N Ala-Ser-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N UCDOXFBTMLKASE-HERUPUMHSA-N 0.000 description 4
- YXXPVUOMPSZURS-ZLIFDBKOSA-N Ala-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 YXXPVUOMPSZURS-ZLIFDBKOSA-N 0.000 description 4
- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 description 4
- UAOSDDXCTBIPCA-QXEWZRGKSA-N Arg-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UAOSDDXCTBIPCA-QXEWZRGKSA-N 0.000 description 4
- FNXCAFKDGBROCU-STECZYCISA-N Arg-Ile-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FNXCAFKDGBROCU-STECZYCISA-N 0.000 description 4
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 4
- YNSUUAOAFCVINY-OSUNSFLBSA-N Arg-Thr-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YNSUUAOAFCVINY-OSUNSFLBSA-N 0.000 description 4
- SUEIIIFUBHDCCS-PBCZWWQYSA-N Asn-His-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SUEIIIFUBHDCCS-PBCZWWQYSA-N 0.000 description 4
- JXMREEPBRANWBY-VEVYYDQMSA-N Asn-Thr-Arg Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JXMREEPBRANWBY-VEVYYDQMSA-N 0.000 description 4
- KVMPVNGOKHTUHZ-GCJQMDKQSA-N Asp-Ala-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KVMPVNGOKHTUHZ-GCJQMDKQSA-N 0.000 description 4
- IJHUZMGJRGNXIW-CIUDSAMLSA-N Asp-Glu-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IJHUZMGJRGNXIW-CIUDSAMLSA-N 0.000 description 4
- YNCHFVRXEQFPBY-BQBZGAKWSA-N Asp-Gly-Arg Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N YNCHFVRXEQFPBY-BQBZGAKWSA-N 0.000 description 4
- LTCKTLYKRMCFOC-KKUMJFAQSA-N Asp-Phe-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LTCKTLYKRMCFOC-KKUMJFAQSA-N 0.000 description 4
- ZKAOJVJQGVUIIU-GUBZILKMSA-N Asp-Pro-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZKAOJVJQGVUIIU-GUBZILKMSA-N 0.000 description 4
- DINOVZWPTMGSRF-QXEWZRGKSA-N Asp-Pro-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O DINOVZWPTMGSRF-QXEWZRGKSA-N 0.000 description 4
- 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 4
- SSNJZBGOMNLSLA-CIUDSAMLSA-N Cys-Leu-Asn Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O SSNJZBGOMNLSLA-CIUDSAMLSA-N 0.000 description 4
- WKKKNGNJDGATNS-QEJZJMRPSA-N Cys-Trp-Glu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKKKNGNJDGATNS-QEJZJMRPSA-N 0.000 description 4
- SHERTACNJPYHAR-ACZMJKKPSA-N Gln-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O SHERTACNJPYHAR-ACZMJKKPSA-N 0.000 description 4
- HVQCEQTUSWWFOS-WDSKDSINSA-N Gln-Gly-Cys Chemical compound C(CC(=O)N)[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N HVQCEQTUSWWFOS-WDSKDSINSA-N 0.000 description 4
- RONJIBWTGKVKFY-HTUGSXCWSA-N Gln-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O RONJIBWTGKVKFY-HTUGSXCWSA-N 0.000 description 4
- SBYVDRJAXWSXQL-AVGNSLFASA-N Glu-Asn-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SBYVDRJAXWSXQL-AVGNSLFASA-N 0.000 description 4
- RDPOETHPAQEGDP-ACZMJKKPSA-N Glu-Asp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RDPOETHPAQEGDP-ACZMJKKPSA-N 0.000 description 4
- GFLQTABMFBXRIY-GUBZILKMSA-N Glu-Gln-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GFLQTABMFBXRIY-GUBZILKMSA-N 0.000 description 4
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 4
- WKJKBELXHCTHIJ-WPRPVWTQSA-N Gly-Arg-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N WKJKBELXHCTHIJ-WPRPVWTQSA-N 0.000 description 4
- TZOVVRJYUDETQG-RCOVLWMOSA-N Gly-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CN TZOVVRJYUDETQG-RCOVLWMOSA-N 0.000 description 4
- HFXJIZNEXNIZIJ-BQBZGAKWSA-N Gly-Glu-Gln Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFXJIZNEXNIZIJ-BQBZGAKWSA-N 0.000 description 4
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 4
- YNIMVVJTPWCUJH-KBPBESRZSA-N Gly-His-Tyr Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YNIMVVJTPWCUJH-KBPBESRZSA-N 0.000 description 4
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 4
- WDXLKVQATNEAJQ-BQBZGAKWSA-N Gly-Pro-Asp Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WDXLKVQATNEAJQ-BQBZGAKWSA-N 0.000 description 4
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 description 4
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 4
- RCHFYMASWAZQQZ-ZANVPECISA-N Gly-Trp-Ala Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CN)=CNC2=C1 RCHFYMASWAZQQZ-ZANVPECISA-N 0.000 description 4
- TVRMJKNELJKNRS-GUBZILKMSA-N His-Glu-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N TVRMJKNELJKNRS-GUBZILKMSA-N 0.000 description 4
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 4
- WSXNWASHQNSMRX-GVXVVHGQSA-N His-Val-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N WSXNWASHQNSMRX-GVXVVHGQSA-N 0.000 description 4
- SYPULFZAGBBIOM-GVXVVHGQSA-N His-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N SYPULFZAGBBIOM-GVXVVHGQSA-N 0.000 description 4
- VAXBXNPRXPHGHG-BJDJZHNGSA-N Ile-Ala-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)O)N VAXBXNPRXPHGHG-BJDJZHNGSA-N 0.000 description 4
- QSPLUJGYOPZINY-ZPFDUUQYSA-N Ile-Asp-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N QSPLUJGYOPZINY-ZPFDUUQYSA-N 0.000 description 4
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 4
- MTFVYKQRLXYAQN-LAEOZQHASA-N Ile-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O MTFVYKQRLXYAQN-LAEOZQHASA-N 0.000 description 4
- 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 4
- 108010065920 Insulin Lispro Proteins 0.000 description 4
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 4
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 4
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 4
- YOZCKMXHBYKOMQ-IHRRRGAJSA-N Leu-Arg-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOZCKMXHBYKOMQ-IHRRRGAJSA-N 0.000 description 4
- BPANDPNDMJHFEV-CIUDSAMLSA-N Leu-Asp-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O BPANDPNDMJHFEV-CIUDSAMLSA-N 0.000 description 4
- QCSFMCFHVGTLFF-NHCYSSNCSA-N Leu-Asp-Val Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O QCSFMCFHVGTLFF-NHCYSSNCSA-N 0.000 description 4
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 4
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-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
- YNNPKXBBRZVIRX-IHRRRGAJSA-N Lys-Arg-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O YNNPKXBBRZVIRX-IHRRRGAJSA-N 0.000 description 4
- DRCILAJNUJKAHC-SRVKXCTJSA-N Lys-Glu-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DRCILAJNUJKAHC-SRVKXCTJSA-N 0.000 description 4
- ITWQLSZTLBKWJM-YUMQZZPRSA-N Lys-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCCN ITWQLSZTLBKWJM-YUMQZZPRSA-N 0.000 description 4
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 4
- LNMKRJJLEFASGA-BZSNNMDCSA-N Lys-Phe-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LNMKRJJLEFASGA-BZSNNMDCSA-N 0.000 description 4
- CENKQZWVYMLRAX-ULQDDVLXSA-N Lys-Phe-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(O)=O CENKQZWVYMLRAX-ULQDDVLXSA-N 0.000 description 4
- LOGFVTREOLYCPF-RHYQMDGZSA-N Lys-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-RHYQMDGZSA-N 0.000 description 4
- LBSWWNKMVPAXOI-GUBZILKMSA-N Met-Val-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O LBSWWNKMVPAXOI-GUBZILKMSA-N 0.000 description 4
- WYBVBIHNJWOLCJ-UHFFFAOYSA-N N-L-arginyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCCN=C(N)N WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0.000 description 4
- LBSARGIQACMGDF-WBAXXEDZSA-N Phe-Ala-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 LBSARGIQACMGDF-WBAXXEDZSA-N 0.000 description 4
- GDBOREPXIRKSEQ-FHWLQOOXSA-N Phe-Gln-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GDBOREPXIRKSEQ-FHWLQOOXSA-N 0.000 description 4
- CDQCFGOQNYOICK-IHRRRGAJSA-N Phe-Glu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDQCFGOQNYOICK-IHRRRGAJSA-N 0.000 description 4
- DRVIASBABBMZTF-GUBZILKMSA-N Pro-Ala-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@@H]1CCCN1 DRVIASBABBMZTF-GUBZILKMSA-N 0.000 description 4
- XUSDDSLCRPUKLP-QXEWZRGKSA-N Pro-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 XUSDDSLCRPUKLP-QXEWZRGKSA-N 0.000 description 4
- QEWBZBLXDKIQPS-STQMWFEESA-N Pro-Gly-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QEWBZBLXDKIQPS-STQMWFEESA-N 0.000 description 4
- FMLRRBDLBJLJIK-DCAQKATOSA-N Pro-Leu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FMLRRBDLBJLJIK-DCAQKATOSA-N 0.000 description 4
- GURGCNUWVSDYTP-SRVKXCTJSA-N Pro-Leu-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GURGCNUWVSDYTP-SRVKXCTJSA-N 0.000 description 4
- FKYKZHOKDOPHSA-DCAQKATOSA-N Pro-Leu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FKYKZHOKDOPHSA-DCAQKATOSA-N 0.000 description 4
- QMABBZHZMDXHKU-FKBYEOEOSA-N Pro-Tyr-Trp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QMABBZHZMDXHKU-FKBYEOEOSA-N 0.000 description 4
- DGDCSVGVWWAJRS-AVGNSLFASA-N Pro-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 DGDCSVGVWWAJRS-AVGNSLFASA-N 0.000 description 4
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 description 4
- MTMJNKFZDQEVSY-BZSNNMDCSA-N Pro-Val-Trp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O MTMJNKFZDQEVSY-BZSNNMDCSA-N 0.000 description 4
- WTWGOQRNRFHFQD-JBDRJPRFSA-N Ser-Ala-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WTWGOQRNRFHFQD-JBDRJPRFSA-N 0.000 description 4
- NRCJWSGXMAPYQX-LPEHRKFASA-N Ser-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N)C(=O)O NRCJWSGXMAPYQX-LPEHRKFASA-N 0.000 description 4
- WXUBSIDKNMFAGS-IHRRRGAJSA-N Ser-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WXUBSIDKNMFAGS-IHRRRGAJSA-N 0.000 description 4
- HBOABDXGTMMDSE-GUBZILKMSA-N Ser-Arg-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O HBOABDXGTMMDSE-GUBZILKMSA-N 0.000 description 4
- DJACUBDEDBZKLQ-KBIXCLLPSA-N Ser-Ile-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O DJACUBDEDBZKLQ-KBIXCLLPSA-N 0.000 description 4
- ZIFYDQAFEMIZII-GUBZILKMSA-N Ser-Leu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZIFYDQAFEMIZII-GUBZILKMSA-N 0.000 description 4
- HEUVHBXOVZONPU-BJDJZHNGSA-N Ser-Leu-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HEUVHBXOVZONPU-BJDJZHNGSA-N 0.000 description 4
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 4
- FBLNYDYPCLFTSP-IXOXFDKPSA-N Ser-Phe-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FBLNYDYPCLFTSP-IXOXFDKPSA-N 0.000 description 4
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 description 4
- DKDHTRVDOUZZTP-IFFSRLJSSA-N Thr-Gln-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DKDHTRVDOUZZTP-IFFSRLJSSA-N 0.000 description 4
- IGGFFPOIFHZYKC-PBCZWWQYSA-N Thr-His-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O IGGFFPOIFHZYKC-PBCZWWQYSA-N 0.000 description 4
- SPVHQURZJCUDQC-VOAKCMCISA-N Thr-Lys-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O SPVHQURZJCUDQC-VOAKCMCISA-N 0.000 description 4
- SCCKSNREWHMKOJ-SRVKXCTJSA-N Tyr-Asn-Ser Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O SCCKSNREWHMKOJ-SRVKXCTJSA-N 0.000 description 4
- GIOBXJSONRQHKQ-RYUDHWBXSA-N Tyr-Gly-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O GIOBXJSONRQHKQ-RYUDHWBXSA-N 0.000 description 4
- QFXVAFIHVWXXBJ-AVGNSLFASA-N Tyr-Ser-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O QFXVAFIHVWXXBJ-AVGNSLFASA-N 0.000 description 4
- UEOOXDLMQZBPFR-ZKWXMUAHSA-N Val-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N UEOOXDLMQZBPFR-ZKWXMUAHSA-N 0.000 description 4
- CFSSLXZJEMERJY-NRPADANISA-N Val-Gln-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CFSSLXZJEMERJY-NRPADANISA-N 0.000 description 4
- JXGWQYWDUOWQHA-DZKIICNBSA-N Val-Gln-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N JXGWQYWDUOWQHA-DZKIICNBSA-N 0.000 description 4
- XBRMBDFYOFARST-AVGNSLFASA-N Val-His-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N XBRMBDFYOFARST-AVGNSLFASA-N 0.000 description 4
- BMOFUVHDBROBSE-DCAQKATOSA-N Val-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N BMOFUVHDBROBSE-DCAQKATOSA-N 0.000 description 4
- JVGHIFMSFBZDHH-WPRPVWTQSA-N Val-Met-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)O)N JVGHIFMSFBZDHH-WPRPVWTQSA-N 0.000 description 4
- MNSSBIHFEUUXNW-RCWTZXSCSA-N Val-Thr-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N MNSSBIHFEUUXNW-RCWTZXSCSA-N 0.000 description 4
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 4
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 4
- 108010041407 alanylaspartic acid Proteins 0.000 description 4
- 108010008355 arginyl-glutamine Proteins 0.000 description 4
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 4
- 108010038850 arginyl-isoleucyl-tyrosine Proteins 0.000 description 4
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 4
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 4
- 108010047857 aspartylglycine Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 108010016616 cysteinylglycine Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 108010049041 glutamylalanine 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
- 108010010147 glycylglutamine Proteins 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 4
- 108010078274 isoleucylvaline Proteins 0.000 description 4
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 4
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 108010085203 methionylmethionine Proteins 0.000 description 4
- 108010084572 phenylalanyl-valine Proteins 0.000 description 4
- 108010051242 phenylalanylserine Proteins 0.000 description 4
- 108010070643 prolylglutamic acid Proteins 0.000 description 4
- 108010080629 tryptophan-leucine Proteins 0.000 description 4
- 108010084932 tryptophyl-proline Proteins 0.000 description 4
- 108010020532 tyrosyl-proline Proteins 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 108010073969 valyllysine Proteins 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 3
- 238000003028 enzyme activity measurement method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- -1 nitrile compounds Chemical class 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 2
- DVJSJDDYCYSMFR-ZKWXMUAHSA-N Ala-Ile-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O DVJSJDDYCYSMFR-ZKWXMUAHSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- RAQMSGVCGSJKCL-FOHZUACHSA-N Asn-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(N)=O RAQMSGVCGSJKCL-FOHZUACHSA-N 0.000 description 2
- SVABRQFIHCSNCI-FOHZUACHSA-N Asp-Gly-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SVABRQFIHCSNCI-FOHZUACHSA-N 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
- IFPBAGJBHSNYPR-ZKWXMUAHSA-N Ser-Ile-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O IFPBAGJBHSNYPR-ZKWXMUAHSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000001961 anticonvulsive agent Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000006167 equilibration buffer Substances 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- AYXYPKUFHZROOJ-SSDOTTSWSA-N pregabalin Chemical compound CC(C)C[C@@H](CN)CC(O)=O AYXYPKUFHZROOJ-SSDOTTSWSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- QAILMFURFUYAJA-UHFFFAOYSA-N 2-(6-methoxynaphthalen-2-yl)propanenitrile Chemical compound C1=C(C(C)C#N)C=CC2=CC(OC)=CC=C21 QAILMFURFUYAJA-UHFFFAOYSA-N 0.000 description 1
- IFASJKOGQCARIN-UHFFFAOYSA-N 3-(2-methylpropyl)pentanedinitrile Chemical compound CC(C)CC(CC#N)CC#N IFASJKOGQCARIN-UHFFFAOYSA-N 0.000 description 1
- GZPHSAQLYPIAIN-UHFFFAOYSA-N 3-pyridinecarbonitrile Chemical compound N#CC1=CC=CN=C1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 1
- CCVSLUOUOXLAKA-QMMMGPOBSA-N C(C(C)C)[C@@H](CC(=O)O)CC#N Chemical compound C(C(C)C)[C@@H](CC(=O)O)CC#N CCVSLUOUOXLAKA-QMMMGPOBSA-N 0.000 description 1
- 241000316922 Caldicoprobacter faecalis Species 0.000 description 1
- 102000016917 Complement C1 Human genes 0.000 description 1
- 108010028774 Complement C1 Proteins 0.000 description 1
- 108050009160 DNA polymerase 1 Proteins 0.000 description 1
- 208000001654 Drug Resistant Epilepsy Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 238000007167 Hofmann rearrangement reaction Methods 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 208000008238 Muscle Spasticity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009529 body temperature measurement Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 108010011035 endodeoxyribonuclease DpnI Proteins 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- CMWTZPSULFXXJA-VIFPVBQESA-N naproxen Chemical compound C1=C([C@H](C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-VIFPVBQESA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 229960001233 pregabalin Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- FVSKHRXBFJPNKK-UHFFFAOYSA-N propionitrile Chemical compound CCC#N FVSKHRXBFJPNKK-UHFFFAOYSA-N 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 1
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
- 208000018198 spasticity Diseases 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- 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
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
- C12Y305/05001—Nitrilase (3.5.5.1)
Abstract
本发明公开一种产物耐受型腈水解酶突变体及其应用,所述突变体是将SEQ ID No.2所示氨基酸序列的第29位,第145位氨基酸中的一位或多位进行突变获得的。腈水解酶突变体AcN‑S29A/N145D的比酶活提高了1.36倍,最适催化温度从50℃提高到了55℃,且55℃下半衰期达到了37.3h,较原来提高了1.71倍;同时产物耐受性及耐酸性显著提高,可减小高产物浓度和产物生成导致反应体系pH下降的影响,能直接以水作为反应介质进行催化反应,底物转化率显著提高,10g/L湿细胞催化剂在35℃下水解1M底物7h可完全转化,最终转化率达到99%以上,具有更好的工业应用前景。
Description
(一)技术领域
本发明属于生物技术领域,涉及一种来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044的腈水解酶突变体,以及其在加巴喷丁中间体1-氰基环己基乙酸合成中的应用。
(二)背景技术
腈水解酶(nitrilase;EC 3.5.5.1)是腈水解酶超家族中一种重要的生物催化剂,能将腈类化合物水解成氨和相应的羧酸,按照催化底物性质的不同,腈水解酶可以大致分为脂肪腈水解酶、芳香腈水解酶和芳基乙腈水解酶三类。自20世纪60年代Thiman和Mahadevan首次在大麦叶中发现腈水解酶以来,更多不同来源的腈水解酶被发现,包括细菌、真菌和一些植物均能提取出腈水解酶。腈水解酶的催化过程效率高,反应条件温和且绿色安全,因此在医药、环保、化工等领域获得广泛应用。由于腈水解酶具有高度的化学、区域及立体选择性等特点,因此在医药等精细化学品的生产中展现了极大的应用潜力,例如:德国巴斯夫建成投产了腈水解酶合成扁桃酸的工艺,最终提取的产品化学纯度可达99%;Franz等采用分枝杆菌来源腈水解酶水解2-(6-甲氧基-2-萘基)-丙腈得到(S)-萘普生,ee值高达99%;徐美珍等以筛选获得的赤霉菌(G.intermedia)WX12为催化剂,以3-异丁基戊二腈为底物进行转化所得产物(R)-3-氰基甲基-5-甲基己酸,经霍夫曼重排反应可制备获得(R)-普瑞巴林,ee值大于99%等。
加巴喷丁是γ-氨基丁酸(GABA)的衍生物,由美国Warner-Lamber公司首先开发为抗癫痫药,研究表明其通过改变γ-氨基丁酸代谢实现药理效果。加巴喷丁除了预防癫痫外,在治疗痉挛、镇痛方面也显示出了一定作用。与其它在售抗癫痫药相比,其具有易口服吸收,不易耐受,心血管副作用小,疗效好,在体内不参与代谢等特点,可以作为追加治疗难治性癫痫的特效药。1-氰基环己基乙酸是合成加巴喷丁的重要前体,在合成路线上处于关键位置。目前,合成加巴喷丁及其重要前体1-氰基环己基乙酸基本采用化学合成技术,这些技术存在反应条件严苛、污染严重、设备要求高等缺陷。Zheng等人开发了一系列利用敏捷食酸菌(Acidovorax facilis)来源的腈水解酶生物催化合成1-氰基环己基乙酸及加巴喷丁的方法(专利号CN108486088B、CN104212784A、CN107235850B),相比而言,能更好地解决化学合成法的缺陷,满足绿色可持续工业生产的要求。
腈水解酶能够用于催化加巴喷丁重要前体1-氰基环己基乙酸。但是,天然的腈水解酶往往存在稳定性差、催化活力不高、生成副产物等缺陷,难以满足工业生产的复杂要求。蛋白质工程是解决上述问题常见且十分有效的方式,通过各种设计实施定点突变或者利用易错PCR、饱和突变再结合高通量筛选实施定向进化等分子改造手段重构蛋白结构可显著提高腈水解酶的活性、稳定性及催化专一性等性能。Gong等通过定点突变构建了双突变体I128L/N161用于催化3-氰基吡啶,催化活力提高了1.98倍,产物中羧酸含量提高至99.1%。Schreiner等利用易错PCR方法获取了一个A.faecalis JM3腈水解酶优良的突变株,其在pH 7.5的条件下对苯丙腈比酶活提高了8倍,序列分析显示该突变体共包含7处突变。Zhang等通过定点饱和突变,获得了高活力和高立体选择性的腈水解酶突变体BaNIT/L223Q/H263D/Q279E,合成普瑞巴林的底物转化率可达46%,产物光学纯度大于99%。
在之前的研究中,来自敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044克隆的腈水解酶已经在大肠杆菌(Escherichia coli)BL21(DE3)中成功重组表达,并实现了催化1-氰基环己基乙腈专一生成1-氰基环己基乙酸。在此背景下,本发明通过定点改造技术,进一步提高该酶的稳定性和催化活力,使其能更高效地实现1-氰基环己基乙腈转化,满足工业生产的复杂要求。
(三)发明内容
本发明目的是提供一种产物耐受型腈水解酶突变体、编码基因、重组载体、工程菌以及在催化1-氰基环己基乙腈合成加巴喷丁中间体1-氰基环己基乙酸中的应用,所述的腈水解酶突变体稳定性、产物耐受性和耐酸性均有显著提升,且具有更高的酶活,1-氰基环己基乙酸合成效率明显提高。产物耐受性和耐酸性提高使其能耐受高产物浓度和产物生成导致的pH下降,可直接以水作为反应介质,更能适应工业生产环境。
本发明采用的技术方案是:
本发明提供一种源自敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 20904的产物耐受型腈水解酶突变体,所述腈水解酶突变体是将SEQ ID NO.2所示氨基酸序列的第29位或第145位氨基酸中的一位或多位进行突变获得的。
进一步,优选所述腈水解酶突变体是将SEQ ID NO.2所示氨基酸序列进行下列之一突变:(1)第29位丝氨酸突变为丙氨酸(S29A),氨基酸序列为SEQ ID NO.4所示,编码基因核苷酸序列为SEQ ID NO.3所示;(2)第145位天冬酰胺突变为天冬氨酸(N145D),氨基酸序列为SEQ ID NO.6所示,编码基因核苷酸序列为SEQ ID NO.5所示;(3)第29位的丝氨酸突变为丙氨酸,同时将第145位天冬酰胺突变为天冬氨酸,氨基酸序列为SEQ ID NO.8所示,编码基因核苷酸序列为SEQ ID NO.7所示。
本发明同时提供了一种提高氨基酸序列为SEQ ID NO.2的腈水解酶活性的方法,该方法是通过在SEQ ID NO.8的氨基酸序列第29位的丝氨酸突变为丙氨酸,同时将第145位天冬酰胺突变为天冬氨酸以改变SEQ ID NO.2所示腈水解酶的氨基酸序列。
本发明同时提供了一种提高氨基酸序列为SEQ ID NO.2的腈水解酶产物耐受性和酸耐受性的方法,该方法是通过在SEQ ID NO.8的氨基酸序列第29位的丝氨酸突变为丙氨酸,同时将第145位天冬酰胺突变为天冬氨酸以改变SEQ ID NO.2所示腈水解酶的氨基酸序列。
本发明又提供了所述腈水解酶突变体的编码基因,含所述编码基因的重组载体以及由所述编码基因构建的工程菌。所述的载体包括但不限于原核表达载体(pET28b、pGEX4T1、pTrC99A)、真核表达载体(pPIC9K、pYD1)、克隆载体pUC18/19,本发明优选原核表达载体pET-28b(+)。所述的工程菌包括但不限于本领域的各种常规工程菌(E.coli BL21(DE3)、E.coli Rosetta-gami(DE3)),本发明优选大肠杆菌E.coli BL21(DE3)。
本发明还提供了一种所述腈水解酶突变体在催化1-氰基环己基乙腈制备1-氰基环己基乙酸中的应用,具体所述的应用包括:以包含腈水解酶突变体编码基因的工程菌经诱导表达后离心获得的湿菌体(静息细胞)、湿菌体超声破碎后提取的粗酶或者粗酶提取纯化的纯酶为催化剂,以1-氰基环己基乙腈为底物,以pH为5.0-10.0的缓冲液或超纯水(pH自然)为反应介质构成反应体系,在20-55℃、200-400rpm条件下恒温水浴反应,反应完全后,将反应液分离纯化,获得1-氰基环己基乙酸。作为优选,所述反应体系中底物终浓度为0.5-1M(优选1M),催化剂用量以提取前的湿菌体重量计为10-50g/L(优选10g/L),反应介质为pH值为7.5、浓度为200mM的Na2HPO4-NaH2PO4缓冲液,催化反应条件优选为35℃、150rpm。
本发明所述的具有生物活性的腈水解酶突变体可以工程菌湿菌体形式使用,也可以是粗酶液形式使用,也可以是经过分离纯化的纯酶液形式使用。同时还可以利用本领域已知的固定化方法将本发明所述的腈水解酶突变体制成固定化细胞或者固定化酶使用。
本发明所述湿菌体按如下方法制备:将含腈水解酶突变体编码基因的工程菌接种到含终浓度50μg/mL卡那霉素的LB液体培养基中,于37℃培养8-10h,培养液按体积浓度2%的接种量接种至含终浓度50μg/mL卡那霉素的新鲜LB液体培养基中,37℃培养至菌体浓度OD600为0.6-0.8之间,加入终浓度为0.1mM的IPTG,28℃、180rpm诱导表达10-12h,4℃、8000rpm离心10min,收集菌体,用生理盐水清洗后,得到湿菌体。LB液体培养基成分为:酵母粉5g/L,蛋白胨10g/L,NaCl 10g/L,溶剂为水,pH自然。
所述粗酶或纯酶按如下步骤制备:
(1)粗酶液:将含腈水解酶突变体编码基因的工程菌经诱导培养获得的湿菌体用200mM Na2HPO4-NaH2PO4缓冲液(pH 7)重悬成菌悬液,超声波破碎(40W,30min,1s破碎,1s暂停),破碎混合液离心(4℃,12000rpm,10min)后,取上清液作为粗酶液;
(2)纯酶:将Ni-NTA柱(MC/20,16mmD×100mmL,美国Applied Biosystems公司)用平衡缓冲液冲洗后,将步骤(1)粗酶液以1mL/min的流速上样,上样量为2个柱体积,上样结束后用平衡缓冲液洗脱弱吸附的杂蛋白,流速为2mL/min,洗脱5个柱体积;再用洗脱缓冲液洗脱3-5个柱体积,根据检测器信号(190nm~700nm全波长扫描吸光度>0.2)收集目的蛋白,流速为1mL/min;最后将收集的目的蛋白分装透析袋(截留分子量为1000D),以50mMNa2HPO4-NaH2PO4(pH 7)缓冲液为透析液室温透析10h,取截留液即为纯酶液;平衡缓冲液为含终浓度300mM NaCl+50mM咪唑的pH8、50mM Na2HPO4-NaH2PO4。洗脱缓冲液为含终浓度300mM NaCl+500mM咪唑的pH8、50mM Na2HPO4-NaH2PO4。
与现有技术相比,本发明的有益效果主要体现在:本发明经过定点突变对蛋白质进行分子改造,使得腈水解酶突变体的稳定性、产物耐受性和耐酸性均有显著提升,而产物耐受性和耐酸性提高使其能耐受高产物浓度和产物生成导致的pH下降,可直接以水作为反应介质,更能适应工业生产环境,具有更高的酶活,1-氰基环己基乙酸合成效率明显提高,催化效率显著提高,具有更好的工业应用前景。其中,腈水解酶突变体AcN-S29A/N145D的比酶活提高了1.36倍,最适催化温度从50℃提高到了55℃,且55℃下半衰期达到了37.3h,较原来提高了1.71倍。利用10g/L含有腈水解酶突变体的重组大肠杆菌静息细胞在35℃下水解1M底物1-氰基环己基乙腈7h可完全转化,最终转化率达到99%以上,而原始酶需要9h。在极高底物浓度(2M)下,10g/L前述湿细胞在35℃下水解2M底物20h可完全转化,最终转化率达到95%以上,而原始酶在相同条件下的转化率仅为63%左右。
(四)附图说明
图1为腈水解酶突变体粗酶液的SDS-PAGE电泳图。泳道1为蛋白MAKER,泳道2为AcN-M,泳道3为AcN-S29A,泳道4为AcN-N145D,泳道5为AcN-S29A/N145D。
图2为腈水解酶突变体在55℃下的失活曲线示意图。
图3为组合突变体AcN-S29A/N145D的最适反应温度示意图。
图4为组合突变体AcN-S29A/N145D的最适反应pH示意图。
图5为含有腈水解酶突变体的重组大肠杆菌静息细胞转化1M 1-氰基环己基乙腈反应进程图,其反应介质分别为超纯水和pH值为7.5,浓度为200mM的Na2HPO4-NaH2PO4缓冲液。
(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:
实施例1:定点突变
以含有克隆于敏捷食酸菌(A.facilis)CCTCC NO:M 209044腈水解酶基因AcN-T201F/Q339K/Q343K(简称AcN-M,核苷酸序列SEQ ID NO.1所示,编码蛋白氨基酸序列为SEQID No.2所示,已在专利申请ZL201710325569.9中公开)的原始重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-M的pET-28b(+)-AcN-M质粒为模版,再进行全质粒定点突变(表1)PCR扩增。
SEQ ID NO.1:
SEQ ID NO.2:
MVSYNSKFLAATVQAEPVWLDADATIDKSIGIIEEAAQKGASLIAFPEVFIPGYPYWAWLGDVKYSLSFTSRYHENSLELGDDRMRRLQLAARRNKIALVMGYSEREAGSRYLSQVFIDERGEIVANRRKLKPTHVERTIYGEGNGTDFLTHDFAFGRVGGLNCWEHVQPLSKFMMYSLGEQVHVASWPAMSPLQPDVFQFSIEANATVTRSYAIEGQTFVLCSTQVIGPSAIETFCLNDEQRALLPQGCGWARIYGPDGSELAKPLAEDAEGILYAEIDLEQILLAKAGADPVGHYSRPDVLSVQFDPRNHTPVHRIGIDGRLDVNTRSRVENFRLRKAAEKERQASKRLGTKLFEQSLLAEEPVPAKLEHHHHHH。
PCR反应体系(50μL):模版DNA0.5~20ng,2×Phanta max Buffer 25μL,10mMdNTP 1μL,上下游引物各1μL,Phanta Max Super-Fidelity DNA Polymerase 1μL,加无菌水补至50μL。
PCR反应条件:(1)95℃预变性5min;(2)95℃变性15s;(3)60℃退火15s;(4)72℃延伸5.5min,步骤(2)~(4)共30个循环;(5)最后72℃延伸10min,16℃保存。
PCR产物经过琼脂糖凝胶电泳验证为阳性后,加入2μL内切酶DpnI消化去除模板质粒,之后42℃热击90s转化到E.coli BL21(DE3)感受态细胞中。感受态细胞于37℃复苏1h后涂布至含有50μg/mL卡那霉素的LB平板,37℃培养1d,挑取单克隆至LB液体培养基,37℃培养8-10h,取菌液进行测序,测序结果正确者即为含腈水解酶突变体AcN-S29A(其对应的核苷酸序列如SEQ ID NO.3所示,对应的氨基酸序列如SEQ ID NO.4所示)的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-S29A,含腈水解酶突变体AcN-N145D(其对应的核苷酸序列如SEQ ID NO.5所示,对应的氨基酸序列如SEQ ID NO.6所示)的重组大肠杆菌E.coliBL21(DE3)/pET28b(+)-AcN-N145D。
表1突变位点的引物设计
以突变体S29A(核苷酸序列SEQ ID NO.3)的pET-28b(+)-AcN-S29A质粒为模板,利用同样的方法得到双突变体,即获得含组合突变体AcN-S29A/N145D(其对应的核苷酸序列如SEQ ID NO.7所示,对应的氨基酸序列如SEQ ID NO.8所示)的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-S29A/N145D。
实施例2:腈水解酶的诱导表达
将实施例1中得到的含腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-S29A、E.coli BL21(DE3)/pET28b(+)-AcN-N145D、E.coli BL21(DE3)/pET28b(+)-AcN-S29A/N145D,以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M接种到LB液体培养基中,37℃培养8-10h,培养液以体积浓度2%接种量接种至100mL含有卡那霉素(终浓度50mg/L)的LB新鲜培养基中,37℃培养至菌体浓度OD600为0.6-0.8之间,加入IPTG(终浓度0.1mM)诱导,28℃、180rpm发酵培养10-12h。培养液离心(4℃、8000r/min)10min收集菌体,用生理盐水清洗2次,得到相应的湿菌体(静息细胞)。
实施例3:腈水解酶的纯化
(1)粗酶液:收集实施例2中的湿菌体用200mM Na2HPO4-NaH2PO4缓冲液(pH 7)重悬成30g/L的菌悬液,超声波破碎(40W,30min,1s破碎,1s暂停),破碎混合液离心(4℃,12000rpm,10min)后,取上清液作为粗酶液。通过SDS-PAGE电泳分析粗酶液,检测腈水解酶是否成功表达,结果见图1。图1表明所有腈水解酶突变体均已成功表达。
(2)纯酶:将Ni-NTA柱(MC/20,16mmD×100mmL,美国Applied Biosystems公司)用平衡缓冲液冲洗后,将步骤(1)粗酶液以1mL/min的流速上样,上样量为2个柱体积,上样结束后用平衡缓冲液洗脱弱吸附的杂蛋白,流速为2mL/min,洗脱5个柱体积;再用洗脱缓冲液洗脱5个柱体积,根据检测器信号(190nm~700nm全波长扫描吸光度>0.2)收集目的蛋白,流速为1mL/min。最后将收集的目的蛋白分装透析袋(截留分子量为1000D),以50mMNa2HPO4-NaH2PO4(pH 7)缓冲液为透析液室温透析10h,取截留液即为纯酶液,分别获得原始纯酶AcN-M,突变体纯酶AcN-S29A、突变体纯酶AcN-N145D、突变体纯酶AcN-S29A/N145D,用BCA试剂盒检测蛋白浓度,分别为3.8mg/mL、4.2mg/mL、3.5mg/mL、4.6mg/mL、4.0mg/mL。
平衡缓冲液为含终浓度300mM NaCl+50mM咪唑的pH8、50mM Na2HPO4-NaH2PO4。洗脱缓冲液为含终浓度300mM NaCl+500mM咪唑的pH8、50mM Na2HPO4-NaH2PO4。
实施例4:腈水解酶及其突变体的酶活
对实施例3方法制备的腈水解酶纯酶液进行酶活的测定:
反应体系:50μL纯酶液、终浓度200mM 1-氰基环己基乙腈,50mM Na2HPO4-NaH2PO4缓冲液(pH=7.0)作为反应介质补足至1mL,具体操作如下:
(1)取50μL纯酶液置于2mL EP管内,并用50mM Na2HPO4-NaH2PO4缓冲液(pH=7.0)作为反应介质补足至1mL,反应液于35℃金属浴(600rpm)预热10min,使其达到反应温度35℃。
(2)向步骤(1)EP管内加入终浓度200mM 1-氰基环己基乙腈,35℃金属浴(600rpm)反应10min,加入5μL 6M的HCl终止反应。
(3)步骤(2)反应液离心(12000rpm、1min),取上清稀释至合适浓度,利用液相色谱(Agilent)外标法测定转化液1-氰基环己基乙酸转化率。
液相色谱柱为J&K Scientific C18-H柱(4.6×250mm,5μm,),流动相为缓冲液(0.58g/L磷酸氢二铵,1.83g/L高氯酸钠,高氯酸调节pH为1.8,溶剂为超纯水):乙腈=76:24(v:v),流速为1mL/min,紫外检测波长215nm,柱温40℃。各个突变体的比酶活结果见表2。
酶活定义(U):在酶活测定条件下,每分钟催化生成1μmol1-氰基环己基乙酸所需要的酶量定义为一个活力单位,比酶活(U/mg protein)定义为:1mg纯酶含有的酶活单位。
表2腈水解酶突变体比酶活比较
实施例5:腈水解酶及其突变体的热稳定性
取5mL实施例3制备的腈水解酶于10mL无菌聚丙烯离心管,保存于55℃恒温水浴锅中,每隔10h取出蛋白,按实施例4步骤(3)的方法,对纯酶的残余酶活进行活力检测。以未水浴保温的纯酶初始活力(Ai)为对照(设AcN-M初始酶活为100%),计算腈水解酶及其突变体在55℃下保温不同时间后的相对残余活力(Ar),结果见图2,所有突变体酶的热稳定性相较于原始酶均有提高,原始酶在保温40h后相对残余活力便降至20%左右,而突变体酶AcN-S29A/N145D、AcN-N145D在80h左右才下降至该水平。
以Ln(Ar/Ai)为纵坐标、保温时间为横坐标作图,进行线性拟合,根据一级失活动力学公式-kd(失活常数)t=Ln(Ar/Ai),可以得到酶蛋白的半衰期t1/2=Ln(2)/kd,结果见表3,组合突变体的半衰期达到了37.3h,是原始酶的1.71倍。
表3腈水解酶突变体在55℃下半衰期
实施例6:腈水解酶及其突变体的产物耐受性和耐酸性
1、产物耐受性:
酶活测定反应体系:纯酶液50μL,终浓度2M的产物1-氰基环己基乙酸,pH 7的磷酸缓冲液作为反应介质补足至1mL。
将实施例3方法制备的腈水解酶纯酶液按照实施例4的方法进行酶活测定,以不含产物1-氰基环己基乙酸的pH 7缓冲液下腈水解酶的活力为对照,计算在含有终浓度2M产物条件下蛋白的相对活力,结果见表4。
2、耐酸性
酶活测定反应体系:纯酶液50μL,pH 4、5、7的磷酸盐缓冲液作为反应介质补足至1mL,构成反应体系。将实施例3方法制备的腈水解酶纯酶液按照实施例4的方法进行酶活测定,以中性pH条件下(pH 7)腈水解酶的活力为对照,计算在酸性pH条件下(pH 4、5)蛋白的相对活力,结果见表4。
突变体AcN-S29A、ACN-N145D、组合突变体AcN-S29A/N145D在低pH下的活力均好于AcN-M,而组合突变体AcN-S29A/N145D具有最优秀的表现;同时,在反应体系中含有2M产物的条件下,原始酶活力受到明显的抑制,仅为正常活力的10%左右,而所有突变体几乎都能保持一半以上的酶活。综合比较各项性能,组合突变体AcN-S29A/N145D均为最佳。
表4腈水解酶突变体产物耐受性及耐酸性比较(每次实验设置3个平行)
实施例7:腈水解酶突变体的最适反应温度与最适反应pH
1、最适反应温度
将实施例3方法制备的腈水解酶AcN-S29A/N145D纯酶液按照实施例4的方法在不同的温度下(25-65℃,以5℃为一梯度)进行活力测定,其他相同。设原始酶AcN-M最适反应温度下的活力为100%,计算各温度下的相对活力,结果见图3,组合突变体的最适反应温度为55℃,较原始酶提高了5℃。
2、最适pH
将实施例3方法制备的腈水解酶AcN-S29A/N145D纯酶液按照实施例4的方法以不同pH的50mM磷酸盐缓冲液(pH 4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10)作为反应介质进行活力测定,其他相同。设原始酶AcN-M最适反应pH下的活力为100%,计算各pH下的相对活力,结果见图4,组合突变体及原始酶的最适反应pH均为7.5,但组合突变体在酸性环境下的残余活力显著提高。
实施例8:含腈水解酶突变体的重组大肠杆菌最适催化温度
将实施例2制备的含腈水解酶突变体AcN-S29A/N145D的重组大肠杆菌E.coliBL21(DE3)/pET28b(+)-AcN-S29A/N145D静息细胞进行最适催化温度的测定。
反应体系如下:静息细胞10g/L,底物1-氰基环己基乙腈1M,20mL的200mM磷酸盐缓冲液(pH 7.5)为反应介质构成反应体系。将反应瓶分别置于不同温度(25、35、45、55℃)的水浴摇床中(150rpm)进行催化反应,反应8h后加入100μL 6M HCl终止反应,反应液离心(12000rpm、1min)取上清,稀释至合适浓度后,同实施例4所述的HPLC分析底物转化率。根据不同温度下转化率的高低,判断最适催化温度,结果见表5。35℃下的反应过程拥有最高转化率,该温度为最适催化温度。
表5各催化温度下的底物转化率
实施例9:含腈水解酶突变体的重组大肠杆菌催化底物1-氰基环己基乙腈
将实施例2制备的含腈水解酶AcN-M的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-M及其突变体AcN-S29A/N145D的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-S29A/N145D静息细胞作为催化剂。
反应体系如下:静息细胞10g/L,底物1-氰基环己基乙腈1M或2M,20mL200mM磷酸盐缓冲液(pH 7.5)或者超纯水(pH自然)为反应介质构成反应体系。将反应瓶置于35℃的水浴摇床中(150rpm)进行催化反应。间隔1h,取样200μL反应液加入5μL 6M HCl终止反应,反应液离心(12000rpm、1min)取上清,稀释至合适浓度后,同实施例4所述的HPLC分析底物转化率,催化反应进程见图5。
在极高底物浓度(2M)下,使用10g/L静息细胞(突变体AcN-S29A/N145D)在上述条件下水解底物20h可完全转化,最终转化率达到95%以上,而原始酶在相同条件下的转化率仅为63%左右。
结果表明,利用10g/L静息细胞(突变体AcN-S29A/N145D)在35℃下水解1M底物,7h可完全转化,最终转化率达到99%以上,而未改造菌株需要9h。静息细胞催化的时空产率达到了576g/L/day,催化剂产率为16.8(g产物/g催化剂)。而以水为反应介质也能在8h内实现完全转化,原始菌株在8h的转化率却不足90%,静息细胞催化高浓度底物能力更强,催化过程受pH变化影响较小。
序列表
<110> 浙江工业大学
<120> 产物耐受型腈水解酶突变体及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1134
<212> DNA
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 1
atggtatctt acaactccaa atttctggct gctaccgtac aggctgaacc ggtttggctg 60
gacgcggacg caactatcga taaatctatt ggtatcatcg aggaggcggc ccagaaaggt 120
gcgtctctga ttgccttccc ggaagttttc atccctggtt acccgtattg ggcctggctg 180
ggtgacgtaa agtactccct gtccttcacc tcccgttacc acgaaaactc cctggaactg 240
ggtgacgacc gtatgcgccg tctgcaactg gctgcgcgtc gtaacaaaat cgcgctggtt 300
atgggttaca gcgagcgtga ggcaggcagc cgctacctgt cccaggtctt tatcgacgaa 360
cgtggtgaaa tcgttgctaa ccgtcgtaaa ctgaaaccaa ctcacgttga acgtacgatt 420
tatggtgaag gcaacggtac cgactttctg acgcatgact tcgcatttgg tcgtgttggt 480
ggtctgaact gctgggagca cgttcagccg ctgtccaaat tcatgatgta ctccctgggt 540
gaacaggtac acgtcgcttc ttggccggct atgtccccgc tgcaaccgga cgtgtttcaa 600
ttttccatcg aggctaatgc gaccgtaacc cgctcctatg ctattgaagg ccaaaccttc 660
gttctgtgct ctacgcaggt tatcggtccg tctgcaattg aaaccttctg tctgaacgat 720
gagcaacgtg cactgctgcc gcagggttgc ggttgggcgc gtatctacgg cccggacggc 780
agcgaactgg ccaagccgct ggctgaagac gcagagggta ttctgtacgc agaaatcgat 840
ctggaacaga ttctgctggc caaggctggc gctgatccgg ttggtcacta cagccgccct 900
gatgtcctgt ccgtgcagtt cgacccgcgt aaccacaccc cggtacaccg cattggtatc 960
gatggccgtc tggatgttaa cacgcgttcc cgtgtagaaa actttcgcct gcgtaaagca 1020
gcagaaaaag aacgtcaggc cagcaaacgt ctgggcacga aactgtttga acagtctctg 1080
ctggcggagg agccggtacc agccaaactc gagcaccacc accaccacca ctga 1134
<210> 2
<211> 377
<212> PRT
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 2
Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu
1 5 10 15
Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile
20 25 30
Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu
35 40 45
Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys
50 55 60
Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu
65 70 75 80
Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys
85 90 95
Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr
100 105 110
Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg
115 120 125
Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly
130 135 140
Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly
145 150 155 160
Gly Leu Asn Cys Trp Glu His Val Gln Pro Leu Ser Lys Phe Met Met
165 170 175
Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser
180 185 190
Pro Leu Gln Pro Asp Val Phe Gln Phe Ser Ile Glu Ala Asn Ala Thr
195 200 205
Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser
210 215 220
Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp
225 230 235 240
Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr
245 250 255
Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu
260 265 270
Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys
275 280 285
Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser
290 295 300
Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile
305 310 315 320
Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg
325 330 335
Leu Arg Lys Ala Ala Glu Lys Glu Arg Gln Ala Ser Lys Arg Leu Gly
340 345 350
Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala
355 360 365
Lys Leu Glu His His His His His His
370 375
<210> 3
<211> 1134
<212> DNA
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 3
atggtatctt acaactccaa atttctggct gctaccgtac aggctgaacc ggtttggctg 60
gacgcggacg caactatcga taaagccatt ggtatcatcg aggaggcggc ccagaaaggt 120
gcgtctctga ttgccttccc ggaagttttc atccctggtt acccgtattg ggcctggctg 180
ggtgacgtaa agtactccct gtccttcacc tcccgttacc acgaaaactc cctggaactg 240
ggtgacgacc gtatgcgccg tctgcaactg gctgcgcgtc gtaacaaaat cgcgctggtt 300
atgggttaca gcgagcgtga ggcaggcagc cgctacctgt cccaggtctt tatcgacgaa 360
cgtggtgaaa tcgttgctaa ccgtcgtaaa ctgaaaccaa ctcacgttga acgtacgatt 420
tatggtgaag gcaacggtac cgactttctg acgcatgact tcgcatttgg tcgtgttggt 480
ggtctgaact gctgggagca cgttcagccg ctgtccaaat tcatgatgta ctccctgggt 540
gaacaggtac acgtcgcttc ttggccggct atgtccccgc tgcaaccgga cgtgtttcaa 600
ttttccatcg aggctaatgc gaccgtaacc cgctcctatg ctattgaagg ccaaaccttc 660
gttctgtgct ctacgcaggt tatcggtccg tctgcaattg aaaccttctg tctgaacgat 720
gagcaacgtg cactgctgcc gcagggttgc ggttgggcgc gtatctacgg cccggacggc 780
agcgaactgg ccaagccgct ggctgaagac gcagagggta ttctgtacgc agaaatcgat 840
ctggaacaga ttctgctggc caaggctggc gctgatccgg ttggtcacta cagccgccct 900
gatgtcctgt ccgtgcagtt cgacccgcgt aaccacaccc cggtacaccg cattggtatc 960
gatggccgtc tggatgttaa cacgcgttcc cgtgtagaaa actttcgcct gcgtaaagca 1020
gcagaaaaag aacgtcaggc cagcaaacgt ctgggcacga aactgtttga acagtctctg 1080
ctggcggagg agccggtacc agccaaactc gagcaccacc accaccacca ctga 1134
<210> 4
<211> 377
<212> PRT
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 4
Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu
1 5 10 15
Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ala Ile Gly Ile
20 25 30
Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu
35 40 45
Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys
50 55 60
Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu
65 70 75 80
Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys
85 90 95
Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr
100 105 110
Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg
115 120 125
Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly
130 135 140
Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly
145 150 155 160
Gly Leu Asn Cys Trp Glu His Val Gln Pro Leu Ser Lys Phe Met Met
165 170 175
Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser
180 185 190
Pro Leu Gln Pro Asp Val Phe Gln Phe Ser Ile Glu Ala Asn Ala Thr
195 200 205
Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser
210 215 220
Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp
225 230 235 240
Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr
245 250 255
Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu
260 265 270
Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys
275 280 285
Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser
290 295 300
Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile
305 310 315 320
Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg
325 330 335
Leu Arg Lys Ala Ala Glu Lys Glu Arg Gln Ala Ser Lys Arg Leu Gly
340 345 350
Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala
355 360 365
Lys Leu Glu His His His His His His
370 375
<210> 5
<211> 1134
<212> DNA
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 5
atggtatctt acaactccaa atttctggct gctaccgtac aggctgaacc ggtttggctg 60
gacgcggacg caactatcga taaatctatt ggtatcatcg aggaggcggc ccagaaaggt 120
gcgtctctga ttgccttccc ggaagttttc atccctggtt acccgtattg ggcctggctg 180
ggtgacgtaa agtactccct gtccttcacc tcccgttacc acgaaaactc cctggaactg 240
ggtgacgacc gtatgcgccg tctgcaactg gctgcgcgtc gtaacaaaat cgcgctggtt 300
atgggttaca gcgagcgtga ggcaggcagc cgctacctgt cccaggtctt tatcgacgaa 360
cgtggtgaaa tcgttgctaa ccgtcgtaaa ctgaaaccaa ctcacgttga acgtacgatt 420
tatggtgaag gcgacggtac cgactttctg acgcatgact tcgcatttgg tcgtgttggt 480
ggtctgaact gctgggagca cgttcagccg ctgtccaaat tcatgatgta ctccctgggt 540
gaacaggtac acgtcgcttc ttggccggct atgtccccgc tgcaaccgga cgtgtttcaa 600
ttttccatcg aggctaatgc gaccgtaacc cgctcctatg ctattgaagg ccaaaccttc 660
gttctgtgct ctacgcaggt tatcggtccg tctgcaattg aaaccttctg tctgaacgat 720
gagcaacgtg cactgctgcc gcagggttgc ggttgggcgc gtatctacgg cccggacggc 780
agcgaactgg ccaagccgct ggctgaagac gcagagggta ttctgtacgc agaaatcgat 840
ctggaacaga ttctgctggc caaggctggc gctgatccgg ttggtcacta cagccgccct 900
gatgtcctgt ccgtgcagtt cgacccgcgt aaccacaccc cggtacaccg cattggtatc 960
gatggccgtc tggatgttaa cacgcgttcc cgtgtagaaa actttcgcct gcgtaaagca 1020
gcagaaaaag aacgtcaggc cagcaaacgt ctgggcacga aactgtttga acagtctctg 1080
ctggcggagg agccggtacc agccaaactc gagcaccacc accaccacca ctga 1134
<210> 6
<211> 377
<212> PRT
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 6
Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu
1 5 10 15
Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile
20 25 30
Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu
35 40 45
Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys
50 55 60
Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu
65 70 75 80
Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys
85 90 95
Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr
100 105 110
Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg
115 120 125
Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly
130 135 140
Asp Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly
145 150 155 160
Gly Leu Asn Cys Trp Glu His Val Gln Pro Leu Ser Lys Phe Met Met
165 170 175
Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser
180 185 190
Pro Leu Gln Pro Asp Val Phe Gln Phe Ser Ile Glu Ala Asn Ala Thr
195 200 205
Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser
210 215 220
Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp
225 230 235 240
Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr
245 250 255
Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu
260 265 270
Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys
275 280 285
Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser
290 295 300
Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile
305 310 315 320
Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg
325 330 335
Leu Arg Lys Ala Ala Glu Lys Glu Arg Gln Ala Ser Lys Arg Leu Gly
340 345 350
Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala
355 360 365
Lys Leu Glu His His His His His His
370 375
<210> 7
<211> 1134
<212> DNA
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 7
atggtatctt acaactccaa atttctggct gctaccgtac aggctgaacc ggtttggctg 60
gacgcggacg caactatcga taaagccatt ggtatcatcg aggaggcggc ccagaaaggt 120
gcgtctctga ttgccttccc ggaagttttc atccctggtt acccgtattg ggcctggctg 180
ggtgacgtaa agtactccct gtccttcacc tcccgttacc acgaaaactc cctggaactg 240
ggtgacgacc gtatgcgccg tctgcaactg gctgcgcgtc gtaacaaaat cgcgctggtt 300
atgggttaca gcgagcgtga ggcaggcagc cgctacctgt cccaggtctt tatcgacgaa 360
cgtggtgaaa tcgttgctaa ccgtcgtaaa ctgaaaccaa ctcacgttga acgtacgatt 420
tatggtgaag gcgacggtac cgactttctg acgcatgact tcgcatttgg tcgtgttggt 480
ggtctgaact gctgggagca cgttcagccg ctgtccaaat tcatgatgta ctccctgggt 540
gaacaggtac acgtcgcttc ttggccggct atgtccccgc tgcaaccgga cgtgtttcaa 600
ttttccatcg aggctaatgc gaccgtaacc cgctcctatg ctattgaagg ccaaaccttc 660
gttctgtgct ctacgcaggt tatcggtccg tctgcaattg aaaccttctg tctgaacgat 720
gagcaacgtg cactgctgcc gcagggttgc ggttgggcgc gtatctacgg cccggacggc 780
agcgaactgg ccaagccgct ggctgaagac gcagagggta ttctgtacgc agaaatcgat 840
ctggaacaga ttctgctggc caaggctggc gctgatccgg ttggtcacta cagccgccct 900
gatgtcctgt ccgtgcagtt cgacccgcgt aaccacaccc cggtacaccg cattggtatc 960
gatggccgtc tggatgttaa cacgcgttcc cgtgtagaaa actttcgcct gcgtaaagca 1020
gcagaaaaag aacgtcaggc cagcaaacgt ctgggcacga aactgtttga acagtctctg 1080
ctggcggagg agccggtacc agccaaactc gagcaccacc accaccacca ctga 1134
<210> 8
<211> 377
<212> PRT
<213> 敏捷食酸菌(Acidovorax facilis)
<400> 8
Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu
1 5 10 15
Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ala Ile Gly Ile
20 25 30
Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu
35 40 45
Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys
50 55 60
Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu
65 70 75 80
Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys
85 90 95
Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr
100 105 110
Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg
115 120 125
Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly
130 135 140
Asp Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly
145 150 155 160
Gly Leu Asn Cys Trp Glu His Val Gln Pro Leu Ser Lys Phe Met Met
165 170 175
Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser
180 185 190
Pro Leu Gln Pro Asp Val Phe Gln Phe Ser Ile Glu Ala Asn Ala Thr
195 200 205
Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser
210 215 220
Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp
225 230 235 240
Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr
245 250 255
Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu
260 265 270
Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys
275 280 285
Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser
290 295 300
Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile
305 310 315 320
Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg
325 330 335
Leu Arg Lys Ala Ala Glu Lys Glu Arg Gln Ala Ser Lys Arg Leu Gly
340 345 350
Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala
355 360 365
Lys Leu Glu His His His His His His
370 375
Claims (9)
1.一种产物耐受型腈水解酶突变体,其特征在于,所述突变体为下列之一:(1)将SEQID No.2所示氨基酸序列的第29位丝氨酸突变为丙氨酸;(2)将SEQ ID No.2所示氨基酸序列第145位天冬酰胺突变为天冬氨酸;(3)将SEQ ID No.2所示氨基酸序列第29位的丝氨酸突变为丙氨酸,同时将第145位天冬酰胺突变为天冬氨酸。
2.一种权利要求1所述腈水解酶突变体的编码基因。
3.一种含权利要求2所述编码基因的重组质粒。
4.一种含有权利要求2所述编码基因的重组基因工程菌。
5.一种权利要求1所述产物耐受型腈水解酶突变体在生物催化1-氰基环己基乙腈制备1-氰基环己基乙酸中的应用。
6.如权利要求5所述的应用,其特征在于,所述的应用以含所述腈水解酶突变体编码基因的重组基因工程菌经诱导表达获得的湿菌体、湿菌体超声破碎后提取的粗酶或粗酶提取纯化的纯酶为催化剂,以1-氰基环己基乙腈为底物,以pH为5.0-10.0的缓冲液或超纯水为反应介质构成反应体系,在20-55℃、200-400rpm条件下恒温水浴反应,反应完全后,将反应液分离纯化,获得1-氰基环己基乙酸。
7.如权利要求6所述的应用,其特征在于,反应体系中底物浓度为500-1000 mM;所述催化剂用量以湿菌体重量计为10-50 g/L。
8.如权利要求6所述的应用,其特征在于,所述湿菌体按如下方法制备:将含腈水解酶突变体编码基因的重组基因工程菌接种到含终浓度50µg/mL卡那霉素的LB液体培养基中,于37℃培养8-10h,培养液按体积浓度2%的接种量接种至含终浓度50µg/mL卡那霉素的新鲜LB液体培养基中,37℃培养至菌体浓度OD600为0.6-0.8之间,加入终浓度为0.1mM的IPTG,28℃、180rpm诱导表达10-12h,4℃、8000rpm离心10min,收集菌体,用生理盐水清洗后,得到湿菌体。
9.如权利要求6所述的应用,其特征在于,所述粗酶或纯酶按如下步骤制备:
(1)粗酶液:将含腈水解酶突变体编码基因的工程菌经诱导培养获得的湿菌体用pH 7、200 mM Na2HPO4-NaH2PO4缓冲液重悬成菌悬液,超声波破碎,破碎混合液离心后,取上清液作为粗酶液;所述超声波破碎条件为:40 W,30 min,1 s破碎,1 s暂停;
(2)纯酶:将Ni-NTA柱用平衡缓冲液冲洗后,将步骤(1)粗酶液以1 mL/min的流速上样,上样量为2个柱体积,上样结束后用平衡缓冲液洗脱弱吸附的杂蛋白,流速为2 mL/min,洗脱5个柱体积;再用洗脱缓冲液洗脱3-5个柱体积,收集目的蛋白,流速为1 mL/min;最后将收集的目的蛋白分装透析袋,以pH 7、50 mM Na2HPO4-NaH2PO4缓冲液为透析液室温透析10h,取截留液即为纯酶液;平衡缓冲液为含终浓度300 mM NaCl+50 mM咪唑的pH8、50 mMNa2HPO4-NaH2PO4;洗脱缓冲液为含终浓度300 mM NaCl+500 mM咪唑的pH8、50 mM Na2HPO4-NaH2PO4。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210260482.9A CN114958801B (zh) | 2022-03-16 | 2022-03-16 | 产物耐受型腈水解酶突变体及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210260482.9A CN114958801B (zh) | 2022-03-16 | 2022-03-16 | 产物耐受型腈水解酶突变体及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114958801A CN114958801A (zh) | 2022-08-30 |
CN114958801B true CN114958801B (zh) | 2024-05-03 |
Family
ID=82975635
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210260482.9A Active CN114958801B (zh) | 2022-03-16 | 2022-03-16 | 产物耐受型腈水解酶突变体及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114958801B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107177576A (zh) * | 2017-05-10 | 2017-09-19 | 浙江工业大学 | 腈水解酶突变体及其应用 |
CN111172140A (zh) * | 2020-01-21 | 2020-05-19 | 浙江工业大学 | 一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用 |
-
2022
- 2022-03-16 CN CN202210260482.9A patent/CN114958801B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107177576A (zh) * | 2017-05-10 | 2017-09-19 | 浙江工业大学 | 腈水解酶突变体及其应用 |
CN111172140A (zh) * | 2020-01-21 | 2020-05-19 | 浙江工业大学 | 一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用 |
Also Published As
Publication number | Publication date |
---|---|
CN114958801A (zh) | 2022-08-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11001823B2 (en) | Nitrilase mutants and application thereof | |
US20210009981A1 (en) | Nitrilase mutant, construction method therefor, and application thereof | |
CN107177576B (zh) | 腈水解酶突变体及其应用 | |
CN111172140B (zh) | 一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用 | |
CN108467860B (zh) | 一种高产γ-氨基丁酸的方法 | |
CN111471668B (zh) | 一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用 | |
US10829755B2 (en) | Genetically engineered arginine deiminase modified by site-directed mutagenesis | |
CN112877307B (zh) | 一种氨基酸脱氢酶突变体及其应用 | |
CN113151237A (zh) | 一种稳定性提高的蔗糖异构酶突变体及其构建方法 | |
CN113025542B (zh) | 产l-谷氨酰胺的重组大肠杆菌及其构建方法与应用 | |
CN109929822B (zh) | 一种米曲霉脂肪酶突变体及其应用 | |
CN112746067B (zh) | 用于制备d-鸟氨酸的赖氨酸脱羧酶突变体 | |
CN110129305B (zh) | 一种用于制备7-aca的头孢菌素c酰化酶突变体 | |
CN110592035B (zh) | 一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用 | |
CN109593739B (zh) | 重组酮酸还原酶突变体、基因、工程菌及其应用 | |
CN114958801B (zh) | 产物耐受型腈水解酶突变体及其应用 | |
CN114277020B (zh) | 一种腈水解酶突变体、工程菌及其应用 | |
CN113512544B (zh) | 一种热稳定性提高的甘露糖异构酶突变体 | |
CN109762801B (zh) | 卤醇脱卤酶突变体及其在合成手性药物中间体中的应用 | |
CN113564151A (zh) | 一种提高ce酶结构异构催化活性的方法及其突变体 | |
CN114196659B (zh) | 酰胺酶突变体、编码基因、工程菌及其应用 | |
CN109280651B (zh) | 一种乳酸脱氢酶突变体基因LbLDH1及其在大肠杆菌中高效表达的发酵方法 | |
CN115261366B (zh) | 一种耐高温纤维二糖差向异构酶突变体、工程菌及其应用 | |
CN113444702B (zh) | 烯酮还原酶突变体及其应用 | |
CN111500564B (zh) | 青霉素g酰化酶突变体及其在酶法合成头孢孟多中的应用 |
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 |