CN113832125A - 一种烟酰胺核糖激酶突变体及其编码基因和应用 - Google Patents
一种烟酰胺核糖激酶突变体及其编码基因和应用 Download PDFInfo
- Publication number
- CN113832125A CN113832125A CN202111217419.9A CN202111217419A CN113832125A CN 113832125 A CN113832125 A CN 113832125A CN 202111217419 A CN202111217419 A CN 202111217419A CN 113832125 A CN113832125 A CN 113832125A
- Authority
- CN
- China
- Prior art keywords
- seq
- mutant
- amino acid
- nicotinamide
- acid sequence
- 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.)
- Granted
Links
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 title claims abstract description 137
- 235000005152 nicotinamide Nutrition 0.000 title claims abstract description 69
- 239000011570 nicotinamide Substances 0.000 title claims abstract description 69
- 229960003966 nicotinamide Drugs 0.000 title claims abstract description 69
- 101001076781 Fructilactobacillus sanfranciscensis (strain ATCC 27651 / DSM 20451 / JCM 5668 / CCUG 30143 / KCTC 3205 / NCIMB 702811 / NRRL B-3934 / L-12) Ribose-5-phosphate isomerase A Proteins 0.000 title claims abstract description 68
- 102000046755 Ribokinases Human genes 0.000 title claims abstract description 68
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 37
- 230000035772 mutation Effects 0.000 claims abstract description 33
- JLEBZPBDRKPWTD-TURQNECASA-O N-ribosylnicotinamide Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 JLEBZPBDRKPWTD-TURQNECASA-O 0.000 claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 235000018417 cysteine Nutrition 0.000 claims description 60
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 60
- 150000001413 amino acids Chemical group 0.000 claims description 57
- 239000002773 nucleotide Substances 0.000 claims description 20
- 125000003729 nucleotide group Chemical group 0.000 claims description 20
- DAYLJWODMCOQEW-TURQNECASA-O NMN(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(O)=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-O 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 19
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 16
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 16
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 14
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 14
- 235000009582 asparagine Nutrition 0.000 claims description 14
- 229960001230 asparagine Drugs 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 9
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 9
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 9
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 7
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 7
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 7
- 235000013922 glutamic acid Nutrition 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 7
- 239000004474 valine Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 claims description 5
- 235000019982 sodium hexametaphosphate Nutrition 0.000 claims description 5
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims description 5
- 235000020956 nicotinamide riboside Nutrition 0.000 claims description 4
- 239000011618 nicotinamide riboside Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 241000235058 Komagataella pastoris Species 0.000 claims description 2
- 241000187747 Streptomyces Species 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 31
- 108090000790 Enzymes Proteins 0.000 abstract description 31
- 230000000694 effects Effects 0.000 abstract description 11
- 239000000758 substrate Substances 0.000 abstract description 8
- 239000011942 biocatalyst Substances 0.000 abstract description 2
- 230000035484 reaction time Effects 0.000 abstract description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 3
- DAYLJWODMCOQEW-TURQNECASA-N NMN zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)([O-])=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-N 0.000 abstract 2
- 108020004414 DNA Proteins 0.000 description 24
- 241000282414 Homo sapiens Species 0.000 description 15
- 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 11
- 108010081551 glycylphenylalanine Proteins 0.000 description 11
- 239000000243 solution Substances 0.000 description 8
- KVWLTGNCJYDJET-LSJOCFKGSA-N Ala-Arg-His Chemical compound C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N KVWLTGNCJYDJET-LSJOCFKGSA-N 0.000 description 7
- LRPZJPMQGKGHSG-XGEHTFHBSA-N Arg-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N)O LRPZJPMQGKGHSG-XGEHTFHBSA-N 0.000 description 7
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 7
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 7
- SVFOIXMRMLROHO-SRVKXCTJSA-N Asp-Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SVFOIXMRMLROHO-SRVKXCTJSA-N 0.000 description 7
- KBJVTFWQWXCYCQ-IUKAMOBKSA-N Asp-Thr-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KBJVTFWQWXCYCQ-IUKAMOBKSA-N 0.000 description 7
- MKMKILWCRQLDFJ-DCAQKATOSA-N Cys-Lys-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MKMKILWCRQLDFJ-DCAQKATOSA-N 0.000 description 7
- HNAUFGBKJLTWQE-IFFSRLJSSA-N Gln-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)N)N)O HNAUFGBKJLTWQE-IFFSRLJSSA-N 0.000 description 7
- AUTNXSQEVVHSJK-YVNDNENWSA-N Glu-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O AUTNXSQEVVHSJK-YVNDNENWSA-N 0.000 description 7
- BPLNJYHNAJVLRT-ACZMJKKPSA-N Glu-Ser-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O BPLNJYHNAJVLRT-ACZMJKKPSA-N 0.000 description 7
- GMVCSRBOSIUTFC-FXQIFTODSA-N Glu-Ser-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMVCSRBOSIUTFC-FXQIFTODSA-N 0.000 description 7
- HAXARWKYFIIHKD-ZKWXMUAHSA-N Gly-Ile-Ser Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HAXARWKYFIIHKD-ZKWXMUAHSA-N 0.000 description 7
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 7
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 description 7
- SKOKHBGDXGTDDP-MELADBBJSA-N His-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N SKOKHBGDXGTDDP-MELADBBJSA-N 0.000 description 7
- MTFVYKQRLXYAQN-LAEOZQHASA-N Ile-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O MTFVYKQRLXYAQN-LAEOZQHASA-N 0.000 description 7
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 7
- XMYURPUVJSKTMC-KBIXCLLPSA-N Ile-Ser-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N XMYURPUVJSKTMC-KBIXCLLPSA-N 0.000 description 7
- KWHFUMYCSPJCFQ-NGTWOADLSA-N Ile-Thr-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N KWHFUMYCSPJCFQ-NGTWOADLSA-N 0.000 description 7
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 7
- 241000880493 Leptailurus serval Species 0.000 description 7
- RIMMMMYKGIBOSN-DCAQKATOSA-N Leu-Asn-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O RIMMMMYKGIBOSN-DCAQKATOSA-N 0.000 description 7
- FQZPTCNSNPWHLJ-AVGNSLFASA-N Leu-Gln-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O FQZPTCNSNPWHLJ-AVGNSLFASA-N 0.000 description 7
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 7
- 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 7
- SSYOBDBNBQBSQE-SRVKXCTJSA-N Lys-Cys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O SSYOBDBNBQBSQE-SRVKXCTJSA-N 0.000 description 7
- INMBONMDMGPADT-AVGNSLFASA-N Lys-Met-Met Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCCCN)N INMBONMDMGPADT-AVGNSLFASA-N 0.000 description 7
- HYSVGEAWTGPMOA-IHRRRGAJSA-N Lys-Pro-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O HYSVGEAWTGPMOA-IHRRRGAJSA-N 0.000 description 7
- HLQWFLJOJRFXHO-CIUDSAMLSA-N Met-Glu-Ser Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O HLQWFLJOJRFXHO-CIUDSAMLSA-N 0.000 description 7
- LCPUWQLULVXROY-RHYQMDGZSA-N Met-Lys-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LCPUWQLULVXROY-RHYQMDGZSA-N 0.000 description 7
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 7
- 108010066427 N-valyltryptophan Proteins 0.000 description 7
- GXDPQJUBLBZKDY-IAVJCBSLSA-N Phe-Ile-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GXDPQJUBLBZKDY-IAVJCBSLSA-N 0.000 description 7
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 7
- BNRFQGLWLQESBG-YESZJQIVSA-N Phe-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O BNRFQGLWLQESBG-YESZJQIVSA-N 0.000 description 7
- VZKBJNBZMZHKRC-XUXIUFHCSA-N Pro-Ile-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O VZKBJNBZMZHKRC-XUXIUFHCSA-N 0.000 description 7
- FHZJRBVMLGOHBX-GUBZILKMSA-N Pro-Pro-Asp Chemical compound OC(=O)C[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1)C(O)=O FHZJRBVMLGOHBX-GUBZILKMSA-N 0.000 description 7
- WTWGOQRNRFHFQD-JBDRJPRFSA-N Ser-Ala-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WTWGOQRNRFHFQD-JBDRJPRFSA-N 0.000 description 7
- GRRAECZXRONTEE-UBHSHLNASA-N Ser-Cys-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O GRRAECZXRONTEE-UBHSHLNASA-N 0.000 description 7
- UOLGINIHBRIECN-FXQIFTODSA-N Ser-Glu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UOLGINIHBRIECN-FXQIFTODSA-N 0.000 description 7
- RHAPJNVNWDBFQI-BQBZGAKWSA-N Ser-Pro-Gly Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O RHAPJNVNWDBFQI-BQBZGAKWSA-N 0.000 description 7
- DCCGCVLVVSAJFK-NUMRIWBASA-N Thr-Asp-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O DCCGCVLVVSAJFK-NUMRIWBASA-N 0.000 description 7
- YJCVECXVYHZOBK-KNZXXDILSA-N Thr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H]([C@@H](C)O)N YJCVECXVYHZOBK-KNZXXDILSA-N 0.000 description 7
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 7
- XOSGQKFEIOCPIJ-SZMVWBNQSA-N Trp-Pro-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC2=CNC3=CC=CC=C32)N XOSGQKFEIOCPIJ-SZMVWBNQSA-N 0.000 description 7
- HKIUVWMZYFBIHG-KKUMJFAQSA-N Tyr-Arg-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O HKIUVWMZYFBIHG-KKUMJFAQSA-N 0.000 description 7
- UABYBEBXFFNCIR-YDHLFZDLSA-N Tyr-Asp-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UABYBEBXFFNCIR-YDHLFZDLSA-N 0.000 description 7
- WVRUKYLYMFGKAN-IHRRRGAJSA-N Tyr-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 WVRUKYLYMFGKAN-IHRRRGAJSA-N 0.000 description 7
- BSCBBPKDVOZICB-KKUMJFAQSA-N Tyr-Leu-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BSCBBPKDVOZICB-KKUMJFAQSA-N 0.000 description 7
- PRONOHBTMLNXCZ-BZSNNMDCSA-N Tyr-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PRONOHBTMLNXCZ-BZSNNMDCSA-N 0.000 description 7
- LMKKMCGTDANZTR-BZSNNMDCSA-N Tyr-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=C(O)C=C1 LMKKMCGTDANZTR-BZSNNMDCSA-N 0.000 description 7
- SCZJKZLFSSPJDP-ACRUOGEOSA-N Tyr-Phe-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O SCZJKZLFSSPJDP-ACRUOGEOSA-N 0.000 description 7
- DOBHJKVVACOQTN-DZKIICNBSA-N Val-Tyr-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 DOBHJKVVACOQTN-DZKIICNBSA-N 0.000 description 7
- 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 7
- 108010005233 alanylglutamic acid Proteins 0.000 description 7
- 108010068380 arginylarginine Proteins 0.000 description 7
- 108010047857 aspartylglycine Proteins 0.000 description 7
- 108010054812 diprotin A Proteins 0.000 description 7
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 7
- 108010015792 glycyllysine Proteins 0.000 description 7
- 108010087823 glycyltyrosine Proteins 0.000 description 7
- 108010018006 histidylserine Proteins 0.000 description 7
- 108010047926 leucyl-lysyl-tyrosine Proteins 0.000 description 7
- 108010056582 methionylglutamic acid Proteins 0.000 description 7
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 7
- 108010079317 prolyl-tyrosine Proteins 0.000 description 7
- 108010009962 valyltyrosine Proteins 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 6
- AJBVYEYZVYPFCF-CIUDSAMLSA-N Ala-Lys-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O AJBVYEYZVYPFCF-CIUDSAMLSA-N 0.000 description 5
- POOCJCRBHHMAOS-FXQIFTODSA-N Asn-Arg-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O POOCJCRBHHMAOS-FXQIFTODSA-N 0.000 description 5
- XNOWYPDMSLSRKP-GUBZILKMSA-N Glu-Met-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(O)=O XNOWYPDMSLSRKP-GUBZILKMSA-N 0.000 description 5
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 5
- LXVFHIBXOWJTKZ-BZSNNMDCSA-N Phe-Asn-Tyr Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O LXVFHIBXOWJTKZ-BZSNNMDCSA-N 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 108010051110 tyrosyl-lysine Proteins 0.000 description 5
- ALOBJFDJTMQQPW-ONGXEEELSA-N Gly-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)CN ALOBJFDJTMQQPW-ONGXEEELSA-N 0.000 description 4
- RSPUIENXSJYZQO-JYJNAYRXSA-N Phe-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 RSPUIENXSJYZQO-JYJNAYRXSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 4
- 229960002064 kanamycin sulfate Drugs 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- HKALUUKHYNEDRS-GUBZILKMSA-N Cys-Leu-Gln Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HKALUUKHYNEDRS-GUBZILKMSA-N 0.000 description 3
- PEZZSFLFXXFUQD-XPUUQOCRSA-N Gly-Cys-Val Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O PEZZSFLFXXFUQD-XPUUQOCRSA-N 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- 206010039966 Senile dementia Diseases 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 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
- 238000002360 preparation method Methods 0.000 description 3
- IAUSCRHURCZUJP-CIUDSAMLSA-N Ala-Lys-Cys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CS)C(O)=O IAUSCRHURCZUJP-CIUDSAMLSA-N 0.000 description 2
- YJRORCOAFUZVKA-FXQIFTODSA-N Asn-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N YJRORCOAFUZVKA-FXQIFTODSA-N 0.000 description 2
- PGBLJHDDKCVSTC-CIUDSAMLSA-N Cys-Met-Gln Chemical compound CSCC[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O PGBLJHDDKCVSTC-CIUDSAMLSA-N 0.000 description 2
- CSMHMEATMDCQNY-DZKIICNBSA-N Gln-Val-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CSMHMEATMDCQNY-DZKIICNBSA-N 0.000 description 2
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 2
- FKGNJUCQKXQNRA-NRPADANISA-N Glu-Cys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCC(O)=O FKGNJUCQKXQNRA-NRPADANISA-N 0.000 description 2
- HOLOYAZCIHDQNS-YVNDNENWSA-N Ile-Gln-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HOLOYAZCIHDQNS-YVNDNENWSA-N 0.000 description 2
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 2
- PPBKJAQJAUHZKX-SRVKXCTJSA-N Leu-Cys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CC(C)C PPBKJAQJAUHZKX-SRVKXCTJSA-N 0.000 description 2
- 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 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- DHZOGDVYRQOGAC-BZSNNMDCSA-N Phe-Cys-Tyr Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N DHZOGDVYRQOGAC-BZSNNMDCSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 108010069495 cysteinyltyrosine Proteins 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 229950006238 nadide Drugs 0.000 description 2
- 238000005580 one pot reaction Methods 0.000 description 2
- 101150040316 ppk2 gene Proteins 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101150077431 Nrk gene Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- FZAQROFXYZPAKI-UHFFFAOYSA-N anthracene-2-sulfonyl chloride Chemical compound C1=CC=CC2=CC3=CC(S(=O)(=O)Cl)=CC=C3C=C21 FZAQROFXYZPAKI-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 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 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- -1 nicotinamide riboses Chemical class 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 108020000161 polyphosphate kinase Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01022—Ribosylnicotinamide kinase (2.7.1.22)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明具体涉及一种烟酰胺核糖激酶突变体及其编码基因和应用。所述的烟酰胺核糖激酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比,在SEQ ID NO:2所示的氨基酸序列的第22位、第178位、第104位、第157位、第115位、第165位、第52位、第146位进行单突变、两两联合突变、三个联合突变或四个联合突变中的任意一种突变。该烟酰胺核糖激酶突变体与野生型酶相比,酶活和热稳定性均得到了显著提高,可作为生物催化剂,转化底物烟酰胺核糖生成烟酰胺单核苷酸,能显著减少反应时间和酶的使用量,降低生产成本,提高NMN收率,具有大规模工业应用前景。
Description
技术领域
本发明涉及生物酶工程技术领域,具体涉及一种烟酰胺核糖激酶突变体及其编码基因和应用。
背景技术
烟酰胺单核苷酸(Nicotinamide mononuclotide,NMN),是一种自然存在的生物活性核苷酸,是哺乳动物体内烟酰胺腺嘌呤二核苷酸(Nicotinamide adeninedinucleotide,NAD+,又称辅酶I)合成途径的重要中间体。近年来在Science、Nature、Cell等国际权威学术杂志上的相关研究报道表明,补充NMN可有效地增加和恢复体内辅酶I水平,大幅延缓衰老和防止老年痴呆症等多种神经元退化疾病,并由此从根本上调理和改善衰老的各种症状。因此以NMN为活性成分的功能性保健食品有着很大的开发潜力和市场前景。
目前NMN的生产方法主要是采用安全绿色的体外酶催化工艺。以烟酰胺核糖(nicotinamide riboside,NR)为起始原料,在烟酰胺核糖激酶(NR kinase,NrK)和ATP的作用下,一步反应得到NMN,收率高,产品纯度高,为目前的主流生产方法。
如中国专利CN110373398A公开了一种烟酰胺核糖激酶突变体及其应用,该新型烟酰胺核糖激酶突变体可用于β-烟酰胺单核苷酸的合成制备,具有酶成本低,转化时间短、工艺操作简单等特点,但对于酶活性和热稳定性并没有明显提高。
目前关于烟酰胺核糖激酶的研究仍然较少,限制了生物酶催化一步反应法制备NMN工业化生产中的应用。通过定向突变方法提高烟酰胺核糖激酶的酶活性和热稳定性,将十分有助于实现工业上减少酶量,降低生产成本。
发明内容
针对现有技术存在的不足,本发明要解决的技术问题是提供一种烟酰胺核糖激酶突变体及其编码基因和应用,以解决现有野生型烟酰胺核糖激酶活性、热稳定性不理想,难以实现工业化生产的问题。
为解决上述技术问题,本发明提供以下技术方案:
来源于Homo sapiens的野生型烟酰胺核糖激酶的氨基酸序列如SEQ ID NO:2所示,其编码基因的核苷酸序列如SEQ ID NO:1所示。
本发明针对野生型NrK,通过大分子建模技术模拟三维结构,利用能量最低原理和分子对接技术预测出可能的与催化活性和稳定性相关的一个或多个位点,进行定点突变后,从中筛选出活性有显著提高的突变体。
具体地,本发明通过大分子建模技术预测出可能与二硫键形成相关的位点,并组合出三个两两联合突变组合,分别为N22和F178、N104和E157、S115和V165。分别对这三个组合的位点进行定点突变,同时与前期筛选到的突变体F52C/H146C进行组合突变,利用高压液相色谱法(HPLC)来进行突变体的筛选。更为具体的为:1、当位点22的天冬酰胺(N)突变为半胱氨酸(C),位点178的苯丙氨酸(F)突变为半胱氨酸(C)时,突变体的催化活性相对于野生型酶来说变化不大,热稳定性得到显著提高;2、当位点104的天冬酰胺(N)突变为半胱氨酸(C),位点157的谷氨酸(E)突变为半胱氨酸(C)时,突变体单位酶活略微升高,热稳定性得到显著提供;3、当位点115的丝氨酸(S)突变为半胱氨酸(C),位点165的缬氨酸(V)突变为半胱氨酸(C)时,突变体的催化活性相对于野生型酶来说变化不大,热稳定性得到显著提高;4、当上述三组突变体分别与F52C/H146C突变进行四个位点组合突变时,突变体酶活和热稳定性均得到了显著提高。
野生型NRK的基因序列通过常州基宇生物技术有限公司全基因合成所得,在编码区两端分别添加NdeI和HindIII限制性内切酶位点。目的基因片段通过限制性内切酶NdeI和HindIII酶切后,与经过同样双酶切的pET28a(+)载体(Novagen公司)进行连接、转化和筛选,筛选得到的阳性质粒NRK-pET28a(+)转入BL21(DE3)宿主菌中,从而构建NRK的体外异源表达体系。
因此,一方面,本发明请求保护一种烟酰胺核糖激酶突变体,其氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比,在SEQ ID NO:2所示的氨基酸序列的第22位、第178位、第104位、第157位、第115位、第165位、第52位、第146位进行单突变、两两联合突变、三个联合突变或四个联合突变中的任意一种突变。
具体地,所述的两两联合突变为:
当SEQ ID NO:2所示的氨基酸序列的第22位由天冬酰胺突变为半胱氨酸,第178位由苯丙氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:4所示;
或,当SEQ ID NO:2所示的氨基酸序列的第104位由天冬酰胺突变为半胱氨酸,第157位由谷氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ IDNO:6所示;
或,当SEQ ID NO:2所示的氨基酸序列的第115位由丝氨酸突变为半胱氨酸,第165位由缬氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:8所示。
具体地,所述的四个联合突变为:
当SEQ ID NO:2所示的氨基酸序列的第22位由天冬酰胺突变为半胱氨酸,第178位由苯丙氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:10所示;
或,当SEQ ID NO:2所示的氨基酸序列的第104位由天冬酰胺突变为半胱氨酸,第157位由谷氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:12所示;
或,当SEQ ID NO:2所示的氨基酸序列的第115位由丝氨酸突变为半胱氨酸,第165位由缬氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:14所示。
另一方面,本发明还请求保护上述烟酰胺核糖激酶突变体的编码基因。
其中,氨基酸序列如SEQ ID NO:4所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:3所示;
或,氨基酸序列如SEQ ID NO:6所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示;
或,氨基酸序列如SEQ ID NO:8所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示;
或,氨基酸序列如SEQ ID NO:10所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:9所示;
或,氨基酸序列如SEQ ID NO:12所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:11所示;
或,氨基酸序列如SEQ ID NO:14所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:13所示。
又一方面,本发明还请求保护含有上述编码基因的载体。
具体地,所述的载体可以为各种表达载体,包括但不限于pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体中的任意一种表达载体。
又一方面,本发明还请求保护含有上述编码基因的宿主细胞。
具体地,所述的宿主细胞可以为任一种合适的宿主细胞,包括但不限于大肠杆菌、枯草芽孢杆菌、链霉菌或毕赤酵母。
又一方面,本发明还请求保护上述烟酰胺核糖激酶突变体、编码基因、载体、宿主细胞在制备烟酰胺单核苷酸中的应用。
再一方面,本发明还提供了一种制备烟酰胺单核苷酸的方法,包括以下步骤:
S1.配置反应体系,包含:1-10g/L上述的烟酰胺核糖激酶突变体,50mM pH6.0-8.0磷酸钠缓冲液,5mM ATP或ADP,10-50g/L烟酰胺核糖,50mM六偏磷酸钠,50mM七水硫酸镁;控制反应体系温度在35-40℃,进行搅拌反应;
S2.反应4h后进行HPLC检测;经过纯化后得烟酰胺单核苷酸。
优选地,所述的方法包括以下步骤:
S1.配置反应体系,包含:0.5-1g/L上述的烟酰胺核糖激酶突变体,50mM pH6.5磷酸钠缓冲液,5mM ATP,10-50g/L烟酰胺核糖,50mM六偏磷酸钠,50mM七水硫酸镁;控制反应体系温度在40℃,进行搅拌反应;
S2.反应4h后进行HPLC检测;经过纯化后得烟酰胺单核苷酸。
反应产物经HPLC检测,反应转化率>90%。烟酰胺单核苷酸的生成率可达到95%。
相对于现有技术,本发明具有以下有益效果:
本发明构建的烟酰胺核糖激酶突变体与野生型酶相比,酶活和热稳定性均得到了显著提高,可作为生物催化剂,转化底物烟酰胺核糖生成烟酰胺单核苷酸,能显著减少反应时间和酶的使用量,降低生产成本,提高NMN收率,具有大规模工业应用前景。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例中,未注明具体条件的实验方法,通常按常规条件,如《分子克隆实验指南》(J.萨姆布鲁克,D.W.拉塞尔著,黄培堂,汪嘉玺,朱厚础等译,第三版,北京:科学出版社,2002)中所述的方法进行。
实施例一原核表达体系的构建
NrK基因片段由常州基宇生物技术有限公司合成,并重组到PUC57载体上。经限制性内切酶NdeI和HindIII(购自New England Biolabs公司,NEB)在37℃双酶切4h后,1%琼脂糖凝胶电泳分离并进行切胶回收(胶回收试剂盒购自天根生化科技(北京)有限公司)。随后与同样经过双酶切的表达载体pET28a(+)(Novagen公司),在T4 DNA连接酶(购自Takara公司)作用下置于低温连接仪里连接过夜。连接液转化DH5a感受态细胞,并进行菌落PCR筛选和测序验证,从而得到阳性重组质粒NrK-pET28a(+)。
将阳性重组质粒NrK-pET28a(+)转化表达宿主菌BL21(DE3)(购自天根生化科技(北京)有限公司),得到原核表达菌株NrK-pET28a(+)/BL21(DE3),作为后续定向突变和发酵的原代菌株。
用于ATP再生的多聚磷酸激酶(PPK2,来源于E.coli)由常州基宇生物技术有限公司合成,后续重组表达质粒的构建同NrK-pET28a(+)质粒的构建,转入BL21(DE3)中后得到表达菌株。
实施例二酶的摇瓶发酵制备酶冻干粉
上述构建的表达菌株NrK-pET28a(+)/BL21(DE3),在加有终浓度为30μg/mL硫酸卡那霉素的5mL LB液体培养基【10g/L胰蛋白胨(OXIOD),5g/L酵母粉(OXIOD),10g/L氯化钠(国药试剂)】中于37℃、200rpm振荡培养过夜后,按1%(V/V)比例接种于含有终浓度为30μg/mL硫酸卡那霉素的400mL LB液体培养基中,于37℃、200rpm振荡培养。待OD600在0.8-1.0之间时,加入终浓度为0.1mM的诱导剂IPTG(异丙基-β-D-硫代半乳糖苷),并在25℃诱导过夜。菌体在4℃、8000rpm条件下离心收集,然后悬浮于50mM pH7.0磷酸钠缓冲液中,超声破碎(200W,3s/5s,30min),4℃、12000rpm离心20min,取上清进行镍柱亲和层析纯化,经咪唑洗脱、除盐后冷冻干燥,即得酶冻干粉。
实施例三突变体的构建和筛选
突变体的构建:采用大分子建模技术预测出可能有益的突变位点为N22和F178、N104和E157、S115和V165三个两两联合突变组合。随后以NrK-pET28a(+)重组质粒为模板,使用合成的相应引物,第一次PCR扩增突变DNA片段,然后以PCR将所得片段作为模板,第二次PCR扩增全长得到NrK的突变基因。
其中:
22位点突变
正向引物(SEQ ID NO:15):5'GACCCTGGCGAAATGCCTGCAAAAACAT 3',
反向引物(SEQ ID NO:16):5'ATGTTTTTGCAGGCATTTCGCCAGGGTC 3';
178位点突变
正向引物(SEQ ID NO:17):5'GAAGAAGACCTGTGCCTGCAAGTGTATG 3',
反向引物(SEQ ID NO:18):5'CATACACTTGCAGGCACAGGTCTTCTTC 3';
104位点突变
正向引物(SEQ ID NO:19):5'TTTCTGCTGTTCTGCTACAAACCGCT 3',
反向引物(SEQ ID NO:20):5'AGCGGTTTGTAGCAGAACAGCAGAAA 3';
157位点突变
正向引物(SEQ ID NO:21):5'GAAATACCGTCAGTGTATGCAAGATATCACC 3',
反向引物(SEQ ID NO:22):5'GGTGATATCTTGCATACACTGACGGTATTTC 3';
115位点突变
正向引物(SEQ ID NO:23):5'CATTTGGAATCGCTGCTATTTTCTGACG 3',
反向引物(SEQ ID NO:24):5'CGTCAGAAAATAGCAGCGATTCCAAATG 3';
165位点突变
正向引物(SEQ ID NO:25):5'ATCACCTGGGAATGCGTGTATCTGGA 3',
反向引物(SEQ ID NO:26):5'TCCAGATACACGCATTCCCAGGTGAT 3';
52位点突变
正向引物(SEQ ID NO:27):5'TAAAAACGGCTGCCTGCAATATGAC 3',
反向引物(SEQ ID NO:28):5'GTCATATTGCAGGCAGCCGTTTTTA 3';
146位点突变
正向引物(SEQ ID NO:29):5'TACTTCGACGGTTGCGTCTGGCCGAT 3',
反向引物(SEQ ID NO:30):5'ATCGGCCAGACGCAACCGTCGAAGTA 3'。
突变体培养:将上述突变得到的质粒转化BL21(DE3)宿主菌后,涂布于含30μg/mL卡那霉素的LB固体培养基上,37℃倒置培养过夜,随后从平板上挑取单克隆,置于含有30μg/mL硫酸卡那霉素的5mL LB液体培养基中进行培养。过夜培养的菌液再按1%(V/V)比例接种于含有30μg/mL硫酸卡那霉素的100mL LB液体培养基中,37℃、200rpm振荡培养4h后加入终浓度为0.1mM的IPTG进行诱导,25℃培养过夜。4℃、8000rpm离心10min收集菌体,用50mMpH7.0磷酸钠缓冲液悬浮后超声破碎(200W,3s/5s,30min),4℃、12000rpm离心20min,取上清进行单位酶活测定。
突变体的筛选:底物NR浓度10g/L,ATP 30mM,10mM七水硫酸镁,加入适量上述制备的上清液,用10mM pH7.0磷酸钠缓冲液补充体积至2mL,置于37℃恒温磁力搅拌器下搅拌反应。反应20min后取样进行HPLC检测。
热稳定性筛选:将野生型酶粉和突变体酶粉溶于50mM pH7.0的磷酸钠缓冲液后,置于45℃水浴锅中温育20min,离心取上清进行残留的酶活测定。
实验结果如下表1所示。
表1野生型和不同突变组合的酶活
*1U定义为单位时间内(1min)生产1μmol产物NMN所需的酶量。
因此,当SEQ ID NO:2所示的氨基酸序列的第22位由天冬酰胺突变为半胱氨酸,第178位由苯丙氨酸突变为半胱氨酸,烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:4所示。相应地,所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:3所示。
当SEQ ID NO:2所示的氨基酸序列的第104位由天冬酰胺突变为半胱氨酸,第157位由谷氨酸突变为半胱氨酸,烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:6所示。相应地,所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示。
当SEQ ID NO:2所示的氨基酸序列的第115位由丝氨酸突变为半胱氨酸,第165位由缬氨酸突变为半胱氨酸,烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:8所示。相应地,所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示。
当SEQ ID NO:2所示的氨基酸序列的第22位由天冬酰胺突变为半胱氨酸,第178位由苯丙氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:10所示。相应地,所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:9所示。
当SEQ ID NO:2所示的氨基酸序列的第104位由天冬酰胺突变为半胱氨酸,第157位由谷氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:12所示。相应地,所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:11所示。
当SEQ ID NO:2所示的氨基酸序列的第115位由丝氨酸突变为半胱氨酸,第165位由缬氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:14所示。相应地,所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:13所示。
实施例四突变体的生物催化
将160mg底物NR溶解于1.6mL 50mM pH6.5磷酸钠缓冲液中,待底物完全溶解后加入50mM六偏磷酸钠、5mM ATP、50mM七水硫酸镁、烟酰胺核糖激酶突变体和PPK2酶量分别是1g/L和0.5g/L,在40℃恒温磁力搅拌器下搅拌反应,反应4h后进行HPLC检测;不同突变体的底物转化率和产物生成率见下表2。
表2野生型与不同突变体的底物转化率和产物生成率
氨基酸编号 | 野生型/突变体名称 | 底物转化率(%) | 产物生成率(%) |
SEQ ID NO:2 | 野生型NrK | 90.5 | 90.1 |
SEQ ID NO:4 | N22C/F178C | 91.4 | 90.8 |
SEQ ID NO:6 | N104C/E157C | 91.6 | 91.5 |
SEQ ID NO:8 | S115C/V165C | 90.9 | 90.6 |
SEQ ID NO:10 | F52C/H146C/N22C/F178C | 95.8 | 95.5 |
SEQ ID NO:12 | F52C/H146C/N104C/E157C | 95.6 | 95.2 |
SEQ ID NO:14 | F52C/H146C/S115C/V165C | 98.1 | 97.8 |
最后应当说明的是,以上内容仅用以说明本发明的技术方案,而非对本发明保护范围的限制,本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换,均不脱离本发明技术方案的实质和范围。
序列表
<110> 中山百灵生物技术股份有限公司
<120> 一种烟酰胺核糖激酶突变体及其编码基因和应用
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 1
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctttctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggtcacgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 2
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 2
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly His Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 3
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 3
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaatgcctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctttctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggtcacgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gtgcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 4
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 4
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Cys Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly His Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Cys Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 5
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 5
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctttctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttct gctacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggtcacgt ctggccgatg tatctgaaat accgtcagtg tatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 6
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 6
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Cys Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly His Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Cys Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 7
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 7
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctttctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gctgctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggtcacgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aatgcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 8
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 8
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Cys Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly His Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Cys Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 9
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 9
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaatgcctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctgcctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggttgcgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gtgcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 10
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 10
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Cys Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Cys Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly Cys Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Cys Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 11
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 11
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctgcctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttct gctacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggttgcgt ctggccgatg tatctgaaat accgtcagtg tatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 12
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 12
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Cys Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Cys Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly Cys Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Cys Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 13
<211> 600
<212> DNA
<213> 智人(Homo sapiens)
<400> 13
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctgcctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gctgctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggttgcgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aatgcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 14
<211> 199
<212> PRT
<213> 智人(Homo sapiens)
<400> 14
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Cys Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Cys Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly Cys Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Cys Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 15
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gaccctggcg aaatgcctgc aaaaacat 28
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgtttttgc aggcatttcg ccagggtc 28
<210> 17
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gaagaagacc tgtgcctgca agtgtatg 28
<210> 18
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
catacacttg caggcacagg tcttcttc 28
<210> 19
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tttctgctgt tctgctacaa accgct 26
<210> 20
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
agcggtttgt agcagaacag cagaaa 26
<210> 21
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gaaataccgt cagtgtatgc aagatatcac c 31
<210> 22
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ggtgatatct tgcatacact gacggtattt c 31
<210> 23
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
catttggaat cgctgctatt ttctgacg 28
<210> 24
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cgtcagaaaa tagcagcgat tccaaatg 28
<210> 25
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atcacctggg aatgcgtgta tctgga 26
<210> 26
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tccagataca cgcattccca ggtgat 26
<210> 27
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
taaaaacggc tgcctgcaat atgac 25
<210> 28
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gtcatattgc aggcagccgt tttta 25
<210> 29
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tacttcgacg gttgcgtctg gccgat 26
<210> 30
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atcggccaga cgcaaccgtc gaagta 26
Claims (10)
1.一种烟酰胺核糖激酶突变体,其特征在于,所述的烟酰胺核糖激酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比,在SEQ ID NO:2所示的氨基酸序列的第22位、第178位、第104位、第157位、第115位、第165位、第52位、第146位进行单突变、两两联合突变、三个联合突变或四个联合突变中的任意一种突变。
2.根据权利要求1所述的烟酰胺核糖激酶突变体,其特征在于,所述的两两联合突变为:
当SEQ ID NO:2所示的氨基酸序列的第22位由天冬酰胺突变为半胱氨酸,第178位由苯丙氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:4所示;
或,当SEQ ID NO:2所示的氨基酸序列的第104位由天冬酰胺突变为半胱氨酸,第157位由谷氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:6所示;
或,当SEQ ID NO:2所示的氨基酸序列的第115位由丝氨酸突变为半胱氨酸,第165位由缬氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:8所示。
3.根据权利要求1所述的烟酰胺核糖激酶突变体,其特征在于,所述的四个联合突变为:
当SEQ ID NO:2所示的氨基酸序列的第22位由天冬酰胺突变为半胱氨酸,第178位由苯丙氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:10所示;
或,当SEQ ID NO:2所示的氨基酸序列的第104位由天冬酰胺突变为半胱氨酸,第157位由谷氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:12所示;
或,当SEQ ID NO:2所示的氨基酸序列的第115位由丝氨酸突变为半胱氨酸,第165位由缬氨酸突变为半胱氨酸,第52位由苯丙氨酸突变为半胱氨酸,第146位的组氨酸突变为半胱氨酸,所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:14所示。
4.权利要求2或3所述的烟酰胺核糖激酶突变体的编码基因,其特征在于,氨基酸序列如SEQ ID NO:4所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:3所示;
或,氨基酸序列如SEQ ID NO:6所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示;
或,氨基酸序列如SEQ ID NO:8所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示;
或,氨基酸序列如SEQ ID NO:10所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:9所示;
或,氨基酸序列如SEQ ID NO:12所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:11所示;
或,氨基酸序列如SEQ ID NO:14所示的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:13所示。
5.含有权利要求4所述的编码基因的载体。
6.根据权利要求5所述的载体,其特征在于,所述的载体为pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体。
7.含有权利要求4所述的编码基因的宿主细胞,其特征在于,所述的宿主细胞为大肠杆菌、枯草芽孢杆菌、链霉菌或毕赤酵母。
8.权利要求1-3任一项所述的烟酰胺核糖激酶突变体、权利要求4所述的编码基因、权利要求5或6所述的载体、权利要求7所述的宿主细胞在制备烟酰胺单核苷酸中的应用。
9.一种制备烟酰胺单核苷酸的方法,其特征在于,所述的方法包括以下步骤:
S1.配置反应体系,包含:0.5-10g/L权利要求1-3任一项所述的烟酰胺核糖激酶突变体,50mM pH6.0-8.0磷酸钠缓冲液,5mM ATP或ADP,10-50g/L烟酰胺核糖,50mM六偏磷酸钠,50mM七水硫酸镁;控制反应体系温度在35-40℃,进行搅拌反应;
S2.反应4h后进行HPLC检测;经过纯化后得烟酰胺单核苷酸。
10.根据权利要求9所述的方法,其特征在于,所述的方法包括以下步骤:
S1.配置反应体系,包含:0.5-1g/L权利要求1-3任一项所述的烟酰胺核糖激酶突变体,50mM pH6.5磷酸钠缓冲液,5mM ATP,10-50g/L烟酰胺核糖,50mM六偏磷酸钠,50mM七水硫酸镁;控制反应体系温度在40℃,进行搅拌反应;
S2.反应4h后进行HPLC检测;经过纯化后得烟酰胺单核苷酸。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111217419.9A CN113832125B (zh) | 2021-10-19 | 2021-10-19 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111217419.9A CN113832125B (zh) | 2021-10-19 | 2021-10-19 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113832125A true CN113832125A (zh) | 2021-12-24 |
CN113832125B CN113832125B (zh) | 2023-09-26 |
Family
ID=78965571
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111217419.9A Active CN113832125B (zh) | 2021-10-19 | 2021-10-19 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113832125B (zh) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114044800A (zh) * | 2015-08-26 | 2022-02-15 | 艾其林医药公司 | 用于治疗医学障碍的芳基、杂芳基和杂环化合物 |
CN114107160A (zh) * | 2021-12-27 | 2022-03-01 | 浙江工业大学 | 一种烟酰胺核糖激酶基因工程菌及其应用 |
CN114606213A (zh) * | 2022-01-28 | 2022-06-10 | 浙江工业大学 | 一种多聚磷酸激酶突变体、工程菌及其应用 |
CN114990088A (zh) * | 2022-06-24 | 2022-09-02 | 中食都庆(山东)生物技术有限公司 | 烟酰胺核糖激酶突变体及其重组菌与nmn的制备方法 |
CN115058402A (zh) * | 2022-06-08 | 2022-09-16 | 中山俊凯生物技术开发有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108424901A (zh) * | 2018-05-14 | 2018-08-21 | 天津市湖滨盘古基因科学发展有限公司 | 一种人的烟酰胺核苷激酶1突变蛋白及其应用 |
US20190153407A1 (en) * | 2017-11-17 | 2019-05-23 | Bontac Bio-Engineering(Shenzhen) Co.,Ltd | Nicotinamide phosphoribosyltransferase (nampt) mutant and use thereof |
CN110373397A (zh) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | 一种烟酰胺磷酸核糖转移酶突变体及其应用 |
CN111718915A (zh) * | 2020-06-16 | 2020-09-29 | 仙居两山生物科技有限公司 | 一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用 |
CN112280762A (zh) * | 2020-11-13 | 2021-01-29 | 中山俊凯生物技术开发有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
CN112437811A (zh) * | 2019-06-27 | 2021-03-02 | 邦泰生物工程(深圳)有限公司 | 一种重组nad合成酶及其基因和应用 |
CN112553178A (zh) * | 2020-12-25 | 2021-03-26 | 中山俊凯生物技术开发有限公司 | 热稳定性和活性增强的烟酰胺核糖激酶突变体及其编码基因和应用 |
CN113106080A (zh) * | 2021-03-31 | 2021-07-13 | 深圳希吉亚生物技术有限公司 | 烟酰胺磷酸核糖转移酶突变体及其应用 |
-
2021
- 2021-10-19 CN CN202111217419.9A patent/CN113832125B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190153407A1 (en) * | 2017-11-17 | 2019-05-23 | Bontac Bio-Engineering(Shenzhen) Co.,Ltd | Nicotinamide phosphoribosyltransferase (nampt) mutant and use thereof |
CN108424901A (zh) * | 2018-05-14 | 2018-08-21 | 天津市湖滨盘古基因科学发展有限公司 | 一种人的烟酰胺核苷激酶1突变蛋白及其应用 |
CN112437811A (zh) * | 2019-06-27 | 2021-03-02 | 邦泰生物工程(深圳)有限公司 | 一种重组nad合成酶及其基因和应用 |
CN110373397A (zh) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | 一种烟酰胺磷酸核糖转移酶突变体及其应用 |
CN111718915A (zh) * | 2020-06-16 | 2020-09-29 | 仙居两山生物科技有限公司 | 一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用 |
CN112280762A (zh) * | 2020-11-13 | 2021-01-29 | 中山俊凯生物技术开发有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
CN112553178A (zh) * | 2020-12-25 | 2021-03-26 | 中山俊凯生物技术开发有限公司 | 热稳定性和活性增强的烟酰胺核糖激酶突变体及其编码基因和应用 |
CN113106080A (zh) * | 2021-03-31 | 2021-07-13 | 深圳希吉亚生物技术有限公司 | 烟酰胺磷酸核糖转移酶突变体及其应用 |
Non-Patent Citations (1)
Title |
---|
LI J.等: "Q9NPI5 · NRK2_HUMAN", EMBL, pages 1 - 2 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114044800A (zh) * | 2015-08-26 | 2022-02-15 | 艾其林医药公司 | 用于治疗医学障碍的芳基、杂芳基和杂环化合物 |
CN114107160A (zh) * | 2021-12-27 | 2022-03-01 | 浙江工业大学 | 一种烟酰胺核糖激酶基因工程菌及其应用 |
CN114606213A (zh) * | 2022-01-28 | 2022-06-10 | 浙江工业大学 | 一种多聚磷酸激酶突变体、工程菌及其应用 |
CN114606213B (zh) * | 2022-01-28 | 2024-05-03 | 浙江工业大学 | 一种多聚磷酸激酶突变体、工程菌及其应用 |
CN115058402A (zh) * | 2022-06-08 | 2022-09-16 | 中山俊凯生物技术开发有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
CN114990088A (zh) * | 2022-06-24 | 2022-09-02 | 中食都庆(山东)生物技术有限公司 | 烟酰胺核糖激酶突变体及其重组菌与nmn的制备方法 |
Also Published As
Publication number | Publication date |
---|---|
CN113832125B (zh) | 2023-09-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112280762B (zh) | 一种烟酰胺核糖激酶突变体及其编码基因和应用 | |
CN113832125B (zh) | 一种烟酰胺核糖激酶突变体及其编码基因和应用 | |
CN112553178B (zh) | 热稳定性和活性增强的烟酰胺核糖激酶突变体及其编码基因和应用 | |
CN110373398B (zh) | 一种烟酰胺核糖激酶突变体及其应用 | |
CN110373397B (zh) | 一种烟酰胺磷酸核糖转移酶突变体及其应用 | |
US11203742B2 (en) | Sucrose phosphorylase mutant with improved enzyme activity and construction method thereof and use thereof | |
US10174298B2 (en) | Nicotinamide phosphoribosyltransferase (NAMPT) mutant and use thereof | |
CN111718915B (zh) | 一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用 | |
KR102132381B1 (ko) | 아스로박터 글로비포미스에 의해 생산되는 케토오스 3-에피머라제 | |
US20180162895A1 (en) | Method for preparing nicotinamide mononucleotide (nmn) | |
CN110358750B (zh) | 新型蔗糖磷酸化酶突变体及其在合成甘油葡糖苷中的应用 | |
US10519429B2 (en) | Nicotinamide phosphoribosyltransferase (NAMPT) mutant and use thereof | |
AU2019297508A1 (en) | Methods and compositions for preparing tagatose from fructose | |
CN113832122A (zh) | 一种7β-HSDH酶突变体及其编码基因和应用 | |
US8852891B2 (en) | N-acetylglucosamine-2-epimerase and method for producing CMP-neuraminic acid using the same | |
WO2023035584A1 (zh) | 发酵生产肝素的酵母工程菌的构建及其应用 | |
CN110804602B (zh) | 一种L-天冬氨酸β-脱羧酶突变体及其应用 | |
CN115058402B (zh) | 一种烟酰胺核糖激酶突变体及其编码基因和应用 | |
CN113699131A (zh) | 一种α-环糊精葡萄糖基转移酶突变体及其应用 | |
CN108034649B (zh) | 一种葡萄糖异构酶突变体及其应用 | |
CN111057698B (zh) | L-阿拉伯糖异构酶、突变体及其应用 | |
CN110951717B (zh) | 一种l-阿拉伯糖异构酶异构体及其应用 | |
CN110904087B (zh) | L-阿拉伯糖差向异构酶突变体及其应用 | |
CN117230049B (zh) | 一种热稳定性提高的褐藻胶裂解酶突变体及其用途 | |
CN115725538A (zh) | 一种烟酰胺核糖激酶突变体、编码基因及应用 |
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 |