CN102559642B - 一种β-葡萄糖苷酶、编码基因、载体及其应用 - Google Patents
一种β-葡萄糖苷酶、编码基因、载体及其应用 Download PDFInfo
- Publication number
- CN102559642B CN102559642B CN2012100178575A CN201210017857A CN102559642B CN 102559642 B CN102559642 B CN 102559642B CN 2012100178575 A CN2012100178575 A CN 2012100178575A CN 201210017857 A CN201210017857 A CN 201210017857A CN 102559642 B CN102559642 B CN 102559642B
- Authority
- CN
- China
- Prior art keywords
- beta
- glucosidase
- gene
- gly
- carrier
- 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.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 54
- 108010047754 beta-Glucosidase Proteins 0.000 title claims abstract description 47
- 102000006995 beta-Glucosidase Human genes 0.000 title claims abstract description 37
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 claims abstract description 38
- 241000894006 Bacteria Species 0.000 claims abstract description 16
- 235000018991 trans-resveratrol Nutrition 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 230000007062 hydrolysis Effects 0.000 claims description 9
- 238000006460 hydrolysis reaction Methods 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000007979 citrate buffer Substances 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- HSTZMXCBWJGKHG-UHFFFAOYSA-N (E)-piceid Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC(C=CC=2C=CC(O)=CC=2)=C1 HSTZMXCBWJGKHG-UHFFFAOYSA-N 0.000 abstract description 3
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 abstract description 3
- 229960003764 polydatin Drugs 0.000 abstract description 3
- 235000021283 resveratrol Nutrition 0.000 abstract description 3
- 229940016667 resveratrol Drugs 0.000 abstract description 3
- HSTZMXCBWJGKHG-CUYWLFDKSA-N trans-piceid Polymers O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(\C=C\C=2C=CC(O)=CC=2)=C1 HSTZMXCBWJGKHG-CUYWLFDKSA-N 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 26
- 238000000034 method Methods 0.000 description 25
- 235000018102 proteins Nutrition 0.000 description 22
- 102000004169 proteins and genes Human genes 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- 229940088598 enzyme Drugs 0.000 description 19
- 108090000765 processed proteins & peptides Proteins 0.000 description 19
- 108091033319 polynucleotide Proteins 0.000 description 18
- 102000040430 polynucleotide Human genes 0.000 description 18
- 239000002157 polynucleotide Substances 0.000 description 18
- 150000001413 amino acids Chemical group 0.000 description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 150000008495 β-glucosides Chemical class 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- -1 alkyl glycoside Chemical class 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 8
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 7
- 235000008696 isoflavones Nutrition 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 238000006555 catalytic reaction Methods 0.000 description 6
- 229930182478 glucoside Natural products 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 244000068988 Glycine max Species 0.000 description 5
- 235000010469 Glycine max Nutrition 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 150000008131 glucosides Chemical class 0.000 description 4
- 229930182470 glycoside Natural products 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 3
- 206010027336 Menstruation delayed Diseases 0.000 description 3
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- BEZTUFWTPVOROW-KJEVXHAQSA-N Thr-Tyr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O BEZTUFWTPVOROW-KJEVXHAQSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 229930182494 ginsenoside Natural products 0.000 description 3
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 150000002515 isoflavone derivatives Chemical class 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CKUVNOCSBYYHIS-UHFFFAOYSA-N (20R)-ginsenoside Rg3 Natural products CC(C)=CCCC(C)(O)C1CCC(C2(CCC3C4(C)C)C)(C)C1C(O)CC2C3(C)CCC4OC1OC(CO)C(O)C(O)C1O CKUVNOCSBYYHIS-UHFFFAOYSA-N 0.000 description 2
- CKUVNOCSBYYHIS-IRFFNABBSA-N (20S)-ginsenoside Rh2 Chemical compound O([C@H]1CC[C@]2(C)[C@H]3C[C@@H](O)[C@H]4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@@H]4[C@@](C)(O)CCC=C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O CKUVNOCSBYYHIS-IRFFNABBSA-N 0.000 description 2
- CKUVNOCSBYYHIS-LGYUXIIVSA-N 20(R)-Ginsenoside Rh2 Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@H]1C(C)(C)[C@H]2[C@@](C)([C@H]3[C@](C)([C@@]4(C)[C@H]([C@H](O)C3)[C@@H]([C@](O)(CC/C=C(\C)/C)C)CC4)CC2)CC1 CKUVNOCSBYYHIS-LGYUXIIVSA-N 0.000 description 2
- IFBHRQDFSNCLOZ-RMPHRYRLSA-N 4-nitrophenyl beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-RMPHRYRLSA-N 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000208340 Araliaceae Species 0.000 description 2
- OGZBJJLRKQZRHL-KJEVXHAQSA-N Arg-Thr-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OGZBJJLRKQZRHL-KJEVXHAQSA-N 0.000 description 2
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 description 2
- LTCKTLYKRMCFOC-KKUMJFAQSA-N Asp-Phe-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LTCKTLYKRMCFOC-KKUMJFAQSA-N 0.000 description 2
- MFDPBZAFCRKYEY-LAEOZQHASA-N Asp-Val-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MFDPBZAFCRKYEY-LAEOZQHASA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 229930183217 Genin Natural products 0.000 description 2
- PIUPHASDUFSHTF-CIUDSAMLSA-N Gln-Pro-Asn Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O PIUPHASDUFSHTF-CIUDSAMLSA-N 0.000 description 2
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 2
- PYFIQROSWQERAS-LBPRGKRZSA-N Gly-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(=O)NCC(O)=O)=CNC2=C1 PYFIQROSWQERAS-LBPRGKRZSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- ADJWHHZETYAAAX-SRVKXCTJSA-N Leu-Ser-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ADJWHHZETYAAAX-SRVKXCTJSA-N 0.000 description 2
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- LCPUWQLULVXROY-RHYQMDGZSA-N Met-Lys-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LCPUWQLULVXROY-RHYQMDGZSA-N 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- AAERWTUHZKLDLC-IHRRRGAJSA-N Phe-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O AAERWTUHZKLDLC-IHRRRGAJSA-N 0.000 description 2
- XROLYVMNVIKVEM-BQBZGAKWSA-N Pro-Asn-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O XROLYVMNVIKVEM-BQBZGAKWSA-N 0.000 description 2
- ZPPVJIJMIKTERM-YUMQZZPRSA-N Pro-Gln-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)N)NC(=O)[C@@H]1CCCN1 ZPPVJIJMIKTERM-YUMQZZPRSA-N 0.000 description 2
- FHJQROWZEJFZPO-SRVKXCTJSA-N Pro-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 FHJQROWZEJFZPO-SRVKXCTJSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- QWZIOCFPXMAXET-CIUDSAMLSA-N Ser-Arg-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O QWZIOCFPXMAXET-CIUDSAMLSA-N 0.000 description 2
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- URPSJRMWHQTARR-MBLNEYKQSA-N Thr-Ile-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O URPSJRMWHQTARR-MBLNEYKQSA-N 0.000 description 2
- JVTHMUDOKPQBOT-NSHDSACASA-N Trp-Gly-Gly Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O)=CNC2=C1 JVTHMUDOKPQBOT-NSHDSACASA-N 0.000 description 2
- 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 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 108010077515 glycylproline Proteins 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 229960001866 silicon dioxide Drugs 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002594 sorbent Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- JAMAWBXXKFGFGX-KZVJFYERSA-N Ala-Arg-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JAMAWBXXKFGFGX-KZVJFYERSA-N 0.000 description 1
- ZEXDYVGDZJBRMO-ACZMJKKPSA-N Ala-Asn-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N ZEXDYVGDZJBRMO-ACZMJKKPSA-N 0.000 description 1
- UHMQKOBNPRAZGB-CIUDSAMLSA-N Ala-Glu-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N UHMQKOBNPRAZGB-CIUDSAMLSA-N 0.000 description 1
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 1
- CBCCCLMNOBLBSC-XVYDVKMFSA-N Ala-His-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O CBCCCLMNOBLBSC-XVYDVKMFSA-N 0.000 description 1
- HQJKCXHQNUCKMY-GHCJXIJMSA-N Ala-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C)N HQJKCXHQNUCKMY-GHCJXIJMSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- CYBJZLQSUJEMAS-LFSVMHDDSA-N Ala-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C)N)O CYBJZLQSUJEMAS-LFSVMHDDSA-N 0.000 description 1
- SFPRJVVDZNLUTG-OWLDWWDNSA-N Ala-Trp-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SFPRJVVDZNLUTG-OWLDWWDNSA-N 0.000 description 1
- ZJLORAAXDAJLDC-CQDKDKBSSA-N Ala-Tyr-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O ZJLORAAXDAJLDC-CQDKDKBSSA-N 0.000 description 1
- VRZDJJWOFXMFRO-ZFWWWQNUSA-N Arg-Gly-Trp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O VRZDJJWOFXMFRO-ZFWWWQNUSA-N 0.000 description 1
- OFIYLHVAAJYRBC-HJWJTTGWSA-N Arg-Ile-Phe Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O OFIYLHVAAJYRBC-HJWJTTGWSA-N 0.000 description 1
- IIAXFBUTKIDDIP-ULQDDVLXSA-N Arg-Leu-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IIAXFBUTKIDDIP-ULQDDVLXSA-N 0.000 description 1
- XSPKAHFVDKRGRL-DCAQKATOSA-N Arg-Pro-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XSPKAHFVDKRGRL-DCAQKATOSA-N 0.000 description 1
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 1
- XRNXPIGJPQHCPC-RCWTZXSCSA-N Arg-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)O)C(O)=O XRNXPIGJPQHCPC-RCWTZXSCSA-N 0.000 description 1
- NVPHRWNWTKYIST-BPNCWPANSA-N Arg-Tyr-Ala Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 NVPHRWNWTKYIST-BPNCWPANSA-N 0.000 description 1
- QCTOLCVIGRLMQS-HRCADAONSA-N Arg-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O QCTOLCVIGRLMQS-HRCADAONSA-N 0.000 description 1
- CPTXATAOUQJQRO-GUBZILKMSA-N Arg-Val-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O CPTXATAOUQJQRO-GUBZILKMSA-N 0.000 description 1
- SRUUBQBAVNQZGJ-LAEOZQHASA-N Asn-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N SRUUBQBAVNQZGJ-LAEOZQHASA-N 0.000 description 1
- WLVLIYYBPPONRJ-GCJQMDKQSA-N Asn-Thr-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O WLVLIYYBPPONRJ-GCJQMDKQSA-N 0.000 description 1
- PDECQIHABNQRHN-GUBZILKMSA-N Asp-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O PDECQIHABNQRHN-GUBZILKMSA-N 0.000 description 1
- YFSLJHLQOALGSY-ZPFDUUQYSA-N Asp-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N YFSLJHLQOALGSY-ZPFDUUQYSA-N 0.000 description 1
- USNJAPJZSGTTPX-XVSYOHENSA-N Asp-Phe-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O USNJAPJZSGTTPX-XVSYOHENSA-N 0.000 description 1
- KGHLGJAXYSVNJP-WHFBIAKZSA-N Asp-Ser-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O KGHLGJAXYSVNJP-WHFBIAKZSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- ABLJDBFJPUWQQB-DCAQKATOSA-N Cys-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N ABLJDBFJPUWQQB-DCAQKATOSA-N 0.000 description 1
- HBHMVBGGHDMPBF-GARJFASQSA-N Cys-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N HBHMVBGGHDMPBF-GARJFASQSA-N 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- 101150103203 D11 gene Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- SGVGIVDZLSHSEN-RYUDHWBXSA-N Gln-Tyr-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O SGVGIVDZLSHSEN-RYUDHWBXSA-N 0.000 description 1
- RSUVOPBMWMTVDI-XEGUGMAKSA-N Glu-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCC(O)=O)C)C(O)=O)=CNC2=C1 RSUVOPBMWMTVDI-XEGUGMAKSA-N 0.000 description 1
- DYFJZDDQPNIPAB-NHCYSSNCSA-N Glu-Arg-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O DYFJZDDQPNIPAB-NHCYSSNCSA-N 0.000 description 1
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- AIGROOHQXCACHL-WDSKDSINSA-N Glu-Gly-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O AIGROOHQXCACHL-WDSKDSINSA-N 0.000 description 1
- QXDXIXFSFHUYAX-MNXVOIDGSA-N Glu-Ile-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O QXDXIXFSFHUYAX-MNXVOIDGSA-N 0.000 description 1
- 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 1
- UMHRCVCZUPBBQW-GARJFASQSA-N Glu-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N UMHRCVCZUPBBQW-GARJFASQSA-N 0.000 description 1
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- GGAPHLIUUTVYMX-QWRGUYRKSA-N Gly-Phe-Ser Chemical compound OC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)C[NH3+])CC1=CC=CC=C1 GGAPHLIUUTVYMX-QWRGUYRKSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- WZPIKDWQVRTATP-SYWGBEHUSA-N Ile-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)=CNC2=C1 WZPIKDWQVRTATP-SYWGBEHUSA-N 0.000 description 1
- TVYWVSJGSHQWMT-AJNGGQMLSA-N Ile-Leu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N TVYWVSJGSHQWMT-AJNGGQMLSA-N 0.000 description 1
- RMNMUUCYTMLWNA-ZPFDUUQYSA-N Ile-Lys-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N RMNMUUCYTMLWNA-ZPFDUUQYSA-N 0.000 description 1
- ANTFEOSJMAUGIB-KNZXXDILSA-N Ile-Thr-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N ANTFEOSJMAUGIB-KNZXXDILSA-N 0.000 description 1
- ZSESFIFAYQEKRD-CYDGBPFRSA-N Ile-Val-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(=O)O)N ZSESFIFAYQEKRD-CYDGBPFRSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 1
- ZDBMWELMUCLUPL-QEJZJMRPSA-N Leu-Phe-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 ZDBMWELMUCLUPL-QEJZJMRPSA-N 0.000 description 1
- ZAVCJRJOQKIOJW-KKUMJFAQSA-N Leu-Phe-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=CC=C1 ZAVCJRJOQKIOJW-KKUMJFAQSA-N 0.000 description 1
- 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 1
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 1
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 1
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- GKFNXYMAMKJSKD-NHCYSSNCSA-N Lys-Asp-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GKFNXYMAMKJSKD-NHCYSSNCSA-N 0.000 description 1
- HEWWNLVEWBJBKA-WDCWCFNPSA-N Lys-Gln-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCCN HEWWNLVEWBJBKA-WDCWCFNPSA-N 0.000 description 1
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 1
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 1
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 1
- XIZQPFCRXLUNMK-BZSNNMDCSA-N Lys-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCCCN)N XIZQPFCRXLUNMK-BZSNNMDCSA-N 0.000 description 1
- MSSJJDVQTFTLIF-KBPBESRZSA-N Lys-Phe-Gly Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O MSSJJDVQTFTLIF-KBPBESRZSA-N 0.000 description 1
- NYTDJEZBAAFLLG-IHRRRGAJSA-N Lys-Val-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O NYTDJEZBAAFLLG-IHRRRGAJSA-N 0.000 description 1
- LQMHZERGCQJKAH-STQMWFEESA-N Met-Gly-Phe Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 LQMHZERGCQJKAH-STQMWFEESA-N 0.000 description 1
- VSJAPSMRFYUOKS-IUCAKERBSA-N Met-Pro-Gly Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O VSJAPSMRFYUOKS-IUCAKERBSA-N 0.000 description 1
- NSMXRFMGZYTFEX-KJEVXHAQSA-N Met-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCSC)N)O NSMXRFMGZYTFEX-KJEVXHAQSA-N 0.000 description 1
- 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 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- JVTMTFMMMHAPCR-UBHSHLNASA-N Phe-Ala-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JVTMTFMMMHAPCR-UBHSHLNASA-N 0.000 description 1
- KIAWKQJTSGRCSA-AVGNSLFASA-N Phe-Asn-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KIAWKQJTSGRCSA-AVGNSLFASA-N 0.000 description 1
- HOYQLNNGMHXZDW-KKUMJFAQSA-N Phe-Glu-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O HOYQLNNGMHXZDW-KKUMJFAQSA-N 0.000 description 1
- VJLLEKDQJSMHRU-STQMWFEESA-N Phe-Gly-Met Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O VJLLEKDQJSMHRU-STQMWFEESA-N 0.000 description 1
- RYQWALWYQWBUKN-FHWLQOOXSA-N Phe-Phe-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O RYQWALWYQWBUKN-FHWLQOOXSA-N 0.000 description 1
- NHHZWPNMYQUNEH-ACRUOGEOSA-N Phe-Tyr-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N NHHZWPNMYQUNEH-ACRUOGEOSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 1
- DMNANGOFEUVBRV-GJZGRUSLSA-N Pro-Trp-Gly Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)O)C(=O)[C@@H]1CCCN1 DMNANGOFEUVBRV-GJZGRUSLSA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 244000153955 Reynoutria sachalinensis Species 0.000 description 1
- 235000003202 Reynoutria sachalinensis Nutrition 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- DSSOYPJWSWFOLK-CIUDSAMLSA-N Ser-Cys-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O DSSOYPJWSWFOLK-CIUDSAMLSA-N 0.000 description 1
- 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 1
- ZOPISOXXPQNOCO-SVSWQMSJSA-N Ser-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CO)N ZOPISOXXPQNOCO-SVSWQMSJSA-N 0.000 description 1
- XXNYYSXNXCJYKX-DCAQKATOSA-N Ser-Leu-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O XXNYYSXNXCJYKX-DCAQKATOSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 1
- PXQUBKWZENPDGE-CIQUZCHMSA-N Thr-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)O)N PXQUBKWZENPDGE-CIQUZCHMSA-N 0.000 description 1
- MQBTXMPQNCGSSZ-OSUNSFLBSA-N Thr-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)O)CCCN=C(N)N MQBTXMPQNCGSSZ-OSUNSFLBSA-N 0.000 description 1
- 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 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- TWJDQTTXXZDJKV-BPUTZDHNSA-N Trp-Arg-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O TWJDQTTXXZDJKV-BPUTZDHNSA-N 0.000 description 1
- RPVDDQYNBOVWLR-HOCLYGCPSA-N Trp-Gly-Leu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O RPVDDQYNBOVWLR-HOCLYGCPSA-N 0.000 description 1
- UKWSFUSPGPBJGU-VFAJRCTISA-N Trp-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O UKWSFUSPGPBJGU-VFAJRCTISA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- MICSYKFECRFCTJ-IHRRRGAJSA-N Tyr-Arg-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O MICSYKFECRFCTJ-IHRRRGAJSA-N 0.000 description 1
- AKXBNSZMYAOGLS-STQMWFEESA-N Tyr-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AKXBNSZMYAOGLS-STQMWFEESA-N 0.000 description 1
- WDIJBEWLXLQQKD-ULQDDVLXSA-N Tyr-Arg-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O WDIJBEWLXLQQKD-ULQDDVLXSA-N 0.000 description 1
- BARBHMSSVWPKPZ-IHRRRGAJSA-N Tyr-Asp-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BARBHMSSVWPKPZ-IHRRRGAJSA-N 0.000 description 1
- FMOSEWZYZPMJAL-KKUMJFAQSA-N Tyr-Glu-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N FMOSEWZYZPMJAL-KKUMJFAQSA-N 0.000 description 1
- JLKVWTICWVWGSK-JYJNAYRXSA-N Tyr-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JLKVWTICWVWGSK-JYJNAYRXSA-N 0.000 description 1
- NVJCMGGZHOJNBU-UFYCRDLUSA-N Tyr-Val-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N NVJCMGGZHOJNBU-UFYCRDLUSA-N 0.000 description 1
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 1
- LTFLDDDGWOVIHY-NAKRPEOUSA-N Val-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N LTFLDDDGWOVIHY-NAKRPEOUSA-N 0.000 description 1
- VVZDBPBZHLQPPB-XVKPBYJWSA-N Val-Glu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VVZDBPBZHLQPPB-XVKPBYJWSA-N 0.000 description 1
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 1
- MBGFDZDWMDLXHQ-GUBZILKMSA-N Val-Met-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N MBGFDZDWMDLXHQ-GUBZILKMSA-N 0.000 description 1
- LJSZPMSUYKKKCP-UBHSHLNASA-N Val-Phe-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 LJSZPMSUYKKKCP-UBHSHLNASA-N 0.000 description 1
- YQMILNREHKTFBS-IHRRRGAJSA-N Val-Phe-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N YQMILNREHKTFBS-IHRRRGAJSA-N 0.000 description 1
- PGQUDQYHWICSAB-NAKRPEOUSA-N Val-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N PGQUDQYHWICSAB-NAKRPEOUSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 description 1
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].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 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000007233 catalytic pyrolysis Methods 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 108010068404 exorphin B4 Proteins 0.000 description 1
- 229960004642 ferric ammonium citrate Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940089161 ginsenoside Drugs 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010084389 glycyltryptophan Proteins 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 235000020688 green tea extract Nutrition 0.000 description 1
- 229940094952 green tea extract Drugs 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000004313 iron ammonium citrate Substances 0.000 description 1
- 235000000011 iron ammonium citrate Nutrition 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供了一种新的水解虎杖苷制备白藜芦醇的β-葡萄糖苷酶、编码基因、载体、工程菌及其应用。该β-葡萄糖苷酶氨基酸序列如SEQ ID No.1所示。本发明的有益效果主要体现在：本发明提供了一种β-葡萄糖苷酶、编码基因、载体、工程菌及其应用，该β-葡萄糖苷酶能够水解虎杖苷制备白藜芦醇，环境友好，产率高，应用前景广阔。
Description
(一)技术领域
本发明涉及一种新的β-葡萄糖苷酶、编码基因、载体及其应用。
(二)背景技术
β-葡萄糖苷酶可以催化水解多种β-葡萄糖苷健，具有底物广谱性，和多种生物功能：
(1)β-葡萄糖苷酶用于纤维素的水解
纤维素是自然界最丰富的碳资源，从纤维素分解为葡萄糖需要3种酶协同作用。β-葡萄糖苷酶可以将纤维二糖水解为葡萄糖，而纤维素酶组分中β-葡萄糖苷酶含量少，活力低，因此成为纤维素酶解的瓶颈。因此筛选或者通过基因工程改造得到高活性的β-葡萄糖苷酶对纤维素有效降解具有重要意义。
(2)β-葡萄糖苷酶用于烷基糖苷和芳香基糖苷的合成
烷基糖苷、苄基糖苷和烯基糖苷不仅是糖化学中重要的中间体，同时也是一类可生物降解的非离子表面活性剂，广泛应用于化妆品、制药、食品和去污剂行业。酶法合成烷基糖苷和芳香基糖苷具有很大的潜在市场。其特点是选择性好，产品纯度高，收率高，制备条件温和。
(3)β-葡萄糖苷酶制备高生物活性苷元的应用
研究发现，自然界多种具有高生物活性的物质，由于大部分以糖苷形式存在而降低了其的活性。利用β-葡萄糖苷酶水解掉葡萄糖后可以得到高活性的苷元。
比如，人参皂甙是人参中的主要有效成分，含量约4％，但并非所有的人参皂甙都有很高的生物活性。如具有强烈抗癌作用的人参皂甙Rh2，在人参中的质量比仅为10-5。而以低活性的人参二醇类皂甙Rg3为底物，β-葡萄糖苷酶可以水解掉一个葡萄糖后得到人参皂甙Rh2。
比如，大豆异黄酮具有的很多重要的生理功能，研究发现大豆异黄酮苷元的生理活性远比其相应糖苷的活性高。大豆异黄酮中97％～99％是以大豆异黄酮糖苷形式存在，苷元形式仅为大豆异黄酮总量的1％～3％。利用β-葡萄糖苷酶可以水解大豆异黄酮糖苷为高生物活性的大豆异黄酮苷元。
据报道白藜芦醇具有抑制肿瘤、抗氧化、抗自由基、抗血栓、抗过敏和具有冠心病、缺血性心脏病的防治作用，白藜芦醇已被列为抗心血管、抗癌最有前途的药物之一。干燥虎杖根茎中白藜芦醇含量仅为0.1％～0.2％，而虎杖苷含量约为2％左右。酶法水解虎杖苷制备白藜芦醇具有反应温和、环境友好、工艺简单等优点，是值得深入研究的制备方法。
(三)发明内容
本发明目的是提供一种新的水解虎杖苷制备白藜芦醇的β-葡萄糖苷酶、编码基因、载体、工程菌及其应用。
本发明采用的技术方案是：
一种β-葡萄糖苷酶，其氨基酸序列如SEQ ID No.1所示：
Met Lys Thr Phe Pro Asp Asp Phe Leu Trp Gly Gly Ala Val Ala
Ala Asn Gln Val Glu Gly Ala Tyr Leu Glu Glu Gly Lys Gly Leu
Ser Thr Ser Asp Val Gln Pro Gln Gly Val Phe Gly Pro Val Val
Glu Arg Val Ala Gly Asp Ser Gly Ile Lys Asp Val Ala Ile Asp
Phe Tyr His Arg Tyr Pro Glu Asp Ile Lys Leu Phe Ala Glu Met
Gly Phe Ser Cys Leu Arg Val Ser Ile Ala Trp Thr Arg Ile Phe
Pro Asn Gly Asp Glu Gln Gln Pro Asn Glu Ala Gly Leu Ala Phe
Tyr Asp Arg Leu Phe Asp Glu Leu Ala Ala His Ser Ile Thr Pro
Leu Val Thr Leu Ser His Tyr Glu Met Pro Trp Gly Leu Val Lys
Gln Tyr Gly Gly Trp Gly Ser Arg Gln Thr Ile Gly Phe Phe Glu
Arg Tyr Ala Arg Thr Val Phe Ala Arg Tyr Lys Glu Lys Val Lys
Leu Trp Leu Thr Phe Asn Glu Ile Leu Lys Gly Ser Ser Leu Met
Tyr Val Phe Cys Leu Pro Asn Thr Ala Ile Val Met Ala Leu Ser
Pro Arg Gly Trp Arg Ser Lys Phe Gly Met Pro Gly Glu Ala Trp
Phe。
由于氨基酸序列的特殊性，任何含有SEQ ID NO.1所示氨基酸序列的肽蛋白的片段或其变体，如其保守性变体、生物活性片段或衍生物，只要该肽蛋白的片段或肽蛋白变体与前述氨基酸序列同源性在95％以上，均属于本发明保护范围之列。具体的所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换；其中，对于变体的保守性改变，所替换的氨基酸具有与原氨基酸相似的结构或化学性质，如用亮氨酸替换异亮氨酸，变体也可具有非保守性改变，如用色氨酸替换甘氨酸。本发明所述肽蛋白的片段、衍生物或类似物是指基本上保持本发明所述的β-葡萄糖苷酶相同的生物学功能或活性的肽蛋白，可以是下列情形：(I)一个或多个氨基酸残基被保守或非保守氨基酸残基(优选的是保守氨基酸残基)取代，并且取代的氨基酸可以是也可以不是由遗传密码子编码的；(II)一个或多个氨基酸残基上的某个基团被其它基团取代；(III)成熟肽蛋白与另一种化合物(比如延长肽蛋白半衰期的化合物，例如聚乙二醇)融合；(IV)附加的氨基酸序列融合进成熟的肽蛋白而形成的肽蛋白序列(如用来纯化此肽蛋白的序列或蛋白原序列)。
所述肽蛋白可以是重组蛋白、天然蛋白或合成蛋白，可以是纯天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如：细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的肽蛋白可以是糖基化的。本发明的肽蛋白还可以包括或不包括起始的甲硫氨酸残基。
本发明还涉及编码所述β-葡萄糖苷酶的基因。
具体的，所述基因序列可如SEQ ID No.2所示：
atgaaaactt tcccggacga ttttttatgg ggcggcgcgg ttgccgcgaa tcaggtagaa
ggcgcgtatc tggaggaggg aaaagggctg tccacctcag acgttcagcc gcagggtgtc
ttcggcccgg tggttgagcg cgtggcgggc gacagcggca tcaaggacgt cgccatcgac
ttctatcacc gctacccgga agacatcaaa ctgttcgctg agatgggctt tagctgcctg
cgcgtctcca ttgcctggac ccgcattttt ccgaacggcg acgagcagca gcctaacgag
gcggggctgg cgttttacga ccggctgttt gacgagctgg ccgcgcacag cattaccccg
ctggtgacgc tctcgcatta cgaaatgccg tgggggctgg tgaagcagta cggcgggtgg
ggcagccgcc agaccattgg gttcttcgag cgctacgccc gtaccgtgtt tgcgcgctac
aaggagaagg tgaagctctg gctgaccttc aacgagatct taaagggttc gagcctgatg
tacgtatttt gcttaccaaa tacagcaata gtaatggctc tcagtccccg tggatggagg
agcaaattcg ggatgcctgg ggaagcatgg ttctaa 。
由于核苷酸序列的特殊性，任何SEQ ID NO：2所示多核苷酸的变体，只要其与该多核苷酸具有70％以上同源性，均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以使生的变位变异体或非生的变异体，包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个多核苷酸的取代、缺失或插入，但不会从实质上改变其编码的肽蛋白的功能。
另外，可与SEQ ID NO：2所示多核苷酸序列杂交的多核苷酸(至少具有50％同源性，优选至少具有70％同源性)，也在本发明保护范围之列，特别是在严格条件下可与本发明所述核苷酸序列杂交的多核苷酸。所述“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2 SSC，0.1％SDS，60℃；或(2)杂交时加用变性剂，如50％(v/v)甲酰胺，0.1％小牛血清，0.1％Ficoll，42℃；或(3)仅在两条序列之间的同源性至少在95％以上，更好是97％以上时才发生杂交。并且，可杂交的多核苷酸编码的肽蛋白与SEQ ID NO：1所示的肽蛋白有相同的生物学功能和活性。
编码本发明所述β-葡萄糖苷酶的多核苷酸序列能用多种方法获得。例如，用本领域熟知的杂交技术分离多核苷酸。这些技术包括但不局限于：(1)用探针与基因或cDNA文库杂交以检出同源性的多核苷酸序列(2)表达文库的活性筛选以检出具有共同结构特征的克隆的多核苷酸片段，该表达文库可以包括环境宏基因组文库。本发明的DNA片段序列也能用下列方法获得：(1)从基因组DNA分离双链DNA序列；(2)化学合成DNA序列以获得所述肽蛋白的双链DNA。
可用常规方法从这些cNDA文库中筛选本发明的基因。这些方法包括(但不限于)：(1)DNA-DNA或DNA-RNA杂交；(2)标志基因功能的出现或丧失；(3)通过测定生物学活性，来检测基因表达的蛋白产物。上述方法可单用，也可多种方法联合应用。
如上所述得到的本发明所述的β-葡萄糖苷酶基因，或者各种DNA片段等的多核苷酸序列可用常规方法双脱氧链终止法测定。这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的cDNA序列测序需反复进行。有时需要测定多个克隆的cDNA序列，才能拼接全长的cDNA序列。
本发明的β-葡萄糖苷酶序列经蛋白质数据库进行搜寻比较，未发现有任何相同肽蛋白序列，而本发明的β-葡萄糖苷酶编码基因经基因数据库进行搜寻比较，也未发现有任何相同基因。
本发明还涉及含有所述基因的重组载体、以及由所述重组载体转化得到的基因工程菌。
所述的基因可用于转化制备重组β-葡萄糖苷酶。具体方法如下：构建含β-葡萄糖苷酶基因的重组载体，将所述重组载体转化至宿主体内，将获得的重组基因工程菌进行诱导培养，培养液分离得到含有重组β-葡萄糖苷酶基因。
本发明所述β-葡萄糖苷酶基因可插入到载体中，以构成含有本发明所述β-葡萄糖苷酶基因的重组载体。“载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。在本发明中适用的载体还包括但不限于：在细菌中表达的基于T7启动子的表达载体；在哺乳动物细胞中表达的pcDNA3.1载体和在昆虫细胞中表达的来源于杆状病毒的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用于构建重组表达载体，优选pET载体系列以其其它原核表达载体系列。表达载体一个重要特征是通常含有复制起始点、启动子、标记基因和翻译调控元件。
本领域的技术人员熟知的方法能用于构建含β-葡萄糖苷酶基因和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA序列、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA的合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；噬菌体的PL启动子；真核启动子包括CMV早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得到增强。增强子是DNA表达顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
此外，表达载体优选包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白，或用于大肠杆菌的卡那霉素或氨苄青霉素等。
本发明所述含β-葡萄糖苷酶基因或含有β-葡萄糖苷酶基因的重组载体可转化或转导入宿主细胞，以构成含有该基因或重组载体的基因工程菌。“宿主细胞”指原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表例子有：大肠杆菌，链霉菌属；细菌细胞如鼠伤寒沙门氏菌；真菌细胞如酵母；植物细胞；昆虫细胞如果蝇S2或Sf9；动物细胞如CHO、COS或Bowes黑素瘤细胞等。
用本发明所述的基因(DNA序列)或含有所述基因(DNA序列)的重组载体转化宿主细胞，可用本领域技术熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的方法为本领域众所周知，可供选择的是用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，或者常规机械方法如显微注射、电穿孔、脂质体包装等。
通过常规的重组DNA技术，利用本发明的多核苷酸序列可用来表达或生产重组的β-葡萄糖苷酶。一般来说有以下步骤：
(1)用本发明的β-葡萄糖苷酶基因(或变异体)，或用含有该基因的重组载体转化或转染合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
在步骤(2)中，根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞的条件下进行培养。当宿主细胞生长在适当的细胞密度后，用合适的方法诱导选择的启动子，将细胞再培养一段时间。
在步骤(3)中，重组肽蛋白可包被于细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法包括但不限于：常规的复性处理、蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声波处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的要点在于提供了SEQ ID NO：1所示的氨基酸序列和SEQ IDNO：2所示的核苷酸序列，在已知该氨基酸序列和核苷酸序列的情况下，该氨基酸序列和核苷酸序列的获得，以及相关载体、宿主细胞的获得，对于本领域技术人员来说均是显而易见的。
本发明还涉及所述的β-葡萄糖苷酶在水解虎杖苷制备白藜芦醇中的应用。
具体的，所述应用为：在pH为3～7的0.1～0.5mol/L的Na2HPO4-柠檬酸缓冲液中，加入等体积的0.5～1mg/ml虎杖苷水溶液，混合均匀，以β-葡萄糖苷酶为催化剂，所述的β-葡萄糖苷酶的终浓度为0.01～0.1mg/ml，混匀后于25～40℃摇床反应10～20小时，取反应液3000～6000rpm离心15分钟，取沉淀经纯化得到所述白藜芦醇。
具体的，所述的应用可按如下步骤进行：(1)配制pH5的0.2mol/L的Na2HPO4-柠檬酸缓冲液；(2)取一支试管，加入上述缓冲液20ml，加入1mg/ml虎杖苷溶液20ml，混合均匀，加入1mg/ml的β-葡萄糖苷酶3ml，混匀后于37℃摇床反应16小时，取反应液5000rpm离心15分钟，沉淀物即为白藜芦醇粗制品，虎杖苷以及白藜芦醇粗品分别用薄层扫描分析(TLC)，方法如下：以硅胶板G为薄层吸附剂，氯仿∶丙酮∶乙酸∶水(4∶4∶1∶0.2)为展开剂，在紫外灯(365nm)下观察荧光斑点，并在365nm处进行扫描测定。结果表明，用此方法可以简便，准确度高，重复性好的检测虎杖苷中白藜芦醇的生成量。
与现有技术相比，本发明的有益效果主要体现在：本发明提供了一种β-葡萄糖苷酶、编码基因、载体、工程菌及其应用，该β-葡萄糖苷酶能够水解虎杖苷制备白藜芦醇，环境友好，产率高，应用前景广阔。
(四)附图说明
图1为β-葡萄糖苷酶降解七叶苷的结果；1-空白组；2-加样组；
图2为β-葡萄糖苷酶催化对硝基苯-β-D-葡萄糖苷(ONPG)最适pH的测定；
图3为β-葡萄糖苷酶催化对硝基苯-β-D-葡萄糖苷(ONPG)最适温度的测定；
图4为薄层层析(TLC)检测β-葡萄糖苷酶催化虎杖苷中白藜芦醇的生成(1-无酶(虎杖苷+缓冲液)样品、2-虎杖苷标准品、3-白藜芦醇标准品、4-D11反应样品、5-苦杏仁酶(β-glucosidase购买)反应样品)；
图5为pH对β-葡萄糖苷酶水解虎杖苷的影响。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：β-葡萄糖苷酶D11基因的七叶苷筛选方法
LB固体培养基的配制。LB固体培养基的终浓度组成为：胰化蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、琼脂粉15g/L、用5mol/L NaOH溶液调pH至7.0，溶剂为水，在15psi(1.05kg/cm2)高压下蒸汽灭菌25min。冷却培养基，在培养基未凝固前加入终浓度为2.5％的柠檬酸铁铵(高压灭菌)和0.1％的七叶苷(过滤除菌)，培养基冷却后，将待筛选的微生物接种于该培养基，37℃培养16小时，微生物菌落周围有黑色斑点的即为阳性(图1)。
实施例2：D11基因全长的cDNA筛选与克隆
构建土壤基因组文库。根据实施例1方法筛选阳性克隆并构建亚文库，通过基因序列测定(由上海生工生物工程技术服务有限公司测定)，得到SEQ ID NO.2所示核苷酸序列。根据SEQ ID NO.2所示核苷酸序列，设计扩增出完整编码阅读框的引物，并在上游和下游引物上分别引入限制性内切酶位点(这可视选用的载体而定)，通过体外扩增技术获得SEQ IDNO.2所示的核苷酸序列的β-葡萄糖苷酶基因，将该基因克隆至pET 32a载体(杭州师范大学生物医药与健康中心保存)，在保证阅读框架正确的前提下鉴定好的表达载体，再将其转入E.Coil BL21中，得到工程菌E.Coil BL21/pET 32a/D11。
实施例3：D11蛋白的表达
将实施例2获得的E.Coil BL21/pET 32a/D11在含100mL 50μg/mL氨苄霉素的LB液体培养基中摇床过夜培养。将10ml过夜培养后的菌液倒入1L 50μg/mL氨苄霉素的LB液体培养基培养，直到菌液OD600达到0.6-0.8时加入IPTG终浓度为1.0mmol/L，25℃诱导5h后，离心收集菌体，菌体用25ml磷酸缓冲液pH7.4使其重悬浮，使用超生破碎仪(30min)，使细胞充分破碎。离心(12000g，15min)取上清，含D11蛋白的上清用0.22μm醋酸纤维素滤膜过滤后镍柱亲和层析进行纯化获得D11纯酶(氨基酸序列如SEQ ID NO.1所示)。
实施例4：D11催化对硝基苯-β-葡萄糖苷测定最适催化条件
本发明以对硝基苯-β-葡萄糖苷(ONPG)为底物，D11催化裂解一分子ONPG得到一份子对硝基苯酚，通过测定对硝基苯酚在420nm波长的UV吸收来测定动力学曲线以分析测定D11最适催化条件。
分别用pH2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、7.5、8.0、8.5、9.0磷酸缓冲液(0.15mol/L)将实施例3取得的D11纯酶配成1mg/ml的酶液，再取各pH值的磷酸缓冲液200ul和对应pH值的酶液100ul，再分别加入200ulONPG(1mg/ml)，30℃保温，测定波长420nm处的动力学曲线，得出D11最适pH值约为4.7(图2)。
用0.15mol/L的磷酸缓冲液(pH4.7)将实施例3取得的D11酶配成1mg/ml的酶液，分别取该酶液100ul，并分别加入200ul磷酸缓冲液(pH4.7)和200ulONPG(1mg/ml)，分别在15、20、25、30、35、45、50、55℃温度条件下(15℃-55℃)，测定波长420nm处的动力学曲线(图3)，得出D11最适温度约为37℃。
实施例6：D11催化虎杖苷方法
分别用pH4.0和pH5.0的Na2HPO4-柠檬酸缓冲液(0.2mol/L)配制成1mg/ml的D11酶液，分别将pH4.0和pH5.0上述缓冲液20ml与1mg/ml虎杖苷溶液20ml混合均匀，再分别加入上述pH4.0和pH5.0的D11酶液3ml，混匀后于37℃摇床反应16小时，反应结束，取反应液10000rpm离心15分钟，弃去上清液，沉淀为白藜芦醇粗品。虎杖苷标准品、白藜芦醇标准品及白藜芦醇粗品分别用薄层层析进行分析(图4)。以硅胶板G为薄层吸附剂，氯仿∶丙酮∶乙酸∶水(4∶4∶1∶0.2)为展开剂，在紫外灯(365nm)下观察荧光斑点，并在365nm处进行扫描测定。
结论：该D11酶能催化虎杖苷为白藜芦醇，转化率约为40％。
SEQUENCE LISTING
<110> 杭州师范大学
<120> 一种β-葡萄糖苷酶、编码基因、载体及其应用
<130>
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 229
<212> PRT
<213> Unknown
<220>
<223> 人工序列
<400> 1
Met Lys Thr Phe Pro Asp Asp Phe Leu Trp Gly Gly Ala Val Ala Ala
1 5 10 15
Asn Gln Val Glu Gly Ala Thr Tyr Arg Leu Glu Glu Gly Lys Gly Leu
20 25 30
Ser Thr Ser Asp Val Gln Pro Gln Gly Val Phe Gly Pro Val Val Glu
35 40 45
Arg Val Ala Gly Asp Ser Gly Ile Lys Asp Val Ala Ile Asp Phe Thr
50 55 60
Tyr Arg His Arg Thr Tyr Arg Pro Glu Asp Ile Lys Leu Phe Ala Glu
65 70 75 80
Met Gly Phe Ser Cys Leu Arg Val Ser Ile Ala Trp Thr Arg Ile Phe
85 90 95
Pro Asn Gly Asp Glu Gln Gln Pro Asn Glu Ala Gly Leu Ala Phe Thr
100 105 110
Tyr Arg Asp Arg Leu Phe Asp Glu Leu Ala Ala His Ser Ile Thr Pro
115 120 125
Leu Val Thr Leu Ser His Thr Tyr Arg Glu Met Pro Trp Gly Leu Val
130 135 140
Lys Gln Thr Tyr Arg Gly Gly Trp Gly Ser Arg Gln Thr Ile Gly Phe
145 150 155 160
Phe Glu Arg Thr Tyr Arg Ala Arg Thr Val Phe Ala Arg Thr Tyr Arg
165 170 175
Lys Glu Lys Val Lys Leu Trp Leu Thr Phe Asn Glu Ile Leu Lys Gly
180 185 190
Ser Ser Leu Met Thr Tyr Arg Val Phe Cys Leu Pro Asn Thr Ala Ile
195 200 205
Val Met Ala Leu Ser Pro Arg Gly Trp Arg Ser Lys Phe Gly Met Pro
210 215 220
Gly Glu Ala Trp Phe
225
<210> 2
<211> 636
<212> DNA
<213> Unknown
<220>
<223> 人工序列
<400> 2
atgaaaactt tcccggacga ttttttatgg ggcggcgcgg ttgccgcgaa tcaggtagaa 60
ggcgcgtatc tggaggaggg aaaagggctg tccacctcag acgttcagcc gcagggtgtc 120
ttcggcccgg tggttgagcg cgtggcgggc gacagcggca tcaaggacgt cgccatcgac 180
ttctatcacc gctacccgga agacatcaaa ctgttcgctg agatgggctt tagctgcctg 240
cgcgtctcca ttgcctggac ccgcattttt ccgaacggcg acgagcagca gcctaacgag 300
gcggggctgg cgttttacga ccggctgttt gacgagctgg ccgcgcacag cattaccccg 360
ctggtgacgc tctcgcatta cgaaatgccg tgggggctgg tgaagcagta cggcgggtgg 420
ggcagccgcc agaccattgg gttcttcgag cgctacgccc gtaccgtgtt tgcgcgctac 480
aaggagaagg tgaagctctg gctgaccttc aacgagatct taaagggttc gagcctgatg 540
tacgtatttt gcttaccaaa tacagcaata gtaatggctc tcagtccccg tggatggagg 600
agcaaattcg ggatgcctgg ggaagcatgg ttctaa 636
Claims (7)
1.一种β-葡萄糖苷酶，其氨基酸序列如SEQ ID No.1所示。
2.编码权利要求1所述β-葡萄糖苷酶的基因。
3.如权利要求2所述的基因，其特征在于所述基因序列如SEQ ID No.2所示。
4.含有权利要求2或3所述基因的重组载体。
5.由权利要求4所述重组载体转化得到的基因工程菌。
6.权利要求2或3所述的基因在制备重组β-葡萄糖苷酶中的应用。
7.如权利要求1所述的β-葡萄糖苷酶在水解虎杖苷制备白藜芦醇中的应用，所述应用为：在pH为3~7的0.1~0.5mol/L的Na2HPO4 -柠檬酸缓冲液中，加入等体积的0.5~1mg/ml虎杖苷水溶液，混合均匀，以β-葡萄糖苷酶为催化剂，所述的β-葡萄糖苷酶的终浓度为0.01~0.1mg/ml，混匀后于25~40℃摇床反应10~20小时，取反应液3000~6000rpm离心15分钟，取沉淀经纯化得到所述白藜芦醇。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100178575A CN102559642B (zh) | 2012-01-19 | 2012-01-19 | 一种β-葡萄糖苷酶、编码基因、载体及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100178575A CN102559642B (zh) | 2012-01-19 | 2012-01-19 | 一种β-葡萄糖苷酶、编码基因、载体及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559642A CN102559642A (zh) | 2012-07-11 |
| CN102559642B true CN102559642B (zh) | 2013-04-24 |
Family
ID=46406250
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100178575A Expired - Fee Related CN102559642B (zh) | 2012-01-19 | 2012-01-19 | 一种β-葡萄糖苷酶、编码基因、载体及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559642B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110372770A (zh) * | 2018-04-12 | 2019-10-25 | 耿兆翔 | 天然含羟基或/和羧基化合物的高效提取方法 |
| CN116751721B (zh) * | 2023-07-18 | 2024-02-13 | 陕西省微生物研究所 | 一株植物乳杆菌121-5及其在转化虎杖苷制备白藜芦醇中的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805755A (zh) * | 2010-04-14 | 2010-08-18 | 卢昶年 | 一种从虎杖中提取纯化白藜芦醇的工艺 |
| CN102321647A (zh) * | 2011-09-08 | 2012-01-18 | 杭州师范大学 | 一种β-葡萄糖苷酶、编码基因、载体、工程菌及其应用 |
-
2012
- 2012-01-19 CN CN2012100178575A patent/CN102559642B/zh not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805755A (zh) * | 2010-04-14 | 2010-08-18 | 卢昶年 | 一种从虎杖中提取纯化白藜芦醇的工艺 |
| CN102321647A (zh) * | 2011-09-08 | 2012-01-18 | 杭州师范大学 | 一种β-葡萄糖苷酶、编码基因、载体、工程菌及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| 田天丽.虎杖中虎杖苷的微生物发酵转化研究.《四川大学学报：自然科学版》.2008,第45卷(第2期),437-440. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559642A (zh) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109593126B (zh) | 多肽、其生产方法和用途 | |
| KR101200571B1 (ko) | 테라박터 속 유래의 신규한 진세노시드 글리코시다제 및 이의 용도 | |
| CN108699559A (zh) | 包括CYP76AD1-β进化枝多肽的组合物及其用途 | |
| CN104017794B (zh) | 用于生产(+)-客烯的方法 | |
| CN102321647B (zh) | 一种β-葡萄糖苷酶、编码基因、载体、工程菌及其应用 | |
| CN105087739A (zh) | 一种新的制备稀有人参皂苷的催化体系及其应用 | |
| KR101710853B1 (ko) | 감초속 식물 유래 트리테르펜 산화 효소, 그것을 코딩하는 유전자 및 그의 이용법 | |
| CN113136373A (zh) | 新型碳苷糖基转移酶及其应用 | |
| CN109477126A (zh) | 罗汉果醇或罗汉果醇糖苷的生产方法 | |
| CN101928716A (zh) | 丹参法呢基焦磷酸合酶(SmFPS)基因及其编码的蛋白和应用 | |
| CN102559642B (zh) | 一种β-葡萄糖苷酶、编码基因、载体及其应用 | |
| CN109715182A (zh) | 工程化β-葡糖苷酶和葡糖基化方法 | |
| CN113862319A (zh) | 人参糖基转移酶在合成甜菊糖中的应用 | |
| CN101928715A (zh) | 丹参一细胞色素P450(SmP450)基因及其编码的蛋白和应用 | |
| CN109402080B (zh) | 蛋白质ugt142及其编码基因与应用 | |
| KR20130055217A (ko) | 신규한 β-글루코시다제 단백질 및 이의 용도 | |
| KR20240032944A (ko) | 람노스가 고도로 특이적인 글리코실트랜스퍼라제 및 이의 응용 | |
| CN101619320A (zh) | 麻疯树鲨烯合酶蛋白编码序列及其在植物中的应用 | |
| CN110878288B (zh) | 多肽、核酸及其在合成橙花叔醇糖苷上的应用 | |
| CN114854722A (zh) | 一种季也蒙念珠菌外切β-1,3-葡聚糖酶及其突变体及应用 | |
| CN111620934B (zh) | 蛋白GmHSFB2b在调控植物黄酮类化合物积累中应用 | |
| CN108998428B (zh) | 滇重楼胆甾醇c22羟化酶及其编码基因和应用 | |
| KR20130055224A (ko) | 신규한 β-글라이코시다제 단백질 및 이의 용도 | |
| JP2008220304A (ja) | 膜結合性プレニルトランスフェラーゼ | |
| CN108410905A (zh) | 调节棉花的棉酚性状的基因以及调节方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130424 Termination date: 20160119 |
|
| EXPY | Termination of patent right or utility model |