CN115029327A - 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 - Google Patents
葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 Download PDFInfo
- Publication number
- CN115029327A CN115029327A CN202210432858.XA CN202210432858A CN115029327A CN 115029327 A CN115029327 A CN 115029327A CN 202210432858 A CN202210432858 A CN 202210432858A CN 115029327 A CN115029327 A CN 115029327A
- Authority
- CN
- China
- Prior art keywords
- ala
- gly
- leu
- val
- thr
- 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.)
- Pending
Links
- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 123
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 104
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 99
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 99
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 23
- 230000000694 effects Effects 0.000 claims abstract description 57
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 74
- 238000006467 substitution reaction Methods 0.000 claims description 66
- 150000001413 amino acids Chemical group 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 30
- 102200031292 rs62508588 Human genes 0.000 claims description 25
- 102220274636 rs144712084 Human genes 0.000 claims description 19
- 102220417870 c.87T>A Human genes 0.000 claims description 15
- 102200099125 rs137853176 Human genes 0.000 claims description 15
- 102220042823 rs35942089 Human genes 0.000 claims description 13
- 102220511094 Endothelial cell-specific molecule 1_L14A_mutation Human genes 0.000 claims description 11
- 101001105586 Xenopus laevis 60S ribosomal protein L18-A Proteins 0.000 claims description 11
- 102200155647 c.151A>G Human genes 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 7
- 102220540191 Serine/threonine-protein kinase WNK1_N88Q_mutation Human genes 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 6
- 102220313168 rs1553262439 Human genes 0.000 claims description 5
- 102220285240 rs766788652 Human genes 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 102220222651 rs113217267 Human genes 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 24
- 108090000790 Enzymes Proteins 0.000 abstract description 24
- 229940088598 enzyme Drugs 0.000 abstract description 24
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000035772 mutation Effects 0.000 description 22
- 108010005233 alanylglutamic acid Proteins 0.000 description 18
- 108010050848 glycylleucine Proteins 0.000 description 18
- 108010037850 glycylvaline Proteins 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 13
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 12
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 12
- 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 12
- 108010089804 glycyl-threonine Proteins 0.000 description 12
- 108010034529 leucyl-lysine Proteins 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 241000235058 Komagataella pastoris Species 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 6
- KUDREHRZRIVKHS-UWJYBYFXSA-N Ala-Asp-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KUDREHRZRIVKHS-UWJYBYFXSA-N 0.000 description 6
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 6
- GRPHQEMIFDPKOE-HGNGGELXSA-N Ala-His-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O GRPHQEMIFDPKOE-HGNGGELXSA-N 0.000 description 6
- PIXQDIGKDNNOOV-GUBZILKMSA-N Ala-Lys-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O PIXQDIGKDNNOOV-GUBZILKMSA-N 0.000 description 6
- VQAVBBCZFQAAED-FXQIFTODSA-N Ala-Pro-Asn Chemical compound C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)N)C(=O)O)N VQAVBBCZFQAAED-FXQIFTODSA-N 0.000 description 6
- OMCKWYSDUQBYCN-FXQIFTODSA-N Ala-Ser-Met Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O OMCKWYSDUQBYCN-FXQIFTODSA-N 0.000 description 6
- VQBULXOHAZSTQY-GKCIPKSASA-N Ala-Trp-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VQBULXOHAZSTQY-GKCIPKSASA-N 0.000 description 6
- YCTIYBUTCKNOTI-UWJYBYFXSA-N Ala-Tyr-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCTIYBUTCKNOTI-UWJYBYFXSA-N 0.000 description 6
- QRIYOHQJRDHFKF-UWJYBYFXSA-N Ala-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 QRIYOHQJRDHFKF-UWJYBYFXSA-N 0.000 description 6
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 6
- SGYSTDWPNPKJPP-GUBZILKMSA-N Arg-Ala-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SGYSTDWPNPKJPP-GUBZILKMSA-N 0.000 description 6
- MCYJBCKCAPERSE-FXQIFTODSA-N Arg-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N MCYJBCKCAPERSE-FXQIFTODSA-N 0.000 description 6
- QPOARHANPULOTM-GMOBBJLQSA-N Arg-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N QPOARHANPULOTM-GMOBBJLQSA-N 0.000 description 6
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 6
- ZZZWQALDSQQBEW-STQMWFEESA-N Arg-Gly-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZZZWQALDSQQBEW-STQMWFEESA-N 0.000 description 6
- NKNILFJYKKHBKE-WPRPVWTQSA-N Arg-Gly-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O NKNILFJYKKHBKE-WPRPVWTQSA-N 0.000 description 6
- HJDNZFIYILEIKR-OSUNSFLBSA-N Arg-Ile-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HJDNZFIYILEIKR-OSUNSFLBSA-N 0.000 description 6
- STHNZYKCJHWULY-AVGNSLFASA-N Arg-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O STHNZYKCJHWULY-AVGNSLFASA-N 0.000 description 6
- FMYQECOAIFGQGU-CYDGBPFRSA-N Arg-Val-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FMYQECOAIFGQGU-CYDGBPFRSA-N 0.000 description 6
- PNHQRQTVBRDIEF-CIUDSAMLSA-N Asn-Leu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)N)N PNHQRQTVBRDIEF-CIUDSAMLSA-N 0.000 description 6
- UXHYOWXTJLBEPG-GSSVUCPTSA-N Asn-Thr-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UXHYOWXTJLBEPG-GSSVUCPTSA-N 0.000 description 6
- WSNSZZGIMVHDHF-TUUVXOQKSA-N Asn-Trp-Asp-Ser Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(N)=O)N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 WSNSZZGIMVHDHF-TUUVXOQKSA-N 0.000 description 6
- CBWCQCANJSGUOH-ZKWXMUAHSA-N Asn-Val-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O CBWCQCANJSGUOH-ZKWXMUAHSA-N 0.000 description 6
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 6
- VZNOVQKGJQJOCS-SRVKXCTJSA-N Asp-Asp-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VZNOVQKGJQJOCS-SRVKXCTJSA-N 0.000 description 6
- SNDBKTFJWVEVPO-WHFBIAKZSA-N Asp-Gly-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SNDBKTFJWVEVPO-WHFBIAKZSA-N 0.000 description 6
- LDGUZSIPGSPBJP-XVYDVKMFSA-N Asp-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)O)N LDGUZSIPGSPBJP-XVYDVKMFSA-N 0.000 description 6
- GGRSYTUJHAZTFN-IHRRRGAJSA-N Asp-Pro-Tyr Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O GGRSYTUJHAZTFN-IHRRRGAJSA-N 0.000 description 6
- JJQGZGOEDSSHTE-FOHZUACHSA-N Asp-Thr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JJQGZGOEDSSHTE-FOHZUACHSA-N 0.000 description 6
- KACWACLNYLSVCA-VHWLVUOQSA-N Asp-Trp-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KACWACLNYLSVCA-VHWLVUOQSA-N 0.000 description 6
- SBDVXRYCOIEYNV-YUMQZZPRSA-N Cys-His-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CS)N SBDVXRYCOIEYNV-YUMQZZPRSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- INFBPLSHYFALDE-ACZMJKKPSA-N Gln-Asn-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O INFBPLSHYFALDE-ACZMJKKPSA-N 0.000 description 6
- RKAQZCDMSUQTSS-FXQIFTODSA-N Gln-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RKAQZCDMSUQTSS-FXQIFTODSA-N 0.000 description 6
- HYPVLWGNBIYTNA-GUBZILKMSA-N Gln-Leu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HYPVLWGNBIYTNA-GUBZILKMSA-N 0.000 description 6
- XKPACHRGOWQHFH-IRIUXVKKSA-N Gln-Thr-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XKPACHRGOWQHFH-IRIUXVKKSA-N 0.000 description 6
- RBSKVTZUFMIWFU-XEGUGMAKSA-N Gln-Trp-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O RBSKVTZUFMIWFU-XEGUGMAKSA-N 0.000 description 6
- VAIWPXWHWAPYDF-FXQIFTODSA-N Glu-Asp-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O VAIWPXWHWAPYDF-FXQIFTODSA-N 0.000 description 6
- CGOHAEBMDSEKFB-FXQIFTODSA-N Glu-Glu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O CGOHAEBMDSEKFB-FXQIFTODSA-N 0.000 description 6
- GGJOGFJIPPGNRK-JSGCOSHPSA-N Glu-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)N)C(O)=O)=CNC2=C1 GGJOGFJIPPGNRK-JSGCOSHPSA-N 0.000 description 6
- BIHMNDPWRUROFZ-JYJNAYRXSA-N Glu-His-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BIHMNDPWRUROFZ-JYJNAYRXSA-N 0.000 description 6
- PJBVXVBTTFZPHJ-GUBZILKMSA-N Glu-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N PJBVXVBTTFZPHJ-GUBZILKMSA-N 0.000 description 6
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 6
- MRWYPDWDZSLWJM-ACZMJKKPSA-N Glu-Ser-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O MRWYPDWDZSLWJM-ACZMJKKPSA-N 0.000 description 6
- RGJKYNUINKGPJN-RWRJDSDZSA-N Glu-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(=O)O)N RGJKYNUINKGPJN-RWRJDSDZSA-N 0.000 description 6
- 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 6
- 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 6
- PHONXOACARQMPM-BQBZGAKWSA-N Gly-Ala-Met Chemical compound [H]NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O PHONXOACARQMPM-BQBZGAKWSA-N 0.000 description 6
- GGEJHJIXRBTJPD-BYPYZUCNSA-N Gly-Asn-Gly Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GGEJHJIXRBTJPD-BYPYZUCNSA-N 0.000 description 6
- CEXINUGNTZFNRY-BYPYZUCNSA-N Gly-Cys-Gly Chemical compound [NH3+]CC(=O)N[C@@H](CS)C(=O)NCC([O-])=O CEXINUGNTZFNRY-BYPYZUCNSA-N 0.000 description 6
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 6
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 6
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 6
- OLPPXYMMIARYAL-QMMMGPOBSA-N Gly-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)CN OLPPXYMMIARYAL-QMMMGPOBSA-N 0.000 description 6
- YNIMVVJTPWCUJH-KBPBESRZSA-N Gly-His-Tyr Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YNIMVVJTPWCUJH-KBPBESRZSA-N 0.000 description 6
- DHNXGWVNLFPOMQ-KBPBESRZSA-N Gly-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)CN DHNXGWVNLFPOMQ-KBPBESRZSA-N 0.000 description 6
- FKYQEVBRZSFAMJ-QWRGUYRKSA-N Gly-Ser-Tyr Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FKYQEVBRZSFAMJ-QWRGUYRKSA-N 0.000 description 6
- ZKJZBRHRWKLVSJ-ZDLURKLDSA-N Gly-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)O ZKJZBRHRWKLVSJ-ZDLURKLDSA-N 0.000 description 6
- RHRLHXQWHCNJKR-PMVVWTBXSA-N Gly-Thr-His Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 RHRLHXQWHCNJKR-PMVVWTBXSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- NTXIJPDAHXSHNL-ONGXEEELSA-N His-Gly-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O NTXIJPDAHXSHNL-ONGXEEELSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- NKVZTQVGUNLLQW-JBDRJPRFSA-N Ile-Ala-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)O)N NKVZTQVGUNLLQW-JBDRJPRFSA-N 0.000 description 6
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 6
- WZDCVAWMBUNDDY-KBIXCLLPSA-N Ile-Glu-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C)C(=O)O)N WZDCVAWMBUNDDY-KBIXCLLPSA-N 0.000 description 6
- WIZPFZKOFZXDQG-HTFCKZLJSA-N Ile-Ile-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O WIZPFZKOFZXDQG-HTFCKZLJSA-N 0.000 description 6
- SJLVSMMIFYTSGY-GRLWGSQLSA-N Ile-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SJLVSMMIFYTSGY-GRLWGSQLSA-N 0.000 description 6
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 6
- UAELWXJFLZBKQS-WHOFXGATSA-N Ile-Phe-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O UAELWXJFLZBKQS-WHOFXGATSA-N 0.000 description 6
- FQYQMFCIJNWDQZ-CYDGBPFRSA-N Ile-Pro-Pro Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 FQYQMFCIJNWDQZ-CYDGBPFRSA-N 0.000 description 6
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 6
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 241000880493 Leptailurus serval Species 0.000 description 6
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 6
- FJUKMPUELVROGK-IHRRRGAJSA-N Leu-Arg-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N FJUKMPUELVROGK-IHRRRGAJSA-N 0.000 description 6
- WUFYAPWIHCUMLL-CIUDSAMLSA-N Leu-Asn-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O WUFYAPWIHCUMLL-CIUDSAMLSA-N 0.000 description 6
- OIARJGNVARWKFP-YUMQZZPRSA-N Leu-Asn-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIARJGNVARWKFP-YUMQZZPRSA-N 0.000 description 6
- JKGHDYGZRDWHGA-SRVKXCTJSA-N Leu-Asn-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JKGHDYGZRDWHGA-SRVKXCTJSA-N 0.000 description 6
- FIJMQLGQLBLBOL-HJGDQZAQSA-N Leu-Asn-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FIJMQLGQLBLBOL-HJGDQZAQSA-N 0.000 description 6
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 6
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 6
- LLBQJYDYOLIQAI-JYJNAYRXSA-N Leu-Glu-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LLBQJYDYOLIQAI-JYJNAYRXSA-N 0.000 description 6
- AUBMZAMQCOYSIC-MNXVOIDGSA-N Leu-Ile-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O AUBMZAMQCOYSIC-MNXVOIDGSA-N 0.000 description 6
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 6
- HDHQQEDVWQGBEE-DCAQKATOSA-N Leu-Met-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O HDHQQEDVWQGBEE-DCAQKATOSA-N 0.000 description 6
- DRWMRVFCKKXHCH-BZSNNMDCSA-N Leu-Phe-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=CC=C1 DRWMRVFCKKXHCH-BZSNNMDCSA-N 0.000 description 6
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 6
- XXXXOVFBXRERQL-ULQDDVLXSA-N Leu-Pro-Phe Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XXXXOVFBXRERQL-ULQDDVLXSA-N 0.000 description 6
- 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 6
- JCFYLFOCALSNLQ-GUBZILKMSA-N Lys-Ala-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JCFYLFOCALSNLQ-GUBZILKMSA-N 0.000 description 6
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 6
- 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 6
- KKFVKBWCXXLKIK-AVGNSLFASA-N Lys-His-Glu Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCCCN)N KKFVKBWCXXLKIK-AVGNSLFASA-N 0.000 description 6
- SLQJJFAVWSZLBL-BJDJZHNGSA-N Lys-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN SLQJJFAVWSZLBL-BJDJZHNGSA-N 0.000 description 6
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 6
- GHKXHCMRAUYLBS-CIUDSAMLSA-N Lys-Ser-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O GHKXHCMRAUYLBS-CIUDSAMLSA-N 0.000 description 6
- QEVRUYFHWJJUHZ-DCAQKATOSA-N Met-Ala-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(C)C QEVRUYFHWJJUHZ-DCAQKATOSA-N 0.000 description 6
- DLAFCQWUMFMZSN-GUBZILKMSA-N Met-Arg-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CCCN=C(N)N DLAFCQWUMFMZSN-GUBZILKMSA-N 0.000 description 6
- CGUYGMFQZCYJSG-DCAQKATOSA-N Met-Lys-Ser Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O CGUYGMFQZCYJSG-DCAQKATOSA-N 0.000 description 6
- ZBLSZPYQQRIHQU-RCWTZXSCSA-N Met-Thr-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O ZBLSZPYQQRIHQU-RCWTZXSCSA-N 0.000 description 6
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 6
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 6
- 108010066427 N-valyltryptophan Proteins 0.000 description 6
- FPTXMUIBLMGTQH-ONGXEEELSA-N Phe-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 FPTXMUIBLMGTQH-ONGXEEELSA-N 0.000 description 6
- FRPVPGRXUKFEQE-YDHLFZDLSA-N Phe-Asp-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O FRPVPGRXUKFEQE-YDHLFZDLSA-N 0.000 description 6
- KBVJZCVLQWCJQN-KKUMJFAQSA-N Phe-Leu-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KBVJZCVLQWCJQN-KKUMJFAQSA-N 0.000 description 6
- RVEVENLSADZUMS-IHRRRGAJSA-N Phe-Pro-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RVEVENLSADZUMS-IHRRRGAJSA-N 0.000 description 6
- QTDBZORPVYTRJU-KKXDTOCCSA-N Phe-Tyr-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O QTDBZORPVYTRJU-KKXDTOCCSA-N 0.000 description 6
- MLQVJYMFASXBGZ-IHRRRGAJSA-N Pro-Asn-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O MLQVJYMFASXBGZ-IHRRRGAJSA-N 0.000 description 6
- KTFZQPLSPLWLKN-KKUMJFAQSA-N Pro-Gln-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KTFZQPLSPLWLKN-KKUMJFAQSA-N 0.000 description 6
- VWXGFAIZUQBBBG-UWVGGRQHSA-N Pro-His-Gly Chemical compound C([C@@H](C(=O)NCC(=O)[O-])NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 VWXGFAIZUQBBBG-UWVGGRQHSA-N 0.000 description 6
- FXGIMYRVJJEIIM-UWVGGRQHSA-N Pro-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FXGIMYRVJJEIIM-UWVGGRQHSA-N 0.000 description 6
- XQPHBAKJJJZOBX-SRVKXCTJSA-N Pro-Lys-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O XQPHBAKJJJZOBX-SRVKXCTJSA-N 0.000 description 6
- CHYAYDLYYIJCKY-OSUNSFLBSA-N Pro-Thr-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CHYAYDLYYIJCKY-OSUNSFLBSA-N 0.000 description 6
- AJJDPGVVNPUZCR-RHYQMDGZSA-N Pro-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1)O AJJDPGVVNPUZCR-RHYQMDGZSA-N 0.000 description 6
- FUOGXAQMNJMBFG-WPRPVWTQSA-N Pro-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 FUOGXAQMNJMBFG-WPRPVWTQSA-N 0.000 description 6
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 6
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 6
- RDFQNDHEHVSONI-ZLUOBGJFSA-N Ser-Asn-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDFQNDHEHVSONI-ZLUOBGJFSA-N 0.000 description 6
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 6
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 6
- QSHKTZVJGDVFEW-GUBZILKMSA-N Ser-Met-Met Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CO)N QSHKTZVJGDVFEW-GUBZILKMSA-N 0.000 description 6
- FKYWFUYPVKLJLP-DCAQKATOSA-N Ser-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FKYWFUYPVKLJLP-DCAQKATOSA-N 0.000 description 6
- NVNPWELENFJOHH-CIUDSAMLSA-N Ser-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)N NVNPWELENFJOHH-CIUDSAMLSA-N 0.000 description 6
- RKDFEMGVMMYYNG-WDCWCFNPSA-N Thr-Gln-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O RKDFEMGVMMYYNG-WDCWCFNPSA-N 0.000 description 6
- CQNFRKAKGDSJFR-NUMRIWBASA-N Thr-Glu-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O CQNFRKAKGDSJFR-NUMRIWBASA-N 0.000 description 6
- HJOSVGCWOTYJFG-WDCWCFNPSA-N Thr-Glu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N)O HJOSVGCWOTYJFG-WDCWCFNPSA-N 0.000 description 6
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 6
- VUSAEKOXGNEYNE-PBCZWWQYSA-N Thr-His-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VUSAEKOXGNEYNE-PBCZWWQYSA-N 0.000 description 6
- XIULAFZYEKSGAJ-IXOXFDKPSA-N Thr-Leu-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 XIULAFZYEKSGAJ-IXOXFDKPSA-N 0.000 description 6
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 6
- KZURUCDWKDEAFZ-XVSYOHENSA-N Thr-Phe-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O KZURUCDWKDEAFZ-XVSYOHENSA-N 0.000 description 6
- IEZVHOULSUULHD-XGEHTFHBSA-N Thr-Ser-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O IEZVHOULSUULHD-XGEHTFHBSA-N 0.000 description 6
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 6
- 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 6
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 6
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 6
- CURFABYITJVKEW-QTKMDUPCSA-N Thr-Val-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O CURFABYITJVKEW-QTKMDUPCSA-N 0.000 description 6
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 6
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 6
- GTNCSPKYWCJZAC-XIRDDKMYSA-N Trp-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N GTNCSPKYWCJZAC-XIRDDKMYSA-N 0.000 description 6
- SNJAPSVIPKUMCK-NWLDYVSISA-N Trp-Glu-Thr Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SNJAPSVIPKUMCK-NWLDYVSISA-N 0.000 description 6
- LNGFWVPNKLWATF-ZVZYQTTQSA-N Trp-Val-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LNGFWVPNKLWATF-ZVZYQTTQSA-N 0.000 description 6
- TZXFLDNBYYGLKA-BZSNNMDCSA-N Tyr-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 TZXFLDNBYYGLKA-BZSNNMDCSA-N 0.000 description 6
- ARPONUQDNWLXOZ-KKUMJFAQSA-N Tyr-Gln-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ARPONUQDNWLXOZ-KKUMJFAQSA-N 0.000 description 6
- LOOCQRRBKZTPKO-AVGNSLFASA-N Tyr-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LOOCQRRBKZTPKO-AVGNSLFASA-N 0.000 description 6
- GIOBXJSONRQHKQ-RYUDHWBXSA-N Tyr-Gly-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O GIOBXJSONRQHKQ-RYUDHWBXSA-N 0.000 description 6
- OHOVFPKXPZODHS-SJWGOKEGSA-N Tyr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N OHOVFPKXPZODHS-SJWGOKEGSA-N 0.000 description 6
- QFXVAFIHVWXXBJ-AVGNSLFASA-N Tyr-Ser-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O QFXVAFIHVWXXBJ-AVGNSLFASA-N 0.000 description 6
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 6
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 6
- CGGVNFJRZJUVAE-BYULHYEWSA-N Val-Asp-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CGGVNFJRZJUVAE-BYULHYEWSA-N 0.000 description 6
- YODDULVCGFQRFZ-ZKWXMUAHSA-N Val-Asp-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O YODDULVCGFQRFZ-ZKWXMUAHSA-N 0.000 description 6
- XEYUMGGWQCIWAR-XVKPBYJWSA-N Val-Gln-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)NCC(=O)O)N XEYUMGGWQCIWAR-XVKPBYJWSA-N 0.000 description 6
- GBESYURLQOYWLU-LAEOZQHASA-N Val-Glu-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N GBESYURLQOYWLU-LAEOZQHASA-N 0.000 description 6
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 description 6
- YTPLVNUZZOBFFC-SCZZXKLOSA-N Val-Gly-Pro Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N1CCC[C@@H]1C(O)=O YTPLVNUZZOBFFC-SCZZXKLOSA-N 0.000 description 6
- DLMNFMXSNGTSNJ-PYJNHQTQSA-N Val-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N DLMNFMXSNGTSNJ-PYJNHQTQSA-N 0.000 description 6
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 6
- CKTMJBPRVQWPHU-JSGCOSHPSA-N Val-Phe-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)O)N CKTMJBPRVQWPHU-JSGCOSHPSA-N 0.000 description 6
- DLLRRUDLMSJTMB-GUBZILKMSA-N Val-Ser-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)O)N DLLRRUDLMSJTMB-GUBZILKMSA-N 0.000 description 6
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 6
- QPJSIBAOZBVELU-BPNCWPANSA-N Val-Tyr-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N QPJSIBAOZBVELU-BPNCWPANSA-N 0.000 description 6
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 6
- ZLNYBMWGPOKSLW-LSJOCFKGSA-N Val-Val-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLNYBMWGPOKSLW-LSJOCFKGSA-N 0.000 description 6
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 6
- 108010041407 alanylaspartic acid Proteins 0.000 description 6
- 108010047495 alanylglycine Proteins 0.000 description 6
- 108010013835 arginine glutamate Proteins 0.000 description 6
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 6
- 108010060035 arginylproline Proteins 0.000 description 6
- 108010036533 arginylvaline Proteins 0.000 description 6
- 108010077245 asparaginyl-proline Proteins 0.000 description 6
- 108010093581 aspartyl-proline Proteins 0.000 description 6
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 6
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 6
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 6
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 6
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 6
- 108010092114 histidylphenylalanine Proteins 0.000 description 6
- 108010031424 isoleucyl-prolyl-proline Proteins 0.000 description 6
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 6
- 108010054155 lysyllysine Proteins 0.000 description 6
- 108010025488 pinealon Proteins 0.000 description 6
- 108700042769 prolyl-leucyl-glycine Proteins 0.000 description 6
- 108010004914 prolylarginine Proteins 0.000 description 6
- 108010070643 prolylglutamic acid Proteins 0.000 description 6
- WSWYMRLTJVKRCE-ZLUOBGJFSA-N Asp-Ala-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O WSWYMRLTJVKRCE-ZLUOBGJFSA-N 0.000 description 5
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 5
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 description 5
- JLJLBWDKDRYOPA-RYUDHWBXSA-N Gly-Gln-Tyr Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JLJLBWDKDRYOPA-RYUDHWBXSA-N 0.000 description 5
- SPQWWEZBHXHUJN-KBIXCLLPSA-N Ile-Glu-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O SPQWWEZBHXHUJN-KBIXCLLPSA-N 0.000 description 5
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 5
- ULIWFCCJIOEHMU-BQBZGAKWSA-N Pro-Gly-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 ULIWFCCJIOEHMU-BQBZGAKWSA-N 0.000 description 5
- SYOMXKPPFZRELL-ONGXEEELSA-N Val-Gly-Lys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N SYOMXKPPFZRELL-ONGXEEELSA-N 0.000 description 5
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 5
- 108010078144 glutaminyl-glycine Proteins 0.000 description 5
- 238000013537 high throughput screening Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 108010061238 threonyl-glycine Proteins 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- WMYJZJRILUVVRG-WDSKDSINSA-N Ala-Gly-Gln Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O WMYJZJRILUVVRG-WDSKDSINSA-N 0.000 description 4
- XWGJDUSDTRPQRK-ZLUOBGJFSA-N Asn-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O XWGJDUSDTRPQRK-ZLUOBGJFSA-N 0.000 description 4
- 241000228245 Aspergillus niger Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- XWYXZPHPYKRYPA-GMOBBJLQSA-N Pro-Asn-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XWYXZPHPYKRYPA-GMOBBJLQSA-N 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 3
- HFPXZWPUVFVNLL-GUBZILKMSA-N Asn-Leu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFPXZWPUVFVNLL-GUBZILKMSA-N 0.000 description 3
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 3
- DTPOVRRYXPJJAZ-FJXKBIBVSA-N Gly-Arg-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N DTPOVRRYXPJJAZ-FJXKBIBVSA-N 0.000 description 3
- PMNHJLASAAWELO-FOHZUACHSA-N Gly-Asp-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PMNHJLASAAWELO-FOHZUACHSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 108010084455 Zeocin Proteins 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 3
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 3
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 3
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 3
- 239000000174 gluconic acid Substances 0.000 description 3
- 235000012208 gluconic acid Nutrition 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- ASQKVGRCKOFKIU-KZVJFYERSA-N Arg-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ASQKVGRCKOFKIU-KZVJFYERSA-N 0.000 description 2
- 241000351920 Aspergillus nidulans Species 0.000 description 2
- 240000006439 Aspergillus oryzae Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 241000194107 Bacillus megaterium Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 2
- DLISPGXMKZTWQG-IFFSRLJSSA-N Glu-Thr-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O DLISPGXMKZTWQG-IFFSRLJSSA-N 0.000 description 2
- UROVZOUMHNXPLZ-AVGNSLFASA-N His-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=CN=CN1 UROVZOUMHNXPLZ-AVGNSLFASA-N 0.000 description 2
- 241001138401 Kluyveromyces lactis Species 0.000 description 2
- AVEGDIAXTDVBJS-XUXIUFHCSA-N Leu-Ile-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AVEGDIAXTDVBJS-XUXIUFHCSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 241000320412 Ogataea angusta Species 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 101150051118 PTM1 gene Proteins 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- GDXZRWYXJSGWIV-GMOBBJLQSA-N Pro-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 GDXZRWYXJSGWIV-GMOBBJLQSA-N 0.000 description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 241000499912 Trichoderma reesei Species 0.000 description 2
- 241000235015 Yarrowia lipolytica Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 101150094958 gox gene Proteins 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000001471 micro-filtration Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 description 1
- QHRTZMDBPOUHPU-UHFFFAOYSA-N 4-(4-amino-3-methoxyphenyl)-2-methoxyaniline;hydrochloride Chemical compound Cl.C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 QHRTZMDBPOUHPU-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- YNDLOUMBVDVALC-ZLUOBGJFSA-N Asn-Ala-Ala Chemical compound C[C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC(=O)N)N YNDLOUMBVDVALC-ZLUOBGJFSA-N 0.000 description 1
- RZVVKNIACROXRM-ZLUOBGJFSA-N Asn-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N RZVVKNIACROXRM-ZLUOBGJFSA-N 0.000 description 1
- HZPSDHRYYIORKR-WHFBIAKZSA-N Asn-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O HZPSDHRYYIORKR-WHFBIAKZSA-N 0.000 description 1
- HAFCJCDJGIOYPW-WDSKDSINSA-N Asp-Gly-Gln Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O HAFCJCDJGIOYPW-WDSKDSINSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241001674013 Chrysosporium lucknowense Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 241000252867 Cupriavidus metallidurans Species 0.000 description 1
- 208000002064 Dental Plaque Diseases 0.000 description 1
- 241000702224 Enterobacteria phage M13 Species 0.000 description 1
- 241000701867 Enterobacteria phage T7 Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- QPTNELDXWKRIFX-YFKPBYRVSA-N Gly-Gly-Gln Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O QPTNELDXWKRIFX-YFKPBYRVSA-N 0.000 description 1
- CVFOYJJOZYYEPE-KBPBESRZSA-N Gly-Lys-Tyr Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CVFOYJJOZYYEPE-KBPBESRZSA-N 0.000 description 1
- 241000579664 Grateloupia proteus Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 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 1
- 241000194036 Lactococcus Species 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- IRNSXVOWSXSULE-DCAQKATOSA-N Lys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN IRNSXVOWSXSULE-DCAQKATOSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000219470 Mirabilis Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 208000025157 Oral disease Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- UUHXBJHVTVGSKM-BQBZGAKWSA-N Pro-Gly-Asn Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O UUHXBJHVTVGSKM-BQBZGAKWSA-N 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 241000589614 Pseudomonas stutzeri Species 0.000 description 1
- 241000232299 Ralstonia Species 0.000 description 1
- 241000191965 Staphylococcus carnosus Species 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- VIBXMCZWVUOZLA-OLHMAJIHSA-N Thr-Asn-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VIBXMCZWVUOZLA-OLHMAJIHSA-N 0.000 description 1
- YLXAMFZYJTZXFH-OLHMAJIHSA-N Thr-Asn-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O YLXAMFZYJTZXFH-OLHMAJIHSA-N 0.000 description 1
- 206010044029 Tooth deposit Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 108091006055 affinity-tagged proteins Proteins 0.000 description 1
- 150000001447 alkali salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000001023 centrifugal evaporation Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002036 drum drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 108010006664 gamma-glutamyl-glycyl-glycine Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000003093 intracellular space Anatomy 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- XNCMOUSLNOHBKY-UHFFFAOYSA-H iron(3+);trisulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XNCMOUSLNOHBKY-UHFFFAOYSA-H 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 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 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 208000030194 mouth disease Diseases 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 102220179898 rs866437643 Human genes 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000176 sodium gluconate Substances 0.000 description 1
- 235000012207 sodium gluconate Nutrition 0.000 description 1
- 229940005574 sodium gluconate Drugs 0.000 description 1
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Polymers & Plastics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Animal Husbandry (AREA)
- Physics & Mathematics (AREA)
- Nutrition Science (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及基因工程领域，特别涉及葡萄糖氧化酶突变体GOx‑MUT7～11及其编码基因和应用。本发明提供葡萄糖氧化酶突变体，与亲本SEQ ID NO：1所示的葡萄糖氧化酶相比，比活以及耐热性有了显著的提升。有利于该酶在工业化生产中应用。
Description
技术领域
本发明涉及基因工程领域，特别涉及葡萄糖氧化酶突变体GOx-MUT7～11及其编码基因和应用。
背景技术
葡萄糖氧化酶(GOD，EC 1.1.3.4)是一种氧化还原酶，由两个亚基组成二聚体，含有2个黄素腺嘌呤二核苷酸(FAD)结合位点。每个单体含有2个不同的区域：一部分与FAD非共价紧密结合，主要为β折叠；另一个与底物β-D-葡萄糖结合，由4 个α-螺旋支撑1个反平行的β-折叠。不同来源的GOD理化性质存在差异，分子量在 130～175KDa范围内，在有氧条件下，能专一性地催化β-D-葡糖生成葡萄糖酸和过氧化氢。
GOD被广泛应用于食品、化工、生物医疗等领域。在食品行业，GOD可被用于催化葡萄糖耗尽真空袋中氧气，抑制微生物生长和繁殖，延长食品保质期，还可提升面制品的口感。在化工领域，GOD不仅常被应用于漂白脱色工艺，还是葡萄糖酸及其衍生物生产中的关键酶。医疗领域中，GOD是葡萄糖检测试剂盒的关键原料，也被加入牙膏中用于降低口腔疾病发生率，还能作为生物电池的电极，为生物传感器和人造器官提供持续的能源。因此，大规模高效生产GOD具有重要的经济价值。然而，GOD在进一步的加工生产过程中，往往需要经历高温的过程，从而不可避免地使得GOD的酶活下降甚至失活。例如，在饲料加工过程中的短暂高温过程会造成葡萄糖氧化酶的失活，影响葡萄糖氧化酶的应用效果，在葡萄糖氧化酶产量得到保证的前提下，葡萄糖氧化酶的耐温性能越来越受到关注。
GOD广泛分布于动植物和微生物体内。目前工业水平的GOD生产，主要以黑曲霉和青霉作为生产菌株，相对青霉GOD而言，黑曲霉表达的GOD热稳定性较好，但都存在酶活水平低、分离纯化复杂的问题。毕赤酵母(Pichiapastoris)遗传背景清楚，易于进行遗传操作，分泌的蛋白糖基化适中，是一种进行外源蛋白表达的常用宿主，尤其是利用P.pastoris进行蛋白分泌表达，可大大简化分离纯化的过程，具有重要的应用价值。
发明内容
本发明的目的是提供热稳定性和比活提高的葡萄糖氧化酶突变体。
本发明的再一目的是提供编码上述热稳定性和比活提高的葡萄糖氧化酶突变体的基因。
本发明的再一目的是提供包含上述葡萄糖氧化酶突变体的基因重组载体。
本发明的再一目的是提供包含上述葡萄糖氧化酶突变体的基因重组菌株。
本发明的再一目的是提供一种提高葡萄糖氧化酶的热稳定性和比活提高的方法。
本发明的再一目的是提供上述热稳定性和比活提高的葡萄糖氧化酶突变体的应用。
根据本发明的葡萄糖氧化酶突变体具有以下氨基酸序列：如SEQ ID NO：1所示的氨基酸序列的第360位氨基酸被取代。
根据本发明的葡萄糖氧化酶突变体进一步具有以下氨基酸序列如SEQ ID NO：1所示的氨基酸序列的第243位或第492位氨基酸被取代。
根据本发明的葡萄糖氧化酶突变体，所述葡萄糖氧化酶突变体通过对氨基酸序列如SEQ ID NO：1所示亲本葡萄糖氧化酶的第14位、第16位、第18位、第43 位、第51位、第87位、第88位、第89位、第163位、第241位、第243位、第 343位、第360位、第492位和第497位中的至少一位氨基酸进行取代获得。
SEQ ID NO：1：
SNGIEASLLKDPKLVAGRTYDYIIAGGGLAGLTVAEKLTENPNITVLVIESGSYE SDRGPIIEDLNAYGEIFGTSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWT RPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNAAC HGLNGTVHVGPRDTGDDYSPLMRALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRADAAREWLLPNYQRPNLRVLTGQYVGKVLLSQNATTPRAVGVEFGT HKSNTHNVYAKHEVLLSAGSTVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQ DQTTSTVRSRITSAGAGQGQAAWFATFNETFGKYTEKAHELLNTKLEQWAEEAVA RGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTGGVASFDVWDLLPFTRGYVH ILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGNN LAYDADLSAWVEYIPEHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRV IDGSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQ。
根据本发明的葡萄糖氧化酶突变体，其中，
第14位氨基酸的取代为L14G或L14A；
第16位氨基酸的取代为A16N或A16F；
第18位氨基酸的取代为R18D；
第43位氨基酸的取代为N43D、N43G、N43H或N43Q；
第51位氨基酸的取代为S51G或S51W；
第87位氨基酸的取代为T87A或T87V；
第88位氨基酸的取代为N88D或N88Q；
第89位氨基酸的取代为N89D；
第163位氨基酸的取代为A163S或A163G；
第241位氨基酸的取代为N241H；
第243位氨基酸的取代为R243Q；
第343位氨基酸的取代为A343G、A343D或A343N；
第360位氨基酸的取代为K360D或K360H；
第492位氨基酸的取代为N492D或N492E；
第497位氨基酸的取代为D497N、D497M或D497E。
根据本发明的葡萄糖氧化酶突变体，其中，所述葡萄糖氧化酶突变体通过对氨基酸序列如SEQ ID NO：1所示亲本葡萄糖氧化酶进行以下取代获得：
突变体GOx-MUT7：A16N、N43D、T87A、N88D、A163S、R243Q、K360D、 N492D(SEQ IDNO：2)；
突变体GOx-MUT8：L14A、T87V、N88D、A163S、R243Q、K360D、N492D (SEQ ID NO：3)；
突变体GOx-MUT9：A16N、R18D、N43D、T87V、N88D、A163S、R243Q、 K360D、N492D(SEQID NO：4)；
突变体GOx-MUT10包含的突变位点为：R18D、S51G、N89D、A163S、N241H、 R243Q、A343G、K360D、N492D(SEQ ID NO：5)；
突变体GOx-MUT11包含的突变位点为：R18D、T87A、N89D、A163G、N241H、 R243Q、A343D、K360D、N492D、D497N(SEQ ID NO：6)。
本发明提供了编码上述葡萄糖氧化酶突变体的基因。
根据本申请的技术方案，亲本葡萄糖氧化酶的基因序列如下所示：
SEQ ID NO：7：
tctaatggtattgaggcttccttgttgaaagacccaaaacttgtcgccggtagaacctacgactacatcattgccggtggtgg tttggctggtttgaccgttgctgagaagttgaccgagaatcctaacatcactgttttggttattgagtccggttcctacgagtctgaccg tggtccaattattgaggatttgaatgcctacggtgaaatcttcggaacttctgtcgaccacgcctatgagaccgttgagttggctacta acaatagaactgctttgatccgttccggtaacggtttgggaggatccactttgattaacggtggaacctggactagaccacataaag cccaagtcgactcctgggagactgtcttcggaaacgaaggttggaactgggactctgttgctgcttactcccttcaggctgaaaga gctcgtgccccaaatgctaagcagatcgccgctggtcactactttaacgccgcatgccacggtttgaacggtactgttcacgttgga ccacgtgatactggtgatgactactctccattgatgagagccttgatgtctgctgtcgaagatcgtggagtccctaccaagaaggac ttgggttgcggagaccctcatggtgtctccatgttcccaaacaccttgcacgaggaccaagttcgtgctgacgctgccagagaatg gttgcttcctaactaccagagaccaaacttgagggtcttgactggtcagtacgtcggtaaggtcttgttgtctcagaacgctaccacc ccaagagctgttggtgtcgagttcggtactcacaagtctaacacccacaacgtctacgctaagcatgaggtccttttgtccgccggt tctactgtttccccaaccatcttggagtattctggaattggtatgaaatctattttggagcctttgggaatcgacaccgttgttgaccttc cagttggtttgaacttgcaggaccagaccacctccactgtccgttctcgtattacttccgctggtgctggacaaggtcaagctgcctg gttcgctaccttcaatgagacctttggtaagtacaccgagaaggcccacgagttgttgaacaccaagttggagcaatgggctgaag aggctgtcgctagaggtggattccataataccaccgccttgttgatccaatacgaaaattatagagattggattgttaaggacaatgtt gcttactccgagttgtttttggataccggtggagtcgcttcctttgacgtctgggacttgttgcctttcacccgtggttacgttcacatttt ggacaaagatccttacttgcgtcacttcgcctacgacccacagtacttcttgaacgagttggacttgttgggtcaagctgctgctact cagttggcccgtaacatttctaactctggtgccatgcaaacctacttcgctggagagaccattccaggaaacaacttggcctacgat gccgacttgtctgcctgggtcgagtacatccctgaacatttccgtccaaactatcacggtgtcggaacctgctccatgatgccaaag gaaatgggtggagtcgtcgacaatgccgctcgtgtttacggagtccagggtttgagagtcatcgacggttctatcccaccaaccca attgtcctcccacgtcatgactgtcttctacgctatggccttgaagatcgctgacgctgttcttgctgactacgcttctatgcagtaa。
根据本发明的具体实施例获得的氧化酶突变体的编码基因如下：SEQ ID NO：8 所示序列编码突变体GOx-MUT7；SEQ ID NO：9所示序列编码突变体GOx-MUT8； SEQ ID NO：10所示序列编码突变体GOx-MUT9；SEQ ID NO：11所示序列编码突变体GOx-MUT10；SEQ ID NO：12所示序列编码突变体GOx-MUT11。
本发明提供了包含上述葡萄糖氧化酶突变体基因的重组载体。
本发明提供了包含上述葡萄糖氧化酶突变体基因的重组菌株。特别适合本发明葡萄糖氧化酶突变体的表达宿主为细菌，包括埃希氏菌属(Escherichia)(如大肠杆菌(E.coli))、假单胞菌属(Pseudomonas)(如荧光假单胞菌(P.fluorescens)或施氏假单胞菌(P.stutzerei))、变形杆菌属(Proteus)(如奇异变形杆菌(Proteus mirabilis))、罗尔斯通菌属(Ralstonia)(如富养罗尔斯通氏菌(R.eutropha))、链霉菌属(Streptomyces)、葡萄球菌属(Staphylococcus)(如肉葡萄球菌(S.carnosus))、乳球菌属(Lactococcus)(如乳酸乳球菌 (L.lactis))、和芽孢杆菌书(Bacillus)(如枯草芽孢杆菌(B.subtilis)、巨大芽孢杆菌(B.megaterium)、地衣芽孢杆菌(B.licheniformis))。另一特别适合的是酵母表达宿主如酿酒酵母(Saccharomycescerevisiae)、粟酒裂殖酵母(Schizosaccharomycespombe)、解脂耶氏酵母(Yarrowialipolytica)、多形汉逊酵母(Hansenulapolymorpha)、乳酸克鲁维酵母(Kluyveromyceslactis)或毕赤酵母(Pichiapastoris)。又一种特别适用的是真菌表达宿主，例如金孢子菌(Chrysosporium lucknowense)、曲霉属(Aspergillus)(如米曲霉 (A.oryzae)、黑曲霉(A.niger)、构巢曲霉(A.nidulans))或里氏木霉(Trichodermareesei)。还适用的是哺乳动物表达宿主，例如小鼠(如NSO)、中国仓鼠卵巢(Chinese hamster ovary，CHO))或幼仓鼠肾(baby hamster kidney，BHK)细胞系，转基因哺乳动物体系例如兔、山羊或牛，其它真核宿主例如昆虫细胞或植物，或病毒性表达体系例如噬菌体M13、T7或λ，或真核病毒如杆状病毒。优选地，酵母细胞或丝状真菌细胞，更优选地，毕赤酵母。
根据本发明的提高葡萄糖氧化酶的热稳定性和比活的方法，包括对氨基酸序列如SEQ ID NO：1所示亲本葡萄糖氧化酶的第14位、第16位、第18位、第43位、第51位、第87位、第88位、第89位、第163位、第241位、第243位、第343 位、第360位、第492位和第497位中的至少一位氨基酸进行取代的步骤。
根据本发明的提高葡萄糖氧化酶的热稳定性和比活的方法，其中，
第14位氨基酸的取代为L14G或L14A；
第16位氨基酸的取代为A16N或A16F；
第18位氨基酸的取代为R18D；
第43位氨基酸的取代为N43D、N43G、N43H或N43Q；
第51位氨基酸的取代为S51G或S51W；
第87位氨基酸的取代为T87A或T87V；
第88位氨基酸的取代为N88D或N88Q；
第89位氨基酸的取代为N89D；
第163位氨基酸的取代为A163S或A163G；
第241位氨基酸的取代为N241H；
第243位氨基酸的取代为R243Q；
第343位氨基酸的取代为A343G、A343D或A343N；
第360位氨基酸的取代为K360D或K360H；
第492位氨基酸的取代为N492D或N492E；
第497位氨基酸的取代为D497N、D497M或D497E。
根据本发明的提高葡萄糖氧化酶的热稳定性和比活的方法，对氨基酸序列如 SEQID NO：1所示亲本葡萄糖氧化酶进行以下取代：
A16N、N43D、T87A、N88D、A163S、R243Q、K360D、N492D；或
L14A、T87V、N88D、A163S、R243Q、K360D、N492D；或
A16N、R18D、N43D、T87V、N88D、A163S、R243Q、K360D、N492D；或
R18D、S51G、N89D、A163S、N241H、R243Q、A343G、K360D、N492D；或
R18D、T87A、N89D、A163G、N241H、R243Q、A343D、K360D、N492D、 D497N。
本发明提供了上述葡萄糖氧化酶突变体在饲料生产、食品加工及医药中的应用。包括但不限于，在饲料生产中作为家禽饲料添加剂、霉菌毒素解毒剂等方面的应用；在食品工业中用于脱氧、改良面粉、去葡萄糖、测定葡萄糖含量、延长食品的保质和保鲜期等，例如，可用于葡萄糖酸钠或葡萄糖酸钙的生产中；在医药行业中，可用于除去或缓解牙斑、牙垢和龋齿的形成，可用于对H2O2敏感的淋巴瘤的治疗，作为试剂盒、酶电极等用于血清(浆)、尿液及脑脊液中葡萄糖体外定量分析等。
本发明提供的所述葡萄糖氧化酶突变体与SEQ ID NO：1氨基酸序列具有至少97％且小于100％的序列一致性，并且其中所述葡萄糖氧化酶变体具有葡萄糖氧化酶活性。
本发明还提供一种制备热稳定性和比活提高的葡萄糖氧化酶突变体的方法，包括以下步骤：
1)构建包含编码所述的葡萄糖氧化酶突变体的基因的重组表达载体；
2)将所述重组表达载体导入宿主细胞中；
3)诱导所述宿主细胞表达葡萄糖氧化酶突变体。
本发明提供葡萄糖氧化酶突变体，与亲本SEQ ID NO：1所示的葡萄糖氧化酶相比，比活以及耐热性有了显著的提升。有利于该酶在工业化生产中应用。
附图说明
图1显示本申请的葡萄糖氧化酶突变体的最适反应pH；
图2显示本申请的葡萄糖氧化酶突变体的最适反应温度；
图3显示本申请的葡萄糖氧化酶突变体的热稳定性。
具体实施方式
下述实施方法是为了更好的解释本发明，而不应该被解释为限制本发明的目的。以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
本发明提供的葡萄糖氧化酶突变体是基于氨基酸序列如SEQ ID NO：1(核酸序列为SEQ ID NO.7)所示的亲本经过多次突变和高通量筛选获得。提高的比活和耐热性，意指相对于氨基酸序列SEQ ID NO：1所示葡萄糖氧化酶(文后简称为GOx) 的比活和热稳定性有显著的提高。
本发明还提供了一种制备热稳定性和比活提高的葡萄糖氧化酶的方法，包括以下步骤：
a)构建包含编码本发明的葡萄糖氧化酶突变体的基因的重组表达载体；
b)将所述重组表达载体导入宿主细胞中；
c)诱导所述宿主细胞表达葡萄糖氧化酶突变体。
该葡萄糖氧化酶突变体被分泌到该营养培养基中，则可直接从培养基中回收。如果该葡萄糖氧化酶突变体没有分泌，则可从细胞裂解液中回收。
葡萄糖氧化酶蛋白质可在多种表达体系中表达，且相应地必须选择适当的下游加工和纯化步骤。表达葡萄糖氧化酶变体的细胞通过任何本领域技术人员所公知的方法，在本发明的一些实施方案中，葡萄糖氧化酶变体可以在细菌宿主中表达，且蛋白质分泌至周质或细胞外间隙。表达生物体的培养根据标准发酵法以适当体积制备。在优选的实施方案中，细胞在发酵罐中生长，且任选地控制生长条件如pH、温度、氧和/或营养素供给。纯化的第一步包括使用几种技术如沉降、微滤、离心或絮凝中的一种或多种从上清液分离细胞。在优选的实施方案中，适用的方法为微滤。如果在细胞内表达，对细胞进行处理，从细胞内间隙释放蛋白质。这些处理可包括例如加压、酶促、渗透压休克、冷冻、超声或其它处理，从而产生细胞提取物，其可以进行进一步的纯化，或可以不进行。
在本发明的一些实施方案中，葡萄糖氧化酶通过诱导培养后，分泌至上清液，进一步由上清液或浓缩的上清液的蛋白纯化可利用包括下述的几种方法中的一种或多种进行：提取或分级法例如硫酸铵或乙醇或酸沉淀，或层析法，包括但不限于离子交换、疏水相互作用、羟磷灰石、通过凝胶过滤的粒径分级、磷酸纤维素或凝集素层析和亲和层析、或其任意组合。在一些优选的方法中，亲和性标记蛋白通过金属螯合剂亲和层析纯化，从而获得高纯度目标蛋白。在另一些优选的实施例中，通过HPLC纯化获得搞纯度的目标蛋白。
在本发明的另一些实施方案中，上清液、或通过超滤来部分纯化的上清液、或浓缩和/或二次过滤(diafiltrated)的上清液，继续通过下述几种技术方法中的任一种干燥：所述技术方法例如，但不限于，喷雾干燥、冷冻干燥、回流式蒸发(down-draughtevaporation)、薄层蒸发、离心蒸发、输送器干燥或其任意组合。
在本发明进一步的实施方案中，使用例如但不限于流化床干燥、输送器干燥、喷雾干燥或转鼓干燥或其任意组合等方法将包括表达的葡萄糖氧化酶的发酵细胞悬浮液作为整体干燥。
术语“活性”或“催化活性”定量描述了在规定反应条件下给定底物的转化。术语“比活”定量描述了在规定反应条件下相对于酶量的催化活性。
在本发明的实施例中，所述葡萄糖氧化酶突变体在对应于SEQ ID NO：1所示序列的第14位、第16位、第18位、第43位、第51位、第87位、第88位、第89 位、第163位、第241位、第243位、第343位、第360位、第492位和第497位中的至少一位氨基酸进行取代获得。
在一些实施例中，所述葡萄糖氧化酶突变体包括在对应于SEQ ID NO：1所示序列的第360位取代，以及第14位、第16位、第18位、第43位、第51位、第87 位、第88位、第89位、第163位、第241位、第243位、第343位、第492位和第497位中的至少一位氨基酸进行取代获得。
在另一些优选的实施例中，所述葡萄糖氧化酶突变体包括对应于SEQ ID NO：1 所示序列的第243位、第360位和第492位取代，以及第14位、第16位、第18位、第43位、第51位、第87位、第88位、第89位、第163位、第241位、第343位和第497位中的至少一位氨基酸进行取代获得。
在另一些优选的实施例中，所述葡萄糖氧化酶突变体包括在对应于SEQ ID NO： 1所示序列的第163位、第243位、第360位和第492位取代，以及第14位、第16 位、第18位、第43位、第51位、第87位、第88位、第89位、第241位、第343 位和第497位中的至少一位氨基酸进行取代获得。
在另一些优选的实施例中，所述葡萄糖氧化酶突变体包括在对应于SEQ ID NO： 1所示序列的第163位、第243位、第360位和第492位取代，第18位、第87位、第88位和第89位至少一位氨基酸进行取代，以及第14位、第16位、第43位、第 51位、第241位、第343位和第497位中的至少一位氨基酸进行取代获得。
更具体地，在优选的实施例中，所述葡萄糖氧化酶突变体选自下组取代中的一个或多个：第14位氨基酸的取代为L14G或L14A；第16位氨基酸的取代为A16N 或A16F；第18位氨基酸的取代为R18D；第43位氨基酸的取代为N43D、N43G、 N43H或N43Q；第51位氨基酸的取代为S51G或S51W；第87位氨基酸的取代为 T87A或T87V；第88位氨基酸的取代为N88D或N88Q；第89位氨基酸的取代为N89D；第163位氨基酸的取代为A163S或A163G；第241位氨基酸的取代为N241H；第243位氨基酸的取代为R243Q；第343位氨基酸的取代为A343G、A343D或A343N；第360位氨基酸的取代为K360D或K360H；第492位氨基酸的取代为N492D或 N492E；以及第497位氨基酸的取代为D497N、D497M或D497E。
更具体地，相对于亲本葡萄糖氧化酶具有提高的比活和耐热性的葡萄糖氧化酶突变体的突变位点为：
A16N、N43D、T87A、N88D、A163S、R243Q、K360D、N492D；或
L14A、T87V、N88D、A163S、R243Q、K360D、N492D；或
A16N、R18D、N43D、T87V、N88D、A163S、R243Q、K360D、N492D；或
R18D、S51G、N89D、A163S、N241H、R243Q、A343G、K360D、N492D；或
R18D、T87A、N89D、A163G、N241H、R243Q、A343D、K360D、N492D、 D497N。
实验材料和试剂：
1、菌株与载体
含有亲本葡萄糖氧化酶GOx基因和表达质粒的菌株，大肠杆菌菌株Top10、毕赤酵母X33、载体pPICZαA、载体pGAPzαA、抗生素Zeocin购自Invitrogen公司。
2、酶与试剂盒
超保真2×Master Mix PCR聚合酶、限制性内切酶，质粒提取试剂盒、纯化试剂盒。
3、培养基
大肠杆菌培养基为LB培养基(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。 LB+Amp培养基为LB培养基加入终浓度为100ug/mL的氨苄青霉素。LB+Zeo培养基为LB培养基加入终浓度为25ug/mL的Zeocin；
酵母培养基为YPD培养基(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPD+Zeo培养基(YPD+Zeo培养基为YPD培养基加入终浓度为100 ug/mL的Zeocin)。酵母诱导培养基BMGY(1％酵母提取物，2％蛋白胨，1.34％YNB， 0.00004％Biotin，1％甘油(v/v))和BMMY(除以0.5％甲醇代替甘油，其余成分与 BMGY相同)；
重组酵母发酵基本盐培养基：磷酸氢二铵5％、磷酸二氢钾0.5％、七水硫酸镁1.5％、硫酸钾1.95％、硫酸钙0.1％、消泡剂0.03％。高压后每升加4.35mlPTM1。PTM1 (微量盐溶液)：硫酸铜0.6％、碘化钾0.018％。一水硫酸锰0.3％、二水钼酸钠0.02％、硼酸0.002％、流水氯化钴0.05％、氯化锌2％、七水硫酸铁6.5％、浓硫酸0.5％、生物素0.02％。
4、化学试剂：
葡萄糖氧化酶标准品、邻联茴香胺盐酸盐及辣根过氧化物、葡萄糖。
5、葡萄糖氧化酶测定方法
葡萄糖氧化酶活性测定采用邻—联茴香胺分光光度法。在葡萄糖氧化酶的作用下，葡萄糖和氧反应，生成葡萄糖酸和过氧化氢，过氧化氢和无色的还原型邻联茴香胺在过氧化物酶的作用下，生成水和红色的氧化型邻联茴香胺。在540nm下测定反应液吸光值，依据标准曲线计算葡萄糖氧化酶的酶活。
实施例1、定点突变及高通量筛选高比活突变菌株
以含亲本葡萄糖氧化酶GOx基因的重组载体pPICzαA-GOx为模板，设计引物构建对应的GOx突变体L14A，A16N，R18D、T87A、N88D、N89D、S51G，K360H 和D497N，进行PCR扩增。
琼脂糖电泳检测PCR扩增结果，纯化回收PCR扩增的目的产物。用限制性内切酶DpnI消解模板，采用化学转化热激法将分解完的产物转入大肠杆菌Top10感受态细胞中，通过菌液PCR验证重组转化子，提取验证正确的转化子的质粒进行测序，从而确定相应的突变体。用PmeI将测序正确的突变体质粒线性化，纯化线性质粒片段，采用电转化法转入毕赤酵母X33感受态细胞中，采用YPD+Zeo培养基筛选。
用牙签逐个将实施例得到的酵母重组转化子挑至24孔板，每个孔中加入1mL 含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mL BMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200 μL至96孔板，进行葡萄糖氧化酶酶活测定。
实施例2、定点饱和突变及筛选
以pPICzαA-GOx为模板，分别对实施例1中的9个位点进行饱和突变，具体构建方法参照实施案例1中的构建方法进行突变体构建，经过高通量筛选方法得到17 个有效突变位点，分别测定相应的酶活并计算出比活。以突变体比活除以原始葡萄糖氧化酶GOx比活，来计算突变体的相对比活。计算结果显示，筛选得到的17个突变体L14G、L14A、A16F、A16N、R18D、T87A、T87V、N88D、N88Q、N89D、 S51G、S51W、K360D、K360H、D497N、D497E和D497M的相对比活都有不同幅度的提升。
表1 GOx和GOx突变体相对比活比较
实施例3、半饱和突变与筛选
以pPICzαA-GOx为模板，设计半饱和突变引物，分别对第43位、第163位、第241位、第243位、第343位和第492位这6个位点进行突变，具体构建方法参照实施案例1中的构建方法进行突变体构建，并通过高通量筛选方法得到13个有效突变位点，分别测定相应的酶活并计算出比活。以突变体比活除以原始葡萄糖氧化酶GOx比活，来计算突变体的相对比活。计算结果显示，筛选得到的13个突变体 N43D、N43G、N43H、N43Q、A163S、A163G、N241H、R243Q、A343G、A343D、 A343N、N492E和N492D的相对比活也有显著的提升。
表2 GOx和GOx突变体相对比活比较
实施例4、高通量筛选耐热提升的葡萄糖氧化酶突变体
将实施例2和实施例3得到的与亲本GOx酶活接近或提升的酵母重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mL BMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200μL至96孔板，进行葡萄糖氧化酶酶活测定和热处理残余酶活测定。通过多轮的筛选比较，最终筛选到12个能显著提高耐热性的有效突变位点，分别为L14A、A16N、R18D、T87A、T87V、N88Q、N88D、 N89D、N241H、A343N、D497E和D497N。这12个突变体的耐热比较如表1所示。
表3亲本葡萄糖氧化酶GOx和GOx突变体耐热比较
实施例5、组合突变与筛选
在实施例2～4中相对比活提升或耐热能力提升的正向突变位点的基础上，进行双位点或者多位点组合突变。采用镍亲和层析纯化法分别将原始葡萄糖氧化酶GOx 和葡萄糖氧化酶GOx的突变体进行纯化，采用高通量方法进行筛选，分别测定相应的酶活和耐热性，并计算出比活。以突变体比活除以原始葡萄糖氧化酶GOx比活，来计算突变体的相对比活。通过多轮组合突变和筛选，实验最终筛选到5个组合突变，分别为：
GOx-MUT7包含的突变位点为：A16N、N43D、T87A、N88D、A163S、R243Q、 K360D、N492D；
GOx-MUT8包含的突变位点为：L14A、T87V、N88D、A163S、R243Q、K360D、 N492D；
GOx-MUT9包含的突变位点为：A16N、R18D、N43D、T87V、N88D、A163S、 R243Q、K360D、N492D；
GOx-MUT10包含的突变位点为：R18D、S51G、N89D、A163S、N241H、R243Q、 A343G、K360D、N492D；
GOx-MUT11包含的突变位点为：R18D、T87A、N89D、A163G、N241H、R243Q、 A343D、K360D、N492D、D497N。
表4原始葡萄糖氧化酶GOx和组合突变体比活力比较
表5原始葡萄糖氧化酶GOx和组合突变体耐热比较
实施例6原始葡萄糖氧化酶GOx及突变体GOx-MUT7、GOx-MUT8、GOx-MUT9、 GOx-MUT10和GOx-MUT11的最适反应pH
在温度37℃的条件下，分别在pH3，pH3.5，pH4，pH4.5，pH5，pH5.5，pH6， pH6.5，pH7，pH7.5条件下测定葡萄糖氧化酶的酶活力，结果如图1所示。从图1 可知，GOx-MUT7、GOx-MUT8、GOx-MUT9、GOx-MUT10和GOx-MUT11在不同pH条件下的相对酶活(pH5.5)与原始葡萄糖氧化酶GOx基本一致，原始葡萄糖氧化酶的最适pH为5.5，突变体的最适反应pH范围是pH5.0-pH6.0。
实施例7原始葡萄糖氧化酶GOx及突变GOx-MUT7、GOx-MUT8、GOx-MUT9、 GOx-MUT10和GOx-MUT11的最适反应温度及耐热性
在pH5.5的条件下，分别在25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、 65℃、70℃，测定葡萄糖氧化酶的酶活力，其中，以37℃条件下测得葡萄糖氧化酶的酶活力为对照，计算酶各自在不同温度条件下的相对酶活。结果如图2显示，亲本及突变体葡萄糖氧化酶的最适反应温度范围是25℃-50℃。
为了研究葡萄糖氧化酶GOx和突变体GOx-MUT7至GOx-MUT11在不同温度下的稳定性，分别将上清液在35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、 75℃、85℃，静置5min，然后在37℃、pH5.5的条件下测定酶活，以未热处理的样品的相对酶活性为100％，结果如图3所示。从图3可知，突变体GOx-MUT7至 GOx-MUT11在75℃处理5min后，酶活保留率都在79％以上。并且，在85℃处理 5min后，突变体GOx-MUT7至GOx-MUT11的残余酶活大于亲本葡萄糖氧化酶GOx，其中仅GOx-MUT11的表现稍显逊色。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
序列表
<110> 广东溢多利生物科技股份有限公司
<120> 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ala Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Arg Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Lys Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asn Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 2
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Asn
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Ala Asp Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 3
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Ala Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Val Asp Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 4
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Asn
1 5 10 15
Gly Asp Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Val Asp Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 5
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Ala
1 5 10 15
Gly Asp Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Gly Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asp Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
His Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Gly Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 6
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Ala
1 5 10 15
Gly Asp Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Ala Asn Asp Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Gly Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
His Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Asp Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asn Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 7
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgccgcat gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgaggg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtaag 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggaaacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 8
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcaacgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctgaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctgc agacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 9
<211> 3504
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tctaatggta ttgaggcttc cttgttgaaa gacccaaaag cagtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctgt cgacaacaga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aatctaatgg tattgaggct tccttgttga aagacccaaa agcagtcgcc 1800
ggtagaacct acgactacat cattgccggt ggtggtttgg ctggtttgac cgttgctgag 1860
aagttgaccg agaatcctaa catcactgtt ttggttattg agtccggttc ctacgagtct 1920
gaccgtggtc caattattga ggatttgaat gcctacggtg aaatcttcgg aacttctgtc 1980
gaccacgcct atgagaccgt tgagttggct gtcgacaaca gaactgcttt gatccgttcc 2040
ggtaacggtt tgggaggatc cactttgatt aacggtggaa cctggactag accacataaa 2100
gcccaagtcg actcctggga gactgtcttc ggaaacgaag gttggaactg ggactctgtt 2160
gctgcttact cccttcaggc tgaaagagct cgtgccccaa atgctaagca gatcgccgct 2220
ggtcactact ttaacgcctc ttgccacggt ttgaacggta ctgttcacgt tggaccacgt 2280
gatactggtg atgactactc tccattgatg agagccttga tgtctgctgt cgaagatcgt 2340
ggagtcccta ccaagaagga cttgggttgc ggagaccctc atggtgtctc catgttccca 2400
aacaccttgc acgaggacca agttcgtgct gacgctgcca gagaatggtt gcttcctaac 2460
taccagagac caaacttgca ggtcttgact ggtcagtacg tcggtaaggt cttgttgtct 2520
cagaacgcta ccaccccaag agctgttggt gtcgagttcg gtactcacaa gtctaacacc 2580
cacaacgtct acgctaagca tgaggtcctt ttgtccgccg gttctactgt ttccccaacc 2640
atcttggagt attctggaat tggtatgaaa tctattttgg agcctttggg aatcgacacc 2700
gttgttgacc ttccagttgg tttgaacttg caggaccaga ccacctccac tgtccgttct 2760
cgtattactt ccgctggtgc tggacaaggt caagctgcct ggttcgctac cttcaatgag 2820
acctttggtg attacaccga gaaggcccac gagttgttga acaccaagtt ggagcaatgg 2880
gctgaagagg ctgtcgctag aggtggattc cataatacca ccgccttgtt gatccaatac 2940
gaaaattata gagattggat tgttaaggac aatgttgctt actccgagtt gtttttggat 3000
accggtggag tcgcttcctt tgacgtctgg gacttgttgc ctttcacccg tggttacgtt 3060
cacattttgg acaaagatcc ttacttgcgt cacttcgcct acgacccaca gtacttcttg 3120
aacgagttgg acttgttggg tcaagctgct gctactcagt tggcccgtaa catttctaac 3180
tctggtgcca tgcaaaccta cttcgctgga gagaccattc caggagacaa cttggcctac 3240
gatgccgact tgtctgcctg ggtcgagtac atccctgaac atttccgtcc aaactatcac 3300
ggtgtcggaa cctgctccat gatgccaaag gaaatgggtg gagtcgtcga caatgccgct 3360
cgtgtttacg gagtccaggg tttgagagtc atcgacggtt ctatcccacc aacccaattg 3420
tcctcccacg tcatgactgt cttctacgct atggccttga agatcgctga cgctgttctt 3480
gctgactacg cttctatgca gtaa 3504
<210> 10
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcaacgg tgacacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctgaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctgt cgacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 11
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcgccgg tgacacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag ggaggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac tgacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
cacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtggag gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 12
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcgccgg tgacacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgccgcat gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
cacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtaacg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacaa cgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
Claims (10)
1.葡萄糖氧化酶突变体，其特征在于，所述葡萄糖氧化酶突变体具有以下氨基酸序列：
如SEQ ID NO：1所示的氨基酸序列的第360位氨基酸被取代。
2.根据权利要求1所述的葡萄糖氧化酶突变体，其特征在于，进一步，如SEQ ID NO：1所示的氨基酸序列的第243位或第492位氨基酸被取代。
3.根据权利要求2所述的葡萄糖氧化酶突变体，其特征在于，进一步，如SEQ ID NO：1所示的氨基酸序列的第14位、第16位、第18位、第43位、第51位、第87位、第88位、第89位、第163位、第241位、第343位和第497位中的至少一位氨基酸被取代。
4.根据权利要求3所述的葡萄糖氧化酶突变体，其特征在于，
第14位氨基酸的取代为L14G或L14A；
第16位氨基酸的取代为A16N或A16F；
第18位氨基酸的取代为R18D；
第43位氨基酸的取代为N43D、N43G、N43H或N43Q；
第51位氨基酸的取代为S51G或S51W；
第87位氨基酸的取代为T87A或T87V；
第88位氨基酸的取代为N88D或N88Q；
第89位氨基酸的取代为N89D；
第163位氨基酸的取代为A163S或A163G；
第241位氨基酸的取代为N241H；
第243位氨基酸的取代为R243Q；
第343位氨基酸的取代为A343G、A343D或A343N；
第360位氨基酸的取代为K360D或K360H；
第492位氨基酸的取代为N492D或N492E；
第497位氨基酸的取代为D497N、D497M或D497E。
5.根据权利要求1所述的葡萄糖氧化酶突变体，其特征在于，所述葡萄糖氧化酶突变体的氨基酸序列如SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5或SEQ ID NO：6所示。
6.一种葡萄糖氧化酶基因，其特征在于，编码权利要求1至5任一项所述的葡萄糖氧化酶突变体。
7.包含权利要求6所述基因的重组载体。
8.包含权利要求6所述基因的重组菌株。
9.一种提高葡萄糖氧化酶的热稳定性和比活的方法，其特征在于，所述方法包括取代氨基酸序列如SEQ ID NO：1所示亲本葡萄糖氧化酶的第360位氨基酸的步骤。
10.权利要求1所述的葡萄糖氧化酶突变体在饲料生产、食品加工及医药中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210432858.XA CN115029327A (zh) | 2022-04-24 | 2022-04-24 | 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210432858.XA CN115029327A (zh) | 2022-04-24 | 2022-04-24 | 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115029327A true CN115029327A (zh) | 2022-09-09 |
Family
ID=83119142
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210432858.XA Pending CN115029327A (zh) | 2022-04-24 | 2022-04-24 | 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115029327A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120244565A1 (en) * | 2009-12-05 | 2012-09-27 | Amano Enzyme Inc. | Mutant enzyme and application thereof |
| CN111004786A (zh) * | 2019-12-25 | 2020-04-14 | 广东溢多利生物科技股份有限公司 | 一种葡萄糖氧化酶及其载体与应用 |
| CN112143717A (zh) * | 2019-06-26 | 2020-12-29 | 青岛蔚蓝生物集团有限公司 | 比活力提高的葡萄糖氧化酶突变体 |
| US20210230561A1 (en) * | 2018-06-04 | 2021-07-29 | Institute Of Animal Science Of Chinese Academy Of Agricultural Sciences | Glucose oxidase god mutant and gene and application thereof |
-
2022
- 2022-04-24 CN CN202210432858.XA patent/CN115029327A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120244565A1 (en) * | 2009-12-05 | 2012-09-27 | Amano Enzyme Inc. | Mutant enzyme and application thereof |
| US20210230561A1 (en) * | 2018-06-04 | 2021-07-29 | Institute Of Animal Science Of Chinese Academy Of Agricultural Sciences | Glucose oxidase god mutant and gene and application thereof |
| CN112143717A (zh) * | 2019-06-26 | 2020-12-29 | 青岛蔚蓝生物集团有限公司 | 比活力提高的葡萄糖氧化酶突变体 |
| CN111004786A (zh) * | 2019-12-25 | 2020-04-14 | 广东溢多利生物科技股份有限公司 | 一种葡萄糖氧化酶及其载体与应用 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108251392B (zh) | 提高比活和热稳定性的葡萄糖氧化酶突变体及其编码基因和应用 | |
| CN105543201B (zh) | 一种头孢菌素c酰化酶突变体 | |
| US20230193215A1 (en) | Mutant glucose oxidase (god) having improved thermal stability and gene and application thereof | |
| CN114395541B (zh) | 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 | |
| CN113862233B (zh) | 提高葡萄糖氧化酶的酸稳定性的方法及突变体q241e/r499e、基因和应用 | |
| CN113151198B (zh) | 一种γ-谷酰胺甲胺合成酶的突变体，其编码基因、氨基酸序列及其应用 | |
| CN108118037A (zh) | 一种耐热性提高的葡萄糖氧化酶突变体 | |
| CN108251389A (zh) | 一种耐热性提高的葡萄糖氧化酶突变体 | |
| CN108251391A (zh) | 新型葡萄糖氧化酶突变体 | |
| CN107177607A (zh) | 枯草芽孢杆菌bs04的尿酸氧化酶基因及其用途 | |
| CN114277004B (zh) | 一种耐高温重组突变型sod及其编码基因和应用 | |
| CN112280755A (zh) | 一种突变酶及其应用和酶催化法制备三胜肽的工艺 | |
| CN106939315B (zh) | 一种草酸脱羧酶的制备方法及应用 | |
| CN115029327A (zh) | 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用 | |
| CN115029328B (zh) | 葡萄糖氧化酶突变体GOx-MUT1~6及其编码基因和应用 | |
| CN113604445B (zh) | 一种酪氨酸酶及其制备与应用 | |
| CN113403290B (zh) | 热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用 | |
| CN109666657B (zh) | 提升耐热性的葡萄糖氧化酶 | |
| CN108251390A (zh) | 一种葡萄糖氧化酶突变体 | |
| CN110129305B (zh) | 一种用于制备7-aca的头孢菌素c酰化酶突变体 | |
| CN112143717B (zh) | 比活力提高的葡萄糖氧化酶突变体 | |
| CN108034642B (zh) | 葡萄糖氧化酶CnGOD19及其改良酶、基因和应用 | |
| CN111944790A (zh) | 中性蛋白酶基因、中性蛋白酶及其制备方法和应用 | |
| CN114349831B (zh) | aspA基因突变体、重组菌及制备L-缬氨酸的方法 | |
| CN113493799A (zh) | 一株高产酸性乳糖酶的黑曲霉菌株 |
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 |