CN114875006B - 转氨酶突变体及手性胺化合物的制备方法 - Google Patents
转氨酶突变体及手性胺化合物的制备方法 Download PDFInfo
- Publication number
- CN114875006B CN114875006B CN202210707289.5A CN202210707289A CN114875006B CN 114875006 B CN114875006 B CN 114875006B CN 202210707289 A CN202210707289 A CN 202210707289A CN 114875006 B CN114875006 B CN 114875006B
- Authority
- CN
- China
- Prior art keywords
- substituted
- unsubstituted
- alkylene
- gly
- ala
- 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
- 102000003929 Transaminases Human genes 0.000 title claims abstract description 71
- 108090000340 Transaminases Proteins 0.000 title claims abstract description 71
- -1 amine compound Chemical class 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 230000035772 mutation Effects 0.000 claims abstract description 46
- 150000001413 amino acids Chemical class 0.000 claims abstract description 45
- 102200109205 rs200857997 Human genes 0.000 claims description 233
- 102220561378 4-galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase FUT5_N86H_mutation Human genes 0.000 claims description 209
- 102200129877 rs587777365 Human genes 0.000 claims description 167
- 102200068606 rs281865223 Human genes 0.000 claims description 99
- 102220038185 rs144829356 Human genes 0.000 claims description 87
- 102220644448 Protein phosphatase 1 regulatory subunit 3G_V64M_mutation Human genes 0.000 claims description 62
- 239000000758 substrate Substances 0.000 claims description 53
- 125000002947 alkylene group Chemical group 0.000 claims description 48
- 125000003107 substituted aryl group Chemical group 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 37
- 235000001014 amino acid Nutrition 0.000 claims description 25
- 125000001118 alkylidene group Chemical group 0.000 claims description 24
- 125000003118 aryl group Chemical group 0.000 claims description 21
- 125000000732 arylene group Chemical group 0.000 claims description 21
- 125000001424 substituent group Chemical group 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 18
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 15
- 229910052736 halogen Inorganic materials 0.000 claims description 15
- 150000002367 halogens Chemical class 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 14
- ISYORFGKSZLPNW-UHFFFAOYSA-N propan-2-ylazanium;chloride Chemical compound [Cl-].CC(C)[NH3+] ISYORFGKSZLPNW-UHFFFAOYSA-N 0.000 claims description 14
- 108020004414 DNA Proteins 0.000 claims description 12
- 125000004432 carbon atom Chemical group C* 0.000 claims description 12
- 125000005549 heteroarylene group Chemical group 0.000 claims description 12
- 125000005346 substituted cycloalkyl group Chemical group 0.000 claims description 12
- 150000002576 ketones Chemical class 0.000 claims description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 11
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 10
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 6
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 claims description 6
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 6
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 6
- 102220520845 Dynein light chain Tctex-type 3_V64S_mutation Human genes 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 5
- 102200059965 rs61753185 Human genes 0.000 claims description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 4
- 235000004279 alanine Nutrition 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 claims description 3
- 125000005842 heteroatom Chemical group 0.000 claims description 3
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 229910052717 sulfur Inorganic materials 0.000 claims description 3
- 125000005649 substituted arylene group Chemical group 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 36
- 102000004169 proteins and genes Human genes 0.000 abstract description 30
- 102000004190 Enzymes Human genes 0.000 abstract description 29
- 108090000790 Enzymes Proteins 0.000 abstract description 29
- 230000000694 effects Effects 0.000 abstract description 24
- 238000006555 catalytic reaction Methods 0.000 abstract description 11
- 229920001184 polypeptide Polymers 0.000 abstract description 7
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 5
- 102220237697 rs72650666 Human genes 0.000 description 80
- 238000006243 chemical reaction Methods 0.000 description 53
- 235000018102 proteins Nutrition 0.000 description 24
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 16
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 15
- 238000003752 polymerase chain reaction Methods 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000008363 phosphate buffer Substances 0.000 description 11
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 11
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 11
- 102220553419 Ral guanine nucleotide dissociation stimulator-like 2_Y89E_mutation Human genes 0.000 description 9
- 230000003197 catalytic effect Effects 0.000 description 8
- 239000010802 sludge Substances 0.000 description 8
- 230000002194 synthesizing effect Effects 0.000 description 7
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 6
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 6
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 6
- 102220626227 DNA-directed primase/polymerase protein_Y89S_mutation Human genes 0.000 description 6
- NCVJJAJVWILAGI-SRVKXCTJSA-N Met-Gln-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N NCVJJAJVWILAGI-SRVKXCTJSA-N 0.000 description 6
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 6
- 102220620962 Protein pitchfork_P83A_mutation Human genes 0.000 description 6
- YFOCMOVJBQDBCE-NRPADANISA-N Val-Ala-Glu Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N YFOCMOVJBQDBCE-NRPADANISA-N 0.000 description 6
- QPPZEDOTPZOSEC-RCWTZXSCSA-N Val-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N)O QPPZEDOTPZOSEC-RCWTZXSCSA-N 0.000 description 6
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 6
- 108010047495 alanylglycine Proteins 0.000 description 6
- 150000001412 amines Chemical class 0.000 description 6
- 108010078144 glutaminyl-glycine Proteins 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 231100000350 mutagenesis Toxicity 0.000 description 6
- 102200012570 rs111033666 Human genes 0.000 description 6
- 238000002741 site-directed mutagenesis Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000009776 industrial production Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 102220473482 Alpha-1B-glycoprotein_K90A_mutation Human genes 0.000 description 4
- 102220519283 Conserved oligomeric Golgi complex subunit 3_T91V_mutation Human genes 0.000 description 4
- 102220626230 DNA-directed primase/polymerase protein_Y89F_mutation Human genes 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 102220534514 Protein quaking_K90S_mutation Human genes 0.000 description 4
- 102220617269 Protein-glutamine gamma-glutamyltransferase K_Y89G_mutation Human genes 0.000 description 4
- 102220483111 TLC domain-containing protein 4_Y85F_mutation Human genes 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 4
- 102220355138 c.268_269delAAinsGT Human genes 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 229960001327 pyridoxal phosphate Drugs 0.000 description 4
- 102220333221 rs1465223718 Human genes 0.000 description 4
- 102220184840 rs199513213 Human genes 0.000 description 4
- 102200150982 rs28939717 Human genes 0.000 description 4
- 102220056395 rs730880037 Human genes 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 3
- ZIBWKCRKNFYTPT-ZKWXMUAHSA-N Ala-Asn-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZIBWKCRKNFYTPT-ZKWXMUAHSA-N 0.000 description 3
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 3
- OMMDTNGURYRDAC-NRPADANISA-N Ala-Glu-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OMMDTNGURYRDAC-NRPADANISA-N 0.000 description 3
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 3
- YHKANGMVQWRMAP-DCAQKATOSA-N Ala-Leu-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YHKANGMVQWRMAP-DCAQKATOSA-N 0.000 description 3
- USNSOPDIZILSJP-FXQIFTODSA-N Arg-Asn-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O USNSOPDIZILSJP-FXQIFTODSA-N 0.000 description 3
- IGULQRCJLQQPSM-DCAQKATOSA-N Arg-Cys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IGULQRCJLQQPSM-DCAQKATOSA-N 0.000 description 3
- BJNUAWGXPSHQMJ-DCAQKATOSA-N Arg-Gln-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O BJNUAWGXPSHQMJ-DCAQKATOSA-N 0.000 description 3
- NKBQZKVMKJJDLX-SRVKXCTJSA-N Arg-Glu-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NKBQZKVMKJJDLX-SRVKXCTJSA-N 0.000 description 3
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 3
- HCIUUZGFTDTEGM-NAKRPEOUSA-N Arg-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N HCIUUZGFTDTEGM-NAKRPEOUSA-N 0.000 description 3
- MJINRRBEMOLJAK-DCAQKATOSA-N Arg-Lys-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N MJINRRBEMOLJAK-DCAQKATOSA-N 0.000 description 3
- QBQVKUNBCAFXSV-ULQDDVLXSA-N Arg-Lys-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QBQVKUNBCAFXSV-ULQDDVLXSA-N 0.000 description 3
- ZEBDYGZVMMKZNB-SRVKXCTJSA-N Arg-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCN=C(N)N)N ZEBDYGZVMMKZNB-SRVKXCTJSA-N 0.000 description 3
- UZSQXCMNUPKLCC-FJXKBIBVSA-N Arg-Thr-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UZSQXCMNUPKLCC-FJXKBIBVSA-N 0.000 description 3
- QUBKBPZGMZWOKQ-SZMVWBNQSA-N Arg-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 QUBKBPZGMZWOKQ-SZMVWBNQSA-N 0.000 description 3
- JYHIVHINLJUIEG-BVSLBCMMSA-N Arg-Tyr-Trp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JYHIVHINLJUIEG-BVSLBCMMSA-N 0.000 description 3
- WOZDCBHUGJVJPL-AVGNSLFASA-N Arg-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N WOZDCBHUGJVJPL-AVGNSLFASA-N 0.000 description 3
- FXGMURPOWCKNAZ-JYJNAYRXSA-N Arg-Val-Phe Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 FXGMURPOWCKNAZ-JYJNAYRXSA-N 0.000 description 3
- FAEFJTCTNZTPHX-ACZMJKKPSA-N Asn-Gln-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FAEFJTCTNZTPHX-ACZMJKKPSA-N 0.000 description 3
- 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 3
- TVVYVAUGRHNTGT-UGYAYLCHSA-N Asp-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O TVVYVAUGRHNTGT-UGYAYLCHSA-N 0.000 description 3
- VFUXXFVCYZPOQG-WDSKDSINSA-N Asp-Glu-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VFUXXFVCYZPOQG-WDSKDSINSA-N 0.000 description 3
- LTXGDRFJRZSZAV-CIUDSAMLSA-N Asp-Glu-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)N LTXGDRFJRZSZAV-CIUDSAMLSA-N 0.000 description 3
- TVIZQBFURPLQDV-DJFWLOJKSA-N Asp-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)O)N TVIZQBFURPLQDV-DJFWLOJKSA-N 0.000 description 3
- HKEZZWQWXWGASX-KKUMJFAQSA-N Asp-Leu-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 HKEZZWQWXWGASX-KKUMJFAQSA-N 0.000 description 3
- IDDMGSKZQDEDGA-SRVKXCTJSA-N Asp-Phe-Asn Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=CC=C1 IDDMGSKZQDEDGA-SRVKXCTJSA-N 0.000 description 3
- UEFODXNXUAVPTC-VEVYYDQMSA-N Asp-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UEFODXNXUAVPTC-VEVYYDQMSA-N 0.000 description 3
- MFDPBZAFCRKYEY-LAEOZQHASA-N Asp-Val-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MFDPBZAFCRKYEY-LAEOZQHASA-N 0.000 description 3
- 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 3
- GMXSSZUVDNPRMA-FXQIFTODSA-N Cys-Arg-Asp Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GMXSSZUVDNPRMA-FXQIFTODSA-N 0.000 description 3
- ZGERHCJBLPQPGV-ACZMJKKPSA-N Cys-Ser-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N ZGERHCJBLPQPGV-ACZMJKKPSA-N 0.000 description 3
- JRZMCSIUYGSJKP-ZKWXMUAHSA-N Cys-Val-Asn Chemical compound SC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O JRZMCSIUYGSJKP-ZKWXMUAHSA-N 0.000 description 3
- 108010090461 DFG peptide Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- DTMLKCYOQKZXKZ-HJGDQZAQSA-N Gln-Arg-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DTMLKCYOQKZXKZ-HJGDQZAQSA-N 0.000 description 3
- VGUYMZGLJUJRBV-YVNDNENWSA-N Glu-Ile-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VGUYMZGLJUJRBV-YVNDNENWSA-N 0.000 description 3
- UGSVSNXPJJDJKL-SDDRHHMPSA-N Glu-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N UGSVSNXPJJDJKL-SDDRHHMPSA-N 0.000 description 3
- HRBYTAIBKPNZKQ-AVGNSLFASA-N Glu-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O HRBYTAIBKPNZKQ-AVGNSLFASA-N 0.000 description 3
- YRMZCZIRHYCNHX-RYUDHWBXSA-N Glu-Phe-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O YRMZCZIRHYCNHX-RYUDHWBXSA-N 0.000 description 3
- DMYACXMQUABZIQ-NRPADANISA-N Glu-Ser-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O DMYACXMQUABZIQ-NRPADANISA-N 0.000 description 3
- 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 3
- JLCYOCDGIUZMKQ-JBACZVJFSA-N Glu-Trp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CCC(=O)O)N JLCYOCDGIUZMKQ-JBACZVJFSA-N 0.000 description 3
- LERGJIVJIIODPZ-ZANVPECISA-N Gly-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)CN)C)C(O)=O)=CNC2=C1 LERGJIVJIIODPZ-ZANVPECISA-N 0.000 description 3
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 3
- XZRZILPOZBVTDB-GJZGRUSLSA-N Gly-Arg-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)CN)C(O)=O)=CNC2=C1 XZRZILPOZBVTDB-GJZGRUSLSA-N 0.000 description 3
- OCDLPQDYTJPWNG-YUMQZZPRSA-N Gly-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)CN OCDLPQDYTJPWNG-YUMQZZPRSA-N 0.000 description 3
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 3
- BEQGFMIBZFNROK-JGVFFNPUSA-N Gly-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)CN)C(=O)O BEQGFMIBZFNROK-JGVFFNPUSA-N 0.000 description 3
- KAJAOGBVWCYGHZ-JTQLQIEISA-N Gly-Gly-Phe Chemical compound [NH3+]CC(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KAJAOGBVWCYGHZ-JTQLQIEISA-N 0.000 description 3
- ZKLYPEGLWFVRGF-IUCAKERBSA-N Gly-His-Gln Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZKLYPEGLWFVRGF-IUCAKERBSA-N 0.000 description 3
- YFGONBOFGGWKKY-VHSXEESVSA-N Gly-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)CN)C(=O)O YFGONBOFGGWKKY-VHSXEESVSA-N 0.000 description 3
- LRQXRHGQEVWGPV-NHCYSSNCSA-N Gly-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN LRQXRHGQEVWGPV-NHCYSSNCSA-N 0.000 description 3
- NNCSJUBVFBDDLC-YUMQZZPRSA-N Gly-Leu-Ser Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O NNCSJUBVFBDDLC-YUMQZZPRSA-N 0.000 description 3
- WDEHMRNSGHVNOH-VHSXEESVSA-N Gly-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)CN)C(=O)O WDEHMRNSGHVNOH-VHSXEESVSA-N 0.000 description 3
- DBJYVKDPGIFXFO-BQBZGAKWSA-N Gly-Met-Ala Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O DBJYVKDPGIFXFO-BQBZGAKWSA-N 0.000 description 3
- IGOYNRWLWHWAQO-JTQLQIEISA-N Gly-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IGOYNRWLWHWAQO-JTQLQIEISA-N 0.000 description 3
- JNGHLWWFPGIJER-STQMWFEESA-N Gly-Pro-Tyr Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JNGHLWWFPGIJER-STQMWFEESA-N 0.000 description 3
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 3
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 3
- IZVICCORZOSGPT-JSGCOSHPSA-N Gly-Val-Tyr Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IZVICCORZOSGPT-JSGCOSHPSA-N 0.000 description 3
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 3
- BQFGKVYHKCNEMF-DCAQKATOSA-N His-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=CN=CN1 BQFGKVYHKCNEMF-DCAQKATOSA-N 0.000 description 3
- RAVLQPXCMRCLKT-KBPBESRZSA-N His-Gly-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RAVLQPXCMRCLKT-KBPBESRZSA-N 0.000 description 3
- FYTCLUIYTYFGPT-YUMQZZPRSA-N His-Gly-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FYTCLUIYTYFGPT-YUMQZZPRSA-N 0.000 description 3
- PMWSGVRIMIFXQH-KKUMJFAQSA-N His-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1NC=NC=1)C1=CN=CN1 PMWSGVRIMIFXQH-KKUMJFAQSA-N 0.000 description 3
- BSVLMPMIXPQNKC-KBPBESRZSA-N His-Phe-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O BSVLMPMIXPQNKC-KBPBESRZSA-N 0.000 description 3
- ZVKDCQVQTGYBQT-LSJOCFKGSA-N His-Pro-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O ZVKDCQVQTGYBQT-LSJOCFKGSA-N 0.000 description 3
- PBVQWNDMFFCPIZ-ULQDDVLXSA-N His-Pro-Phe Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 PBVQWNDMFFCPIZ-ULQDDVLXSA-N 0.000 description 3
- CYHJCEKUMCNDFG-LAEOZQHASA-N Ile-Gln-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)NCC(=O)O)N CYHJCEKUMCNDFG-LAEOZQHASA-N 0.000 description 3
- LGMUPVWZEYYUMU-YVNDNENWSA-N Ile-Glu-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N LGMUPVWZEYYUMU-YVNDNENWSA-N 0.000 description 3
- PWDSHAAAFXISLE-SXTJYALSSA-N Ile-Ile-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O PWDSHAAAFXISLE-SXTJYALSSA-N 0.000 description 3
- CIDLJWVDMNDKPT-FIRPJDEBSA-N Ile-Phe-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N CIDLJWVDMNDKPT-FIRPJDEBSA-N 0.000 description 3
- KCTIFOCXAIUQQK-QXEWZRGKSA-N Ile-Pro-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O KCTIFOCXAIUQQK-QXEWZRGKSA-N 0.000 description 3
- AUIYHFRUOOKTGX-UKJIMTQDSA-N Ile-Val-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N AUIYHFRUOOKTGX-UKJIMTQDSA-N 0.000 description 3
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 3
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 3
- 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 3
- ZALAVHVPPOHAOL-XUXIUFHCSA-N Leu-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(C)C)N ZALAVHVPPOHAOL-XUXIUFHCSA-N 0.000 description 3
- YUTNOGOMBNYPFH-XUXIUFHCSA-N Leu-Pro-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YUTNOGOMBNYPFH-XUXIUFHCSA-N 0.000 description 3
- LSLUTXRANSUGFY-XIRDDKMYSA-N Leu-Trp-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(O)=O LSLUTXRANSUGFY-XIRDDKMYSA-N 0.000 description 3
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 3
- SSJBMGCZZXCGJJ-DCAQKATOSA-N Lys-Asp-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O SSJBMGCZZXCGJJ-DCAQKATOSA-N 0.000 description 3
- VQXAVLQBQJMENB-SRVKXCTJSA-N Lys-Glu-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O VQXAVLQBQJMENB-SRVKXCTJSA-N 0.000 description 3
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 3
- MUYQDMBLDFEVRJ-LSJOCFKGSA-N Met-Ala-His Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 MUYQDMBLDFEVRJ-LSJOCFKGSA-N 0.000 description 3
- 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 3
- CYZBFPYMSJGBRL-DRZSPHRISA-N Phe-Ala-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CYZBFPYMSJGBRL-DRZSPHRISA-N 0.000 description 3
- IDUCUXTUHHIQIP-SOUVJXGZSA-N Phe-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O IDUCUXTUHHIQIP-SOUVJXGZSA-N 0.000 description 3
- 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 3
- ZLGQEBCCANLYRA-RYUDHWBXSA-N Phe-Gly-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O ZLGQEBCCANLYRA-RYUDHWBXSA-N 0.000 description 3
- AAERWTUHZKLDLC-IHRRRGAJSA-N Phe-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O AAERWTUHZKLDLC-IHRRRGAJSA-N 0.000 description 3
- GTMSCDVFQLNEOY-BZSNNMDCSA-N Phe-Tyr-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N GTMSCDVFQLNEOY-BZSNNMDCSA-N 0.000 description 3
- XALFIVXGQUEGKV-JSGCOSHPSA-N Phe-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 XALFIVXGQUEGKV-JSGCOSHPSA-N 0.000 description 3
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 description 3
- CGSOWZUPLOKYOR-AVGNSLFASA-N Pro-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 CGSOWZUPLOKYOR-AVGNSLFASA-N 0.000 description 3
- MCPXQHVVCPTRIM-HJOGWXRNSA-N Pro-Trp-Trp Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)O)C(=O)[C@@H]1CCCN1 MCPXQHVVCPTRIM-HJOGWXRNSA-N 0.000 description 3
- 108010003201 RGH 0205 Proteins 0.000 description 3
- 108010025216 RVF peptide Proteins 0.000 description 3
- QGMLKFGTGXWAHF-IHRRRGAJSA-N Ser-Arg-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QGMLKFGTGXWAHF-IHRRRGAJSA-N 0.000 description 3
- RNFKSBPHLTZHLU-WHFBIAKZSA-N Ser-Cys-Gly Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)O)N)O RNFKSBPHLTZHLU-WHFBIAKZSA-N 0.000 description 3
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 3
- OQPNSDWGAMFJNU-QWRGUYRKSA-N Ser-Gly-Tyr Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OQPNSDWGAMFJNU-QWRGUYRKSA-N 0.000 description 3
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 3
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 3
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 3
- XDARBNMYXKUFOJ-GSSVUCPTSA-N Thr-Asp-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XDARBNMYXKUFOJ-GSSVUCPTSA-N 0.000 description 3
- URPSJRMWHQTARR-MBLNEYKQSA-N Thr-Ile-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O URPSJRMWHQTARR-MBLNEYKQSA-N 0.000 description 3
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 3
- MECLEFZMPPOEAC-VOAKCMCISA-N Thr-Leu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MECLEFZMPPOEAC-VOAKCMCISA-N 0.000 description 3
- NDXSOKGYKCGYKT-VEVYYDQMSA-N Thr-Pro-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O NDXSOKGYKCGYKT-VEVYYDQMSA-N 0.000 description 3
- CYCGARJWIQWPQM-YJRXYDGGSA-N Thr-Tyr-Ser Chemical compound C[C@@H](O)[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CO)C([O-])=O)CC1=CC=C(O)C=C1 CYCGARJWIQWPQM-YJRXYDGGSA-N 0.000 description 3
- HYNAKPYFEYJMAS-XIRDDKMYSA-N Trp-Arg-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HYNAKPYFEYJMAS-XIRDDKMYSA-N 0.000 description 3
- MXFPBNFKVBHIRW-BZSNNMDCSA-N Tyr-Lys-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O MXFPBNFKVBHIRW-BZSNNMDCSA-N 0.000 description 3
- BIVIUZRBCAUNPW-JRQIVUDYSA-N Tyr-Thr-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O BIVIUZRBCAUNPW-JRQIVUDYSA-N 0.000 description 3
- LYPKCSYAKLTBHJ-ILWGZMRPSA-N Tyr-Trp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC4=CC=C(C=C4)O)N)C(=O)O LYPKCSYAKLTBHJ-ILWGZMRPSA-N 0.000 description 3
- REJBPZVUHYNMEN-LSJOCFKGSA-N Val-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N REJBPZVUHYNMEN-LSJOCFKGSA-N 0.000 description 3
- CWSIBTLMMQLPPZ-FXQIFTODSA-N Val-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N CWSIBTLMMQLPPZ-FXQIFTODSA-N 0.000 description 3
- ROLGIBMFNMZANA-GVXVVHGQSA-N Val-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N ROLGIBMFNMZANA-GVXVVHGQSA-N 0.000 description 3
- PYPZMFDMCCWNST-NAKRPEOUSA-N Val-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N PYPZMFDMCCWNST-NAKRPEOUSA-N 0.000 description 3
- XXWBHOWRARMUOC-NHCYSSNCSA-N Val-Lys-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N)C(=O)O)N XXWBHOWRARMUOC-NHCYSSNCSA-N 0.000 description 3
- DEGUERSKQBRZMZ-FXQIFTODSA-N Val-Ser-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O DEGUERSKQBRZMZ-FXQIFTODSA-N 0.000 description 3
- DVLWZWNAQUBZBC-ZNSHCXBVSA-N Val-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N)O DVLWZWNAQUBZBC-ZNSHCXBVSA-N 0.000 description 3
- 108010087924 alanylproline Proteins 0.000 description 3
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 3
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 3
- 108010094001 arginyl-tryptophyl-arginine Proteins 0.000 description 3
- 108010068380 arginylarginine Proteins 0.000 description 3
- 108010038633 aspartylglutamate Proteins 0.000 description 3
- 108010047857 aspartylglycine Proteins 0.000 description 3
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 3
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 3
- 108010077435 glycyl-phenylalanyl-glycine Proteins 0.000 description 3
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 108010015792 glycyllysine Proteins 0.000 description 3
- 108010081551 glycylphenylalanine Proteins 0.000 description 3
- 108010037850 glycylvaline Proteins 0.000 description 3
- 238000013537 high throughput screening Methods 0.000 description 3
- 108010036413 histidylglycine Proteins 0.000 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 description 3
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 3
- 108010057821 leucylproline Proteins 0.000 description 3
- 108010056582 methionylglutamic acid Proteins 0.000 description 3
- 238000003032 molecular docking Methods 0.000 description 3
- 108010012581 phenylalanylglutamate Proteins 0.000 description 3
- 108010051242 phenylalanylserine Proteins 0.000 description 3
- 108010015796 prolylisoleucine Proteins 0.000 description 3
- 108010053725 prolylvaline Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108010080629 tryptophan-leucine Proteins 0.000 description 3
- 108010051110 tyrosyl-lysine Proteins 0.000 description 3
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 3
- 108010027345 wheylin-1 peptide Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 102220512989 Uncharacterized protein KIAA0040_W60A_mutation Human genes 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000011914 asymmetric synthesis Methods 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005891 transamination reaction Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- PDRMRVHPAQKTLT-NAKRPEOUSA-N Cys-Ile-Val Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O PDRMRVHPAQKTLT-NAKRPEOUSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101001110310 Lentilactobacillus kefiri NADP-dependent (R)-specific alcohol dehydrogenase Proteins 0.000 description 1
- 240000006240 Linum usitatissimum Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- DJQIUOKSNRBTSV-CYDGBPFRSA-N Val-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](C(C)C)N DJQIUOKSNRBTSV-CYDGBPFRSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- PYNXFZCZUAOOQC-UTKZUKDTSA-N sacubitril Chemical compound C1=CC(C[C@H](C[C@@H](C)C(=O)OCC)NC(=O)CCC(O)=O)=CC=C1C1=CC=CC=C1 PYNXFZCZUAOOQC-UTKZUKDTSA-N 0.000 description 1
- 229960003953 sacubitril Drugs 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- MFFMDFFZMYYVKS-SECBINFHSA-N sitagliptin Chemical compound C([C@H](CC(=O)N1CC=2N(C(=NN=2)C(F)(F)F)CC1)N)C1=CC(F)=C(F)C=C1F MFFMDFFZMYYVKS-SECBINFHSA-N 0.000 description 1
- 229960004034 sitagliptin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
-
- 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
- C12P13/001—Amines; Imines
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供了一种转氨酶突变体及手性胺化合物的制备方法。其中,该转氨酶突变体,包括(a)具有SEQ ID NO:1所示的氨基酸序列的蛋白质;或(b)具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列的蛋白质;或(c)在(b)中的氨基酸序列的如下至少一个位点:Y89、L380、N86、Y85、T91、P83、K90、S417、S424、F301、G164、T452等,经过氨基酸突变且具有转氨酶功能的蛋白质;(d)与(a)、(b)或(c)中任一项限定的氨基酸序列具有80%以上同源性且具有转氨酶功能的蛋白质。能够解决现有技术中的转氨酶活性低的问题,适用于酶催化领域。
Description
技术领域
本发明涉及酶催化领域,具体而言,涉及一种转氨酶突变体及手性胺化合物的制备方法。
背景技术
手性胺是指小分子化合物手性中心含有氨基的一类化合物,是许多重要生物活性分子的结构单元和合成很多手性药物的关键中间体。目前,手性胺的制备主要通过化学法、生物拆分法和生物不对称合法实现。化学法中存在反应路线长、条件苛刻、使用有毒的过渡态金属催化剂、产品立体选择性低等缺点;生物拆分法的理论最高收率只有50%,因此在放大生产上都有一定的局限性。
转氨酶催化的不对称合成法由于理论收率高,并且具有高选择性、高转化率及温和的反应条件等优点,已成为合成手性胺的首选方法。但是,大多数野生型酶的缺点是它们的底物范围有限,并且通常难以接受羰基旁边一侧大于甲基取代基的基团(本申请中称之为合成大位阻手性胺化合物),这使得真正能够被工业化应用的转氨酶并不多。转氨酶催化大位阻的手性胺化合物的合成是一个长久以来存在的挑战。
通过定向进化的手段对野生酶进行改造,改善其底物接受范围,特别是在大位阻的手性胺化合物的合成上,是解决这一挑战的很好的方法。两个很好的案例是美国Codexis公司对不同来源的野生型转氨酶进行定向进化,获得的突变体可以用于高效催化Sitagliptin[Science,329(Jul.16TN.5989),305-309]和Sacubitril[ACS Catal.2021,11(6),3762-3770]前体酮,合成相应的大位阻的手性胺化合物。凯莱英生命科学技术(天津)有限公司(Asymchem)的生物合成技术研发中心团队在之前的研究中,筛选到一株来源于Chromobaterium violaceum的转氨酶突变体,可用于催化合成大位阻手性胺。之后,为了进一步适应工业化生产中高底物浓度,低酶量和极端环境的要求,Asymchem继续开发可以合成大位阻的手性胺化合物的转氨酶,以提高生产效率,降低工业生产成本,减少工业三废排放。
发明内容
本发明的主要目的在于提供一种转氨酶突变体及手性胺化合物的制备方法,以解决现有技术中的转氨酶活性低的问题。
为了实现上述目的,根据本发明的第一个方面,提供了一种转氨酶突变体,包括(a)具有SEQ ID NO:1所示的氨基酸序列的蛋白质;或(b)具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列的蛋白质;或(c)在(b)中的氨基酸序列的如下至少一个位点:Y89、L380、N86、Y85、T91、P83、K90、S417、S424、F301、G164、T452、M180、F449、F320、Y322、D315、A31、L295、V64、H154、F409、T402、T126、F364、A433、L379、D416、N151、H274、I311、V327、R77或N317,经过氨基酸突变且具有转氨酶功能的蛋白质;(d)与(a)、(b)或(c)中任一项限定的氨基酸序列具有80%以上同源性且具有转氨酶功能的蛋白质。
进一步地,(c)的氨基酸突变各自独立地选自如下:S424A或S424F或S424Q或S424P或S424R或S424N或S424V或S424Y或S424E或S424I;L380A;S417A或S417Q或S417F或S417I;Y89A或Y89D或Y89S或Y89H或Y89M或Y89G或Y89F;F409A或F409S或F409Q或F409L或F409H或F409P或F409K或F409G或F409V或F409N;N86M或N86P或N86D或N86A或N86V或N86H;Y85M或Y85F或Y85R;P83C或P83A或P83S或P83G或P83R;T91M或T91A或T91V或T91N或T91I;K90Y或K90G或K90A或K90V或K90S;F301S;G164S;T452S;M180V;F449L;F320H;Y322T;D315V或D315C或D315R或D315M;A31V;L295Q或L295M或L295F或L295Q;V64M或V64A或V64S;H154S;T402K或T402S;T126C;F364L;A433T;L379A或L379S;D416A;N151A;H274Y;I311F;V327S;R77Q;N317Y;其中,数字前字母代表原始氨基酸,数字后字母代表突变氨基酸;优选地,(d)中,与(a)、(b)或(c)中限定的氨基酸序列具有85%以上,优选90%以上,更优选95%以上,进一步优选99%以上同源性且具有转氨酶功能的蛋白质。
进一步地,转氨酶突变体的突变包括如下任意一种氨基酸突变:W60A;Y89A;N151A;F166A;E168A;V234A;I262A;L379A;L380A;R405A;D416A;S417A;C418A;S424A;
V234A+S424A;V234A+L380A;V234A+L379A;V234A+D416A;V234A+S417A;
V234A+Y89A;V234A+N151A;V234A+L379A+L380A;V234A+D416A+S417A;
V234A+D416A+S424A;V234A+S417A+S424A;V234A+L380A+Y89D;V234A+L380A+Y89S;
V234A+L380A+Y89H;V234A+L380A+Y89M;V234A+L380A+F409A;V234A+L380A+F409S;
V234A+L380A+F409Q;V234A+L380A+F409L;V234A+L380A+S424F;V234A+L380A+S424Q;
V234A+L380A+S424P;V234A+L380A+S424R;V234A+L380A+S424N;V234A+L380A+N86M;
V234A+L380A+N86P;V234A+L380A+N86D;V234A+L380A+N86A;V234A+L380A+N86V;
V234A+L380A+N86H;V234A+L380A+N86M+Y89D;V234A+L380A+N86M+Y89S;
V234A+L380A+N86M+Y89G;V234A+L380A+N86M+Y89H;V234A+L380A+N86M+Y89F;
V234A+L380A+N86M+Y89A;V234A+L380A+Y89D+F409H;V234A+L380A+Y89D+F409A;
V234A+L380A+Y89D+F409P;V234A+L380A+Y89D+F409K;V234A+L380A+Y89D+F409G;
V234A+L380A+Y89D+S424A;V234A+L380A+Y89D+S424V;V234A+L380A+Y89D+S424Y;
V234A+L380A+Y89D+S424E;V234A+L380A+Y89D+S424Q;V234A+L380A+Y89D+N86H;
V234A+L380A+Y89D+N86A;V234A+L380A+Y89D+N86H+Y85M;
V234A+L380A+Y89D+N86H+Y85F;V234A+L380A+Y89D+N86H+Y85R;
V234A+L380A+Y89D+N86H+Y85M+S417Q;V234A+L380A+Y89D+N86H+Y85M+S417F;
V234A+L380A+Y89D+N86H+Y85M+S417I;V234A+L380A+Y89D+N86H+Y85M+S424I;
V234A+L380A+Y89D+N86H+Y85M+P83C;V234A+L380A+Y89D+N86H+Y85M+P83A;
V234A+L380A+Y89D+N86H+Y85M+P83S;V234A+L380A+Y89D+N86H+Y85M+P83G;
V234A+L380A+Y89D+N86H+Y85M+T91M;V234A+L380A+Y89D+N86H+Y85M+T91A;
V234A+L380A+Y89D+N86H+Y85M+T91V;V234A+L380A+Y89D+N86H+Y85M+T91N;
V234A+L380A+Y89D+N86H+Y85M+T91I;V234A+L380A+Y89D+N86H+Y85M+T91M+P83S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83C;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83G;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83R;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90V;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417A;
V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A;
V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+A239S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y+F409L;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+G164S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+I311F;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315C+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+V327S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295M;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295F;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64A;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N+T402K;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+R77Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+N317Y;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+A433T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y。
为了实现上述目的,根据本发明的第二个方面,提供了一种DNA分子,该DNA分子编码上述的转氨酶突变体。
为了实现上述目的,根据本发明的第三个方面,提供了一种重组质粒,该重组质粒连接有上述DNA分子。
为了实现上述目的,根据本发明的第四个方面,提供了一种宿主细胞,该宿主细胞内转化有上述重组质粒。
进一步地,宿主细胞包括原核细胞;优选地,原核细胞包括大肠杆菌。
为了实现上述目的,根据本发明的第五个方面,提供了一种手性胺化合物的制备方法,该制备方法包括利用上述转氨酶突变体在氨基供体的作用下,对式I所示的酮类底物进行转氨基反应,制备获得手性胺化合物,
Ar1选自第一取代芳基、第一未取代芳基、取代亚芳基或未取代亚芳基、取代杂亚芳基或未取代杂亚芳基;Ar2选自第二取代芳基、第二未取代芳基、取代环烷基、未取代环烷基、烷基或者亚烷基;R选自H、烷基、亚烷基或次烷基,亚烷基或次烷基的C原子数选自1~5,亚烷基或次烷基包括取代亚烷基或次烷基或未取代亚烷基或次烷基;当R选自亚烷基或次烷基时,亚烷基或次烷基与Ar1和/或Ar2连接成环;第一取代芳基、取代亚芳基、取代杂亚芳基、第二取代芳基或取代亚烷基或次烷基中的取代基各自独立选自卤素、羟基、氨基、甲基、乙基或-CH2CH2OH;取代杂亚芳基中的杂原子选自N、O或S。
进一步地,第一取代芳基、第二取代芳基或取代亚芳基的取代基各自独立选自卤素或-CH2CH2OH;优选地,取代基各自独立位于第一取代芳基、第二取代芳基或取代亚芳基的邻位、间位或对位中的任意一个或多个位置;优选地,卤素选自F、Cl或Br。
进一步地,Ar1选自第一取代芳基、取代亚芳基、第一未取代芳基或未取代亚芳基,Ar2选自第二未取代芳基,R选自未取代亚烷基,未取代亚烷基的C原子数选自1~5;未取代亚烷基与Ar1连接成环。
进一步地,Ar1选自未取代杂亚芳基,Ar2选自第二取代芳基,第二取代芳基的取代基选自卤素,R选自取代亚烷基,取代亚烷基的取代基选自羟基,取代亚烷基的C原子数选自1~5,取代亚烷基与Ar1连接成环。
进一步地,Ar1选自取代亚芳基,取代亚芳基的取代基为卤素,Ar2选自取代环烷基或未取代环烷基,取代环烷基或未取代环烷基的C原子数选自3~8;R选自H。
进一步地,Ar1选自第一未取代芳基;Ar2选自第二取代芳基、第二未取代芳基、取代环烷基或未取代环烷基;R选自H、甲基、亚甲基或次甲基。
进一步地,Ar1选自环烷基或第一取代芳基,取代基选自羟基、甲基、乙基或-CH2CH2OH,Ar2选自第二未取代芳基或烷基;R选自H、甲基、亚甲基或次甲基。
进一步地,酮类底物选自
进一步地,氨基供体包括异丙胺、异丙胺盐酸盐、丙氨酸、正丁胺或苯胺。
应用本发明的技术方案,以来源于Chromobaterium violaceum的转氨酶突变体(SEQ ID NO:1)为母本,进行了单点定点突变、饱和突变、组合突变和易错PCR等蛋白质工程改造,获得了底物谱宽、能够催化合成大位阻手性胺化合物、酶活性高、耐受极端环境能力强的转氨酶突变体。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的,转氨酶催化的不对称合成法由于理论收率高,并且具有高选择性、高转化率及温和的反应条件等优点,已成为合成手性胺的首选方法。但是,大多数野生型酶的缺点是它们的底物范围有限,并且通常难以以大位阻酮类底物为底物,合成大位阻手性胺化合物。
因而,在本申请中发明人尝试对于转氨酶进行突变,发现多个关键活性氨基酸位点,从而获得上述多种转氨酶突变体,能够催化合成大位阻手性胺化合物,因而提出了本申请的一系列保护方案。
在本申请第一种典型的实施方式中,提供了一种转氨酶突变体,包括(a)具有SEQID NO:1所示的氨基酸序列的蛋白质;或(b)具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列的蛋白质;或(c)在(b)中的氨基酸序列的如下至少一个位点:Y89、L380、N86、Y85、T91、P83、K90、S417、S424、F301、G164、T452、M180、F449、F320、Y322、D315、A31、L295、V64、H154、F409、T402、T126、F364、A433、L379、D416、N151、H274、I311、V327、R77或N317,经过氨基酸突变且具有转氨酶功能的蛋白质;(d)与(a)、(b)或(c)中任一项限定的氨基酸序列具有80%以上同源性且具有转氨酶功能的蛋白质。
上述SEQ ID NO:1所示的氨基酸序列,是来源于Chromobaterium violaceum的氨基酸突变体。通过对该氨基酸序列进行同源模建、活性位点模拟、定点突变等计算机模拟和分子生物学实验,发现了V234这一关键氨基酸位点,将V进行点突变,突变为A获得SEQ IDNO:2所示的氨基酸序列,或突变为C获得SEQ ID NO:3所示的氨基酸序列。以V234突变的氨基酸为基础,发现Y89、L380、N86、Y85、T91、P83、K90、S417、S424、F301、G164、T452、M180、F449、F320、Y322、D315、A31、L295、V64、H154、F409、T402、T126、F364、A433、L379、D416、N151、H274、I311、V327、R77或N317等活性位点对蛋白质的活性影响也较大,通过对上述氨基酸位点进行突变,能够获得具有转氨酶功能、乃至转氨酶功能增强的蛋白质。对于上述获得的蛋白质,在非关键突变位点和活性位点处可以进行变化,能够获得与上述氨基酸序列有80%以上同源性、且具有转氨酶功能的蛋白质。
SEQ ID NO:1:
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGAWCVNVGYGRKDFAEAARRQMEELPFYNTAYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGFKEMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMDSCGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA。
SEQ ID NO:2:
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGAWCVNVGYGRKDFAEAARRQMEELPFYNTAYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGFKEMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGAIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMDSCGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA。
SEQ ID NO:3:
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGAWCVNVGYGRKDFAEAARRQMEELPFYNTAYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGFKEMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGCIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMDSCGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA。
在一种优选的实施例中,(c)的氨基酸突变各自独立地选自如下:S424A或S424F或S424Q或S424P或S424R或S424N或S424V或S424Y或S424E或S424I;L380A;S417A或S417Q或S417F或S417I;Y89A或Y89D或Y89S或Y89H或Y89M或Y89G或Y89F;F409A或F409S或F409Q或F409L或F409H或F409P或F409K或F409G或F409V或F409N;N86M或N86P或N86D或N86A或N86V或N86H;Y85M或Y85F或Y85R;P83C或P83A或P83S或P83G或P83R;T91M或T91A或T91V或T91N或T91I;K90Y或K90G或K90A或K90V或K90S;F301S;G164S;T452S;M180V;F449L;F320H;Y322T;D315V或D315C或D315R或D315M;A31V;L295Q或L295M或L295F或L295Q;V64M或V64A或V64S;H154S;T402K或T402S;T126C;F364L;A433T;L379A或L379S;D416A;N151A;H274Y;I311F;V327S;R77Q;N317Y;其中,数字前字母代表原始氨基酸,数字后字母代表突变氨基酸;优选地,(d)中,与(a)、(b)或(c)中限定的氨基酸序列具有85%以上,优选90%以上,更优选95%、96%、97%或98%以上,进一步优选99%、99.9%以上同源性且具有转氨酶功能的蛋白质。
在本申请中,申请人对上述活性位点继续进行探究,发现活性位点突变为不同氨基酸,相应的蛋白质活性也有差别,特定的突变能够增强转氨酶活性。通过试验探究,发现对于活性位点进行上述特定的突变,能够获得活性增强的蛋白质。对于转氨酶蛋白质的氨基酸突变位点,可以在上述的突变中进行灵活的选择和组合。
在一种优选的实施例中,转氨酶突变体的突变包括如下任意一种氨基酸突变:
W60A;Y89A;N151A;F166A;E168A;V234A;I262A;L379A;L380A;R405A;D416A;S417A;C418A;S424A;V234A+S424A;V234A+L380A;V234A+L379A;V234A+D416A;V234A+S417A;V234A+Y89A;V234A+N151A;V234A+L379A+L380A;V234A+D416A+S417A;V234A+D416A+S424A;V234A+S417A+S424A;V234A+L380A+Y89D;V234A+L380A+Y89S;V234A+L380A+Y89H;V234A+L380A+Y89M;V234A+L380A+F409A;V234A+L380A+F409S;V234A+L380A+F409Q;V234A+L380A+F409L;V234A+L380A+S424F;V234A+L380A+S424Q;V234A+L380A+S424P;V234A+L380A+S424R;V234A+L380A+S424N;V234A+L380A+N86M;V234A+L380A+N86P;V234A+L380A+N86D;V234A+L380A+N86A;V234A+L380A+N86V;V234A+L380A+N86H;V234A+L380A+N86M+Y89D;V234A+L380A+N86M+Y89S;V234A+L380A+N86M+Y89G;V234A+L380A+N86M+Y89H;V234A+L380A+N86M+Y89F;V234A+L380A+N86M+Y89A;V234A+L380A+Y89D+F409H;V234A+L380A+Y89D+F409A;V234A+L380A+Y89D+F409P;V234A+L380A+Y89D+F409K;V234A+L380A+Y89D+F409G;V234A+L380A+Y89D+S424A;V234A+L380A+Y89D+S424V;V234A+L380A+Y89D+S424Y;V234A+L380A+Y89D+S424E;V234A+L380A+Y89D+S424Q;V234A+L380A+Y89D+N86H;V234A+L380A+Y89D+N86A;V234A+L380A+Y89D+N86H+Y85M;V234A+L380A+Y89D+N86H+Y85F;V234A+L380A+Y89D+N86H+Y85R;V234A+L380A+Y89D+N86H+Y85M+S417Q;V234A+L380A+Y89D+N86H+Y85M+S417F;V234A+L380A+Y89D+N86H+Y85M+S417I;V234A+L380A+Y89D+N86H+Y85M+S424I;V234A+L380A+Y89D+N86H+Y85M+P83C;V234A+L380A+Y89D+N86H+Y85M+P83A;V234A+L380A+Y89D+N86H+Y85M+P83S;V234A+L380A+Y89D+N86H+Y85M+P83G;V234A+L380A+Y89D+N86H+Y85M+T91M;V234A+L380A+Y89D+N86H+Y85M+T91A;V234A+L380A+Y89D+N86H+Y85M+T91V;V234A+L380A+Y89D+N86H+Y85M+T91N;V234A+L380A+Y89D+N86H+Y85M+T91I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83C;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83G;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83R;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90V;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417A;
V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A;
V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+A239S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y+F409L;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+G164S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+T452S;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+I311F;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315C+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+V327S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295M;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295F;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64A;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N+T402K;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+R77Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+N317Y;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+A433T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y。
上述氨基酸突变均在本申请实施例中进行试验探究,均具有转氨酶活性,相较于具有SEQ ID NO:1所示的氨基酸序列的母本,能够获得具有底物谱宽、能够催化合成大位阻手性胺化合物、酶活性高和/或耐受极端环境能力强的转氨酶突变体。
在本申请第二种典型的实施方式中,提供了一种DNA分子,该DNA分子编码上述转氨酶突变体。
在本申请第三种典型的实施方式中,提供了一种重组质粒,该重组质粒连接有上述DNA分子。
上述DNA能够编码上述转氨酶突变体,并能够连接在重组质粒上形成环状DNA。上述DNA和重组质粒均能在RNA聚合酶、核糖体、tRNA等的作用下,进行转录、翻译,获得上述转氨酶突变体。
在本申请第四种典型的实施方式中,提供了一种宿主细胞,该宿主细胞内转化有上述重组质粒。
在一种优选的实施例中,宿主细胞包括原核细胞;优选地,原核细胞包括大肠杆菌。
利用上述宿主细胞,能够在宿主细胞中进行重组质粒的复制,也能够将重组质粒上携带的DNA分子进行转录、翻译,获得大量转氨酶突变体。利用现有技术,对宿主细胞进行破碎蛋白纯化、破碎后粗酶催化或其他方式,能够获得转氨酶突变体,并进行后续对于胺类化合物的催化。该宿主细胞为非植物来源的宿主细胞。
在本申请第五种典型的实施方式中,提供了一种手性胺化合物的制备方法,该制备方法包括利用上述转氨酶突变体在氨基供体的作用下,对式I所示的酮类底物进行转氨基反应,制备获得手性胺化合物;Ar1选自第一取代芳基、第一未取代芳基、取代亚芳基或未取代亚芳基、取代杂亚芳基或未取代杂亚芳基;Ar2选自第二取代芳基、第二未取代芳基、取代环烷基、未取代环烷基、烷基或者亚烷基;R选自H、烷基、亚烷基或次烷基,亚烷基或次烷基的C原子数选自1~5,亚烷基或次烷基包括取代亚烷基或次烷基或未取代亚烷基或次烷基;当R选自亚烷基或次烷基时,亚烷基或次烷基与Ar1和/或Ar2连接成环;第一取代芳基、取代亚芳基、取代杂亚芳基、第二取代芳基或取代亚烷基或次烷基中的取代基各自独立选自卤素、羟基、氨基、甲基、乙基或-CH2CH2OH;取代杂亚芳基中的杂原子选自N、O或S。
利用上述制备方法,在提供的氨基供体的作用下,能够利用上述转氨酶突变体对式I所示的酮类化合物进行催化,将羰基催化为具有手性的氨基,获得手性胺化合物。上述转氨酶突变体,能够对于上述的原料酮类化合物,包括羰基旁边一侧带有大于甲基取代基的基团的酮类化合物,进行手性催化,合成大位阻手性胺。并且能够在高底物浓度、低酶量、极端环境等工业化生产条件下进行催化,能够提高生产效率,降低工业生产成本,减少工业三废排放。
在一种优选的实施例中,第一取代芳基、第二取代芳基或取代亚芳基的取代基各自独立选自卤素或-CH2CH2OH;优选地,取代基各自独立位于第一取代芳基、第二取代芳基或取代亚芳基的邻位、间位或对位中的任意一个或多个位置;优选地,卤素选自F、Cl或Br。
在一种优选的实施例中,Ar1选自第一取代芳基、取代亚芳基、第一未取代芳基或未取代亚芳基,Ar2选自第二未取代芳基,R选自未取代亚烷基,未取代亚烷基的C原子数选自1~5;未取代亚烷基与Ar1连接成环。
在一种优选的实施例中,Ar1选自未取代杂亚芳基,Ar2选自第二取代芳基,第二取代芳基的取代基选自卤素,R选自取代亚烷基,取代亚烷基的取代基选自羟基,取代亚烷基的C原子数选自1~5,取代亚烷基与Ar1连接成环。
在一种优选的实施例中,Ar1选自取代亚芳基,取代亚芳基的取代基为卤素,Ar2选自取代环烷基或未取代环烷基,取代环烷基或未取代环烷基的C原子数选自3~8;R选自H。
在一种优选的实施例中,Ar1选自第一未取代芳基,Ar2选自第二取代芳基、第二未取代芳基、取代环烷基或未取代环烷基;R选自H、甲基、亚甲基或次甲基。
在一种优选的实施例中,Ar1选自环烷基或第一取代芳基,取代基选自羟基、甲基、乙基或-CH2CH2OH,Ar2选自第二未取代芳基或烷基;R选自H、甲基、亚甲基或次甲基。
在一种优选的实施例中,酮类底物选自
上述酮类底物的底物范围较广,在羰基旁边一侧有大于甲基取代基的基团,由于空间位阻的影响,利用现有技术中的转氨酶对上述底物进行手性催化难度较大。且现有技术中,即使有能够进行手性催化的转氨酶,在高浓度底物浓度、低酶量、极端环境等工业化生产中的,也难以进行工业化的大批量生产。利用上述转氨酶突变体,能够对上述酮类底物进行高效的手性催化,将羰基催化为手性氨基,得到具有立体选择性的手性胺类化合物。
在一种优选的实施例中,氨基供体包括但不限于异丙胺、异丙胺盐酸盐、丙氨酸、正丁胺或苯胺中的一种或多种。氨基供体可以在现有技术中常用的氨基供体中进行灵活选择或组合。
本申请中,发明人根据已经解析出的蛋白质的三维结构(PDB:4BA5),对筛出的突变体进行同源建模,根据模型结构对不同的大位阻酮化合物进行分子对接。分析对接结果,选择了18个活性中心附近有可能影响蛋白催化活性的残基,这些残基包括:L59,W60,F88,Y89,N151,Y153,F166,E168,V234,I262,L379,L380,F397,R405,D416,S417,C418,S424。对这些残基进行单点突变,全部突变成丙氨酸,进行“丙氨酸扫描”,观察这些改变对蛋白功能的影响。
定点突变:是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组,也可以是质粒)中引入所需变化(通常是表征有利方向的变化),包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征,是基因研究工作中一种非常有用的手段。利用全质粒PCR引入定点突变的方法简单有效,是目前使用比较多的手段。其原理是,一对包含突变位点的引物(正、反向),和模版质粒退火后用聚合酶“循环延伸”,(所谓的循环延伸是指聚合酶按照模版延伸引物,一圈后回到引物5’端终止,再经过反复加热退火延伸的循环,这个反应区别于滚环扩增,不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。DpnI酶切延伸产物,由于原来的模版质粒来源于常规大肠杆菌,是经dam甲基化修饰的,对DpnI敏感而被切碎,而体外合成的带突变序列的质粒由于没有甲基化而不被切开,因此在随后的转化中得以成功转化,即可得到突变质粒的克隆。
定点突变菌经测序鉴定正确后,在25℃,0.2mM IPTG诱导过夜的条件下,诱导酮还原酶的表达。然后通过超声破碎细胞的方法获得粗酶,用于反应特性检测。经反应特性验证后,能使转氨酶催化特性明显改善的位点来自:Y89A,N151A,V234A,L379A,L380A,D416A,S417A,S424A。
然后,对有益的氨基酸位点进行组合,以获得性状更优的突变体。组合突变中双点突变的构建方法和单点突变的构建方法一样,采用全质粒PCR法构建。同时突变2个及以上位点的多点突变通过采用重叠延伸PCR扩增进行,获得含多点突变的突变基因,两端经限制性内切酶酶切后,连接到表达载体上,转化至大肠杆菌细胞内,涂布于含有100μg/mL氨苄青霉素的LB培养皿中,37℃培养过夜,获得组合突变体,测序鉴定。对获得的正确突变体进行验活,得到催化特性提高的突变体,用作下一轮饱和突变的模板。之后,根据分子对接模型,在底物通道和一段关键loop区的位置,又选择20个点进行饱和突变。
饱和突变是通过对目的蛋白的编码基因进行改造,短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变体的一种方法。此方法不仅是蛋白质定向改造的强有力工具,而且是蛋白质结构-功能关系研究的重要手段。饱和突变往往能获得比单点突变更为理想的进化体。而对于定点突变方法不能解决的这些问题,恰恰是饱和突变方法所擅长的独特之处。饱和突变获得的突变体经测序鉴定,分别测试其对不同底物的活性和高温下的耐受性。
获得活性和耐受性大幅提高的转氨酶突变体后,使用易错PCR的方法对其进行随机突变,构建高质量的突变体库,开发合适的高通量筛选方法,对文库进行筛选,获得活性进一步提高的突变体。
易错PCR:意为易错条件下的PCR,即容易使复制出的DNA序列出现错误的PCR技术,又称错配PCR或倾向错误PCR。具体是指通过利用低保真度TaqDNA聚合酶和改变PCR反应条件,降低DNA复制的保真度,在新DNA链合成过程中增加碱基错配,从而使扩增产物出现较多点突变的一种体外诱导DNA序列变异的方法。
开发如下的高通量筛选方法对易错突变体库进行筛选:
1、突变体培养:96孔板每孔加入300μL LB培养基,将琼脂平板上的单克隆接种到深孔96孔板中,37℃、200rpm过夜培养;利用Qpix转接过夜培养的菌液,至另一块每孔加入800μL LB培养基96孔板中,37℃、200rpm,培养5h后,待96孔板菌液OD600达到0.6-0.9,再次利用Qpix向96孔板中加入IPTG溶液,使孔板中IPTG终浓度为0.1mM,25℃、200rpm过夜诱导约16h;4000rpm,离心5min,弃上清,用全细胞进行反应。
2、96孔板高通量筛选体系:
将8μL PLP母液(1mg/mL)、3.5μL的6M异丙胺盐酸盐(20eq),0.1M Tris-Cl 9.0补到100μL混匀,将混样分装至每孔装有菌泥的96孔板中,最后加入底物母液(0.3mg底物溶于15μL DMSO总体积15%中),混匀,50℃恒温摇床700rpm,反应18h。
易错PCR完成之后,发明人已经获得了小试水平下活性和耐受性都大幅提高的突变体。但是在工业应用时仍然存在酶量较大,耐受性不够的问题。之后,又运用上述相同的突变方法,进行了多轮饱和突变和组合突变的进化,得到了一系列转氨酶突变体,结果发现这些转氨酶突变体对多种酮类底物的催化效率进一步得到极大提升,能够用于多种手性胺化合物的合成,尤其是大位阻手性胺化合物的高效合成,对极端环境的耐受性也有所提高。
下面将结合具体的实施例来进一步详细解释本申请的有益效果。
实施例1
利用上述方法,在母本SEQ ID NO:1的基础上进行定点突变。具体突变位点见表1,并按照如下反应条件对突变体的催化活性进行检测:
1mL反应体系包括2mg底物1或底物2或底物3或底物4,1mg PLP,2mg异丙胺盐酸盐,400μL粗酶液(由200mg湿菌泥制得),pH8.0 100mM磷酸盐buffer,50℃反应42h。上述湿菌泥由相应突变体的大肠杆菌发酵液经过离心所得,粗酶液由得到的湿菌泥加pH8.0100mM磷酸盐buffer,经超声或者匀浆机均质破壁,再经过浓缩得到。
检测结果见表1。
表1:
注:上表中,0代表转化率<1%,+代表转化率大于等于1%且小于5%,++代表转化率大于等于5%且小于等于10%。
实施例2
在实施例1的基础上进行组合突变,并按照与实施例1相同的反应条件对组合突变进行活性筛选,结果见表2。
表2:
注:上表中,0代表转化率<1%,+代表转化率大于等于1%且小于5%,++代表转化率大于等于5%且小于等于10%,+++代表转化率大于等于10%且小于15%,++++代表转化率大于等于15%且小于20%。
实施例3
在实施例2的基础上进行多轮饱和突变,并按照如下反应条件对突变体的催化活性进行检测:
1mL反应体系包括4mg底物1或底物2或底物3或底物4,1mg PLP,2mg异丙胺盐酸盐,400μL粗酶液(由200mg湿菌泥制得),pH8.0 100mM磷酸盐buffer,50℃反应18h。结果见表3。
表3:
注:上表中,0代表转化率<1%,+代表转化率大于等于1%且小于5%,++代表转化率大于等于5%且小于10%,+++代表转化率大于等于10%且小于15%,++++代表转化率大于等于15%且小于20%,+++++代表转化率大于等于20%且小于40%,++++++代表转化率大于等于40%。
实施例4
选择实施例1、2和3中的部分突变体,按照如下反应条件对突变体的耐受性进行检测:
1mL反应体系包括4mg底物1或底物2或底物3或底物4,1mg PLP,2mg异丙胺盐酸盐,经过在70℃下处理1h后的400μL粗酶液(由200mg湿菌泥制得),pH8.0 100mM磷酸盐buffer,50℃反应18h。结果见表4。
表4:
注:上表中,+代表相对残余活力大于等于10%且小于30%,++代表相对残余活力大于等于30%且小于40%,+++代表相对残余活力大于等于40%且小于50%,++++代表相对残余活力大于等于50%且小于60%。
相对残余活力是指经高温、碱性、有机溶剂等极端条件适量处理后的酶液测定的酶活力,与未经过极端环境下处理的酶液在最适条件下的酶活力的百分比。在相同的处理条件下,相对残余活力高,说明该酶在此条件下的稳定越高。
实施例5
在实施例3的基础上进行多轮易错PCR和组合突变,并按照如下反应条件对突变体的催化活性进行检测:
1mL反应体系包括10mg底物1或底物2或底物3或底物4,1mg PLP,2mg异丙胺盐酸盐,100μL粗酶液(由50mg湿菌泥制得),pH8.0 100mM磷酸盐buffer,50℃反应18h。结果见表5。
表5:
注:上表中,0代表转化率<1%,+代表转化率大于等于1%且小于5%,++代表转化率大于等于5%且小于10%,+++代表转化率大于等于10%且小于15%,++++代表转化率大于等于15%且小于20%,+++++代表转化率大于等于20%且小于40%,++++++代表转化率大于等于40%。
实施例6
在实施例5的基础上进行多轮饱和突变和组合突变,并按照如下反应条件对突变体的催化活性进行检测:
1mL反应体系包括50mg底物1或底物2或底物3或底物4,1mg PLP,10mg异丙胺盐酸盐,50μL粗酶液(由25mg湿菌泥制得),pH8.0 100mM磷酸盐buffer,45℃反应18h。结果见表6。
表6:
注:上表中,0代表转化率<1%,+代表转化率大于等于1%且小于5%,++代表转化率大于等于5%且小于10%,+++代表转化率大于等于10%且小于20%,++++代表转化率大于等于20%且小于40%,+++++代表转化率大于等于40%且小于60%,++++++代表转化率大于等于60%。
实施例7
选择实施例5和6中的部分突变体,按照如下反应条件对突变体的耐受性进行检测:
1mL反应体系包括10mg底物1或底物2或底物3或底物4,1mg PLP,2mg异丙胺盐酸盐,经过在70℃下处理1h后的100μL粗酶液(由50mg湿菌泥制得),pH8.0 100mM磷酸盐buffer,50℃反应18h。结果见表7。
表7:
注:上表中,+代表相对残余活力大于等于50%且小于60%,++代表相对残余活力大于等于60%且小于70%,+++代表相对残余活力大于等于70%。
实施例8
室温向250mL四口瓶内加入50mL 100mmol/L磷酸盐缓冲液(5vol),20mL 5mol/L异丙胺盐酸盐溶液(2vol),调pH=8.5~9.0。再加入0.1g磷酸吡哆醛(1wt%),10g(底物1),搅拌均匀,再加入SEQ ID NO:1基础上突变的的转氨酶突变体(V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L)的酶液2mL(0.1wt,0.5g/mL)调pH=8.5~9.0。升温至45℃搅拌反应过夜。反应完全后,将体系调酸至pH=2-3,变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH=12,再用50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T<40℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测,纯度>99%,de值>99%,收率87%
实施例9
室温向250mL四口瓶内加入50mL 100mmol/L磷酸盐缓冲液(5vol),20mL 5mol/L异丙胺盐酸盐溶液(2vol),调pH=8.5~9.0。再加入0.1g磷酸吡哆醛(1wt%),10g搅拌均匀,再加入SEQ ID NO:1基础上突变的转氨酶突变体(V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L)的酶液2mL(0.1wt,0.5g/mL)调pH=8.5~9.0。升温至45℃搅拌反应过夜。反应完全后,将体系调酸至pH=2-3,变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH=12,再用50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T<40℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测,纯度>98%,de值>99%,收率84%.
实施例10
室温向250mL四口瓶内加入50mL 100mmol/L磷酸盐缓冲液(5vol),20mL 5mol/L异丙胺盐酸盐溶液(2vol),调pH=8.5~9.0。再加入0.1g磷酸吡哆醛(1wt%),10g(底物3),搅拌均匀,再加入SEQ ID NO:1基础上突变的CvTA的转氨酶突变体(V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L)的酶液2mL(0.1wt,0.5g/mL)调pH=8.5~9.0。升温至45℃搅拌反应过夜。反应完全后,将体系调酸至pH=2-3,变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH=12,再用50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T<40℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测,纯度>98%,ee值>99%,收率82%。
实施例11
室温向250mL四口瓶内加入50mL 100mmol/L磷酸盐缓冲液(5vol),20mL 5mol/L异丙胺盐酸盐溶液(2vol),调pH=8.5~9.0。再加入0.1g磷酸吡哆醛(1wt%),10g搅拌均匀,再加入SEQ ID NO:1基础上突变的转氨酶突变体(V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L)的酶液2mL(0.1wt,0.5g/mL)调pH=8.5~9.0。升温至45℃搅拌反应过夜。反应完全后,将体系调酸至pH=2-3,变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH=12,再用50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T<40℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测,纯度>99%,de值>99%,收率91%。
实施例12
采用SEQ ID NO:1基础上突变的转氨酶突变体(V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364)的酶液,参照实施例6至实施例9的催化合成步骤,对底物5-16进行催化反应,结果如表8:
表8:
底物 | 纯度 | de值 | 收率 |
5 | >99% | >99% | 87% |
6 | >98% | >99% | 87% |
7 | >98% | >99% | 82% |
8 | >99% | >99% | 90% |
9 | >98% | >99% | 86% |
10 | >99% | >99% | 84% |
11 | >99% | >99% | 89% |
12 | >99% | >99% | 85% |
13 | >98% | >99% | 86% |
14 | >99% | >99% | 90% |
15 | >99% | >99% | 86% |
16 | >99% | >99% | 83% |
从以上的描述中,可以看出,本发明上述的实施例实现了如下技术效果:本申请在获得的具有优良活性的转氨酶母本的基础上,通过探究转氨酶的活性位点,发现了多个能够提高转氨酶活性和/或耐受性的位点,通过对于一个或多个位点的组合探究,获得多种具有优秀性能的转氨酶突变体,上述转氨酶突变体具有底物谱宽、能够催化合成大位阻手性胺化合物、酶活性高、耐受极端环境能力强等优点,能够满足工艺生产的需求。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 凯莱英生命科学技术(天津)有限公司
<120> 转氨酶突变体及手性胺化合物的制备方法
<130> PN173418KLY
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 459
<212> PRT
<213> Artificial Sequence
<220>
<221> VARIANT
<222> (1)..(459)
<223> 来源于Chromobaterium violaceum的氨基酸突变体
<400> 1
Met Gln Lys Gln Arg Thr Cys Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Cys Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Ala Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Ala Tyr Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Phe Lys Glu Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Leu Leu Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Asp
405 410 415
Ser Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
<210> 2
<211> 459
<212> PRT
<213> Artificial Sequence
<220>
<221> VARIANT
<222> (234)..(234)
<223> V234A
<400> 2
Met Gln Lys Gln Arg Thr Cys Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Cys Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Ala Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Ala Tyr Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Phe Lys Glu Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Ala Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Leu Leu Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Asp
405 410 415
Ser Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
<210> 3
<211> 459
<212> PRT
<213> Artificial Sequence
<220>
<221> VARIANT
<222> (234)..(234)
<223> V234C
<400> 3
Met Gln Lys Gln Arg Thr Cys Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Cys Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Ala Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Ala Tyr Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Phe Lys Glu Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Cys Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Leu Leu Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Asp
405 410 415
Ser Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
Claims (17)
1.一种转氨酶突变体,其特征在于,所述转氨酶突变体由SEQ ID NO:1发生氨基酸突变得到,所述氨基酸突变选自如下任意一种:
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+I311F;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315C+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+V327S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295M;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295F;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64A;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N+T402K;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+R77Q;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+N317Y;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+A433T;
V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y。
2.一种DNA分子,其特征在于,所述DNA分子编码权利要求1所述的转氨酶突变体。
3.一种重组质粒,其特征在于,所述重组质粒连接有权利要求2所述的DNA分子。
4.一种宿主细胞,其特征在于,所述宿主细胞内转化有权利要求3所述的重组质粒。
5.根据权利要求4所述的宿主细胞,其特征在于,所述宿主细胞包括原核细胞。
6.根据权利要求5所述的宿主细胞,其特征在于,所述原核细胞包括大肠杆菌。
7.一种手性胺化合物的制备方法,其特征在于,所述制备方法包括利用权利要求1所述的转氨酶突变体在氨基供体的作用下,对式I所示的酮类底物进行转氨基反应,制备获得所述手性胺化合物,
Ar1选自第一取代芳基、第一未取代芳基、取代亚芳基或未取代亚芳基、取代杂亚芳基或未取代杂亚芳基;
Ar2选自第二取代芳基、第二未取代芳基、取代环烷基、未取代环烷基、烷基或者亚烷基;
R选自H、烷基、亚烷基或次烷基,所述亚烷基或次烷基的C原子数选自1~5,所述亚烷基或次烷基包括取代亚烷基或次烷基或未取代亚烷基或次烷基;
当R选自亚烷基或次烷基时,所述亚烷基或次烷基与所述Ar1和/或所述Ar2连接成环;
所述第一取代芳基、所述取代亚芳基、所述取代杂亚芳基、所述第二取代芳基或所述取代亚烷基或次烷基中的取代基各自独立选自卤素、羟基、氨基、甲基、乙基或-CH2CH2OH;
所述取代杂亚芳基中的杂原子选自N、O或S。
8.根据权利要求7所述的制备方法,其特征在于,所述第一取代芳基、所述第二取代芳基或所述取代亚芳基的所述取代基各自独立选自所述卤素或所述-CH2CH2OH。
9.根据权利要求8所述的制备方法,其特征在于,所述取代基各自独立位于所述第一取代芳基、所述第二取代芳基或所述取代亚芳基的邻位、间位或对位中的任意一个或多个位置。
10.根据权利要求9所述的制备方法,其特征在于,所述卤素选自F、Cl或Br。
11.根据权利要求10所述的制备方法,其特征在于,
所述Ar1选自所述第一取代芳基、所述取代亚芳基、所述第一未取代芳基或所述未取代亚芳基,
所述Ar2选自所述第二未取代芳基,
所述R选自所述未取代亚烷基,所述未取代亚烷基的C原子数选自1~5;
所述未取代亚烷基与所述Ar1连接成环。
12.根据权利要求10所述的制备方法,其特征在于,
所述Ar1选自所述未取代杂亚芳基,
所述Ar2选自所述第二取代芳基,所述第二取代芳基的所述取代基选自所述卤素,
所述R选自所述取代亚烷基,所述取代亚烷基的取代基选自所述羟基,所述取代亚烷基的C原子数选自1~5,
所述取代亚烷基与所述Ar1连接成环。
13.根据权利要求10所述的制备方法,其特征在于,
所述Ar1选自所述取代亚芳基,所述取代亚芳基的所述取代基为所述卤素,
所述Ar2选自所述取代环烷基或所述未取代环烷基,所述取代环烷基或所述未取代环烷基的C原子数选自3~8;
所述R选自所述H。
14.根据权利要求10所述的制备方法,其特征在于,
所述Ar1选自所述第一未取代芳基;
所述Ar2选自所述第二取代芳基、所述第二未取代芳基、所述取代环烷基或所述未取代环烷基;
所述R选自所述H、甲基、亚甲基或次甲基。
15.根据权利要求10所述的制备方法,其特征在于,
所述Ar1选自环烷基或所述第一取代芳基,所述取代基选自所述羟基、所述甲基、所述乙基或所述-CH2CH2OH,
所述Ar2选自所述第二未取代芳基或所述烷基;
所述R选自所述H、甲基、亚甲基或次甲基。
16.根据权利要求7至15中任一项所述的制备方法,其特征在于,所述酮类底物选自
17.根据权利要求16所述的制备方法,其特征在于,所述氨基供体包括异丙胺、异丙胺盐酸盐、丙氨酸、正丁胺或苯胺。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210707289.5A CN114875006B (zh) | 2022-06-21 | 2022-06-21 | 转氨酶突变体及手性胺化合物的制备方法 |
PCT/CN2022/109140 WO2023245815A1 (zh) | 2022-06-21 | 2022-07-29 | 转氨酶突变体及手性胺化合物的制备方法 |
EP22947559.5A EP4417690A1 (en) | 2022-06-21 | 2022-07-29 | Transaminase mutant and preparation method for chiral amine compound |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210707289.5A CN114875006B (zh) | 2022-06-21 | 2022-06-21 | 转氨酶突变体及手性胺化合物的制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114875006A CN114875006A (zh) | 2022-08-09 |
CN114875006B true CN114875006B (zh) | 2024-03-12 |
Family
ID=82681323
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210707289.5A Active CN114875006B (zh) | 2022-06-21 | 2022-06-21 | 转氨酶突变体及手性胺化合物的制备方法 |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP4417690A1 (zh) |
CN (1) | CN114875006B (zh) |
WO (1) | WO2023245815A1 (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116083385A (zh) * | 2022-12-02 | 2023-05-09 | 凯莱英生命科学技术(天津)有限公司 | 转氨酶突变体及应用 |
CN118028262B (zh) * | 2024-04-11 | 2024-08-30 | 天津凯莱英生物科技有限公司 | 转氨酶突变体及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112458123A (zh) * | 2021-02-04 | 2021-03-09 | 凯莱英医药集团(天津)股份有限公司 | 手性胺的合成方法 |
CN112501145A (zh) * | 2021-02-04 | 2021-03-16 | 凯莱英医药集团(天津)股份有限公司 | 转氨酶突变体及其应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20230227797A1 (en) * | 2019-10-28 | 2023-07-20 | Asymchem Laboratories (Tianjin) Co., Ltd. | Transaminase mutant and use thereof |
-
2022
- 2022-06-21 CN CN202210707289.5A patent/CN114875006B/zh active Active
- 2022-07-29 EP EP22947559.5A patent/EP4417690A1/en active Pending
- 2022-07-29 WO PCT/CN2022/109140 patent/WO2023245815A1/zh active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112458123A (zh) * | 2021-02-04 | 2021-03-09 | 凯莱英医药集团(天津)股份有限公司 | 手性胺的合成方法 |
CN112501145A (zh) * | 2021-02-04 | 2021-03-16 | 凯莱英医药集团(天津)股份有限公司 | 转氨酶突变体及其应用 |
CN112980810A (zh) * | 2021-02-04 | 2021-06-18 | 凯莱英医药集团(天津)股份有限公司 | 转氨酶突变体及其应用 |
Non-Patent Citations (1)
Title |
---|
aspartate aminotransferase family protein [Chromobacterium violaceum].Genbank:WP_081573061.1.2017,全文. * |
Also Published As
Publication number | Publication date |
---|---|
EP4417690A1 (en) | 2024-08-21 |
WO2023245815A1 (zh) | 2023-12-28 |
CN114875006A (zh) | 2022-08-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN114875006B (zh) | 转氨酶突变体及手性胺化合物的制备方法 | |
CN108048419B (zh) | 转氨酶突变体及其应用 | |
KR101869432B1 (ko) | 케톤에 대한 활성이 증대된 오메가 트랜스아미네이즈의 변이체 및 이것을 이용한 광학활성 아민의 제조방법 | |
CN116083385A (zh) | 转氨酶突变体及应用 | |
WO2019153183A1 (zh) | 一种单加氧酶突变体及其制备方法和应用 | |
CN110592042A (zh) | 转氨酶突变体及其应用 | |
CN110205310B (zh) | 转氨酶突变体及其应用 | |
CN108795893B (zh) | 一种氨基酸脱氢酶突变体及其制备方法和应用 | |
US11952575B2 (en) | Transaminase mutant and use thereof | |
CN115772514A (zh) | 一种腈水合酶底物通道氨基酸基序的改造用于肉桂酰胺的制备 | |
CN116855471B (zh) | 一种嘌呤核苷磷酸化酶突变体及其应用 | |
JP7498244B2 (ja) | モノオキシゲナーゼ突然変異体及びその使用 | |
JP2022533106A (ja) | アミノ基転移酵素突然変異体及びその応用 | |
CN116083406A (zh) | 酰胺酶突变体及其应用 | |
CN113817699B (zh) | 转氨酶突变体及其应用 | |
WO2018127143A1 (zh) | 一种高活性s-氰醇裂解酶及其应用 | |
CN111254134A (zh) | 腈基水解酶突变体及其在(s)-单腈单酸合成中的应用 | |
JP7241845B2 (ja) | トランスアミナーゼ突然変異体及びその使用 | |
CN110938608A (zh) | 醛酮还原酶突变体、编码基因及其在合成(s)-tcpe中的应用 | |
CN115927220A (zh) | 单加氧酶突变体及其应用 | |
CN111454933B (zh) | D-氨甲酰水解酶突变体及其在d-芳香族氨基酸合成中的应用 | |
CN118638755A (zh) | 亚胺还原酶突变体以及手性肼类化合物的制备方法 | |
WO2024044987A1 (zh) | 单加氧酶突变体及其应用 | |
CN116064449A (zh) | 一种转氨酶突变体及其应用 | |
CN117603935A (zh) | 一种ω-转氨酶突变体及其编码基因与应用 |
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 | ||
GR01 | Patent grant |