CN108913641B - 一种重组大肠杆菌及其应用 - Google Patents
一种重组大肠杆菌及其应用 Download PDFInfo
- Publication number
- CN108913641B CN108913641B CN201810696239.5A CN201810696239A CN108913641B CN 108913641 B CN108913641 B CN 108913641B CN 201810696239 A CN201810696239 A CN 201810696239A CN 108913641 B CN108913641 B CN 108913641B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- escherichia coli
- ascorbic acid
- recombinant escherichia
- final concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 35
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 55
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 33
- 239000002211 L-ascorbic acid Substances 0.000 claims abstract description 32
- 235000000069 L-ascorbic acid Nutrition 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 239000000758 substrate Substances 0.000 claims abstract description 15
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 12
- 229930006000 Sucrose Natural products 0.000 claims abstract description 12
- 241001608472 Bifidobacterium longum Species 0.000 claims abstract description 11
- 229940009291 bifidobacterium longum Drugs 0.000 claims abstract description 11
- 239000005720 sucrose Substances 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims description 50
- 230000004151 fermentation Effects 0.000 claims description 50
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 239000008367 deionised water Substances 0.000 claims description 16
- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- 239000003054 catalyst Substances 0.000 claims description 13
- 235000011187 glycerol Nutrition 0.000 claims description 12
- 229930027917 kanamycin Natural products 0.000 claims description 11
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 11
- 229960000318 kanamycin Drugs 0.000 claims description 11
- 229930182823 kanamycin A Natural products 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 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 description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 239000007832 Na2SO4 Substances 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 229930195727 α-lactose Natural products 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- 230000003698 anagen phase Effects 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 3
- 239000002518 antifoaming agent Substances 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000013613 expression plasmid Substances 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 238000012136 culture method Methods 0.000 claims 1
- MLSJBGYKDYSOAE-DCWMUDTNSA-N L-Ascorbic acid-2-glucoside Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)=C1O MLSJBGYKDYSOAE-DCWMUDTNSA-N 0.000 abstract description 26
- 230000003197 catalytic effect Effects 0.000 abstract description 6
- 238000006206 glycosylation reaction Methods 0.000 abstract description 4
- 108020000005 Sucrose phosphorylase Proteins 0.000 abstract description 3
- 238000010170 biological method Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000013595 glycosylation Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 10
- 229960004793 sucrose Drugs 0.000 description 9
- 238000001514 detection method Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 108010047857 aspartylglycine Proteins 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- OQCWXQJLCDPRHV-UWVGGRQHSA-N Arg-Gly-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O OQCWXQJLCDPRHV-UWVGGRQHSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 2
- VTIAEOKFUJJBTC-YDHLFZDLSA-N Val-Tyr-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VTIAEOKFUJJBTC-YDHLFZDLSA-N 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 108010064235 lysylglycine Proteins 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- IMIZPWSVYADSCN-UHFFFAOYSA-N 4-methyl-2-[[4-methyl-2-[[4-methyl-2-(pyrrolidine-2-carbonylamino)pentanoyl]amino]pentanoyl]amino]pentanoic acid Chemical compound CC(C)CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C1CCCN1 IMIZPWSVYADSCN-UHFFFAOYSA-N 0.000 description 1
- 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 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- XCVRVWZTXPCYJT-BIIVOSGPSA-N Ala-Asn-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N XCVRVWZTXPCYJT-BIIVOSGPSA-N 0.000 description 1
- GORKKVHIBWAQHM-GCJQMDKQSA-N Ala-Asn-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GORKKVHIBWAQHM-GCJQMDKQSA-N 0.000 description 1
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-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
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 1
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- NHWYNIZWLJYZAG-XVYDVKMFSA-N Ala-Ser-His Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N NHWYNIZWLJYZAG-XVYDVKMFSA-N 0.000 description 1
- MMLHRUJLOUSRJX-CIUDSAMLSA-N Ala-Ser-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN MMLHRUJLOUSRJX-CIUDSAMLSA-N 0.000 description 1
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 1
- ZCUFMRIQCPNOHZ-NRPADANISA-N Ala-Val-Gln Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N ZCUFMRIQCPNOHZ-NRPADANISA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- 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 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- YBZMTKUDWXZLIX-UWVGGRQHSA-N Arg-Leu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YBZMTKUDWXZLIX-UWVGGRQHSA-N 0.000 description 1
- FKQITMVNILRUCQ-IHRRRGAJSA-N Arg-Phe-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O FKQITMVNILRUCQ-IHRRRGAJSA-N 0.000 description 1
- HGKHPCFTRQDHCU-IUCAKERBSA-N Arg-Pro-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O HGKHPCFTRQDHCU-IUCAKERBSA-N 0.000 description 1
- VUGWHBXPMAHEGZ-SRVKXCTJSA-N Arg-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N VUGWHBXPMAHEGZ-SRVKXCTJSA-N 0.000 description 1
- SYFHFLGAROUHNT-VEVYYDQMSA-N Arg-Thr-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O SYFHFLGAROUHNT-VEVYYDQMSA-N 0.000 description 1
- ANAHQDPQQBDOBM-UHFFFAOYSA-N Arg-Val-Tyr Natural products CC(C)C(NC(=O)C(N)CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O ANAHQDPQQBDOBM-UHFFFAOYSA-N 0.000 description 1
- DDPXDCKYWDGZAL-BQBZGAKWSA-N Asn-Gly-Arg Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N DDPXDCKYWDGZAL-BQBZGAKWSA-N 0.000 description 1
- SXNJBDYEBOUYOJ-DCAQKATOSA-N Asn-His-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)N)N SXNJBDYEBOUYOJ-DCAQKATOSA-N 0.000 description 1
- BXUHCIXDSWRSBS-CIUDSAMLSA-N Asn-Leu-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BXUHCIXDSWRSBS-CIUDSAMLSA-N 0.000 description 1
- NCFJQJRLQJEECD-NHCYSSNCSA-N Asn-Leu-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O NCFJQJRLQJEECD-NHCYSSNCSA-N 0.000 description 1
- UXHYOWXTJLBEPG-GSSVUCPTSA-N Asn-Thr-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UXHYOWXTJLBEPG-GSSVUCPTSA-N 0.000 description 1
- 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 1
- SVABRQFIHCSNCI-FOHZUACHSA-N Asp-Gly-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SVABRQFIHCSNCI-FOHZUACHSA-N 0.000 description 1
- TZOZNVLBTAFJRW-UGYAYLCHSA-N Asp-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](CC(=O)O)N TZOZNVLBTAFJRW-UGYAYLCHSA-N 0.000 description 1
- UJGRZQYSNYTCAX-SRVKXCTJSA-N Asp-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UJGRZQYSNYTCAX-SRVKXCTJSA-N 0.000 description 1
- JXGJJQJHXHXJQF-CIUDSAMLSA-N Asp-Met-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O JXGJJQJHXHXJQF-CIUDSAMLSA-N 0.000 description 1
- IWLZBRTUIVXZJD-OLHMAJIHSA-N Asp-Thr-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O IWLZBRTUIVXZJD-OLHMAJIHSA-N 0.000 description 1
- WAEDSQFVZJUHLI-BYULHYEWSA-N Asp-Val-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O WAEDSQFVZJUHLI-BYULHYEWSA-N 0.000 description 1
- 241000873544 Bifidobacterium longum subsp. longum JCM 1217 Species 0.000 description 1
- 108010025880 Cyclomaltodextrin glucanotransferase Proteins 0.000 description 1
- DEVDFMRWZASYOF-ZLUOBGJFSA-N Cys-Asn-Asp Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O DEVDFMRWZASYOF-ZLUOBGJFSA-N 0.000 description 1
- BSGXXYRIDXUEOM-IHRRRGAJSA-N Cys-Phe-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CS)N BSGXXYRIDXUEOM-IHRRRGAJSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- IXFVOPOHSRKJNG-LAEOZQHASA-N Gln-Asp-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IXFVOPOHSRKJNG-LAEOZQHASA-N 0.000 description 1
- OOLCSQQPSLIETN-JYJNAYRXSA-N Gln-His-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CCC(=O)N)N)O OOLCSQQPSLIETN-JYJNAYRXSA-N 0.000 description 1
- QBLMTCRYYTVUQY-GUBZILKMSA-N Gln-Leu-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QBLMTCRYYTVUQY-GUBZILKMSA-N 0.000 description 1
- KLKYKPXITJBSNI-CIUDSAMLSA-N Gln-Met-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O KLKYKPXITJBSNI-CIUDSAMLSA-N 0.000 description 1
- ZOXBSICWUDAOHX-GUBZILKMSA-N Glu-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O ZOXBSICWUDAOHX-GUBZILKMSA-N 0.000 description 1
- NKLRYVLERDYDBI-FXQIFTODSA-N Glu-Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKLRYVLERDYDBI-FXQIFTODSA-N 0.000 description 1
- HILMIYALTUQTRC-XVKPBYJWSA-N Glu-Gly-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HILMIYALTUQTRC-XVKPBYJWSA-N 0.000 description 1
- QIQABBIDHGQXGA-ZPFDUUQYSA-N Glu-Ile-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QIQABBIDHGQXGA-ZPFDUUQYSA-N 0.000 description 1
- LGYCLOCORAEQSZ-PEFMBERDSA-N Glu-Ile-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O LGYCLOCORAEQSZ-PEFMBERDSA-N 0.000 description 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
- FGSGPLRPQCZBSQ-AVGNSLFASA-N Glu-Phe-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O FGSGPLRPQCZBSQ-AVGNSLFASA-N 0.000 description 1
- CQAHWYDHKUWYIX-YUMQZZPRSA-N Glu-Pro-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O CQAHWYDHKUWYIX-YUMQZZPRSA-N 0.000 description 1
- 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 1
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 1
- XUDLUKYPXQDCRX-BQBZGAKWSA-N Gly-Arg-Asn Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O XUDLUKYPXQDCRX-BQBZGAKWSA-N 0.000 description 1
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 1
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 1
- GAFKBWKVXNERFA-QWRGUYRKSA-N Gly-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 GAFKBWKVXNERFA-QWRGUYRKSA-N 0.000 description 1
- OHUKZZYSJBKFRR-WHFBIAKZSA-N Gly-Ser-Asp Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O OHUKZZYSJBKFRR-WHFBIAKZSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 1
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 1
- NIOPEYHPOBWLQO-KBPBESRZSA-N Gly-Trp-Glu Chemical compound NCC(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(O)=O NIOPEYHPOBWLQO-KBPBESRZSA-N 0.000 description 1
- FULZDMOZUZKGQU-ONGXEEELSA-N Gly-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)CN FULZDMOZUZKGQU-ONGXEEELSA-N 0.000 description 1
- LSQHWKPPOFDHHZ-YUMQZZPRSA-N His-Asp-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N LSQHWKPPOFDHHZ-YUMQZZPRSA-N 0.000 description 1
- PQKCQZHAGILVIM-NKIYYHGXSA-N His-Glu-Thr Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)Cc1cnc[nH]1)C(O)=O PQKCQZHAGILVIM-NKIYYHGXSA-N 0.000 description 1
- HAPWZEVRQYGLSG-IUCAKERBSA-N His-Gly-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O HAPWZEVRQYGLSG-IUCAKERBSA-N 0.000 description 1
- PZAJPILZRFPYJJ-SRVKXCTJSA-N His-Ser-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O PZAJPILZRFPYJJ-SRVKXCTJSA-N 0.000 description 1
- FFKJUTZARGRVTH-KKUMJFAQSA-N His-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FFKJUTZARGRVTH-KKUMJFAQSA-N 0.000 description 1
- JUCZDDVZBMPKRT-IXOXFDKPSA-N His-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O JUCZDDVZBMPKRT-IXOXFDKPSA-N 0.000 description 1
- YERBCFWVWITTEJ-NAZCDGGXSA-N His-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC3=CN=CN3)N)O YERBCFWVWITTEJ-NAZCDGGXSA-N 0.000 description 1
- HIJIJPFILYPTFR-ACRUOGEOSA-N His-Tyr-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)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 HIJIJPFILYPTFR-ACRUOGEOSA-N 0.000 description 1
- SYPULFZAGBBIOM-GVXVVHGQSA-N His-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N SYPULFZAGBBIOM-GVXVVHGQSA-N 0.000 description 1
- WECYRWOMWSCWNX-XUXIUFHCSA-N Ile-Arg-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(O)=O WECYRWOMWSCWNX-XUXIUFHCSA-N 0.000 description 1
- DMHGKBGOUAJRHU-UHFFFAOYSA-N Ile-Arg-Pro Natural products CCC(C)C(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O DMHGKBGOUAJRHU-UHFFFAOYSA-N 0.000 description 1
- YKRIXHPEIZUDDY-GMOBBJLQSA-N Ile-Asn-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKRIXHPEIZUDDY-GMOBBJLQSA-N 0.000 description 1
- BGZIJZJBXRVBGJ-SXTJYALSSA-N Ile-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N BGZIJZJBXRVBGJ-SXTJYALSSA-N 0.000 description 1
- WUKLZPHVWAMZQV-UKJIMTQDSA-N Ile-Glu-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N WUKLZPHVWAMZQV-UKJIMTQDSA-N 0.000 description 1
- VOBYAKCXGQQFLR-LSJOCFKGSA-N Ile-Gly-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O VOBYAKCXGQQFLR-LSJOCFKGSA-N 0.000 description 1
- PMMMQRVUMVURGJ-XUXIUFHCSA-N Ile-Leu-Pro Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O PMMMQRVUMVURGJ-XUXIUFHCSA-N 0.000 description 1
- 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 1
- JHNJNTMTZHEDLJ-NAKRPEOUSA-N Ile-Ser-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O JHNJNTMTZHEDLJ-NAKRPEOUSA-N 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- 150000000996 L-ascorbic acids Chemical class 0.000 description 1
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 1
- HXWALXSAVBLTPK-NUTKFTJISA-N Leu-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(C)C)N HXWALXSAVBLTPK-NUTKFTJISA-N 0.000 description 1
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 description 1
- STAVRDQLZOTNKJ-RHYQMDGZSA-N Leu-Arg-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STAVRDQLZOTNKJ-RHYQMDGZSA-N 0.000 description 1
- 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 1
- CLVUXCBGKUECIT-HJGDQZAQSA-N Leu-Asp-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CLVUXCBGKUECIT-HJGDQZAQSA-N 0.000 description 1
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- XXXXOVFBXRERQL-ULQDDVLXSA-N Leu-Pro-Phe Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XXXXOVFBXRERQL-ULQDDVLXSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 1
- URHJPNHRQMQGOZ-RHYQMDGZSA-N Leu-Thr-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(O)=O URHJPNHRQMQGOZ-RHYQMDGZSA-N 0.000 description 1
- ODRREERHVHMIPT-OEAJRASXSA-N Leu-Thr-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ODRREERHVHMIPT-OEAJRASXSA-N 0.000 description 1
- VHTIZYYHIUHMCA-JYJNAYRXSA-N Leu-Tyr-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O VHTIZYYHIUHMCA-JYJNAYRXSA-N 0.000 description 1
- NTXYXFDMIHXTHE-WDSOQIARSA-N Leu-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 NTXYXFDMIHXTHE-WDSOQIARSA-N 0.000 description 1
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 1
- TVHCDSBMFQYPNA-RHYQMDGZSA-N Lys-Thr-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TVHCDSBMFQYPNA-RHYQMDGZSA-N 0.000 description 1
- XABXVVSWUVCZST-GVXVVHGQSA-N Lys-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN XABXVVSWUVCZST-GVXVVHGQSA-N 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- JCMMNFZUKMMECJ-DCAQKATOSA-N Met-Lys-Asn Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O JCMMNFZUKMMECJ-DCAQKATOSA-N 0.000 description 1
- OVTOTTGZBWXLFU-QXEWZRGKSA-N Met-Val-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O OVTOTTGZBWXLFU-QXEWZRGKSA-N 0.000 description 1
- 108010047562 NGR peptide Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- FPTXMUIBLMGTQH-ONGXEEELSA-N Phe-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 FPTXMUIBLMGTQH-ONGXEEELSA-N 0.000 description 1
- BBDSZDHUCPSYAC-QEJZJMRPSA-N Phe-Ala-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O BBDSZDHUCPSYAC-QEJZJMRPSA-N 0.000 description 1
- AYPMIIKUMNADSU-IHRRRGAJSA-N Phe-Arg-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O AYPMIIKUMNADSU-IHRRRGAJSA-N 0.000 description 1
- YQNBKXUTWBRQCS-BVSLBCMMSA-N Phe-Arg-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 YQNBKXUTWBRQCS-BVSLBCMMSA-N 0.000 description 1
- CSYVXYQDIVCQNU-QWRGUYRKSA-N Phe-Asp-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O CSYVXYQDIVCQNU-QWRGUYRKSA-N 0.000 description 1
- DOXQMJCSSYZSNM-BZSNNMDCSA-N Phe-Lys-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O DOXQMJCSSYZSNM-BZSNNMDCSA-N 0.000 description 1
- AXIOGMQCDYVTNY-ACRUOGEOSA-N Phe-Phe-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=CC=1)C1=CC=CC=C1 AXIOGMQCDYVTNY-ACRUOGEOSA-N 0.000 description 1
- MGLBSROLWAWCKN-FCLVOEFKSA-N Phe-Phe-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MGLBSROLWAWCKN-FCLVOEFKSA-N 0.000 description 1
- RVEVENLSADZUMS-IHRRRGAJSA-N Phe-Pro-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RVEVENLSADZUMS-IHRRRGAJSA-N 0.000 description 1
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 1
- WWAQEUOYCYMGHB-FXQIFTODSA-N Pro-Asn-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1 WWAQEUOYCYMGHB-FXQIFTODSA-N 0.000 description 1
- VJLJGKQAOQJXJG-CIUDSAMLSA-N Pro-Asp-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VJLJGKQAOQJXJG-CIUDSAMLSA-N 0.000 description 1
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-N 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- IBGCFJDLCYTKPW-NAKRPEOUSA-N Pro-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 IBGCFJDLCYTKPW-NAKRPEOUSA-N 0.000 description 1
- AQGUSRZKDZYGGV-GMOBBJLQSA-N Pro-Ile-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O AQGUSRZKDZYGGV-GMOBBJLQSA-N 0.000 description 1
- WFIVLLFYUZZWOD-RHYQMDGZSA-N Pro-Lys-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WFIVLLFYUZZWOD-RHYQMDGZSA-N 0.000 description 1
- AWQGDZBKQTYNMN-IHRRRGAJSA-N Pro-Phe-Asp Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N[C@@H](CC(=O)O)C(=O)O AWQGDZBKQTYNMN-IHRRRGAJSA-N 0.000 description 1
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 1
- 230000010757 Reduction Activity Effects 0.000 description 1
- WTUJZHKANPDPIN-CIUDSAMLSA-N Ser-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N WTUJZHKANPDPIN-CIUDSAMLSA-N 0.000 description 1
- KNZQGAUEYZJUSQ-ZLUOBGJFSA-N Ser-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N KNZQGAUEYZJUSQ-ZLUOBGJFSA-N 0.000 description 1
- CRZRTKAVUUGKEQ-ACZMJKKPSA-N Ser-Gln-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CRZRTKAVUUGKEQ-ACZMJKKPSA-N 0.000 description 1
- WBINSDOPZHQPPM-AVGNSLFASA-N Ser-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)O WBINSDOPZHQPPM-AVGNSLFASA-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
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 1
- BYCVMHKULKRVPV-GUBZILKMSA-N Ser-Lys-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O BYCVMHKULKRVPV-GUBZILKMSA-N 0.000 description 1
- AABIBDJHSKIMJK-FXQIFTODSA-N Ser-Ser-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O AABIBDJHSKIMJK-FXQIFTODSA-N 0.000 description 1
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 1
- SDFUZKIAHWRUCS-QEJZJMRPSA-N Ser-Trp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N SDFUZKIAHWRUCS-QEJZJMRPSA-N 0.000 description 1
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 1
- GKMYGVQDGVYCPC-IUKAMOBKSA-N Thr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)N GKMYGVQDGVYCPC-IUKAMOBKSA-N 0.000 description 1
- VUSAEKOXGNEYNE-PBCZWWQYSA-N Thr-His-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VUSAEKOXGNEYNE-PBCZWWQYSA-N 0.000 description 1
- YDWLCDQXLCILCZ-BWAGICSOSA-N Thr-His-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YDWLCDQXLCILCZ-BWAGICSOSA-N 0.000 description 1
- JRAUIKJSEAKTGD-TUBUOCAGSA-N Thr-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N JRAUIKJSEAKTGD-TUBUOCAGSA-N 0.000 description 1
- LKJCABTUFGTPPY-HJGDQZAQSA-N Thr-Pro-Gln Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O LKJCABTUFGTPPY-HJGDQZAQSA-N 0.000 description 1
- YOPQYBJJNSIQGZ-JNPHEJMOSA-N Thr-Tyr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 YOPQYBJJNSIQGZ-JNPHEJMOSA-N 0.000 description 1
- VEYXZZGMIBKXCN-UBHSHLNASA-N Trp-Asp-Asp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VEYXZZGMIBKXCN-UBHSHLNASA-N 0.000 description 1
- BURPTJBFWIOHEY-UWJYBYFXSA-N Tyr-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BURPTJBFWIOHEY-UWJYBYFXSA-N 0.000 description 1
- CRWOSTCODDFEKZ-HRCADAONSA-N Tyr-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O CRWOSTCODDFEKZ-HRCADAONSA-N 0.000 description 1
- UNUZEBFXGWVAOP-DZKIICNBSA-N Tyr-Glu-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UNUZEBFXGWVAOP-DZKIICNBSA-N 0.000 description 1
- CNLKDWSAORJEMW-KWQFWETISA-N Tyr-Gly-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](C)C(O)=O CNLKDWSAORJEMW-KWQFWETISA-N 0.000 description 1
- DAOREBHZAKCOEN-ULQDDVLXSA-N Tyr-Leu-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O DAOREBHZAKCOEN-ULQDDVLXSA-N 0.000 description 1
- ZOBLBMGJKVJVEV-BZSNNMDCSA-N Tyr-Lys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O ZOBLBMGJKVJVEV-BZSNNMDCSA-N 0.000 description 1
- XOVDRAVPGHTYLP-JYJNAYRXSA-N Tyr-Pro-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(O)=O XOVDRAVPGHTYLP-JYJNAYRXSA-N 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- YFOCMOVJBQDBCE-NRPADANISA-N Val-Ala-Glu Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N YFOCMOVJBQDBCE-NRPADANISA-N 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- WKWJJQZZZBBWKV-JYJNAYRXSA-N Val-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WKWJJQZZZBBWKV-JYJNAYRXSA-N 0.000 description 1
- JLFKWDAZBRYCGX-ZKWXMUAHSA-N Val-Asn-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N JLFKWDAZBRYCGX-ZKWXMUAHSA-N 0.000 description 1
- XQVRMLRMTAGSFJ-QXEWZRGKSA-N Val-Asp-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XQVRMLRMTAGSFJ-QXEWZRGKSA-N 0.000 description 1
- VLOYGOZDPGYWFO-LAEOZQHASA-N Val-Asp-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VLOYGOZDPGYWFO-LAEOZQHASA-N 0.000 description 1
- 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 1
- VCAWFLIWYNMHQP-UKJIMTQDSA-N Val-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N VCAWFLIWYNMHQP-UKJIMTQDSA-N 0.000 description 1
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 1
- QZKVWWIUSQGWMY-IHRRRGAJSA-N Val-Ser-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QZKVWWIUSQGWMY-IHRRRGAJSA-N 0.000 description 1
- 206010047623 Vitamin C deficiency Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- -1 alpha-glycosidase Proteins 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 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
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 1
- 108010018691 arginyl-threonyl-arginine Proteins 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000002587 enol group Chemical group 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 208000010233 scurvy Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种重组大肠杆菌及其应用,所述重组大肠杆菌是将SEQ ID NO.1所示的来源于长双歧杆菌IEF101的L‑抗坏血酸糖基化酶基因转入大肠杆菌宿主细胞获得的。本发明采用生物法一步催化转化高效生产单一糖基化产品AA‑2G;本发明重组大肠杆菌E.coli IEF‑blsp101能够在胞内高效合成蔗糖磷酸化酶,并以L‑抗坏血酸和蔗糖为底物,高效催化L‑抗坏血酸的糖基化反应,45‑72h内催化得到90‑100g/L的AA‑2G,平均生产强度大于1.3g·L‑1·h‑1。
Description
(一)技术领域
本发明涉及一种含L-抗坏血酸糖基化酶基因的重组大肠杆菌及其生产2-O-α-D-葡萄糖基-L-抗坏血酸(AA-2G)的应用。
(二)技术背景
L-抗坏血酸(VC)是一种常见的水溶性维生素和人体所必须的营养元素,其结构如图1所示。缺乏VC会引起坏血病,心脏病,癌症等疾病,由于人体无法合成VC,因此必须从食物中获取。此外,也可作为调味剂,还原剂,抗氧化剂,漂白剂等,广泛应用于食品,化妆品和医药等领域。但是VC本身不稳定,其分子中2位和3位碳原子上的烯醇式羟基极易被氧化解离,使VC丧失还原活性,从而限制了其应用。
L-抗坏血酸衍生物主要有金属盐,酯类衍生物,葡萄糖衍生物,其中2-O-α-D-葡萄糖基-L-抗坏血酸(AA-2G)因其具有强稳定性,高安全性而备受关注。AA-2G进入体内后能被α-葡萄糖苷酶分解为VC和D-葡萄糖,使VC可以在体内保持有活性的烯醇式结构,是VC的最佳替代品,目前广泛应用于化妆品、食品、医疗保健及畜牧业个水产养殖等行业。
(三)发明内容
本发明目的是提供一种含有L-抗坏血酸糖基化酶基因的大肠杆菌及制备2-O-α-D-葡萄糖基-L-抗坏血酸(AA-2G)的应用,实现生物法一步催化转化,高效生产AA-2G。
本发明采用的技术方案是:
第一方面,本发明提供一种重组大肠杆菌,所述重组大肠杆菌是将SEQ ID NO.1所示来源于长双歧杆菌(Bifidobacterium longum)IEF101的L-抗坏血酸糖基化酶基因(GenBank登入号:MH473732)转入大肠杆菌宿主细胞获得的。
进一步,所述L-抗坏血酸糖基化酶基因编码蛋白的氨基酸序列为SEQ ID NO.2。
进一步,所述重组大肠杆菌按如下方法构建:将SEQ ID NO.1所示的L-抗坏血酸糖基化酶基因克隆到pET28a质粒上,构建pET28a-blsp重组表达质粒,并转化到大肠杆菌E.coli BL21(DE3)中,得到重组大肠杆菌E.coli BL21(DE3)(pET28a-blsp)。
第二方面,本发明提供一种所述重组大肠杆菌在制备AA-2G中的应用,具体所述的应用以重组大肠杆菌经发酵培养获得的发酵液、发酵液离心后的湿菌体与缓冲液或去离子水的菌悬液或菌悬液破碎液为催化剂,以L-抗坏血酸为底物,以蔗糖为辅助底物构成反应体系,在30-45℃(优选35-40℃)、pH为4.8-5.5的条件下进行反应,获得含AA-2G的反应液,分离纯化获得AA-2G。所述反应体系中,催化剂用量以湿菌体重量计,所述湿菌体含量为5-100g/L(优选5-10g/L),底物终浓度为50-250g/L(优选100-250g/L),所述蔗糖终浓度为200-400g/L(优选250-300g/L)。
进一步,所述催化剂为发酵液离心后的湿菌体用缓冲液或去离子水的菌悬液破碎后的破碎液,所述催化剂用量以破碎前湿菌体用量计,所述底物以一次投料法加入反应液中。
进一步,所述缓冲液为pH5.2,50mM柠檬酸钠缓冲液。
进一步,反应液的温度控制30-45℃(优选35-40℃),反应过程控制pH4.8-5.5之间,反应48-72h。
进一步,本发明所述湿菌体按如下方法制备:(1)将重组大肠杆菌接种在含50μg/ml卡那霉素的种子培养基中,30-37℃,100-250rpm培养至对数生长中期,获得种子液,所述种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO40.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH7.0;
2)发酵培养:将种子液以体积5%的接种量接种到含卡那霉素50mg/L的发酵培养基中,在30-37℃培养3-6h(优选35℃,5h);加入终浓度为5-22g/L(优选10g/L)的α-乳糖,在22-25℃继续发酵12-18h(优选23℃、12h),获得发酵液;所述发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
所述发酵培养还可以在发酵罐中进行:将新鲜培养的种子液按体积浓度5%的接种量,接种到含质量浓度0.05%消泡剂和50mg/L卡那霉素的发酵培养基中中,32℃培养4h;加入终浓度为5-22g/L(优选15g/L)的α-乳糖,控制发酵温度24℃,溶解氧DO控制大于20%,用25%的氨水控制发酵pH6.8,开始恒速补料甘油溶液,继续发酵15h,获得发酵液;所述甘油溶液终浓度组成:甘油250g/L,生物素4.5mg/L,MgSO4·7H2O 10g/L,溶剂为去离子水。
本发明所述的L-抗坏血酸糖基化酶基因来源于长双歧杆菌(Bifidobacteriumlongum)IEF101,其GenBank登入号为MH473732。
与现有技术相比,本发明有益效果体现在:①采用生物法一步催化转化高效生产单一糖基化产品AA-2G;②本发明重组大肠杆菌E.coli IEF-blsp101能够在胞内高效合成蔗糖磷酸化酶,并以L-抗坏血酸和蔗糖为底物,高效催化L-抗坏血酸的糖基化反应,45-72h内催化得到90-100g/L的AA-2G,平均生产强度大于1.3g·L-1·h-1。
(四)附图说明
图1为AA-2G结构式。
图2为pET28a-blsp载体的结构示意图。
(五)具体实施方法
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:
本发明实施例中如无特殊说明所用方法均为常规方法,所用试剂均可从商业途径获得。
LB培养基:酵母粉5.0g/L,蛋白胨10g/L、NaCl10g/L,溶剂为去离子水,pH7.0。
种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO40.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH7.0。
发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
实施例1含L-抗坏血酸糖基化酶基因的重组大肠杆菌E.coli IEF-blsp101的构建
采用细菌基因组DNA提取试剂盒提取对数生长中期的长双歧杆菌(Bifidobacterium longum)IEF101的基因组DNA,以此为模板,用以下引物进行PCR扩增:
bloSP-F:
GCCTGGTGCCGCGCGGCAGCCATATGAAAAACAAAGTGCAACTCATC;
bloSP-R:
GTCGACGGAGCTCGAATTCGGATCCTTAGTCGATATCGGCAATCGG。
采用南京诺唯赞生物科技有限公司(Vazyme Biotech Co.,Ltd)的高效保真酶Phanta Max Super-Fidelity DNA Polymerase进行PCR扩增,其PCR扩增程序为:95℃3min;95℃15s、58℃15s、72℃1.5min,30个循环;72℃5min。
将所得的PCR产物采用PCR产物回收试剂盒纯化,采用南京诺唯赞生物科技有限公司的一步克隆试剂盒One Step Cloning Kit,克隆到pET28a+质粒的Nde I和BamHI之间。pET28a+质粒用TaKaRa公司的Nde I和BamHI进行双酶切,37℃静置4h后,用DNA胶回收试剂盒纯化酶切后的pET28a+质粒。将纯化的PCR产物和酶切纯化的pET28a+质粒进行连接反应(根据试剂盒说明书操作),转化E.coli BL21(DE3)的高效感受态细胞中,在含终浓度50mg/L卡那霉素的LB平板上筛选。菌落PCR验证阳性克隆子,并提取质粒进行测序分析。阳性克隆子含有的重组表达载体命名为pET28a-blsp(图2),其中L-抗坏血酸糖基化酶基因blsp核苷酸序列为SEQ ID NO.1所示,其基因序列与NCBI数据库中Bifidobacterium longumsubsp.longum JCM 1217的蔗糖磷酸化酶基因(AP010888.1)相似度为99%,编码蛋白氨基酸序列为SEQ ID NO.2所示。
含重组质粒pET28a-blsp的阳性克隆子E.coli BL21(DE3)(pET28a-blsp)记为大肠杆菌(Escherichia coli)IFE-blsp101。
实施例2、制备生产AA-2G的发酵液菌剂
实施例1制备的E.coliIFE-blsp101在含有50μg/m L卡那霉素的种子培养基中,37℃,200rpm培养至对数生长中期,获得种子液。
将新鲜培养的种子液以体积浓度5%的接种量接种到含卡那霉素50mg/L的发酵培养基中,35℃培养5h,加入终浓度为10g/Lα-乳糖,控制发酵温度23℃,溶解氧DO控制大于20%,用25%的氨水控制发酵pH6.8,继续发酵12h,获得湿菌体的含量为30g/L的发酵液,作为催化剂。
将新鲜发酵液,用去离子水稀释3倍,使湿菌体的含量为10g/L,采用高压细胞匀浆仪破碎细胞后,得到粗酶液,需尽快用于催化反应,避免长时间保存。
实施例3菌剂在生产AA-2G中的应用
1、发酵液催化活性的检测
将实施例2方法制备的发酵液离心,取湿菌体细胞0.5g重新悬浮于50mL的pH5.2,50mM柠檬酸钠缓冲液中,用高压匀浆仪将菌体破碎,加入终浓度210g/L的VC和270g/L的蔗糖构成50ml反应体系,在40℃水浴锅中搅拌催化反应12h,反应液用于HPLC分析,测得残留的底物VC浓度为164g/L,形成的产物AA-2G的浓度为30g/L。
液相色谱检测条件:样品前处理,取50μL反应液,加入到950μL的0.01mol/L稀盐酸中,用0.22μm滤膜过滤,将滤液加入液相样品瓶中;色谱柱:C18柱,250×4.6mm;柱温:25℃;流动相:K2HPO4·3H2O:0.57g/L,磷酸调pH=2.00;流速:1mL/min;检测器:紫外检测器;检测波长:240nm;进样量:10μL。底物L-抗坏血酸的出峰时间一般为5.07min。产物AA-2G的出峰时间为6.3min。
2、稀释发酵液直接作为催化剂制备AA-2G
湿菌体含量5g/L的稀释发酵液催化:取20ml实施例2方法制备的发酵液,加入已调pH5.2的VC水溶液,并加入蔗糖,定容至120mL;使VC终浓度210g/L和蔗糖终浓度为270g/L,湿菌体细胞含量为5g/L,再次校正pH5.2;将反应液加入到棕色烧瓶中,在40℃水浴锅中,磁力搅拌,催化反应72h。反应液用于HPLC分析,测得残留的底物VC浓度为134g/L,形成的产物AA-2G的浓度为75g/L。
湿菌体含量10g/L的稀释发酵液催化:取40ml将实施例2方法制备的发酵液,加入已调pH5.2的VC水溶液,并加入蔗糖,定容至120L,湿菌体细胞含量为10g/L,VC终浓度210g/L,蔗糖终浓度为270g/L,调反应也pH5.2,将反应液加入到棕色烧瓶中,在40℃水浴锅中,磁力搅拌,催化反应72h,反应液用于HPLC分析,测得残留的底物VC浓度为102g/L,形成的产物AA-2G的浓度为105g/L。
3、2L发酵罐中催化剂制备及其在15L反应体系中转化的应用
(1)菌种活化
实施例1制备的大肠杆菌E.coliIFE-blsp101接种在含有50μg/mL卡那霉素的种子培养基中,37℃,200rpm培养至对数生长中期,获得种子液。
(2)2L发酵罐中的催化剂制备
将新鲜培养的种子液按体积浓度5%的接种量,接种到1.5L的含质量浓度0.05%消泡剂和50mg/L卡那霉素的发酵培养基中,32℃培养4h;加入终浓度为15g/L的α-乳糖,控制发酵温度24℃,溶解氧DO控制大于20%,用25%的氨水控制发酵pH6.8,开始恒速补料400ml的甘油溶液(甘油250g/L,生物素4.5mg/L,MgSO4·7H2O 10g/L)。继续发酵24h,得到用于生产AA-2G的大肠杆菌E.coliIFE-blsp101发酵液,湿菌体含量为100g/L。
(3)15L发酵液中的转化
取步骤(2)制备的2L大肠杆菌E.coliIFE-blsp101发酵液(湿菌体终浓度20g/L),离心收集菌体后重新悬浮于8L去离子水中,用高压匀浆仪破碎细胞得到细胞破碎液,加入3.9kg蔗糖(终浓度260g/L),并称取3.0kg(终浓度100g/L)L-抗坏血酸用7L去离子水溶解后用NaOH调节pH5.2,将底物混合后安装全自动机械搅拌,在40℃条件下进行催化反应,反应72h后得到AA-2G催化液。
(4)AA-2G产物浓度检测
样品前处理:取50μL反应液,加入950μL的0.01mol/L稀盐酸中,12000×g离心5min,用0.22μm滤膜过滤,将滤液稀释到1/10后加入液相样品瓶中;色谱柱:C18柱,250×4.6mm;柱温:25℃;流动相:K2HPO4·3H2O:0.57g/L;磷酸调pH=2.00;流速:1mL/min;检测器:紫外检测器;检测波长:240nm;进样量:10μL。底物L-抗坏血酸的出峰时间一般为5.07min。产物AA-2G的出峰时间为6.3min。经过72h的转化反应,测得残留的底物L-抗坏血酸浓度为159g/L,形成的产物AA-2G浓度为93g/L,其产量显著高于文献报道的环糊精糖基转移酶、α-糖苷酶、淀粉酶等催化转化AA-2G的水平(Han R etal.ApplMicrobiolBiotechnol,2012,95:313–320)。
序列表
<110> 浙江工业大学
<120> 一种重组大肠杆菌及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1527
<212> DNA
<213> 长双歧杆菌(Bifidobacterium longum)
<400> 1
atgaaaaaca aagtgcaact catcacatac gccgatcgtc tcggcgatgg cactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggcttcg acccgatcga ccataccaaa 180
gtcgacgaac gtctcggcag ctgggacgac gtcgccgaac tctccaagac ccacaacatc 240
atggtcgacg ccatcgtcaa ccacatgagt tgggaatcca agcagttcca agacgtgctt 300
gaaaaaggtg aggaatccga gtattacccg atgttcctga ccatgagctc cgtcttcccg 360
aacggcgcca ccgaagaaga cctggccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacgcgc ttggtctggg tgagcttcac cccgcagcag 480
gtggacatcg acactgattc cgccaagggt tgggaatacc tgatgtcgat cttcgatcag 540
atggccgcca gccacgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggaa 600
gccggcacca gctgcttcat gacccccaag acgttcaagc tcatctcccg cctacgcgag 660
gaaggcgtca agcgaggcct tgaaatcctc atcgaggttc acagctacta caagaagcag 720
gtggaaatcg cctccaaggt ggaccgcgtc tacgatttcg ccctgccgcc gctgcttctg 780
cactcgctgt tcaccggtca cgtcgaaccc gtggcccact ggaccgagat ccgcccgaac 840
aacgccgtca ccgtactcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccatg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgcgtccaac 1020
ctcgacctct accaggtcaa ctccacgtac tactcggccc tcggctgcaa cgaccagcac 1080
tacttggccg cccgcgccgt gcagttcttc ctgccgggcg tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggccgcaa cgacatggaa ctgctgcgcc gcaccaacaa cggccgcgac 1200
atcaaccgcc actactactc caccgccgaa atcgatgaaa acctcgaacg cccggtggtc 1260
aaggccctga acgccctggc caagttccgc aacgagctgt ctgcattcga tggcgagttc 1320
agctacgagg tcgatggcga cacgtccatc accttccgct ggaccgccgc cgacggcacg 1380
tccacggccg ccctcacctt cgagcccgga cgcggcctcg gcacagacaa caccaccccg 1440
gttgccagcc ttgcctggag cgatgccgcc ggcgaccacg aaacccgcga tctgctcgcc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 2
<211> 508
<212> PRT
<213> 长双歧杆菌(Bifidobacterium longum)
<400> 2
Met Lys Asn Lys Val Gln Leu Ile Thr Tyr Ala Asp Arg Leu Gly Asp
1 5 10 15
Gly Thr Leu Ser Ser Met Thr Asp Ile Leu Arg Thr Arg Phe Asp Gly
20 25 30
Val Tyr Asp Gly Val His Ile Leu Pro Phe Phe Thr Pro Phe Asp Gly
35 40 45
Ala Asp Ala Gly Phe Asp Pro Ile Asp His Thr Lys Val Asp Glu Arg
50 55 60
Leu Gly Ser Trp Asp Asp Val Ala Glu Leu Ser Lys Thr His Asn Ile
65 70 75 80
Met Val Asp Ala Ile Val Asn His Met Ser Trp Glu Ser Lys Gln Phe
85 90 95
Gln Asp Val Leu Glu Lys Gly Glu Glu Ser Glu Tyr Tyr Pro Met Phe
100 105 110
Leu Thr Met Ser Ser Val Phe Pro Asn Gly Ala Thr Glu Glu Asp Leu
115 120 125
Ala Gly Ile Tyr Arg Pro Arg Pro Gly Leu Pro Phe Thr His Tyr Lys
130 135 140
Phe Ala Gly Lys Thr Arg Leu Val Trp Val Ser Phe Thr Pro Gln Gln
145 150 155 160
Val Asp Ile Asp Thr Asp Ser Ala Lys Gly Trp Glu Tyr Leu Met Ser
165 170 175
Ile Phe Asp Gln Met Ala Ala Ser His Val Arg Tyr Ile Arg Leu Asp
180 185 190
Ala Val Gly Tyr Gly Ala Lys Glu Ala Gly Thr Ser Cys Phe Met Thr
195 200 205
Pro Lys Thr Phe Lys Leu Ile Ser Arg Leu Arg Glu Glu Gly Val Lys
210 215 220
Arg Gly Leu Glu Ile Leu Ile Glu Val His Ser Tyr Tyr Lys Lys Gln
225 230 235 240
Val Glu Ile Ala Ser Lys Val Asp Arg Val Tyr Asp Phe Ala Leu Pro
245 250 255
Pro Leu Leu Leu His Ser Leu Phe Thr Gly His Val Glu Pro Val Ala
260 265 270
His Trp Thr Glu Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr
275 280 285
His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg
290 295 300
Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn
305 310 315 320
Thr Ile His Ala Asn Thr His Gly Glu Ser Gln Ala Ala Thr Gly Ala
325 330 335
Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser
340 345 350
Ala Leu Gly Cys Asn Asp Gln His Tyr Leu Ala Ala Arg Ala Val Gln
355 360 365
Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala
370 375 380
Gly Arg Asn Asp Met Glu Leu Leu Arg Arg Thr Asn Asn Gly Arg Asp
385 390 395 400
Ile Asn Arg His Tyr Tyr Ser Thr Ala Glu Ile Asp Glu Asn Leu Glu
405 410 415
Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu
420 425 430
Leu Ser Ala Phe Asp Gly Glu Phe Ser Tyr Glu Val Asp Gly Asp Thr
435 440 445
Ser Ile Thr Phe Arg Trp Thr Ala Ala Asp Gly Thr Ser Thr Ala Ala
450 455 460
Leu Thr Phe Glu Pro Gly Arg Gly Leu Gly Thr Asp Asn Thr Thr Pro
465 470 475 480
Val Ala Ser Leu Ala Trp Ser Asp Ala Ala Gly Asp His Glu Thr Arg
485 490 495
Asp Leu Leu Ala Asn Pro Pro Ile Ala Asp Ile Asp
500 505
Claims (10)
1.一种重组大肠杆菌,其特征在于所述重组大肠杆菌是将SEQ ID NO.1所示的来源于长双歧杆菌(Bifidobacterium longum)IEF101的L-抗坏血酸糖基化酶基因转入大肠杆菌宿主细胞获得的。
2.如权利要求1所述重组大肠杆菌,其特征在于所述L-抗坏血酸糖基化酶基因编码蛋白的氨基酸序列为SEQ ID NO.2所示。
3.如权利要求1所述重组大肠杆菌,其特征在于所述重组大肠杆菌按如下方法构建:将SEQ ID NO.1所示的L-抗坏血酸糖基化酶基因克隆到pET28a质粒上,构建pET28a-blsp重组表达质粒,并转化到大肠杆菌E.coli BL21(DE3)中,得到重组大肠杆菌E.coli BL21(DE3)(pET28a-blsp)。
4.一种权利要求1所述重组大肠杆菌在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用。
5.如权利要求4所述的应用,其特征在于所述的应用是将重组大肠杆菌经发酵培养获得的发酵液、发酵液离心后的湿菌体与缓冲液或去离子水的菌悬液或菌悬液破碎液为催化剂,以L-抗坏血酸为底物,以蔗糖为辅助底物构成反应体系,在30-45℃、pH为4.8-5.5的条件下进行反应,获得含2-O-α-D-葡萄糖基-L-抗坏血酸的反应液,分离纯化,获得2-O-α-D-葡萄糖基-L-抗坏血酸。
6.如权利要求5所述的应用,其特征在于所述反应体系中,催化剂用量以湿菌体重量计,所述湿菌体含量为5-100g/L,L-抗坏血酸终浓度为50-250g/L,所述蔗糖终浓度为200-400g/L。
7.如权利要求5所述的应用,其特征在于所述催化剂为发酵液离心后的湿菌体用缓冲液或去离子水的菌悬液破碎后的破碎液,所述催化剂用量以破碎前湿菌体用量计。
8.如权利要求5所述的应用,其特征在于所述缓冲液为pH5.2,50mM柠檬酸钠缓冲液。
9.如权利要求5所述的应用,其特征在于所述发酵液按如下方法制备:(1)将重组大肠杆菌接种在含50mg/L卡那霉素的种子培养基中,30-37℃、180-250rpm培养至对数生长中期,获得种子液;所述种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH6.8-7.0;
(2)发酵培养:将种子液以体积浓度5%的接种量接种到含卡那霉素50mg/L的发酵培养基中,在30-37℃培养4-6h;加入终浓度为5-22g/L的α-乳糖,在22-25℃继续发酵12-18h,得到发酵液;所述发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O0.3g/L,溶剂为去离子水,pH6.8-7.0。
10.如权利要求9所述的应用,其特征在于步骤(2)发酵培养方法为:将新鲜培养的种子液按体积浓度5%的接种量,接种到含质量浓度0.05%消泡剂和50mg/L卡那霉素的发酵培养基中,32℃培养4h;加入终浓度为5-22g/L的α-乳糖,控制发酵温度24℃,溶解氧DO大于20%,pH6.8,恒速补料甘油溶液;继续发酵15h,获得发酵液;所述甘油溶液终浓度组成:甘油250g/L,生物素4.5mg/L,MgSO4·7H2O 10g/L,溶剂为去离子水;所述甘油溶液与发酵培养基体积比为1:3.75。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810696239.5A CN108913641B (zh) | 2018-06-29 | 2018-06-29 | 一种重组大肠杆菌及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810696239.5A CN108913641B (zh) | 2018-06-29 | 2018-06-29 | 一种重组大肠杆菌及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108913641A CN108913641A (zh) | 2018-11-30 |
CN108913641B true CN108913641B (zh) | 2021-02-02 |
Family
ID=64423112
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810696239.5A Active CN108913641B (zh) | 2018-06-29 | 2018-06-29 | 一种重组大肠杆菌及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108913641B (zh) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109988799B (zh) * | 2019-01-24 | 2021-02-02 | 浙江工业大学 | 一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷中的应用 |
CN109988778B (zh) * | 2019-05-14 | 2020-10-27 | 南京工业大学 | 一种蔗糖磷酸化酶基因及其应用 |
CN110343654B (zh) * | 2019-08-15 | 2021-03-30 | 江南大学 | 一种产蔗糖磷酸化酶的基因工程菌 |
CN111172127A (zh) * | 2020-01-17 | 2020-05-19 | 浙江工业大学 | 一种蔗糖磷酸化酶在制备甘油葡萄糖苷中的应用 |
CN111172128A (zh) * | 2020-01-21 | 2020-05-19 | 浙江工业大学 | 一种蔗糖磷酸化酶在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用 |
CN114317478B (zh) * | 2022-01-05 | 2023-10-20 | 北京化工大学 | 一种蔗糖磷酸化酶的应用及利用其制备2-α-甘油葡萄糖苷的方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017050920A1 (en) * | 2015-09-25 | 2017-03-30 | Acib Gmbh | Method for small molecule glycosylation |
CN107058200A (zh) * | 2016-11-09 | 2017-08-18 | 山东格得生物科技有限公司 | 制备l‑抗坏血酸‑2‑葡萄糖苷的方法 |
CN107400653A (zh) * | 2017-08-03 | 2017-11-28 | 浙江工业大学 | 一种含α‑糖苷酶基因的重组大肠杆菌及其应用 |
CN107630057A (zh) * | 2016-07-18 | 2018-01-26 | 中国科学院微生物研究所 | 一种生产2-氧-α-D-吡喃葡糖基抗坏血酸的方法及其专用工程菌 |
-
2018
- 2018-06-29 CN CN201810696239.5A patent/CN108913641B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017050920A1 (en) * | 2015-09-25 | 2017-03-30 | Acib Gmbh | Method for small molecule glycosylation |
CN107630057A (zh) * | 2016-07-18 | 2018-01-26 | 中国科学院微生物研究所 | 一种生产2-氧-α-D-吡喃葡糖基抗坏血酸的方法及其专用工程菌 |
CN107058200A (zh) * | 2016-11-09 | 2017-08-18 | 山东格得生物科技有限公司 | 制备l‑抗坏血酸‑2‑葡萄糖苷的方法 |
CN107400653A (zh) * | 2017-08-03 | 2017-11-28 | 浙江工业大学 | 一种含α‑糖苷酶基因的重组大肠杆菌及其应用 |
Non-Patent Citations (3)
Title |
---|
"Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from Bifidobacterium longum";Taeyeon Kwon et al.;《Biotechnology Letters》;20070110;第29卷(第4期);全文 * |
"蔗糖磷酸化酶全细胞催化 AA-2G 的条件优化";余磊等;《现代生物医学进展》;20170531;第17卷(第14期);全文 * |
"重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷";万月佳等;《生物工程学报》;20121225;第28卷(第12期);第1452页第1.2.2节 * |
Also Published As
Publication number | Publication date |
---|---|
CN108913641A (zh) | 2018-11-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108913641B (zh) | 一种重组大肠杆菌及其应用 | |
CN109988799B (zh) | 一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷中的应用 | |
CN108467860B (zh) | 一种高产γ-氨基丁酸的方法 | |
CN107937365B (zh) | 一种β-半乳糖苷酶的突变体及其制备方法和应用 | |
CN111172127A (zh) | 一种蔗糖磷酸化酶在制备甘油葡萄糖苷中的应用 | |
CN107058200B (zh) | 制备l-抗坏血酸-2-葡萄糖苷的方法 | |
CN112813013B (zh) | 一种生产羟基酪醇的重组大肠杆菌及其应用 | |
CN114410605B (zh) | 一种利用角质酶突变体促进重组蛋白胞外表达的方法 | |
CN112980906B (zh) | 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用 | |
CN112592880A (zh) | 一种产假尿苷工程菌及其应用 | |
CN109022338A (zh) | 一种酶转化苯丙氨酸生产苯丙酮酸的工艺 | |
CN110982865A (zh) | 一种碱性环糊精葡萄糖基转移酶在生产α-葡萄糖基橙皮苷中的应用 | |
CN109762794B (zh) | 一种葡萄糖基转移酶在生产乙基香兰素-α-D-葡萄糖苷中的应用 | |
CN113337495B (zh) | 一种提高唾液酸产量的方法与应用 | |
CN114480465A (zh) | 一种产生2’-岩藻糖基乳糖的枯草芽孢杆菌及其应用 | |
CN107828752B (zh) | 一种蔗糖淀粉酶、制备方法及在生产α-熊果苷中的应用 | |
CN109837261B (zh) | 一种葡萄糖苷酶突变体及其应用 | |
CN114107341A (zh) | 真菌来源的α-L-鼠李糖苷酶在高效生产淫羊藿苷中的应用 | |
CN109576239A (zh) | 耐热磷酸化酶及其应用 | |
CN111172128A (zh) | 一种蔗糖磷酸化酶在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用 | |
CN109929863B (zh) | 一种利用全细胞转化生产异麦芽酮糖的方法 | |
CN111455003A (zh) | 一种微藻制备d-阿洛酮糖的方法 | |
CN116731997A (zh) | 一种定点突变的蔗糖磷酸化酶及其应用 | |
CN107988131B (zh) | 一种高产α-酮-γ-甲硫基丁酸的方法 | |
CN111172089A (zh) | 一种利用重组海藻糖合成酶合成海藻糖的方法 |
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 | ||
TR01 | Transfer of patent right |
Effective date of registration: 20231218 Address after: No.2 building, Chongwen Park, Nanshan Zhiyuan, no.3370 Liuxian Avenue, Fuguang community, Taoyuan Street, Nanshan District, Shenzhen, Guangdong Patentee after: Shenzhen Shanhai Innovation Technology Co.,Ltd. Address before: 310014 No. 18 Chao Wang Road, Xiacheng District, Zhejiang, Hangzhou Patentee before: JIANG University OF TECHNOLOGY |
|
TR01 | Transfer of patent right |