CN110144336B - 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法 - Google Patents
一种s-腺苷甲硫氨酸合成酶突变体及其制备方法 Download PDFInfo
- Publication number
- CN110144336B CN110144336B CN201910436026.3A CN201910436026A CN110144336B CN 110144336 B CN110144336 B CN 110144336B CN 201910436026 A CN201910436026 A CN 201910436026A CN 110144336 B CN110144336 B CN 110144336B
- Authority
- CN
- China
- Prior art keywords
- ala
- gly
- val
- leu
- asp
- 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
- 108010007784 Methionine adenosyltransferase Proteins 0.000 title abstract description 27
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 102000007357 Methionine adenosyltransferase Human genes 0.000 title abstract 3
- 150000001413 amino acids Chemical group 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 5
- 102000003960 Ligases Human genes 0.000 claims 4
- 108090000364 Ligases Proteins 0.000 claims 4
- 150000001875 compounds Chemical class 0.000 claims 1
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 abstract description 36
- 102000004190 Enzymes Human genes 0.000 abstract description 29
- 108090000790 Enzymes Proteins 0.000 abstract description 29
- 238000006243 chemical reaction Methods 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 19
- 238000000034 method Methods 0.000 abstract description 18
- 230000035772 mutation Effects 0.000 abstract description 15
- 238000009825 accumulation Methods 0.000 abstract description 14
- 239000000758 substrate Substances 0.000 abstract description 14
- 230000005764 inhibitory process Effects 0.000 abstract description 10
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 230000004048 modification Effects 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract description 2
- 102100026115 S-adenosylmethionine synthase isoform type-1 Human genes 0.000 description 36
- 229960001570 ademetionine Drugs 0.000 description 34
- 239000000047 product Substances 0.000 description 21
- 102200027762 rs199475643 Human genes 0.000 description 15
- 101710186227 S-adenosylmethionine synthase Proteins 0.000 description 14
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 12
- 229960004452 methionine Drugs 0.000 description 12
- 238000012408 PCR amplification Methods 0.000 description 11
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 10
- 229930195722 L-methionine Natural products 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 8
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 108010005233 alanylglutamic acid Proteins 0.000 description 8
- 108010078144 glutaminyl-glycine Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 239000007788 liquid Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000004474 valine Substances 0.000 description 5
- FXKNPWNXPQZLES-ZLUOBGJFSA-N Ala-Asn-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O FXKNPWNXPQZLES-ZLUOBGJFSA-N 0.000 description 4
- PBAMJJXWDQXOJA-FXQIFTODSA-N Ala-Asp-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PBAMJJXWDQXOJA-FXQIFTODSA-N 0.000 description 4
- YSMPVONNIWLJML-FXQIFTODSA-N Ala-Asp-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(O)=O YSMPVONNIWLJML-FXQIFTODSA-N 0.000 description 4
- XPBVBZPVNFIHOA-UVBJJODRSA-N Ala-Trp-Val Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 XPBVBZPVNFIHOA-UVBJJODRSA-N 0.000 description 4
- PGNNQOJOEGFAOR-KWQFWETISA-N Ala-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 PGNNQOJOEGFAOR-KWQFWETISA-N 0.000 description 4
- RWCLSUOSKWTXLA-FXQIFTODSA-N Arg-Asp-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RWCLSUOSKWTXLA-FXQIFTODSA-N 0.000 description 4
- FEZJJKXNPSEYEV-CIUDSAMLSA-N Arg-Gln-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FEZJJKXNPSEYEV-CIUDSAMLSA-N 0.000 description 4
- QAXCZGMLVICQKS-SRVKXCTJSA-N Arg-Glu-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N QAXCZGMLVICQKS-SRVKXCTJSA-N 0.000 description 4
- OHYQKYUTLIPFOX-ZPFDUUQYSA-N Arg-Glu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OHYQKYUTLIPFOX-ZPFDUUQYSA-N 0.000 description 4
- MSILNNHVVMMTHZ-UWVGGRQHSA-N Arg-His-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CN=CN1 MSILNNHVVMMTHZ-UWVGGRQHSA-N 0.000 description 4
- OWSMKCJUBAPHED-JYJNAYRXSA-N Arg-Pro-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OWSMKCJUBAPHED-JYJNAYRXSA-N 0.000 description 4
- GFFRWIJAFFMQGM-NUMRIWBASA-N Asn-Glu-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GFFRWIJAFFMQGM-NUMRIWBASA-N 0.000 description 4
- BCADFFUQHIMQAA-KKHAAJSZSA-N Asn-Thr-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BCADFFUQHIMQAA-KKHAAJSZSA-N 0.000 description 4
- VPPXTHJNTYDNFJ-CIUDSAMLSA-N Asp-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N VPPXTHJNTYDNFJ-CIUDSAMLSA-N 0.000 description 4
- VHQOCWWKXIOAQI-WDSKDSINSA-N Asp-Gln-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O VHQOCWWKXIOAQI-WDSKDSINSA-N 0.000 description 4
- UJGRZQYSNYTCAX-SRVKXCTJSA-N Asp-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UJGRZQYSNYTCAX-SRVKXCTJSA-N 0.000 description 4
- LIVXPXUVXFRWNY-CIUDSAMLSA-N Asp-Lys-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O LIVXPXUVXFRWNY-CIUDSAMLSA-N 0.000 description 4
- JDDYEZGPYBBPBN-JRQIVUDYSA-N Asp-Thr-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JDDYEZGPYBBPBN-JRQIVUDYSA-N 0.000 description 4
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 4
- RRIJEABIXPKSGP-FXQIFTODSA-N Cys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CS RRIJEABIXPKSGP-FXQIFTODSA-N 0.000 description 4
- CFQVGYWKSLKWFX-KBIXCLLPSA-N Cys-Glu-Ile Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CFQVGYWKSLKWFX-KBIXCLLPSA-N 0.000 description 4
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 4
- ZEEPYMXTJWIMSN-GUBZILKMSA-N Gln-Lys-Ser Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@@H](N)CCC(N)=O ZEEPYMXTJWIMSN-GUBZILKMSA-N 0.000 description 4
- FALJZCPMTGJOHX-SRVKXCTJSA-N Gln-Met-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O FALJZCPMTGJOHX-SRVKXCTJSA-N 0.000 description 4
- FITIQFSXXBKFFM-NRPADANISA-N Gln-Val-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FITIQFSXXBKFFM-NRPADANISA-N 0.000 description 4
- CLROYXHHUZELFX-FXQIFTODSA-N Glu-Gln-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O CLROYXHHUZELFX-FXQIFTODSA-N 0.000 description 4
- PVBBEKPHARMPHX-DCAQKATOSA-N Glu-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCC(O)=O PVBBEKPHARMPHX-DCAQKATOSA-N 0.000 description 4
- CAVMESABQIKFKT-IUCAKERBSA-N Glu-Gly-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)N CAVMESABQIKFKT-IUCAKERBSA-N 0.000 description 4
- LGYCLOCORAEQSZ-PEFMBERDSA-N Glu-Ile-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O LGYCLOCORAEQSZ-PEFMBERDSA-N 0.000 description 4
- ZCOJVESMNGBGLF-GRLWGSQLSA-N Glu-Ile-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZCOJVESMNGBGLF-GRLWGSQLSA-N 0.000 description 4
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 4
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 4
- VHPVBPCCWVDGJL-IRIUXVKKSA-N Glu-Thr-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VHPVBPCCWVDGJL-IRIUXVKKSA-N 0.000 description 4
- HGJREIGJLUQBTJ-SZMVWBNQSA-N Glu-Trp-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(O)=O HGJREIGJLUQBTJ-SZMVWBNQSA-N 0.000 description 4
- MLILEEIVMRUYBX-NHCYSSNCSA-N Glu-Val-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O MLILEEIVMRUYBX-NHCYSSNCSA-N 0.000 description 4
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 4
- KFMBRBPXHVMDFN-UWVGGRQHSA-N Gly-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCNC(N)=N KFMBRBPXHVMDFN-UWVGGRQHSA-N 0.000 description 4
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 4
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 4
- QPCVIQJVRGXUSA-LURJTMIESA-N Gly-Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QPCVIQJVRGXUSA-LURJTMIESA-N 0.000 description 4
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 4
- UTYGDAHJBBDPBA-BYULHYEWSA-N Gly-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN UTYGDAHJBBDPBA-BYULHYEWSA-N 0.000 description 4
- 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 4
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 4
- FHQRLHFYVZAQHU-IUCAKERBSA-N Gly-Lys-Gln Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O FHQRLHFYVZAQHU-IUCAKERBSA-N 0.000 description 4
- FJWSJWACLMTDMI-WPRPVWTQSA-N Gly-Met-Val Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(O)=O FJWSJWACLMTDMI-WPRPVWTQSA-N 0.000 description 4
- GAFKBWKVXNERFA-QWRGUYRKSA-N Gly-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 GAFKBWKVXNERFA-QWRGUYRKSA-N 0.000 description 4
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 4
- UIQGJYUEQDOODF-KWQFWETISA-N Gly-Tyr-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 UIQGJYUEQDOODF-KWQFWETISA-N 0.000 description 4
- DUAWRXXTOQOECJ-JSGCOSHPSA-N Gly-Tyr-Val Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O DUAWRXXTOQOECJ-JSGCOSHPSA-N 0.000 description 4
- DNVDEMWIYLVIQU-RCOVLWMOSA-N Gly-Val-Asp Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O DNVDEMWIYLVIQU-RCOVLWMOSA-N 0.000 description 4
- ZSKJIISDJXJQPV-BZSNNMDCSA-N His-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 ZSKJIISDJXJQPV-BZSNNMDCSA-N 0.000 description 4
- 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 4
- WCHONUZTYDQMBY-PYJNHQTQSA-N His-Pro-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WCHONUZTYDQMBY-PYJNHQTQSA-N 0.000 description 4
- UKTUOMWSJPXODT-GUDRVLHUSA-N Ile-Asn-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N UKTUOMWSJPXODT-GUDRVLHUSA-N 0.000 description 4
- QZZIBQZLWBOOJH-PEDHHIEDSA-N Ile-Ile-Val Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)O QZZIBQZLWBOOJH-PEDHHIEDSA-N 0.000 description 4
- SNHYFFQZRFIRHO-CYDGBPFRSA-N Ile-Met-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)O)N SNHYFFQZRFIRHO-CYDGBPFRSA-N 0.000 description 4
- PZWBBXHHUSIGKH-OSUNSFLBSA-N Ile-Thr-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PZWBBXHHUSIGKH-OSUNSFLBSA-N 0.000 description 4
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 4
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 4
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 4
- KVMULWOHPPMHHE-DCAQKATOSA-N Leu-Glu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KVMULWOHPPMHHE-DCAQKATOSA-N 0.000 description 4
- MJTOYIHCKVQICL-ULQDDVLXSA-N Leu-Met-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N MJTOYIHCKVQICL-ULQDDVLXSA-N 0.000 description 4
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 4
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 4
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 4
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 4
- AAKRWBIIGKPOKQ-ONGXEEELSA-N Leu-Val-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AAKRWBIIGKPOKQ-ONGXEEELSA-N 0.000 description 4
- HQVDJTYKCMIWJP-YUMQZZPRSA-N Lys-Asn-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O HQVDJTYKCMIWJP-YUMQZZPRSA-N 0.000 description 4
- YEIYAQQKADPIBJ-GARJFASQSA-N Lys-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)N)C(=O)O YEIYAQQKADPIBJ-GARJFASQSA-N 0.000 description 4
- WGLAORUKDGRINI-WDCWCFNPSA-N Lys-Glu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WGLAORUKDGRINI-WDCWCFNPSA-N 0.000 description 4
- WAIHHELKYSFIQN-XUXIUFHCSA-N Lys-Ile-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O WAIHHELKYSFIQN-XUXIUFHCSA-N 0.000 description 4
- QBHGXFQJFPWJIH-XUXIUFHCSA-N Lys-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN QBHGXFQJFPWJIH-XUXIUFHCSA-N 0.000 description 4
- QAHFGYLFLVGBNW-DCAQKATOSA-N Met-Ala-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN QAHFGYLFLVGBNW-DCAQKATOSA-N 0.000 description 4
- DGNZGCQSVGGYJS-BQBZGAKWSA-N Met-Gly-Asp Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O DGNZGCQSVGGYJS-BQBZGAKWSA-N 0.000 description 4
- VQILILSLEFDECU-GUBZILKMSA-N Met-Pro-Ala Chemical compound [H]N[C@@H](CCSC)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O VQILILSLEFDECU-GUBZILKMSA-N 0.000 description 4
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 4
- KAJLHCWRWDSROH-BZSNNMDCSA-N Phe-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=CC=C1 KAJLHCWRWDSROH-BZSNNMDCSA-N 0.000 description 4
- BSJCSHIAMSGQGN-BVSLBCMMSA-N Phe-Pro-Trp Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O BSJCSHIAMSGQGN-BVSLBCMMSA-N 0.000 description 4
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 4
- BQMFWUKNOCJDNV-HJWJTTGWSA-N Phe-Val-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BQMFWUKNOCJDNV-HJWJTTGWSA-N 0.000 description 4
- KLSOMAFWRISSNI-OSUNSFLBSA-N Pro-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 KLSOMAFWRISSNI-OSUNSFLBSA-N 0.000 description 4
- OFGUOWQVEGTVNU-DCAQKATOSA-N Pro-Lys-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OFGUOWQVEGTVNU-DCAQKATOSA-N 0.000 description 4
- RMJZWERKFFNNNS-XGEHTFHBSA-N Pro-Thr-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O RMJZWERKFFNNNS-XGEHTFHBSA-N 0.000 description 4
- DBIDZNUXSLXVRG-FXQIFTODSA-N Ser-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N DBIDZNUXSLXVRG-FXQIFTODSA-N 0.000 description 4
- HVKMTOIAYDOJPL-NRPADANISA-N Ser-Gln-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVKMTOIAYDOJPL-NRPADANISA-N 0.000 description 4
- PMCMLDNPAZUYGI-DCAQKATOSA-N Ser-Lys-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMCMLDNPAZUYGI-DCAQKATOSA-N 0.000 description 4
- PJIQEIFXZPCWOJ-FXQIFTODSA-N Ser-Pro-Asp Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O PJIQEIFXZPCWOJ-FXQIFTODSA-N 0.000 description 4
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 4
- GUZGCDIZVGODML-NKIYYHGXSA-N Thr-Gln-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O GUZGCDIZVGODML-NKIYYHGXSA-N 0.000 description 4
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 4
- MEJHFIOYJHTWMK-VOAKCMCISA-N Thr-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)O MEJHFIOYJHTWMK-VOAKCMCISA-N 0.000 description 4
- NCXVJIQMWSGRHY-KXNHARMFSA-N Thr-Leu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O NCXVJIQMWSGRHY-KXNHARMFSA-N 0.000 description 4
- QNCFWHZVRNXAKW-OEAJRASXSA-N Thr-Lys-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QNCFWHZVRNXAKW-OEAJRASXSA-N 0.000 description 4
- PZSDPRBZINDEJV-HTUGSXCWSA-N Thr-Phe-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O PZSDPRBZINDEJV-HTUGSXCWSA-N 0.000 description 4
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 4
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 4
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 4
- SDNVRAKIJVKAGS-LKTVYLICSA-N Tyr-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N SDNVRAKIJVKAGS-LKTVYLICSA-N 0.000 description 4
- DXYWRYQRKPIGGU-BPNCWPANSA-N Tyr-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DXYWRYQRKPIGGU-BPNCWPANSA-N 0.000 description 4
- BEIGSKUPTIFYRZ-SRVKXCTJSA-N Tyr-Asp-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O BEIGSKUPTIFYRZ-SRVKXCTJSA-N 0.000 description 4
- WOCYUGQDXPTQPY-FXQIFTODSA-N Val-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N WOCYUGQDXPTQPY-FXQIFTODSA-N 0.000 description 4
- 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 4
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 4
- XPKCFQZDQGVJCX-RHYQMDGZSA-N Val-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N)O XPKCFQZDQGVJCX-RHYQMDGZSA-N 0.000 description 4
- VENKIVFKIPGEJN-NHCYSSNCSA-N Val-Met-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N VENKIVFKIPGEJN-NHCYSSNCSA-N 0.000 description 4
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 108010044940 alanylglutamine Proteins 0.000 description 4
- 108010087924 alanylproline Proteins 0.000 description 4
- 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 4
- 108010062796 arginyllysine Proteins 0.000 description 4
- 108010093581 aspartyl-proline Proteins 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 108010049041 glutamylalanine Proteins 0.000 description 4
- HPAIKDPJURGQLN-UHFFFAOYSA-N glycyl-L-histidyl-L-phenylalanine Natural products C=1C=CC=CC=1CC(C(O)=O)NC(=O)C(NC(=O)CN)CC1=CN=CN1 HPAIKDPJURGQLN-UHFFFAOYSA-N 0.000 description 4
- 108010075431 glycyl-alanyl-phenylalanine Proteins 0.000 description 4
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 4
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 4
- 108010089804 glycyl-threonine Proteins 0.000 description 4
- 108010048994 glycyl-tyrosyl-alanine Proteins 0.000 description 4
- 108010015792 glycyllysine Proteins 0.000 description 4
- 108010092114 histidylphenylalanine Proteins 0.000 description 4
- 108010018006 histidylserine Proteins 0.000 description 4
- 229960000310 isoleucine Drugs 0.000 description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 4
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 108010034529 leucyl-lysine Proteins 0.000 description 4
- 108010000761 leucylarginine Proteins 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 108010005942 methionylglycine Proteins 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 108010061238 threonyl-glycine Proteins 0.000 description 4
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 4
- PJLLMGWWINYQPB-PEFMBERDSA-N Ile-Asn-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PJLLMGWWINYQPB-PEFMBERDSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101150021948 SAM2 gene Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- SZTTYWIUCGSURQ-AUTRQRHGSA-N Val-Glu-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SZTTYWIUCGSURQ-AUTRQRHGSA-N 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 101150095438 metK gene Proteins 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- KVCGISUBCHHTDD-UHFFFAOYSA-M sodium;4-methylbenzenesulfonate Chemical compound [Na+].CC1=CC=C(S([O-])(=O)=O)C=C1 KVCGISUBCHHTDD-UHFFFAOYSA-M 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 2
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000006957 competitive inhibition Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 230000006959 non-competitive inhibition Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RMFRFTSSEHRKKW-UHFFFAOYSA-N 1,2-bis(diisopropylphosphino)ethane Chemical compound CC(C)P(C(C)C)CCP(C(C)C)C(C)C RMFRFTSSEHRKKW-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 1
- PNTWNAXGBOZMBO-MNXVOIDGSA-N Ile-Lys-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PNTWNAXGBOZMBO-MNXVOIDGSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 101000990108 Streptomyces spectabilis S-adenosylmethionine synthase Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 230000002905 effect on arthritis Effects 0.000 description 1
- 230000002898 effect on depression Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229940002712 malachite green oxalate Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- KNVAYBMMCPLDOZ-UHFFFAOYSA-N propan-2-yl 12-hydroxyoctadecanoate Chemical compound CCCCCCC(O)CCCCCCCCCCC(=O)OC(C)C KNVAYBMMCPLDOZ-UHFFFAOYSA-N 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940005657 pyrophosphoric acid Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 102200128633 rs104893843 Human genes 0.000 description 1
- 102200057424 rs138789658 Human genes 0.000 description 1
- 102200076705 rs373802463 Human genes 0.000 description 1
- 102220077477 rs797044897 Human genes 0.000 description 1
- 102200070454 rs903823830 Human genes 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种S‑腺苷甲硫氨酸合成酶突变体及其制备方法，该突变体是将如SEQ ID NO.1所示氨基酸序列的第65位、第104位、第186位进行单点、双点或三点突变而获得。与野生型S‑腺苷甲硫氨酸合成酶相比，该突变体在10mM底物反应体系中SAM最终积累量增加1.06mM。同时，该突变体比酶活相比于野生型eMAT增加3.3倍，为6.02±0.22U/mg。这进一步为SAM的酶法制备提供了可能性，也为分子改造改善酶的产物抑制现象提供了思路。
Description
技术领域
本发明涉及一种催化活性提高且产物抑制降低的S-腺苷甲硫氨酸合成酶突变体及其制备方法，属于基因工程领域。
背景技术
S-腺苷甲硫氨酸(SAM)是一种重要的生理活性物质，参与生物体内转甲基、转硫基、转氨丙基等多种生化反应。临床上，SAM对治疗肝炎、抑郁症、关节炎等具有显著的治疗效果，因此市场需求量巨大。
目前，SAM主要通过微生物发酵法生产，但该法存在生产周期长、底物转化率低、提取过程复杂、产物易发生自消旋等问题。相比较而言，酶催化法具有反应时间短、立体选择性高、提纯过程较简单等特点。
S-腺苷甲硫氨酸合成酶(S-adenosylmethioninesynthetase，简称MAT，EC2.5.6.1)，催化ATP和L-甲硫氨酸生成SAM，同时生成副产物正磷酸和焦磷酸。通过比较不同来源MAT的酶学性质，我们发现来源于大肠杆菌的MAT(eMAT)具有蛋白表达量较高、比酶活较高、Km较小等优势。但该酶也存在严重的产物抑制现象，导致酶催化效率低，SAM无法有效积累。虽然加入高浓度对甲苯磺酸钠能够克服产物抑制，但SAM对甲苯磺酸钠成品作为药物时存在生物利用率低的问题。因此，通过酶的分子改造而非加入对甲苯磺酸钠来降低eMAT的产物抑制现象在酶法制备SAM中具有重要意义。
目前，已有一些通过分子改造改善MAT催化性能的报道。Dippe等对来源于枯草芽孢杆菌的MAT进行理性设计时发现，将317位异亮氨酸突变为缬氨酸时能增加MAT的催化活性，同时降低产物抑制现象(Chem.Commum(Camb).51(2015)3637-3640)(USPatent20160264945A1)。以此为基础，Yin等对来源于大肠杆菌和枯草芽孢杆菌的MAT进行同源序列对比后，将eMAT的303位异亮氨酸突变为缬氨酸，eMAT的酶活和产物抑制现象均有所改善(Molecules 22(2017)1365-1377)。另外，Hu等人利用DNA shuffling技术对来源于大肠杆菌、酿酒酵母和壮观链霉菌的MAT基因进行重组，获得两株酶活提高、SAM积累量也增加的菌株GS115/DS56和GS115/DS16，并对突变株进行了序列分析(表1)(J.Biotechnol.141(2009)97-103)。其中，GS115/DS56 MAT氨基酸序列与酿酒酵母SAM2(酿酒酵母MAT的特称)氨基酸序列同源率为99％，GS115/DS16 MAT氨基酸序列与壮观链霉菌MAT(sMAT)序列同源率为98％。将原核生物和真核生物的MAT氨基酸序列进行比对发现，MAT属于高保守型蛋白家族，尤其是活性中心的位点。因此，可以通过定点突变和半理性设计对eMAT进行分子改造。
表1 pDS56和pDS16的突变位点
发明内容
本发明的目的是提供一种S-腺苷甲硫氨酸合成酶突变体、编码基因、重组载体、重组菌以及其在酶法制备SAM中的应用。
本发明采用的技术方案是：
本发明提供一种S-腺苷甲硫氨酸合成酶突变体，所述突变体是将SEQ ID NO.1所示氨基酸序列的第65位、第104位、第186位进行突变获得。
进一步，先将303位异亮氨酸突变为缬氨酸(I303V),再在单点突变的基础上将65位异亮氨酸突变为缬氨酸(I303V/I65V),接着在两点突变的基础上将186位亮氨酸突变为缬氨酸(I303V/I65V/L186V),最后在三点突变的基础上将104位天冬酰胺突变为赖氨酸(I303V/I65V/L186V/N104K)。
更进一步，所述突变体氨基酸序列为下列之一：SEQ ID NO.2，SEQ ID NO.3，SEQID NO.4。
本发明还涉及一种编码S-腺苷甲硫氨酸合成酶突变体的基因。
本发明还涉及一种携带所述基因的重组载体。
本发明还涉及一种由重组载体转化制备的重组菌。
本发明提供一种所述S-腺苷甲硫氨酸合成酶突变体在酶法合成SAM中的应用。
与现有技术相比，本发明有益效果主要体现在：
本发明构建了一种高效表达S-腺苷甲硫氨酸合成酶突变体的重组大肠杆菌；该重组大肠杆菌表达的S-腺苷甲硫氨酸合成酶突变体比酶活比野生型eMAT提高3.31倍，为6.02±0.22U/mg；在10mM底物反应体系中，该突变体可以使SAM最终积累量为2.68mM，而野生型eMATSAM积累量仅为1.62mM。
附图说明
图1metK基因片段核酸电泳图，1泳道为DNA Maker，2泳道为metK基因PCR扩增产物。
图2E.coli K12来源MAT与文献报道MAT氨基酸序列比对结果。
图3野生型重组菌SDS-PAGE电泳图，泳道1为蛋白Maker，泳道2为全细胞破胞蛋白，泳道3为破胞液上清蛋白，泳道4为破胞液沉淀蛋白。
图4S-腺苷甲硫氨酸合成酶eMAT野生型与突变体在不同底物浓度下SAM积累量变化曲线。
具体实施方式
下面结合具体实施例对本发明进行描述，但本发明的保护范围并非仅限于此：
实施例1：S-腺苷甲硫氨酸合成酶重组菌的构建
选取Escherichia coli K12来源的S-腺苷甲硫氨酸合成酶，利用PCR技术，以E.coli K12基因组为模板，以metK-F:5’-GATCCGAATTCATGGCAAAACACCTTTTTACG-3’(SEQ IDNO.5)和metK-R:5’-GTGCTCGAGTTACTTCAGACCGGCAGCAT(SEQ ID NO.6)为引物，对metK基因进行扩增，并在其5’端和3’端分别引入EcoR I和Xho I限制性内切酶酶切位点(下划线标出)。PCR反应体系(50μL)为：2×PrimeSTAR Max Premix 25μL，模板DNA1μL，上下游引物各1μL，无菌水22μL。PCR反应条件为：98℃预变性5min；98℃变性10s，60℃退火5s，72℃延伸10s，循环30次；72℃延伸5min。以1％琼脂糖凝胶电泳验证PCR扩增产物，当目的条带大小符合编码SEQ ID NO.1大小时(图1)，用PCR产物回收试剂和进行产物回收。将纯化后的PCR产物和质粒pET-28a(+)分别同时用限制性内切酶EcoR I和Xho I进行双酶切反应。酶切结束后，将酶切产物进行大孔琼脂糖凝胶电泳，并从核酸胶上切下正确的条带用于DNA胶回收。回收后的片段测定浓度后按照摩尔比3：1(目的基因：质粒)进行酶连反应。接着，通过热激法将酶连产物转入到E.coli BL21(DE3)感受态细胞中，获得重组菌E.coli BL21(DE3)/pET-28a(+)-metK。
实施例2：S-腺苷甲硫氨酸合成酶基因的单点突变
将实施例1中的重组菌E.coli BL21(DE3)/pET-28a(+)-metK培养过夜后提取重组质粒pET-28a(+)-metK，并以其为模板，对303位进行定点突变。定点突变用FastMutagenesis system试剂盒，以I303V-F:5’-TCAGGTTTCCTACGCAGTCGGCGTGGC-3’(SEQ IDNO.7)和I303V-R:5’-CCAGAAGGCACTTTCTCAGTACCGAAAG-3’(SEQ ID NO.8)为引物，进行全质粒PCR扩增(94℃ 5min；94℃20s，60℃20s，72℃60s，循环30次；72℃ 10min)。PCR产物经琼脂糖凝胶电泳验证后用限制酶DpnI对模板进行消化，再通过热激法将消化后的产物转入到E.coli BL21(DE3)感受态细胞中，获得单点突变重组菌。单点突变氨基酸序列为SEQ IDNO.1。
实施例3：S-腺苷甲硫氨酸合成酶两点突变的构建
将eMAT与SAM2、sMAT氨基酸序列进行同源比对，找到eMAT与pDS16和pDS56中对应的突变位点，确定可以突变的点有：K18R，L31P，I65V，E324G，L329V(图2，三角形标出)。以突变体I303V编码基因为模板，以K18R-F:5’-TCTGAAGGGCATCCTGACAGAATTGCTGAC-3’(SEQ IDNO.9)和K18R-R:5’-CTGTCAGGATGCCCTTCAGAGACGGAC-3’(SEQ ID NO.10)；I31P-F:5’-TGATGCCGTTTTAGACGCGCCCCTCGAACAG-3’(SEQ ID NO.11)和I31P-R:5’-GGCGCGTCTAAAACGGCATCAGAAATTTGG-3’(SEQ ID NO.12)；I65V-F:5’-ACCAGCGCCTGGGTAGACGTCGAAGAGATC-3’(SEQ ID NO.13)和I65V-R:5’-CGTCTACCCAGGCGCTGGTGGTGATTTCG-3’(SEQ ID NO.14)；E324G-F:5’-TACTGAGAAAGTGCCTTCTGGACAACTGACC-3’(SEQ ID NO.15)和E324G-R:5’-CCAGAAGGCACTTTCTCAGTACCGAAAG-3’(SEQ ID NO.16)；L329V-F:5’-TTCTGAACAACTGACCCTGGTGGTACGTGAG-3’(SEQ ID NO.17)和L329V-R:5’-CCAGGGTCAGTTGTTCAGAAGGCACTTT-3’(SEQ ID NO.18)为引物，用FAST MUTAGENESIS system试剂盒对这些位点进行全质粒PCR。PCR产物经限制酶DpnI消化后，再通过热激法将产物转入到E.coli BL21(DE3)感受态细胞中，获得两点突变重组菌。将所有两点突变重组菌诱导表达、纯化后，测定比酶活和不同浓度L-甲硫氨酸下SAM积累量，发现只有突变体I303V/I65V SAM积累量增加。突变体I303V/I65V氨基酸序列为SEQ ID NO.2。
实施例3：S-腺苷甲硫氨酸合成酶三点突变的构建
MAT属于氨基酸序列高度保守的蛋白家族，尤其是对于活性中心的位点。在实施例2中进行氨基酸序列比对时，发现不仅活性中心的位点高度保守，与活性中心相邻的位点也具有较高的保守性，除了第104位，第165位，第186位，第188位和第240位(图2，圆形标出)。据报道，SAM2在低底物浓度下没有产物抑制。因此，我们推测这些位点可能与产物抑制现象的改善有关，并以突变体I303V/I65V编码基因为模板通过定点突变将它们对应突变为：104A，165T，186I/V，188A和240A。通过检测突变后的比酶活和不同底物浓度下SAM的积累量发现，只有突变体I303V/I65V/L186V能够使酶活和SAM积累量同时提高。突变体I303V/I65V/L186V氨基酸序列为SEQ ID NO.3。
实施例4：S-腺苷甲硫氨酸合成酶四点突变的构建
在对实施例3中的突变体进行酶活和不同底物浓度下SAM的积累量时发现，突变体I303V/I65V/N104A比酶活相比于突变体I303V/I65V提高1.3倍，但SAM积累量严重下降。考虑到该位点位于与产物和底物进出相关的柔性Loop上，可能与SAM的释放相关，因此以突变体I303V/I65V/L186V编码基因为模板对104位进行饱和突变。定点饱和突变以N104-F:5’-CAAACAGTCTCCTGACATCNNKCAGGGCGTTG-3’(SEQ ID NO.19)和N104-R:5’-MNNGATGTCAGGAGACTGTTTGCCGATAGCGC-3’(SEQ ID NO.20)为引物，用Fast Mutagenesis system试剂盒。
构建突变体库后，从突变体库随机挑选单菌落进行高通量筛选。即将突变体库(重组菌转化平板)的单菌落挑入到含有300μL LB液体培养基(含50μg/mL卡那霉素)的96孔浅孔板中，37℃、220rpm培养8h。用移液器吸取50μL浅孔板中的菌液到对应的装有600μL LB液体培养基(含50μg/mL卡那霉素)的96孔深孔板中，37℃、220rpm培养2h，向每个孔中加入终浓度为0.1mM的IPTG，28℃、220rpm诱导培养12h。室温下4000×g离心10min，去培养基。用100mMTris-HCl(pH 8.0)重悬菌体，静置5-10min后，再次重悬3-5次，取最后一次重悬的菌液2μL加入装有98μL预混液(100mMTris-HCl，1mM ATP，1mM L-甲硫氨酸，1mM SAM，20mMMgCl2，150mMKCl，pH 8.0)的96孔酶标板中，混匀，37℃反应5min。反应结束后，向酶标板中加入100μL显色液(将用5M盐酸配制的34mM(NH4)6Mo7O24·4H2O和2.16mM草酸孔雀绿以体积比3：1的比例混匀、过滤而来)，静置3min后，于620nm处测量吸光值。通过对比突变子与野生型重组菌反应后吸光值的大小，从酶标板上挑选出吸光值明显增加的2-3个位点，从浅孔板中找到对应的菌进行复筛，最终得到1个酶活明显提高、SAM积累量略有下降的突变体I303V/I65V/L186V/N104K。突变体I303V/I65V/L186V/N10K氨基酸序列为SEQ ID NO.4。
实施例5：S-腺苷甲硫氨酸合成酶野生型和突变体的诱导表达
将野生型重组菌和突变体重组菌分别加入到5mL含50μg/mL卡那霉素的LB培养基(1％蛋白胨，1％氯化钠，0.5％酵母提取物，pH 7.0)中，37℃、220rpm培养8-12h。再按2％的接种量，转接1mL至50mL含50μg/mL卡那霉素的LB培养基中。待菌体浓度(OD600)达到0.6-0.8时，加入终浓度为0.1mM的IPTG，28℃、220rpm培养12h。4℃、6000×g离心10min收集菌体，100mMTris-HCl离心重悬3次后，用裂解液(100mMTris-HCl，300mMNaCl，10mM咪唑，5％甘油，pH 8.0)将菌体重悬，使OD600浓缩为8。接着，将重悬的菌体置于冰水浴中超声破胞，4℃、12000×g离心10min，分别取全细胞破胞液、破胞上清液和破胞沉淀重悬液进行SDS-PAGE电泳分析(图3)。
取破胞液上清上样至Ni-NTA亲和柱(购于上海生工生物工程技术服务有限公司)，用裂解液平衡柱成分，用洗杂液(100mMTris-HCl，300mMNaCl，50mM咪唑，5％甘油，pH 8.0)洗去杂质，用洗脱液(100mMTris-HCl，300mMNaCl，250mM咪唑，5％甘油，pH 8.0)洗脱目的蛋白，获得纯酶液。
实施例6：S-腺苷甲硫氨酸合成酶野生型和突变体不同底物浓度下SAM积累量的测定
产物抑制现象具体可表现为在一定底物浓度下，即使延长反应时间，底物也不能完全转化，因此测定不同浓度L-甲硫氨酸情况下SAM的积累量变化。
SAM积累量的反应体系为：总体积为0.4mL的预混液含有100mMTris-HCl(pH 8.0)、10mM ATP、0.2-10mM L-甲硫氨酸、20mMMgCl2和150mMKCl，加入0.1mL纯酶液。为了使反应充分进行，于37℃、200rpm恒温反应4h。反应结束后，用0.25mL 20％的高氯酸溶液终止酶反应。4℃、13000rpm离心10min后，用高效液相色谱法测SAM浓度(图4)。
实施例7：S-腺苷甲硫氨酸合成酶野生型和突变体的比酶活测定
酶活的测定方法：总体积为480μL的预混液中含有100mMTris-HCl(pH 8.0)、5mMATP、5mM L-甲硫氨酸、20mM MgCl2，150mMKCl，加入20μL纯酶液，37℃、400rpm金属浴反应5min。用0.25mL 20％的高氯酸溶液终止酶反应。4℃、13000rpm离心10min后，用高效液相色谱法测SAM浓度。1个单位酶活(1U)被定义为每分钟转化生成1μmol SAM的酶量。
高效液相色谱法：采用Hypersil BDS C18(250×4.6mm,5μm)色谱柱，以40mM磷酸二氢铵、2mM庚烷磺酸钠和18％(v/v)甲醇水溶液(pH 3.0)为流动相，流动相流速为0.8mL/min，进样量为10μL，在254nm波长下检测SAM出峰面积。
用PierceTM BCA Protein Assay Kit微孔板法测量蛋白质浓度后，计算野生型和突变体S-腺苷甲硫氨酸合成酶的比酶活，结果见表2。比酶活＝酶活/蛋白含量(U/mg)。突变体I303V/I65V/L186V/N104K相比于野生型eMAT比酶活提高3.3倍。
表2 S-腺苷甲硫氨酸合成酶野生型及其突变体酶活力
实施例8：S-腺苷甲硫氨酸合成酶野生型和突变体的动力学参数测定
eMAT催化ATP和L-甲硫氨酸转化生成SAM是按照双底物反应机制中的序列机制进行。序列机制的动力学方程为：
在测量Km过程，首先将L-甲硫氨酸浓度固定在0.15-0.6mM范围下的某一浓度，测定0-0.2mM ATP的反应速率。再将ATP浓度固定在0.15-0.6mM范围下的某一浓度，测定0-0.2mM L-甲硫氨酸的反应速率。最后根据序列机制Lineweaver-Burk作图法求出Km。
于eMAT而言，SAM是底物ATP的竞争性抑制剂，竞争性抑制的方程为：
SAM是底物L-甲硫氨酸的非竞争性抑制剂，非竞争性抑制的方程为：
在测量Ki过程中，先固定SAM和L-甲硫氨酸的浓度，测量0-0.2mM ATP的反应速率。再固定SAM和ATP的浓度，测量0-0.2mM ATP的反应速率。最后根据双倒数作图法得到竞争性抑制曲线和非竞争性抑制曲线，从而获得Ki。动力学参数见表3。
表3 S-腺苷甲硫氨酸合成酶野生型和突变体的动力学参数
序列表
<110> 浙江大学
<120> 一种S-腺苷甲硫氨酸合成酶突变体及其制备方法
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 384
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro
1 5 10 15
Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu
20 25 30
Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr
35 40 45
Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp
50 55 60
Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His
65 70 75 80
Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile
85 90 95
Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro
100 105 110
Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
115 120 125
Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg
130 135 140
Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp
145 150 155 160
Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly
165 170 175
Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu
180 185 190
Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile
195 200 205
Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe
210 215 220
Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys
225 230 235 240
Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala
245 250 255
Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp
260 265 270
Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala
275 280 285
Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Val Gly
290 295 300
Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys
305 310 315 320
Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu
325 330 335
Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr
340 345 350
Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp
355 360 365
Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys
370 375 380
<210> 2
<211> 384
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro
1 5 10 15
Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu
20 25 30
Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr
35 40 45
Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp
50 55 60
Val Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His
65 70 75 80
Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile
85 90 95
Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro
100 105 110
Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
115 120 125
Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg
130 135 140
Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp
145 150 155 160
Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly
165 170 175
Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu
180 185 190
Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile
195 200 205
Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe
210 215 220
Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys
225 230 235 240
Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala
245 250 255
Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp
260 265 270
Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala
275 280 285
Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Val Gly
290 295 300
Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys
305 310 315 320
Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu
325 330 335
Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr
340 345 350
Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp
355 360 365
Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys
370 375 380
<210> 3
<211> 384
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro
1 5 10 15
Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu
20 25 30
Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr
35 40 45
Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp
50 55 60
Val Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His
65 70 75 80
Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile
85 90 95
Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro
100 105 110
Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
115 120 125
Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg
130 135 140
Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp
145 150 155 160
Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly
165 170 175
Lys Ile Val Gly Ile Asp Ala Val Val Val Ser Thr Gln His Ser Glu
180 185 190
Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile
195 200 205
Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe
210 215 220
Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys
225 230 235 240
Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala
245 250 255
Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp
260 265 270
Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala
275 280 285
Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Val Gly
290 295 300
Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys
305 310 315 320
Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu
325 330 335
Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr
340 345 350
Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp
355 360 365
Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys
370 375 380
<210> 4
<211> 384
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro
1 5 10 15
Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu
20 25 30
Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr
35 40 45
Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp
50 55 60
Val Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His
65 70 75 80
Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile
85 90 95
Gly Lys Gln Ser Pro Asp Ile Lys Gln Gly Val Asp Arg Ala Asp Pro
100 105 110
Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
115 120 125
Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg
130 135 140
Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp
145 150 155 160
Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly
165 170 175
Lys Ile Val Gly Ile Asp Ala Val Val Val Ser Thr Gln His Ser Glu
180 185 190
Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile
195 200 205
Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe
210 215 220
Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys
225 230 235 240
Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala
245 250 255
Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp
260 265 270
Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala
275 280 285
Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Val Gly
290 295 300
Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys
305 310 315 320
Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu
325 330 335
Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr
340 345 350
Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp
355 360 365
Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys
370 375 380
<210> 5
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gatccgaatt catggcaaaa caccttttta cg 32
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtgctcgagt tacttcagac cggcagcat 29
<210> 7
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tcaggtttcc tacgcagtcg gcgtggc 27
<210> 8
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccagaaggca ctttctcagt accgaaag 28
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tctgaagggc atcctgacag aattgctgac 30
<210> 10
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctgtcaggat gcccttcaga gacggac 27
<210> 11
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgatgccgtt ttagacgcgc ccctcgaaca g 31
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggcgcgtcta aaacggcatc agaaatttgg 30
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
accagcgcct gggtagacgt cgaagagatc 30
<210> 14
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgtctaccca ggcgctggtg gtgatttcg 29
<210> 15
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tactgagaaa gtgccttctg gacaactgac c 31
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccagaaggca ctttctcagt accgaaag 28
<210> 17
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttctgaacaa ctgaccctgg tggtacgtga g 31
<210> 18
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccagggtcag ttgttcagaa ggcacttt 28
<210> 19
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
caaacagtct cctgacatcn nkcagggcgt tg 32
<210> 20
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
mnngatgtca ggagactgtt tgccgatagc gc 32
Claims (4)
1.一种S-腺苷甲硫氨酸合成酶突变体，其特征在于，所述S-腺苷甲硫氨酸合成酶的氨基酸序列为下列之一：SEQ ID NO.2所示氨基酸序列，SEQ ID NO.3所示氨基酸序列，SEQ IDNO.4所示氨基酸序列。
2.一种编码如权利要求1所述S-腺苷甲硫氨酸合成酶突变体的基因。
3.一种包含如权利要求2所述的S-腺苷甲硫氨酸合成酶突变体编码基因的重组载体。
4.一种包含如权利要求3所述重组载体的重组菌。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910436026.3A CN110144336B (zh) | 2019-05-23 | 2019-05-23 | 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910436026.3A CN110144336B (zh) | 2019-05-23 | 2019-05-23 | 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110144336A CN110144336A (zh) | 2019-08-20 |
| CN110144336B true CN110144336B (zh) | 2020-09-04 |
Family
ID=67592773
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910436026.3A Active CN110144336B (zh) | 2019-05-23 | 2019-05-23 | 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110144336B (zh) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819605B (zh) * | 2020-01-07 | 2020-04-07 | 中国科学院天津工业生物技术研究所 | 甲硫氨酸合成酶突变体、突变基因及其在制备维生素b12中的应用 |
| CN113444699B (zh) * | 2020-03-26 | 2022-06-03 | 中国科学院青岛生物能源与过程研究所 | 一种提高乙酰丙酮合成效率的乙酰丙酮裂解酶突变体、核苷酸、表达载体、重组菌及应用 |
| CN111235126B (zh) * | 2020-04-01 | 2021-08-20 | 湖南福来格生物技术有限公司 | 一种s-腺苷甲硫氨酸合成酶突变体及利用其的制备方法 |
| CN111235084B (zh) * | 2020-04-01 | 2021-08-20 | 湖南福来格生物技术有限公司 | 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法 |
| CN114107243B (zh) * | 2021-12-09 | 2023-04-07 | 江南大学 | 一种甲硫氨酸腺苷转移酶突变体 |
| CN117551629A (zh) * | 2022-11-11 | 2024-02-13 | 山东理工大学 | 一种基于244位点的s-腺苷蛋氨酸合成酶突变体及其应用 |
| CN116355875B (zh) * | 2023-05-08 | 2023-08-01 | 北京量维生物科技研究院有限公司 | 甲硫氨酸腺苷基转移酶突变体及其在生产s-腺苷甲硫氨酸中的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008034338A1 (fr) * | 2006-08-31 | 2008-03-27 | Bioright Worldwide Company Limited | Mutants de la s-adénosylméthionine synthétase, les adn les codant et les utilisations de ces mutants |
| CN108588047A (zh) * | 2018-04-11 | 2018-09-28 | 苏州汇桢生物技术有限公司 | 同型半胱氨酸甲基转移酶突变体及其应用、以及核酸、表达载体、宿主细胞、试剂 |
-
2019
- 2019-05-23 CN CN201910436026.3A patent/CN110144336B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008034338A1 (fr) * | 2006-08-31 | 2008-03-27 | Bioright Worldwide Company Limited | Mutants de la s-adénosylméthionine synthétase, les adn les codant et les utilisations de ces mutants |
| CN108588047A (zh) * | 2018-04-11 | 2018-09-28 | 苏州汇桢生物技术有限公司 | 同型半胱氨酸甲基转移酶突变体及其应用、以及核酸、表达载体、宿主细胞、试剂 |
Non-Patent Citations (5)
| Title |
|---|
| Biosynthesis of S-Adenosylmethionine by Magnetically Immobilized Escherichia coli Cells Highly Expressing a Methionine Adenosyltransferase Variant;Chunli Yin等;《Molecules》;20170831;第22卷(第8期);第1365-1377页 * |
| Crystal Structure of the S-Adenosylmethionine Synthetase Ternary Complex:A Novel Catalytic Mechanism of S-Adenosylmethionine Synthesis from ATP and Met;Junichi Komoto等;《Biochemistry》;20040127;第43卷(第7期);第1821-1831页 * |
| DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl-l-methionine production in Pichia pastoris;Hui Hu等;《Journal of Biotechnology》;20090520;第141卷(第3-4期);第97-103页 * |
| Rationally Engineered Variants of S-adenosylmethionine (SAM) Synthase: Reduced Product Inhibition and Synthesis of Artificial Cofactor Homologues;M Dippe等;《Chem Commun (Camb).》;20150228;第51卷(第17期);第3637-3640页 * |
| S-adenosylmethionine synthetase [Escherichia coli CFT073];GenBank:AAN81976.1;《GenBank数据库》;20140131;参见序列 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110144336A (zh) | 2019-08-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110144336B (zh) | 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法 | |
| CN109609474B (zh) | 一种氨基酸脱氢酶突变体及其在合成l-草铵膦中的应用 | |
| CN111269900B (zh) | 一种l-氨基酸脱氨酶突变体的制备及其应用 | |
| CN111019878B (zh) | L-苏氨酸产量提高的重组大肠杆菌及其构建方法与应用 | |
| CN110229805B (zh) | 一种通过序列一致性制备的谷氨酸脱羧酶突变体及其应用 | |
| CN108913671B (zh) | 一种ω-转氨酶突变体及其应用 | |
| CN110172454B (zh) | 一种s-腺苷甲硫氨酸合成酶突变体及其高通量筛选方法 | |
| CN110938616B (zh) | 一种温泉热碱芽孢杆菌来源的腈水合酶的突变体 | |
| CN114606213B (zh) | 一种多聚磷酸激酶突变体、工程菌及其应用 | |
| CN109609478B (zh) | α-转氨酶及突变体以及在不对称合成L-草铵膦中的应用 | |
| CN112574970B (zh) | 一种烟酰胺单核苷酸腺苷转移酶突变体及其应用 | |
| CN111808829B (zh) | 一种γ-谷氨酰甲胺合成酶突变体及其应用 | |
| CN111518783B (zh) | 重组(R)-ω-转氨酶、突变体及其在制备西他列汀中的应用 | |
| CN111154746B (zh) | 酰胺酶突变体及其在催化合成2-氯烟酸中的应用 | |
| CN112458073B (zh) | 一种h-蛋白突变体及其应用 | |
| CN110438097B (zh) | 一种热稳定性与酶活提高的l-异亮氨酸羟化酶突变体 | |
| CN109609476B (zh) | α-转氨酶和突变体及其在不对称合成L-草铵膦中的应用 | |
| Lv et al. | A novel approach to cloning and expression of human thymidylate synthase | |
| CN114277020A (zh) | 一种腈水解酶突变体、工程菌及其应用 | |
| CN108624568B (zh) | 变异黄嘌呤脱氢酶及其应用 | |
| CN109370997B (zh) | 一种苯丙氨酸氨基变位酶突变体 | |
| CN112680425B (zh) | 一种醇脱氢酶突变体及其应用 | |
| CN114752576B (zh) | 过氧化氢酶突变体及其应用 | |
| CN113999827B (zh) | 一种亮氨酸脱氢酶突变体及其制备方法与应用 | |
| CN116254253B (zh) | 一种通过dna合成改组组合突变获得的谷氨酸脱羧酶突变体及应用 |
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 |