CN108148824A - 一种耐有机溶剂高效半乳糖苷酶及其应用 - Google Patents
一种耐有机溶剂高效半乳糖苷酶及其应用 Download PDFInfo
- Publication number
- CN108148824A CN108148824A CN201810196588.0A CN201810196588A CN108148824A CN 108148824 A CN108148824 A CN 108148824A CN 201810196588 A CN201810196588 A CN 201810196588A CN 108148824 A CN108148824 A CN 108148824A
- Authority
- CN
- China
- Prior art keywords
- galactosidase
- ala
- gly
- arg
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010093031 Galactosidases Proteins 0.000 title claims abstract description 33
- 102000002464 Galactosidases Human genes 0.000 title claims abstract description 31
- 239000003960 organic solvent Substances 0.000 title claims abstract description 19
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims abstract description 44
- 238000006243 chemical reaction Methods 0.000 claims abstract description 38
- DBLDQZASZZMNSL-QMMMGPOBSA-N L-tyrosinol Natural products OC[C@@H](N)CC1=CC=C(O)C=C1 DBLDQZASZZMNSL-QMMMGPOBSA-N 0.000 claims abstract description 28
- 235000004330 tyrosol Nutrition 0.000 claims abstract description 28
- 238000003259 recombinant expression Methods 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 10
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 9
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 claims abstract description 8
- OOCCDEMITAIZTP-QPJJXVBHSA-N (E)-cinnamyl alcohol Chemical compound OC\C=C\C1=CC=CC=C1 OOCCDEMITAIZTP-QPJJXVBHSA-N 0.000 claims abstract description 7
- BVJSUAQZOZWCKN-UHFFFAOYSA-N p-hydroxybenzyl alcohol Chemical compound OCC1=CC=C(O)C=C1 BVJSUAQZOZWCKN-UHFFFAOYSA-N 0.000 claims abstract description 7
- OOCCDEMITAIZTP-UHFFFAOYSA-N allylic benzylic alcohol Natural products OCC=CC1=CC=CC=C1 OOCCDEMITAIZTP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- CQRYARSYNCAZFO-UHFFFAOYSA-N salicyl alcohol Chemical compound OCC1=CC=CC=C1O CQRYARSYNCAZFO-UHFFFAOYSA-N 0.000 claims abstract 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 51
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 26
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 6
- 230000013595 glycosylation Effects 0.000 claims description 5
- 238000006206 glycosylation reaction Methods 0.000 claims description 5
- 239000008101 lactose Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- MSHFRERJPWKJFX-UHFFFAOYSA-N 4-Methoxybenzyl alcohol Chemical compound COC1=CC=C(CO)C=C1 MSHFRERJPWKJFX-UHFFFAOYSA-N 0.000 claims 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose 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)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims 1
- 238000006555 catalytic reaction Methods 0.000 claims 1
- 239000000243 solution Substances 0.000 claims 1
- -1 glucoside compounds Chemical class 0.000 abstract description 34
- 229930182478 glucoside Natural products 0.000 abstract description 11
- 150000008195 galaktosides Chemical class 0.000 abstract description 10
- 238000000746 purification Methods 0.000 abstract description 8
- 241000894006 Bacteria Species 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 4
- 230000002194 synthesizing effect Effects 0.000 abstract description 3
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 230000002210 biocatalytic effect Effects 0.000 abstract 2
- 238000005918 transglycosylation reaction Methods 0.000 abstract 2
- 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 abstract 1
- 239000013604 expression vector Substances 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 20
- 239000000047 product Substances 0.000 description 20
- 229930182470 glycoside Natural products 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 15
- 238000000034 method Methods 0.000 description 13
- 150000002338 glycosides Chemical class 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 210000004556 brain Anatomy 0.000 description 9
- 230000006378 damage Effects 0.000 description 8
- 239000003814 drug Substances 0.000 description 7
- 229910000397 disodium phosphate Inorganic materials 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 150000008131 glucosides Chemical class 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 4
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 4
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 108010079364 N-glycylalanine Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 241001165494 Rhodiola Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 108010049041 glutamylalanine Proteins 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
- 108010092114 histidylphenylalanine Proteins 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 241000607720 Serratia Species 0.000 description 3
- 241000607715 Serratia marcescens Species 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 230000002490 cerebral effect Effects 0.000 description 3
- 230000001934 delay Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 229930182830 galactose Natural products 0.000 description 3
- 239000000348 glycosyl donor Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 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 2
- SDMAQFGBPOJFOM-GUBZILKMSA-N Ala-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SDMAQFGBPOJFOM-GUBZILKMSA-N 0.000 description 2
- KIUYPHAMDKDICO-WHFBIAKZSA-N Ala-Asp-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KIUYPHAMDKDICO-WHFBIAKZSA-N 0.000 description 2
- IFTVANMRTIHKML-WDSKDSINSA-N Ala-Gln-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O IFTVANMRTIHKML-WDSKDSINSA-N 0.000 description 2
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 2
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 2
- FEGOCLZUJUFCHP-CIUDSAMLSA-N Ala-Pro-Gln Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O FEGOCLZUJUFCHP-CIUDSAMLSA-N 0.000 description 2
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 2
- AENHOIXXHKNIQL-AUTRQRHGSA-N Ala-Tyr-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H]([NH3+])C)CC1=CC=C(O)C=C1 AENHOIXXHKNIQL-AUTRQRHGSA-N 0.000 description 2
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 2
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 2
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 2
- 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 2
- VKKYFICVTYKFIO-CIUDSAMLSA-N Arg-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N VKKYFICVTYKFIO-CIUDSAMLSA-N 0.000 description 2
- NMRHDSAOIURTNT-RWMBFGLXSA-N Arg-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N NMRHDSAOIURTNT-RWMBFGLXSA-N 0.000 description 2
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 description 2
- INOIAEUXVVNJKA-XGEHTFHBSA-N Arg-Thr-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O INOIAEUXVVNJKA-XGEHTFHBSA-N 0.000 description 2
- 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 2
- VLIJAPRTSXSGFY-STQMWFEESA-N Arg-Tyr-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 VLIJAPRTSXSGFY-STQMWFEESA-N 0.000 description 2
- LLQIAIUAKGNOSE-NHCYSSNCSA-N Arg-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N LLQIAIUAKGNOSE-NHCYSSNCSA-N 0.000 description 2
- ULBHWNVWSCJLCO-NHCYSSNCSA-N Arg-Val-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N ULBHWNVWSCJLCO-NHCYSSNCSA-N 0.000 description 2
- QLSRIZIDQXDQHK-RCWTZXSCSA-N Arg-Val-Thr 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)O)C(O)=O QLSRIZIDQXDQHK-RCWTZXSCSA-N 0.000 description 2
- UGXVKHRDGLYFKR-CIUDSAMLSA-N Asn-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(N)=O UGXVKHRDGLYFKR-CIUDSAMLSA-N 0.000 description 2
- OWUCNXMFJRFOFI-BQBZGAKWSA-N Asn-Gly-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O OWUCNXMFJRFOFI-BQBZGAKWSA-N 0.000 description 2
- JQSWHKKUZMTOIH-QWRGUYRKSA-N Asn-Gly-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N JQSWHKKUZMTOIH-QWRGUYRKSA-N 0.000 description 2
- XMHFCUKJRCQXGI-CIUDSAMLSA-N Asn-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O XMHFCUKJRCQXGI-CIUDSAMLSA-N 0.000 description 2
- PIABYSIYPGLLDQ-XVSYOHENSA-N Asn-Thr-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PIABYSIYPGLLDQ-XVSYOHENSA-N 0.000 description 2
- LRCIOEVFVGXZKB-BZSNNMDCSA-N Asn-Tyr-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LRCIOEVFVGXZKB-BZSNNMDCSA-N 0.000 description 2
- XPGVTUBABLRGHY-BIIVOSGPSA-N Asp-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N XPGVTUBABLRGHY-BIIVOSGPSA-N 0.000 description 2
- WBDWQKRLTVCDSY-WHFBIAKZSA-N Asp-Gly-Asp Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O WBDWQKRLTVCDSY-WHFBIAKZSA-N 0.000 description 2
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 2
- KHGPWGKPYHPOIK-QWRGUYRKSA-N Asp-Gly-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KHGPWGKPYHPOIK-QWRGUYRKSA-N 0.000 description 2
- HOBNTSHITVVNBN-ZPFDUUQYSA-N Asp-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N HOBNTSHITVVNBN-ZPFDUUQYSA-N 0.000 description 2
- JUWISGAGWSDGDH-KKUMJFAQSA-N Asp-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=CC=C1 JUWISGAGWSDGDH-KKUMJFAQSA-N 0.000 description 2
- IQCJOIHDVFJQFV-LKXGYXEUSA-N Asp-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O IQCJOIHDVFJQFV-LKXGYXEUSA-N 0.000 description 2
- HCOQNGIHSXICCB-IHRRRGAJSA-N Asp-Tyr-Arg Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)O HCOQNGIHSXICCB-IHRRRGAJSA-N 0.000 description 2
- SBORMUFGKSCGEN-XHNCKOQMSA-N Cys-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N)C(=O)O SBORMUFGKSCGEN-XHNCKOQMSA-N 0.000 description 2
- CIVXDCMSSFGWAL-YUMQZZPRSA-N Cys-Lys-Gly Chemical compound C(CCN)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CS)N CIVXDCMSSFGWAL-YUMQZZPRSA-N 0.000 description 2
- ALTQTAKGRFLRLR-GUBZILKMSA-N Cys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N ALTQTAKGRFLRLR-GUBZILKMSA-N 0.000 description 2
- YJIUYQKQBBQYHZ-ACZMJKKPSA-N Gln-Ala-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YJIUYQKQBBQYHZ-ACZMJKKPSA-N 0.000 description 2
- CYTSBCIIEHUPDU-ACZMJKKPSA-N Gln-Asp-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O CYTSBCIIEHUPDU-ACZMJKKPSA-N 0.000 description 2
- NKCZYEDZTKOFBG-GUBZILKMSA-N Gln-Gln-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NKCZYEDZTKOFBG-GUBZILKMSA-N 0.000 description 2
- PSERKXGRRADTKA-MNXVOIDGSA-N Gln-Leu-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PSERKXGRRADTKA-MNXVOIDGSA-N 0.000 description 2
- JNVGVECJCOZHCN-DRZSPHRISA-N Gln-Phe-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(O)=O JNVGVECJCOZHCN-DRZSPHRISA-N 0.000 description 2
- XUMFMAVDHQDATI-DCAQKATOSA-N Gln-Pro-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUMFMAVDHQDATI-DCAQKATOSA-N 0.000 description 2
- ZJICFHQSPWFBKP-AVGNSLFASA-N Glu-Asn-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZJICFHQSPWFBKP-AVGNSLFASA-N 0.000 description 2
- QPRZKNOOOBWXSU-CIUDSAMLSA-N Glu-Asp-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N QPRZKNOOOBWXSU-CIUDSAMLSA-N 0.000 description 2
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 2
- 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 2
- OAGVHWYIBZMWLA-YFKPBYRVSA-N Glu-Gly-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)NCC(O)=O OAGVHWYIBZMWLA-YFKPBYRVSA-N 0.000 description 2
- ZWABFSSWTSAMQN-KBIXCLLPSA-N Glu-Ile-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O ZWABFSSWTSAMQN-KBIXCLLPSA-N 0.000 description 2
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 2
- WIKMTDVSCUJIPJ-CIUDSAMLSA-N Glu-Ser-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WIKMTDVSCUJIPJ-CIUDSAMLSA-N 0.000 description 2
- JVWPPCWUDRJGAE-YUMQZZPRSA-N Gly-Asn-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JVWPPCWUDRJGAE-YUMQZZPRSA-N 0.000 description 2
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 2
- UPADCCSMVOQAGF-LBPRGKRZSA-N Gly-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)CN)C(O)=O)=CNC2=C1 UPADCCSMVOQAGF-LBPRGKRZSA-N 0.000 description 2
- AAHSHTLISQUZJL-QSFUFRPTSA-N Gly-Ile-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AAHSHTLISQUZJL-QSFUFRPTSA-N 0.000 description 2
- AFWYPMDMDYCKMD-KBPBESRZSA-N Gly-Leu-Tyr Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AFWYPMDMDYCKMD-KBPBESRZSA-N 0.000 description 2
- SSFWXSNOKDZNHY-QXEWZRGKSA-N Gly-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN SSFWXSNOKDZNHY-QXEWZRGKSA-N 0.000 description 2
- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 2
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 2
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 2
- RNMNYMDTESKEAJ-KKUMJFAQSA-N His-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CN=CN1 RNMNYMDTESKEAJ-KKUMJFAQSA-N 0.000 description 2
- ISQOVWDWRUONJH-YESZJQIVSA-N His-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CN=CN3)N)C(=O)O ISQOVWDWRUONJH-YESZJQIVSA-N 0.000 description 2
- FVEWRQXNISSYFO-ZPFDUUQYSA-N Ile-Arg-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FVEWRQXNISSYFO-ZPFDUUQYSA-N 0.000 description 2
- XLDYDEDTGMHUCZ-GHCJXIJMSA-N Ile-Asp-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N XLDYDEDTGMHUCZ-GHCJXIJMSA-N 0.000 description 2
- HGNUKGZQASSBKQ-PCBIJLKTSA-N Ile-Asp-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HGNUKGZQASSBKQ-PCBIJLKTSA-N 0.000 description 2
- NYEYYMLUABXDMC-NHCYSSNCSA-N Ile-Gly-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)O)N NYEYYMLUABXDMC-NHCYSSNCSA-N 0.000 description 2
- IIWQTXMUALXGOV-PCBIJLKTSA-N Ile-Phe-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N IIWQTXMUALXGOV-PCBIJLKTSA-N 0.000 description 2
- ZNOBVZFCHNHKHA-KBIXCLLPSA-N Ile-Ser-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZNOBVZFCHNHKHA-KBIXCLLPSA-N 0.000 description 2
- 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 2
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 2
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 2
- 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 2
- 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 2
- VBZOAGIPCULURB-QWRGUYRKSA-N Leu-Gly-His Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N VBZOAGIPCULURB-QWRGUYRKSA-N 0.000 description 2
- POZULHZYLPGXMR-ONGXEEELSA-N Leu-Gly-Val Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O POZULHZYLPGXMR-ONGXEEELSA-N 0.000 description 2
- OMHLATXVNQSALM-FQUUOJAGSA-N Leu-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(C)C)N OMHLATXVNQSALM-FQUUOJAGSA-N 0.000 description 2
- OVZLLFONXILPDZ-VOAKCMCISA-N Leu-Lys-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OVZLLFONXILPDZ-VOAKCMCISA-N 0.000 description 2
- OZTZJMUZVAVJGY-BZSNNMDCSA-N Leu-Tyr-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N OZTZJMUZVAVJGY-BZSNNMDCSA-N 0.000 description 2
- XFIHDSBIPWEYJJ-YUMQZZPRSA-N Lys-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN XFIHDSBIPWEYJJ-YUMQZZPRSA-N 0.000 description 2
- KYNNSEJZFVCDIV-ZPFDUUQYSA-N Lys-Ile-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O KYNNSEJZFVCDIV-ZPFDUUQYSA-N 0.000 description 2
- OSOLWRWQADPDIQ-DCAQKATOSA-N Met-Asp-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OSOLWRWQADPDIQ-DCAQKATOSA-N 0.000 description 2
- STTRPDDKDVKIDF-KKUMJFAQSA-N Met-Glu-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 STTRPDDKDVKIDF-KKUMJFAQSA-N 0.000 description 2
- JHDNAOVJJQSMMM-GMOBBJLQSA-N Met-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](CCSC)N JHDNAOVJJQSMMM-GMOBBJLQSA-N 0.000 description 2
- SBFPAAPFKZPDCZ-JYJNAYRXSA-N Met-Pro-Tyr Chemical compound [H]N[C@@H](CCSC)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O SBFPAAPFKZPDCZ-JYJNAYRXSA-N 0.000 description 2
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 2
- 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 2
- 206010029350 Neurotoxicity Diseases 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 240000000220 Panda oleosa Species 0.000 description 2
- 235000016496 Panda oleosa Nutrition 0.000 description 2
- ZWJKVFAYPLPCQB-UNQGMJICSA-N Phe-Arg-Thr Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)Cc1ccccc1)C(O)=O ZWJKVFAYPLPCQB-UNQGMJICSA-N 0.000 description 2
- 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 2
- IQXOZIDWLZYYAW-IHRRRGAJSA-N Phe-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N IQXOZIDWLZYYAW-IHRRRGAJSA-N 0.000 description 2
- JEBWZLWTRPZQRX-QWRGUYRKSA-N Phe-Gly-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O JEBWZLWTRPZQRX-QWRGUYRKSA-N 0.000 description 2
- APJPXSFJBMMOLW-KBPBESRZSA-N Phe-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 APJPXSFJBMMOLW-KBPBESRZSA-N 0.000 description 2
- SFKOEHXABNPLRT-KBPBESRZSA-N Phe-His-Gly Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)NCC(O)=O SFKOEHXABNPLRT-KBPBESRZSA-N 0.000 description 2
- WKTSCAXSYITIJJ-PCBIJLKTSA-N Phe-Ile-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O WKTSCAXSYITIJJ-PCBIJLKTSA-N 0.000 description 2
- MJQFZGOIVBDIMZ-WHOFXGATSA-N Phe-Ile-Gly Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)O MJQFZGOIVBDIMZ-WHOFXGATSA-N 0.000 description 2
- XNMYNGDKJNOKHH-BZSNNMDCSA-N Phe-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XNMYNGDKJNOKHH-BZSNNMDCSA-N 0.000 description 2
- CYQQWUPHIZVCNY-GUBZILKMSA-N Pro-Arg-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O CYQQWUPHIZVCNY-GUBZILKMSA-N 0.000 description 2
- YKQNVTOIYFQMLW-IHRRRGAJSA-N Pro-Cys-Tyr Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H]1NCCC1)C1=CC=C(O)C=C1 YKQNVTOIYFQMLW-IHRRRGAJSA-N 0.000 description 2
- PULPZRAHVFBVTO-DCAQKATOSA-N Pro-Glu-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PULPZRAHVFBVTO-DCAQKATOSA-N 0.000 description 2
- DMKWYMWNEKIPFC-IUCAKERBSA-N Pro-Gly-Arg Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O DMKWYMWNEKIPFC-IUCAKERBSA-N 0.000 description 2
- LCUOTSLIVGSGAU-AVGNSLFASA-N Pro-His-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LCUOTSLIVGSGAU-AVGNSLFASA-N 0.000 description 2
- AUQGUYPHJSMAKI-CYDGBPFRSA-N Pro-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 AUQGUYPHJSMAKI-CYDGBPFRSA-N 0.000 description 2
- NFLNBHLMLYALOO-DCAQKATOSA-N Pro-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@@H]1CCCN1 NFLNBHLMLYALOO-DCAQKATOSA-N 0.000 description 2
- VVAWNPIOYXAMAL-KJEVXHAQSA-N Pro-Thr-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VVAWNPIOYXAMAL-KJEVXHAQSA-N 0.000 description 2
- IMNVAOPEMFDAQD-NHCYSSNCSA-N Pro-Val-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IMNVAOPEMFDAQD-NHCYSSNCSA-N 0.000 description 2
- JPIDMRXXNMIVKY-VZFHVOOUSA-N Ser-Ala-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPIDMRXXNMIVKY-VZFHVOOUSA-N 0.000 description 2
- FCRMLGJMPXCAHD-FXQIFTODSA-N Ser-Arg-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O FCRMLGJMPXCAHD-FXQIFTODSA-N 0.000 description 2
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 2
- DYEGLQRVMBWQLD-IXOXFDKPSA-N Ser-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](CO)N)O DYEGLQRVMBWQLD-IXOXFDKPSA-N 0.000 description 2
- ZWSZBWAFDZRBNM-UBHSHLNASA-N Ser-Trp-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O ZWSZBWAFDZRBNM-UBHSHLNASA-N 0.000 description 2
- VGYBYGQXZJDZJU-XQXXSGGOSA-N Thr-Glu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VGYBYGQXZJDZJU-XQXXSGGOSA-N 0.000 description 2
- 206010044221 Toxic encephalopathy Diseases 0.000 description 2
- WCTYCXZYBNKEIV-SXNHZJKMSA-N Trp-Glu-Ile Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)=CNC2=C1 WCTYCXZYBNKEIV-SXNHZJKMSA-N 0.000 description 2
- IQXWAJUIAQLZNX-IHPCNDPISA-N Trp-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N IQXWAJUIAQLZNX-IHPCNDPISA-N 0.000 description 2
- UEFHVUQBYNRNQC-SFJXLCSZSA-N Trp-Phe-Thr Chemical compound C([C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=CC=C1 UEFHVUQBYNRNQC-SFJXLCSZSA-N 0.000 description 2
- HWCBFXAWVTXXHZ-NYVOZVTQSA-N Trp-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)N HWCBFXAWVTXXHZ-NYVOZVTQSA-N 0.000 description 2
- YXSSXUIBUJGHJY-SFJXLCSZSA-N Trp-Thr-Phe Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)[C@H](O)C)C(O)=O)C1=CC=CC=C1 YXSSXUIBUJGHJY-SFJXLCSZSA-N 0.000 description 2
- WNGMGTMSUBARLB-RXVVDRJESA-N Trp-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)N)C(=O)NCC(O)=O)=CNC2=C1 WNGMGTMSUBARLB-RXVVDRJESA-N 0.000 description 2
- CDRYEAWHKJSGAF-BPNCWPANSA-N Tyr-Ala-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O CDRYEAWHKJSGAF-BPNCWPANSA-N 0.000 description 2
- DKKHULUSOSWGHS-UWJYBYFXSA-N Tyr-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N DKKHULUSOSWGHS-UWJYBYFXSA-N 0.000 description 2
- QNJYPWZACBACER-KKUMJFAQSA-N Tyr-Asp-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O QNJYPWZACBACER-KKUMJFAQSA-N 0.000 description 2
- SLCSPPCQWUHPPO-JYJNAYRXSA-N Tyr-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 SLCSPPCQWUHPPO-JYJNAYRXSA-N 0.000 description 2
- FBHBVXUBTYVCRU-BZSNNMDCSA-N Tyr-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CN=CN1 FBHBVXUBTYVCRU-BZSNNMDCSA-N 0.000 description 2
- GITNQBVCEQBDQC-KKUMJFAQSA-N Tyr-Lys-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O GITNQBVCEQBDQC-KKUMJFAQSA-N 0.000 description 2
- ABZWHLRQBSBPTO-RNXOBYDBSA-N Tyr-Trp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC4=CC=C(C=C4)O)N ABZWHLRQBSBPTO-RNXOBYDBSA-N 0.000 description 2
- RZAGEHHVNYESNR-RNXOBYDBSA-N Tyr-Trp-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RZAGEHHVNYESNR-RNXOBYDBSA-N 0.000 description 2
- JFAWZADYPRMRCO-UBHSHLNASA-N Val-Ala-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JFAWZADYPRMRCO-UBHSHLNASA-N 0.000 description 2
- XLDYBRXERHITNH-QSFUFRPTSA-N Val-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)C(C)C XLDYBRXERHITNH-QSFUFRPTSA-N 0.000 description 2
- 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 2
- 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 2
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 108010044940 alanylglutamine Proteins 0.000 description 2
- 108010070944 alanylhistidine Proteins 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 108010080488 arginyl-arginyl-leucine Proteins 0.000 description 2
- 108010062796 arginyllysine Proteins 0.000 description 2
- 108010077245 asparaginyl-proline Proteins 0.000 description 2
- 108010038633 aspartylglutamate Proteins 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 102000055102 bcl-2-Associated X Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 108010006664 gamma-glutamyl-glycyl-glycine Proteins 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 125000003147 glycosyl group Chemical group 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- WVDDGKGOMKODPV-UHFFFAOYSA-N hydroxymethyl benzene Natural products OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 2
- 208000037906 ischaemic injury Diseases 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 108010091871 leucylmethionine Proteins 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 108010064235 lysylglycine Proteins 0.000 description 2
- 108010005942 methionylglycine Proteins 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000007135 neurotoxicity Effects 0.000 description 2
- 231100000228 neurotoxicity Toxicity 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 2
- 108010025488 pinealon Proteins 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108010029020 prolylglycine Proteins 0.000 description 2
- 230000010410 reperfusion Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000011506 response to oxidative stress Effects 0.000 description 2
- 230000000284 resting effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010080629 tryptophan-leucine Proteins 0.000 description 2
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 244000281594 Cassia siamea Species 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 244000042430 Rhodiola rosea Species 0.000 description 1
- 235000003713 Rhodiola rosea Nutrition 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000003293 cardioprotective effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 230000036252 glycation Effects 0.000 description 1
- 239000000937 glycosyl acceptor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 208000003906 hydrocephalus Diseases 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000003680 myocardial damage Effects 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000001202 rhombencephalon Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Saccharide Compounds (AREA)
Abstract
本发明涉及一种耐有机溶剂高效半乳糖苷酶及其应用。本发明所述的半乳糖苷酶具有如SEQ ID NO：2所示的氨基酸序列，编码所述半糖苷酶的基因具有如SEQ ID NO：1所示的核苷酸序列。本发明还提供该半乳糖苷酶的重组表达载体或重组菌。本发明涉及的半乳糖苷酶在生物催化转糖基化反应合成新型红景天苷的应用是通过非水相中生物催化转糖基化反应的调控，建立高效合成糖苷化合物的非水相体系，实现了酪醇、对羟基苯甲醇、水杨醇、肉桂醇等多种类化合物的半乳糖苷类产物的高效制备，反应体系简单，产物转化率较高、易于纯化，大幅度降低了成本，具有很好的工业应用价值。
Description
技术领域
本发明涉及生物制药技术领域，具体涉及一种耐有机溶剂高效半乳糖苷酶及其在非水相反应合成新型红景天苷类似物应用。
背景技术
红景天苷是一种天然的糖苷化合物，是植物红景天(Rhodiola rosea) 的有效成分之一，具有提高免疫力、抗肿瘤、降血糖、抗衰老、抗疲劳、耐缺氧、抗辐射等多方面药理作用。对于高辐射环境工作人员、脑力劳动者、中老年人具有积极的滋补保健作用；是宇航员、飞行员、登山运动员、潜水员提高在恶劣环境中生存能力的保健药品，同时是高原地区重要的抗缺氧药品，是一种极具有开发应用前景的环境适应药物。近年来，由于其独特的抗衰老作用，在化妆品领域应用日趋广泛。
糖苷类化合物结构中糖基的类型对糖苷类药物的生物活性有较大影响，当受体分子中连接不同类型的糖分子，其生物活性也表现出较大的差异。在酪醇的分子上连接不同的糖分子，获得的多种红景天苷衍生物的生物活性有着显著差异，酪醇的半乳糖苷衍生物的作用效果相对较强。酪醇-β-D-半乳糖苷具有清除DPPH自由基和羟基自由基的作用，半乳糖苷是酪醇所有羟基取代中最强的，其抗氧化活性比红景天苷（酪醇-β-D-葡萄糖苷）有着30%左右的差异，并且在生物体内有更多的受体，有更高的生物利用度，是一种潜在的具有高生物活性的新型红景天苷新型衍生物（石力夫，蔡溱，姚斌. 红景天苷衍生物及其制备方法和用途 [P].中国专利，CN1475492A.）。
在心脏保护方面，酪醇-β-D-半乳糖苷能增加心肌SOD 活力，减轻氧化应激反应程度，降低细胞膜脂质过氧化产物MDA的含量以及减少NO 合成，使NO 对细胞的毒性作用降低，从而减少缺血心肌细胞死亡，降低心肌损伤（Patel V C, Yellon D M, Singh K J, etal. Inhibition of nitric oxide limits infarct size in the in situ rabbitheart[J]. Biochemical & Biophysical Research Communications, 1993, 194(1):234-238.）。其作用强于红景天苷，能有效地减少活性氧簇对细胞的损伤。在脑、神经细胞保护方面，红景天苷及其类似物酪醇-β-D-半乳糖苷能预防大脑缺血性损伤，在大鼠实验中能有效地减少氧化损伤，这一作用是通过减少Bax的表达和修复促凋亡、抗凋亡蛋白的平衡实现的（Shi T Y, Feng S F, Xing J H, et al. Neuroprotective effects ofSalidroside and its analogue tyrosol galactoside against focal cerebralischemia in vivo and H2O2-induced neurotoxicity in vitro[J]. NeurotoxicityResearch, 2012, 21(4):358-367.）。缺血/再灌注( I/R)损伤是中风、颅脑外伤以及心、脑外科手术等引起不可逆损伤的共同病理生理过程。研究发现，红景天苷及其类似物酪醇-β-D-半乳糖苷可以保护细胞免受超氧阴离子自由基(O2)和过氧化氢(H2O2)的损伤，具有治疗I/R 损伤的作用，并且酪醇-β-D-半乳糖苷效果更强。红景天苷及其类似物酪醇-β-D-半乳糖苷可以增高脑I/R后脑组织中乙酰胆碱的含量，抑制脑I/R 损伤后脑内白细胞介素1β的表达，可减轻大鼠脑I/R 后脑水肿程度，缓解海马区自由基代谢异常，改善认知功能，可以易化脑I/R损伤后中枢神经系统神经再生和修复的可塑性，抑制APE/Ref21降低（刘晓梅,王淑英, 刘斌. 红景天苷对脑缺血再灌注大鼠脑神经元APE/Ref-1的影响[J]. 中国老年学, 2009, 29(5):518-520.）。
目前，红景天苷主要通过提取法、化学合成法、植物细胞培养和酶法合成等技术获得，这些技术存在各自不同的优缺点。红景天是提取红景天苷的主要植物，但是红景天是高寒地带植物，野生资源匮乏，且人工栽培技术尚不成熟，因此难以保证红景天原料的大量供应。又加之红景天苷的提取工艺复杂，过中需要用到的试剂较多，虽有多次完善，但从天然植物中获得的红景天苷依然很少，不能满足市场的需求。虽然在1969年俄国化学家Troshchenko等利用化学法合成了红景天苷，并且经我国学者不断地优化改良，但是化学合成过程依然需要多步保护和去保护措施，过程复杂且成本较高，副产物多，难以分离。并且要使用价格昂贵的糖基供体和各种重金属催化剂、有机试剂，对环境危害较大，至今仍未投入生产使用。植物细胞培养法因设备昂贵、技术难度大，产物提纯成本高，数代培养后细胞退化不产产物等缺点未能应用到实际生产之中。
酶催化合成红景天苷的方法具有反应条件温和，具有很好的位置选择性和立体专一性，与传统的化学法合成糖苷类化合物相比，本发明开发的生物技术方法生产糖苷类化合物，产物的构型明确，不存在α，β 两种构型的糖苷混合物，产物结构单一，纯度高，是开发成为药物使用的重要安全保证。当前用于红景天苷合成的酶主要有糖基转移酶和糖苷酶。UDP-葡萄糖基转移酶的糖基供体UDP-糖苷价格昂贵，且对糖基受体具有高度专一性，使得合成缺乏灵活性。而糖苷酶催化糖和醇类直接成苷属于逆水解反应，受热力学控制，由于反应平衡的限制，最终产率过低。受反应热力学平衡的影响，该反应需要严格控制反应体系的水含量。但是在有机溶剂中，酶又容易失活，不利于反应的进行，因而需要筛选对亲水有机溶剂高耐受性的糖苷酶进行反应。
发明内容
针对半乳糖苷酶受热力学平衡限制产率过低、糖苷酶在有机相中易失活等问题，本发明提供了一种有机溶剂耐受性高、稳定性好、合成效率高的半乳糖苷酶，含有编码该半乳糖苷酶基因的重组表达载体、重组表达转化体及其高效制备方法，以及利用该酶采用添加亲水有机溶剂的非水相反应体系进行新型红景天苷类似物的合成。
本发明目的之一，在于提供一种耐有机溶剂高效半乳糖苷酶，其氨基酸序列如SEQID NO：2所示。
本发明另一目的在于提供编码上述半乳糖苷酶的基因，其核苷酸序列如SEQ IDNO：1所示。该基因序列从沙雷氏菌属菌株（Serratia marcescens）MH6的总DNA中提取，命名为gal，全长1386bp。编码序列从第一个碱基起至第1386个碱基止，起始密码子为ATG，终止密码子为TAA。
本发明另一目的在于提供含有所述耐有机溶剂高效半乳糖苷酶编码基因的重组载体。所述重组表达载体是将基因插入载体连接后获得重组表达载体；将重组表达载体转化到宿主菌株获得重组表达转化体。
本发明还提供了该酶其在非水相反应合成新型红景天苷类似物的应用。培养前述的重组表达转化体，获得重组表达的耐有机溶剂高效半乳糖苷酶。其中，所述的培养重组表达转化体中所用的培养基为LB/Kana培养基。将培养获得的重组转化体离心收集细胞，并经生理盐水洗涤2次后获得静息细胞。用pH7.5-8.5的PBS缓冲重悬细胞，经超声破碎后离心收集粗酶液，并经镍柱纯化获取纯化后的耐有机溶剂高效半乳糖苷酶。
所述的非水相体系为0%~30%（v/v）有机溶剂体系，醇类浓度为10 g·L-1 -60 g·L-1，乳糖浓度为100~250 g·L-1，反应温度为20~65℃，反应pH为5~11，反应时间15小时。
本发明应用的产物红景天苷类似物具有清除DPPH自由基和羟基自由基的作用，保护细胞免受超氧阴离子自由基(O2)和过氧化氢(H2O2)的损伤。对于心肌细胞，它能增加心肌SOD 活力，减轻氧化应激反应程度。在脑、神经细胞保护方面，红景天苷及其类似物酪醇-β-D-半乳糖苷能预防大脑缺血性损伤，能通过减少Bax的表达和修复促凋亡、抗凋亡蛋白的平衡有效地减少氧化损伤。相对于其他糖基衍生物，其在生物体内有更多的受体，有更高的生物利用度。
本发明开发的生物技术方法生产糖苷类化合物，产物的构型明确，不存在α，β 两种构型的糖苷混合物，产物结构单一，副产物少，纯度高，是开发成为药物使用的重要安全保证。本发明选择添加有机溶剂的非水相体系进行催化，可以克服水相体系高水活度和有机体系糖溶解度较低的问题，与其它生物法制备糖苷类化合物相比具有产量高、时空产率高等优势。该糖苷酶以廉价寡糖为糖基供体，显著降低了糖苷产物的生产成本，反应体系中使用DMSO、DMF 和叔丁醇等绿色无毒性溶剂，也有利于环境保护。多种半乳糖苷产物的高产量制备获得，对于进一步研究开发新型高选择性靶向性药物具有重要的积极意义。
附图说明
图1是gal基因的PCR扩增电泳图；其中M：DNA Marker；1，2：gal基因的PCR扩增产物；
图2是为半乳糖苷酶的聚丙烯酰胺电泳图；其中：(1)空质粒宿主菌E.coliBL21/pET28a(+)的破碎上清对照；(2)重组菌E-pET-gal 诱导表达的破碎上清；(3)纯化后的重组半乳糖苷酶；
图3是实施例4转化液HPLC图谱，N为酪醇，N1为酪醇半乳糖苷，N2为酪醇双糖基化产物；
图4是实施例4质谱分析图谱；
图5是实施例4的13C-NMR图谱；
图6是实施例4的1H-NMR图谱；
图7是实施例4半乳糖苷酶催化酪醇的转糖基化反应的进程曲线；
图8是半乳糖苷酶催化酪醇的转糖基化反应在不同pH下的转化率；
图9是半乳糖苷酶催化酪醇的转糖基化反应在不同温度下的转化率。
具体实施方式
实施例1
本实施例说明本发明的耐有机溶剂高效半乳糖苷酶gal编码基因的分离克隆程序，本发明的半乳糖苷酶gal编码基因从沙雷氏菌属菌株（Serratia marcescens）MH6的总DNA中提取，所述MH6菌株保藏编号为CCTCC NO：M208205，已在申请人在先申请CN101586086B中公开。
采用酚-氯仿法抽提菌体总DNA。通过对全基因组序列分析，Vector NTI 软件分析比对，确定保守区域，获得一个编码半乳糖苷酶的基因，根据该基因核苷酸序列设计引物SF和SR。
SF (SEQ ID NO：3)序列为：CGCCATATGCGAGAAAATTATTATGAAC
SR (SEQ ID NO：4)序列为：GCGAAGCTTTTATTTCATCTCCAAACGAC
其中，引物SF下划线部分为NdeⅠ酶切位点，引物SR下划线部分为Hind Ⅲ酶切位点。以沙雷氏菌属菌株（Serratia marcescens）MH6基因组为模板，进行PCR扩增。PCR体系为：2×Taq Plus Master Mix 25μL，引物SF和SR各2μL，DNA模板2μL和ddH2O 19μL。PCR扩增步骤为：（1）95℃，预变形5min；（2）95℃，变形30S；（3）52℃，退火30S；（4）72℃，延伸2min；步骤（2）～（4）重复30次；（5）72℃彻底延伸10min，冷却至4℃。PCR产物经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收目的条带（图1）。获得一条完整的半乳糖苷酶基因序列，全长1386bp，命名为糖苷酶gal，其碱基序列如序列表中SEQ ID NO:1所示。
实施例2
本实施例说明重组表达载体和重组表达转化体的制备。
将实施例1所得的半乳糖苷酶基因片段在37 ℃用限制性内切酶Hind Ⅲ和NheⅠ双酶切3 h，经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收目标片段。将目标片段在T4 DNA连接酶的作用下，与同样经Hind Ⅲ和NheⅠ酶切后的质粒pET28a，在16 ℃下过夜连接得到重组表达质粒pET-gal。
将上述重组表达质粒转化到大肠埃希氏菌(E.coli)BL21(DE3)感受态细胞中，在含有卡那霉素的抗性平板上对阳性重组体进行筛选，挑选单克隆，菌落PCR验证阳性克隆，即获得阳性重组转化体大肠埃希氏菌(E.coli) BL21 (DE3)/pET-gal（以下简称E-pET-gal）。
实施例3
本实施例说明重组半乳糖苷酶gal诱导表达与纯化过程。
将重组菌 E-pET-gal 接种到LB/Kana 液体培养基中过夜培养，以2% (v/v)的接种量接种至新鲜培养基中，培养至OD600 到0.6-0.8 时，加入诱导剂0.5 mM异丙基硫代-β-D-半乳糖苷(IPTG)，37℃，180 r·min-1 诱导表达4 h。诱导后的发酵液在4℃，8000 r·min-1 条件下离心10 min，菌体细胞用等体积缓冲液(50 mM 的Na2HPO4-KH2PO4， pH 7.0)悬浮，经生理盐水洗涤两遍后用pH7.5-8.5的PBS缓冲重悬细胞，获得静息细胞液在冰浴中超声破碎(300 W；超声3 s，间歇 5 s，总时间5 min)，4℃，12000 r·min-1 离心15 min 去除细胞碎片，得到重组半乳糖苷酶的粗酶液。粗酶液用Ni-NTA Agarose亲和柱（GeneralElectric Company）层析除去杂蛋白，得到纯化后的半乳糖苷酶。得到的纯化后的重组糖苷酶gal从图2可以看到。
实施例4
本实施例说明本发明半乳糖苷酶gal在合成酪醇半乳糖苷中的应用
催化合成酪醇半乳糖苷的反应条件：在15% (v/v) DMSO体系中，pH 7.5 磷酸缓冲(50mMKH2PO4-Na2HPO4)，乳糖200 g·L-1，酶用量2.4 U·mL-1，反应温度50℃。50 g·L-1 酪醇的条件下可获得约33.1 g·L-1 的酪醇单半乳糖苷，占总糖苷产物的91%，时空产率约2.07g/L/h。在本优选的条件下，将酪醇转化液经HPLC、LC-MS、13C-NMR和1H-NMR鉴定，确认产物为酪醇半乳糖苷。检测图谱如图3-6所示。该反应的反应进程曲线如图7所示。
实施例5
本实施例说明本发明半乳糖苷酶gal在合成对羟基苯甲醇半乳糖苷中的应用
催化合成对羟基苯甲醇半乳糖苷的反应条件：25% (v/v)DMSO体系中，pH 7.5 磷酸缓冲(50mMKH2PO4-Na2HPO4)，乳糖200 g·L-1，酶用量2.4 U·mL-1，反应温度50℃。55 g·L-1对羟基苯甲醇的条件下可获得约40.5 g·L-1 的对羟基苯甲醇单半乳糖苷，占总糖苷产物的相对含量99%，时空产率约2.53 g/L/h。
实施例6
本实施例说明本发明半乳糖苷酶gal在合成对甲氧基苯甲醇半乳糖苷中的应用
催化合成甲氧基苯甲醇半乳糖苷的反应条件：10% (v/v)DMSO体系中，pH 7.5 磷酸缓冲(50mMKH2PO4-Na2HPO4)，乳糖200 g·L-1，酶用量2.4 U·mL-1，反应温度50℃。30g·L-1甲氧基苯甲醇的条件下可获得约26.2 g·L-1 的甲氧基苯甲醇半乳糖苷，占总糖苷产物的相对含量95%，时空产率约1.65 g/L/h。
实施例7
本实施例说明本发明半乳糖苷酶gal在合成肉桂醇半乳糖苷中的应用
催化合成肉桂醇半乳糖苷的反应条件：10% (v/v)DMSO体系中，pH 7.5 磷酸缓冲(50mMKH2PO4-Na2HPO4)，乳糖200 g·L-1，酶用量2.4 U·mL-1，反应温度50℃。55 g·L-1肉桂醇的条件下可获得约9.82 g·L-1 的肉桂醇半乳糖苷，占总糖苷产物的相对含量90%，时空产率约0.62g/L/h。
实施例8
本实施例说明本发明半乳糖苷酶gal在合成水杨醇半乳糖苷中的应用
催化合成水杨醇半乳糖苷的反应条件：10% (v/v)DMSO体系中，pH 7.5 磷酸缓冲(50mMKH2PO4-Na2HPO4)，乳糖200 g·L-1，酶用量2.4 U·mL-1，反应温度50℃。60 g·L-1水杨醇的条件下可获得约39.75 g·L-1 的水杨醇半乳糖苷，占总糖苷产物的相对含量87%，时空产率约2.51g/L/h。
实施例9
本实施例说明本发明半乳糖苷酶gal在不同pH下合成酪醇半乳糖苷中的应用
催化合成酪醇半乳糖苷的反应条件：在15% (v/v) DMSO体系中，pH不同的缓冲溶液中，乳糖100 g·L-1，酪醇用量10 g·L-1，酶用量2.4 U·mL-1，反应温度50℃。结果如附图8所示，最适pH为7.5，产率在26.1%。
实施例10
本实施例说明本发明半乳糖苷酶gal在不同温度下合成酪醇半乳糖苷中的应用
催化合成酪醇半乳糖苷的反应条件：在15% (v/v) DMSO体系中，pH7.5的磷酸缓冲，乳糖100 g·L-1，酪醇用量10 g•L-1，酶用量2.4 U·mL-1，反应温度20~65℃。结果如附图9所示，最适温度为50℃，产率在27%。
实施例11
本实施例说明本发明半乳糖苷酶gal在不同种类、浓度有机溶剂合成酪醇半乳糖苷中的应用
催化合成酪醇半乳糖苷的反应条件：在DMSO、DMF、叔丁醇、丙酮体系中，pH 7.5 磷酸缓冲(50mMKH2PO4-Na2HPO4)，乳糖250 g·L-1，酪醇用量25g•L-1酶用量2.4 U·mL-1，反应温度50℃。结果如下表1所示，表1中N1为酪醇半乳糖苷。
表1半乳糖苷酶催化酪醇的转糖基化反应在不同浓度不同有机溶剂下的转化率
| 溶剂系统（v/v） | 产物N1占总糖苷产物的相对含量 | 总转化率 |
| 水 | 78.40% | 32.31% |
| 5%叔丁醇 | 88.10% | 32.54% |
| 10%叔丁醇 | 90.36% | 28.92% |
| 15%叔丁醇 | 91.23% | 26.05% |
| 20%叔丁醇 | 93.45% | 24.47% |
| 25%叔丁醇 | 100% | 16.11% |
| 5%丙酮 | 85.40% | 32.06% |
| 10%丙酮 | 90.21% | 27.20% |
| 15%丙酮 | 94.57% | 19.78% |
| 20%丙酮 | 98.87% | 18.70% |
| 5%DMSO | 85.42% | 33.31% |
| 10%DMSO | 88.73% | 31.02% |
| 15%DMSO | 91.23% | 29.63% |
| 20%DMSO | 92.56% | 24.93% |
| 25%DMSO | 93.70% | 21.21% |
| 30%DMSO | 95.48% | 17.93% |
| 5%DMF | 86.05% | 32.65% |
| 10%DMF | 88.17% | 29.56% |
| 15%DMF | 93.86% | 24.87% |
| 20%DMF | 98.03% | 14.33% |
序列表
<110> 南京工业大学
<120> 一种耐有机溶剂高效半乳糖苷酶及其应用
<130> xb18030903
<141> 2018-03-09
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1386
<212> DNA
<213> Artificial Sequence
<220>
<221> CDS
<222> (1)..(1386)
<400> 1
atg gaa tat caa ttt gcc gac ggg ttc tgg tgg ggc agc gcc acc tcc 48
Met Glu Tyr Gln Phe Ala Asp Gly Phe Trp Trp Gly Ser Ala Thr Ser
1 5 10 15
gcg ccg cag tct gaa ggg gcg gcc gcg cgg gac ggc aag agt cgc aac 96
Ala Pro Gln Ser Glu Gly Ala Ala Ala Arg Asp Gly Lys Ser Arg Asn
20 25 30
atc ttc gac tat tgg tac gag atc gcg ccc gag cgc ttt cac gga cgg 144
Ile Phe Asp Tyr Trp Tyr Glu Ile Ala Pro Glu Arg Phe His Gly Arg
35 40 45
gtg ggg ccg act gag gcc tcg acc ttt tat gac cat ttc cgc acc gat 192
Val Gly Pro Thr Glu Ala Ser Thr Phe Tyr Asp His Phe Arg Thr Asp
50 55 60
atc ggg ctg ctc aaa acg ctg ggc cat aat act ttc aga acc tcg atc 240
Ile Gly Leu Leu Lys Thr Leu Gly His Asn Thr Phe Arg Thr Ser Ile
65 70 75 80
tcg tgg tcg cgg ctg atc ccg gac ggc gac ggt gaa gtg aac ccg cag 288
Ser Trp Ser Arg Leu Ile Pro Asp Gly Asp Gly Glu Val Asn Pro Gln
85 90 95
gcg gtg gcg ttc tat aac gcg atg atc gac gag ctg ctg gcg cag ggc 336
Ala Val Ala Phe Tyr Asn Ala Met Ile Asp Glu Leu Leu Ala Gln Gly
100 105 110
atc acc ccg ttt atc aat ctc tat cac ttc gat atg ccg ctg tgc atg 384
Ile Thr Pro Phe Ile Asn Leu Tyr His Phe Asp Met Pro Leu Cys Met
115 120 125
caa cag cgc ggc ggt tgg gaa agc cgg gcg gtg gtc gaa gcc tat gcg 432
Gln Gln Arg Gly Gly Trp Glu Ser Arg Ala Val Val Glu Ala Tyr Ala
130 135 140
cgt tac gcc gat acc tgt ttc ggc ctg ttt ggc gat cgg gtc acc cac 480
Arg Tyr Ala Asp Thr Cys Phe Gly Leu Phe Gly Asp Arg Val Thr His
145 150 155 160
tgg ttc acc ttc aac gag ccg atc gtg ccg gtg gaa gcc ggt tac ctg 528
Trp Phe Thr Phe Asn Glu Pro Ile Val Pro Val Glu Ala Gly Tyr Leu
165 170 175
aat gac ctg cac tac cct tgc gtg gtg gac ttc aaa cgg gcg gtc acc 576
Asn Asp Leu His Tyr Pro Cys Val Val Asp Phe Lys Arg Ala Val Thr
180 185 190
gtg gcg tat cac agc gtg ctg gcg cac gcc atg gcg gtg cgg cgt ttc 624
Val Ala Tyr His Ser Val Leu Ala His Ala Met Ala Val Arg Arg Phe
195 200 205
aga gcg cgg agg ctg ccg ggg agc atc ggc atc att ctg aat ctg agc 672
Arg Ala Arg Arg Leu Pro Gly Ser Ile Gly Ile Ile Leu Asn Leu Ser
210 215 220
ccg acc tat ccg cgc tcc gac gcg ccg gag gat cgg cag gcc gca cac 720
Pro Thr Tyr Pro Arg Ser Asp Ala Pro Glu Asp Arg Gln Ala Ala His
225 230 235 240
gac gcc gat ctg ctg ctc aac cgc agt ttc ctc gac ccg gtc gcc aaa 768
Asp Ala Asp Leu Leu Leu Asn Arg Ser Phe Leu Asp Pro Val Ala Lys
245 250 255
ggg cgt tac ccg gcc gca ttg ctg cac ctg ttg gag cga cat ggg ctg 816
Gly Arg Tyr Pro Ala Ala Leu Leu His Leu Leu Glu Arg His Gly Leu
260 265 270
atg ccc tat tgc gaa ccg cag gac gcg cag ctg atc gaa ggc ggc gtg 864
Met Pro Tyr Cys Glu Pro Gln Asp Ala Gln Leu Ile Glu Gly Gly Val
275 280 285
gtg gat atc ctt ggc gtc aac tat tac cag ccg cgc agg gtg cag gcg 912
Val Asp Ile Leu Gly Val Asn Tyr Tyr Gln Pro Arg Arg Val Gln Ala
290 295 300
aaa gcg gga cgg cgc gcc gag ggg ccg atc gca tcg ccg gag gat ttg 960
Lys Ala Gly Arg Arg Ala Glu Gly Pro Ile Ala Ser Pro Glu Asp Leu
305 310 315 320
ttc agt tac tat gca atg ccg ggc cgc aaa atc aat ccg cac cgc ggc 1008
Phe Ser Tyr Tyr Ala Met Pro Gly Arg Lys Ile Asn Pro His Arg Gly
325 330 335
tgg gag atc tac gag aag ggg ctg tac gac atc ctg atg gat ctg aaa 1056
Trp Glu Ile Tyr Glu Lys Gly Leu Tyr Asp Ile Leu Met Asp Leu Lys
340 345 350
gag aac tac ggc aat ctg ccc tgt tat att tcg gaa aat ggc atg ggc 1104
Glu Asn Tyr Gly Asn Leu Pro Cys Tyr Ile Ser Glu Asn Gly Met Gly
355 360 365
gtc gag ggc gaa gag gcg ttt atc ggc gcc gat ggg cgg gtg gag gac 1152
Val Glu Gly Glu Glu Ala Phe Ile Gly Ala Asp Gly Arg Val Glu Asp
370 375 380
gat tac cgc atc gac ttt att cgc gag cac ctg aag tgg ctg cac cgc 1200
Asp Tyr Arg Ile Asp Phe Ile Arg Glu His Leu Lys Trp Leu His Arg
385 390 395 400
gcg ctg gcg gaa ggc tcg cag tgc aaa ggc tat cat ctg tgg acc ttt 1248
Ala Leu Ala Glu Gly Ser Gln Cys Lys Gly Tyr His Leu Trp Thr Phe
405 410 415
atc gac tgc tgg tcc tgg ctg aac gcc tac aag aat cgt tat ggg ctg 1296
Ile Asp Cys Trp Ser Trp Leu Asn Ala Tyr Lys Asn Arg Tyr Gly Leu
420 425 430
gtg cgg ctg gat cgg gcg gat cag cgg cgc acc att aaa aaa agc ggt 1344
Val Arg Leu Asp Arg Ala Asp Gln Arg Arg Thr Ile Lys Lys Ser Gly
435 440 445
tac tgg ttt gcc gag gcg gcc aga cgc aac gga ttt gac taa 1386
Tyr Trp Phe Ala Glu Ala Ala Arg Arg Asn Gly Phe Asp
450 455 460
<210> 2
<211> 461
<212> PRT
<213> Artificial Sequence
<400> 2
Met Glu Tyr Gln Phe Ala Asp Gly Phe Trp Trp Gly Ser Ala Thr Ser
1 5 10 15
Ala Pro Gln Ser Glu Gly Ala Ala Ala Arg Asp Gly Lys Ser Arg Asn
20 25 30
Ile Phe Asp Tyr Trp Tyr Glu Ile Ala Pro Glu Arg Phe His Gly Arg
35 40 45
Val Gly Pro Thr Glu Ala Ser Thr Phe Tyr Asp His Phe Arg Thr Asp
50 55 60
Ile Gly Leu Leu Lys Thr Leu Gly His Asn Thr Phe Arg Thr Ser Ile
65 70 75 80
Ser Trp Ser Arg Leu Ile Pro Asp Gly Asp Gly Glu Val Asn Pro Gln
85 90 95
Ala Val Ala Phe Tyr Asn Ala Met Ile Asp Glu Leu Leu Ala Gln Gly
100 105 110
Ile Thr Pro Phe Ile Asn Leu Tyr His Phe Asp Met Pro Leu Cys Met
115 120 125
Gln Gln Arg Gly Gly Trp Glu Ser Arg Ala Val Val Glu Ala Tyr Ala
130 135 140
Arg Tyr Ala Asp Thr Cys Phe Gly Leu Phe Gly Asp Arg Val Thr His
145 150 155 160
Trp Phe Thr Phe Asn Glu Pro Ile Val Pro Val Glu Ala Gly Tyr Leu
165 170 175
Asn Asp Leu His Tyr Pro Cys Val Val Asp Phe Lys Arg Ala Val Thr
180 185 190
Val Ala Tyr His Ser Val Leu Ala His Ala Met Ala Val Arg Arg Phe
195 200 205
Arg Ala Arg Arg Leu Pro Gly Ser Ile Gly Ile Ile Leu Asn Leu Ser
210 215 220
Pro Thr Tyr Pro Arg Ser Asp Ala Pro Glu Asp Arg Gln Ala Ala His
225 230 235 240
Asp Ala Asp Leu Leu Leu Asn Arg Ser Phe Leu Asp Pro Val Ala Lys
245 250 255
Gly Arg Tyr Pro Ala Ala Leu Leu His Leu Leu Glu Arg His Gly Leu
260 265 270
Met Pro Tyr Cys Glu Pro Gln Asp Ala Gln Leu Ile Glu Gly Gly Val
275 280 285
Val Asp Ile Leu Gly Val Asn Tyr Tyr Gln Pro Arg Arg Val Gln Ala
290 295 300
Lys Ala Gly Arg Arg Ala Glu Gly Pro Ile Ala Ser Pro Glu Asp Leu
305 310 315 320
Phe Ser Tyr Tyr Ala Met Pro Gly Arg Lys Ile Asn Pro His Arg Gly
325 330 335
Trp Glu Ile Tyr Glu Lys Gly Leu Tyr Asp Ile Leu Met Asp Leu Lys
340 345 350
Glu Asn Tyr Gly Asn Leu Pro Cys Tyr Ile Ser Glu Asn Gly Met Gly
355 360 365
Val Glu Gly Glu Glu Ala Phe Ile Gly Ala Asp Gly Arg Val Glu Asp
370 375 380
Asp Tyr Arg Ile Asp Phe Ile Arg Glu His Leu Lys Trp Leu His Arg
385 390 395 400
Ala Leu Ala Glu Gly Ser Gln Cys Lys Gly Tyr His Leu Trp Thr Phe
405 410 415
Ile Asp Cys Trp Ser Trp Leu Asn Ala Tyr Lys Asn Arg Tyr Gly Leu
420 425 430
Val Arg Leu Asp Arg Ala Asp Gln Arg Arg Thr Ile Lys Lys Ser Gly
435 440 445
Tyr Trp Phe Ala Glu Ala Ala Arg Arg Asn Gly Phe Asp
450 455 460
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 3
cgccatatgc gagaaaatta ttatgaac 28
<210> 4
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 4
gcgaagcttt tatttcatct ccaaacgac 29
Claims (10)
1.一种耐有机溶剂半乳糖苷酶，其氨基酸序列如SEQ ID NO：2所示。
2.编码权利要求1所述半乳糖苷酶的基因。
3.编码权利要求1所述半乳糖苷酶的基因，其核苷酸序列如SEQ ID NO：1所示。
4.包含权利要求3所述核苷酸序列的重组表达载体。
5.包含权利要求4所述重组表达载体的重组表达转化体。
6.权利要求1所述半乳糖苷酶在非水相中生物催化转糖基化反应合成红景天苷类似物中的应用。
7.根据权利要求6所述应用，其特征在于，将半乳糖苷酶、乳糖、醇类在非水相体系中反应，分离获取红景天苷类似物产物；所述非水相体系为含有磷酸缓冲的有机溶剂。
8.根据权利要求7所述应用，其特征在于，所述非水相体系为含有磷酸缓冲的DMSO、DMF、丙酮或者叔丁醇溶液。
9.根据权利要求7所述应用，其特征在于，所述醇类为酪醇、水杨醇、对羟基苯甲醇、对甲氧基苯甲醇或肉桂醇。
10.根据权利要求7-9任一项所述的应用，其特征在于，所述的非水相体系为5%~30%（v/v）DMSO体系、5%~25%叔丁醇体系、5%~20%丙酮体系或5%~20%DMF体系；
醇类浓度为10g·L-1 -60 g·L-1，乳糖浓度为100~250 g·L-1，反应温度为20~65℃，反应pH为5~11。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810196588.0A CN108148824B (zh) | 2018-03-09 | 2018-03-09 | 一种耐有机溶剂高效半乳糖苷酶及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810196588.0A CN108148824B (zh) | 2018-03-09 | 2018-03-09 | 一种耐有机溶剂高效半乳糖苷酶及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108148824A true CN108148824A (zh) | 2018-06-12 |
| CN108148824B CN108148824B (zh) | 2022-07-29 |
Family
ID=62456142
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810196588.0A Active CN108148824B (zh) | 2018-03-09 | 2018-03-09 | 一种耐有机溶剂高效半乳糖苷酶及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108148824B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778928A (zh) * | 2005-10-11 | 2006-05-31 | 山东大学 | 一种转糖基α一半乳糖苷酶基因 |
| WO2006138005A2 (en) * | 2005-05-10 | 2006-12-28 | Monsanto Technology, Llc | Genes and uses for plant improvement |
| CN101074435A (zh) * | 2006-05-16 | 2007-11-21 | 中国农业科学院饲料研究所 | α－半乳糖苷酶基因、其编码蛋白及其制备方法和应用 |
-
2018
- 2018-03-09 CN CN201810196588.0A patent/CN108148824B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006138005A2 (en) * | 2005-05-10 | 2006-12-28 | Monsanto Technology, Llc | Genes and uses for plant improvement |
| CN1778928A (zh) * | 2005-10-11 | 2006-05-31 | 山东大学 | 一种转糖基α一半乳糖苷酶基因 |
| CN101074435A (zh) * | 2006-05-16 | 2007-11-21 | 中国农业科学院饲料研究所 | α－半乳糖苷酶基因、其编码蛋白及其制备方法和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108148824B (zh) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3979678B2 (ja) | 新規糖転移酵素及びそれをコードする遺伝子並びに該酵素の製造方法 | |
| Kurosawa et al. | Cloning and expression of Gal beta 1, 3GalNAc-specific GalNAc alpha 2, 6-sialyltransferase. | |
| Miyagi et al. | Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase. | |
| CN109750011B (zh) | 一种甘露糖6-磷酸磷酸酶及甘露糖生物制备方法 | |
| Ferrari et al. | Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases | |
| AU2016326518A1 (en) | Cannabinoid glycoside prodrugs and methods of synthesis | |
| KR100387303B1 (ko) | 비환원성 당질로부터 트레할로오스를 유리하는 재조합 열안정성 효소 | |
| CN105087739B (zh) | 一种新的制备稀有人参皂苷的催化体系及其应用 | |
| US7879586B2 (en) | Gene encoding methylated catechin synthase | |
| KR100427529B1 (ko) | 말토오스를 트레할로오스로 변환하는 재조합형 열안정성 효소 | |
| CN108753748B (zh) | 大黄素糖基转移酶蛋白FtUGT75R2及其编码基因与应用 | |
| KR100374449B1 (ko) | 효소를인코우드하는디엔에이(dna),재조합디엔에이(dna)와효소,형질전환체및이들의제조방법과용도 | |
| CN113151211A (zh) | 一种α-1,3-岩藻糖基转移酶突变体及利用该突变体制备3-岩藻糖基乳糖的应用 | |
| DE60035801T2 (de) | Bakterielle stämme von genus klebsiella und deren gen kodierend für isomaltulose synthase | |
| Suh et al. | Cloning, sequencing and overexpression of cob A which encodes ATP: corrinoid adenosyltranferase in Salmonella typhimurium | |
| KR100374448B1 (ko) | 효소를인코우드하는디엔에이(dna),재조합디엔에이(dna)와효소,형질전환체및이들의제조방법과용도 | |
| KR101048148B1 (ko) | 데이노코커스 제오써르말리스로부터 유래한 아밀로수크라아제를 이용한 알파 알부틴의 제조방법 | |
| CN108148824A (zh) | 一种耐有机溶剂高效半乳糖苷酶及其应用 | |
| JP3030431B2 (ja) | キチン脱アセチル化酵素遺伝子、該遺伝子を含むベクター及び形質転換体 | |
| KR100914525B1 (ko) | 신규 ｎ―아세틸글루코사민―２―에피머라아제 및 이를 이용한 ｃｍｐ―ｎ―아세틸뉴라민산의 제조방법 | |
| KR100387302B1 (ko) | 말토오스를 트레할로오스로 변환하는 재조합형 효소 | |
| CN114277024B (zh) | 一种新型三萜合酶及其应用 | |
| KR102084065B1 (ko) | 써모토가 마리티마 유래의 내열성 재조합 셀룰라아제 b 단백질 및 이의 용도 | |
| CN106906192B (zh) | 一种葡萄糖基转移酶及在合成藏红花酸葡萄糖酯中的应用 | |
| CN112626047A (zh) | 一组亚精胺衍生物糖基转移酶及其编码基因和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |