CN103146592A - Microzyme converting ginsenoside Rb1 to generate Rd and application thereof - Google Patents
Microzyme converting ginsenoside Rb1 to generate Rd and application thereof Download PDFInfo
- Publication number
- CN103146592A CN103146592A CN2013100387984A CN201310038798A CN103146592A CN 103146592 A CN103146592 A CN 103146592A CN 2013100387984 A CN2013100387984 A CN 2013100387984A CN 201310038798 A CN201310038798 A CN 201310038798A CN 103146592 A CN103146592 A CN 103146592A
- Authority
- CN
- China
- Prior art keywords
- ginsenoside
- generate
- saccharomycetes
- conversion
- microzyme
- 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
- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 title claims abstract description 21
- TXEWRVNOAJOINC-UHFFFAOYSA-N ginsenoside Rb2 Natural products CC(=CCCC(OC1OC(COC2OCC(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CCC45C)C TXEWRVNOAJOINC-UHFFFAOYSA-N 0.000 title claims abstract description 11
- UFNDONGOJKNAES-UHFFFAOYSA-N Ginsenoside Rb1 Natural products CC(=CCCC(C)(OC1OC(COC2OC(CO)C(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CC(O)C45C)C UFNDONGOJKNAES-UHFFFAOYSA-N 0.000 title claims abstract 10
- 229930182494 ginsenoside Natural products 0.000 claims abstract description 77
- 229940089161 ginsenoside Drugs 0.000 claims abstract description 72
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 29
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 25
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims description 59
- 108090000790 Enzymes Proteins 0.000 claims description 35
- 102000004190 Enzymes Human genes 0.000 claims description 35
- 239000000203 mixture Substances 0.000 claims description 24
- 239000000758 substrate Substances 0.000 claims description 24
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- 239000007974 sodium acetate buffer Substances 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 18
- FBFMBWCLBGQEBU-RXMALORBSA-N (2s,3r,4s,5s,6r)-2-[(2r,3r,4s,5s,6r)-2-[[(3s,5r,6s,8r,9r,10r,12r,13r,14r,17s)-3,12-dihydroxy-4,4,8,10,14-pentamethyl-17-[(2s)-6-methyl-2-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhept-5-en-2-yl]-2,3,5,6,7,9,11,12,13,15,16,17-dodecah Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FBFMBWCLBGQEBU-RXMALORBSA-N 0.000 claims description 17
- FBFMBWCLBGQEBU-GYMUUCMZSA-N 20-gluco-ginsenoside-Rf Natural products O([C@](CC/C=C(\C)/C)(C)[C@@H]1[C@H]2[C@H](O)C[C@H]3[C@](C)([C@]2(C)CC1)C[C@H](O[C@@H]1[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O2)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1C(C)(C)[C@@H](O)CC[C@]31C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FBFMBWCLBGQEBU-GYMUUCMZSA-N 0.000 claims description 17
- HYPFYJBWSTXDAS-UHFFFAOYSA-N Ginsenoside Rd Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C4CCC5C(C)(C)C(CCC5(C)C4CC(O)C23C)OC6OC(CO)C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C HYPFYJBWSTXDAS-UHFFFAOYSA-N 0.000 claims description 17
- 241000235342 Saccharomycetes Species 0.000 claims description 17
- UOJAEODBOCLNBU-UHFFFAOYSA-N vinaginsenoside R4 Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O UOJAEODBOCLNBU-UHFFFAOYSA-N 0.000 claims description 17
- 239000006228 supernatant Substances 0.000 claims description 16
- 238000000502 dialysis Methods 0.000 claims description 15
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 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 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 230000006920 protein precipitation Effects 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000012856 packing Methods 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 239000013049 sediment Substances 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 235000008434 ginseng Nutrition 0.000 abstract description 13
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 abstract description 11
- 235000003140 Panax quinquefolius Nutrition 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 3
- 108010031186 Glycoside Hydrolases Proteins 0.000 abstract description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 abstract description 2
- 238000011161 development Methods 0.000 abstract description 2
- 241000208340 Araliaceae Species 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 230000002503 metabolic effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 31
- 240000004371 Panax ginseng Species 0.000 description 12
- 229930182490 saponin Natural products 0.000 description 11
- 235000017709 saponins Nutrition 0.000 description 11
- 230000009466 transformation Effects 0.000 description 11
- 150000007949 saponins Chemical class 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 125000003147 glycosyl group Chemical group 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000004060 metabolic process Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000233866 Fungi Species 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 102000011040 TRPV Cation Channels Human genes 0.000 description 4
- 108010062740 TRPV Cation Channels Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 108020004463 18S ribosomal RNA Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- QDZOEBFLNHCSSF-PFFBOGFISA-N (2S)-2-[[(2R)-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-1-[(2R)-2-amino-5-carbamimidamidopentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-N-[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]pentanediamide Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CCCNC(N)=N)C1=CC=CC=C1 QDZOEBFLNHCSSF-PFFBOGFISA-N 0.000 description 2
- 108091006146 Channels Proteins 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 235000002789 Panax ginseng Nutrition 0.000 description 2
- 102400000096 Substance P Human genes 0.000 description 2
- 101800003906 Substance P Proteins 0.000 description 2
- 241000269370 Xenopus <genus> Species 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 2
- 230000003502 anti-nociceptive effect Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- YKPUWZUDDOIDPM-SOFGYWHQSA-N capsaicin Chemical compound COC1=CC(CNC(=O)CCCC\C=C\C(C)C)=CC=C1O YKPUWZUDDOIDPM-SOFGYWHQSA-N 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 108010020184 ginsenoside-beta-glucosidase Proteins 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002398 materia medica Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 210000001178 neural stem cell Anatomy 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- -1 panaxadiol saponins Chemical class 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- ADNPLDHMAVUMIW-CUZNLEPHSA-N substance P Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CCCN=C(N)N)C1=CC=CC=C1 ADNPLDHMAVUMIW-CUZNLEPHSA-N 0.000 description 2
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102400000748 Beta-endorphin Human genes 0.000 description 1
- 101800005049 Beta-endorphin Proteins 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- BGXFQDFSVDZUIW-UHFFFAOYSA-N Decursinol Natural products O1C(=O)C=CC2=C1C=C1OC(C)(C)C(O)CC1=C2 BGXFQDFSVDZUIW-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241000180649 Panax notoginseng Species 0.000 description 1
- 235000003143 Panax notoginseng Nutrition 0.000 description 1
- SYFJYASKXNAXKC-UHFFFAOYSA-N Panaxadiol Natural products CC1(C)CCCC(O1)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5CCC34C SYFJYASKXNAXKC-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- VLSMHEGGTFMBBZ-UHFFFAOYSA-N alpha-Kainic acid Natural products CC(=C)C1CNC(C(O)=O)C1CC(O)=O VLSMHEGGTFMBBZ-UHFFFAOYSA-N 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000003288 anthiarrhythmic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- WOPZMFQRCBYPJU-NTXHZHDSSA-N beta-endorphin Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)O)C1=CC=CC=C1 WOPZMFQRCBYPJU-NTXHZHDSSA-N 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- CNHRRMQBWQJRPN-UHFFFAOYSA-N chikusetsusaponin LM5 Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC(C(C(O)C1O)O)OC1COC1OC(CO)C(O)C1O CNHRRMQBWQJRPN-UHFFFAOYSA-N 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- OORMXZNMRWBSTK-LGFJJATJSA-N dammarane Chemical compound C1CCC(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@H](C)CCCC(C)C)[C@H]4CC[C@@H]3[C@]21C OORMXZNMRWBSTK-LGFJJATJSA-N 0.000 description 1
- BGXFQDFSVDZUIW-LBPRGKRZSA-N decursinol Chemical compound O1C(=O)C=CC2=C1C=C1OC(C)(C)[C@@H](O)CC1=C2 BGXFQDFSVDZUIW-LBPRGKRZSA-N 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000003492 excitotoxic effect Effects 0.000 description 1
- 231100000063 excitotoxicity Toxicity 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- JDCPEKQWFDWQLI-LUQKBWBOSA-N ginsenoside Rc Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@@H](CO)[C@H](O)[C@H]1O JDCPEKQWFDWQLI-LUQKBWBOSA-N 0.000 description 1
- SPFXZQZPHXUJSR-UHFFFAOYSA-N ginsenoside-Rc Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1OC2OC(CO)C(O)C2O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CCC45C)C SPFXZQZPHXUJSR-UHFFFAOYSA-N 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- VLSMHEGGTFMBBZ-OOZYFLPDSA-N kainic acid Chemical compound CC(=C)[C@H]1CN[C@H](C(O)=O)[C@H]1CC(O)=O VLSMHEGGTFMBBZ-OOZYFLPDSA-N 0.000 description 1
- 229950006874 kainic acid Drugs 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 150000008130 triterpenoid saponins Chemical class 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses microzyme converting ginsenoside Rb1 to generate Rd and application of the microzyme converting the ginsenoside Rb1 to generate the Rd. The microzyme is saccharomyces cerevisiae GWYS01. The preservation unit is China representative culture collection center. The preservation address is Wuhan University in Wuhan, China. The preservation number is CCTCC NO: M2013038. The preservation data is January, 22ed, 2013. The invention further discloses the application of the microzyme converting the ginsenoside Rb1 to generate the Rd. According to the microzyme converting the ginsenoside Rb1 to generate the Rd and the application of the microzyme converting the ginsenoside Rb1 to generate the Rd, glycosidase which is a microzyme metabolic product is used for converting the ginsenoside Rb1 to the Rd, bioavailability of effective ingredients in ginseng is improved, a conversion rate of the Rb1 in 24 hours reaches 97.5%, and a rate of generation of the Rd through conversion is 62%. The microzyme converting the ginsenoside Rb1 to generate the Rd and the application of the microzyme converting the ginsenoside Rb1 to generate the Rd are low in cost, simple in operation, and short in conversion period, and promote development of modernization and industrialization of traditional Chinese medicine ginsenoside.
Description
Technical field
The present invention relates to a kind of conversion ginsenoside Rb
1generate the yeast of Rd, also relate to this saccharomycetic application simultaneously, belong to technical field of traditional Chinese medicines.
Background technology
Ginseng (Panax ginseng G.A.Meyer) is the traditional Chinese medicine of China's preciousness, is applied to the tcm clinical practice history of existing more than 2,000 year.The main active ingredient of ginseng is ginsenoside (ginsenoside), and wherein, the protopanoxadiol saponins belongs to dammarane type four-ring triterpenoid saponin, mainly comprises Rb
1, Rc, Rd, Rg
3and Rh
2deng.The textural difference of above monomer only is that C3 is different with the glycosyl side chain on the C20 position, but the pharmacologically active demonstrated has difference.As Rb
1show good anti-arrhythmia and anti-aging effects (ginsenoside Rb
1pharmacology activity research progress, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008,33 (12): 1371-1377), Rc can weaken pain (Effects of ginsenosides on vanilloid receptor (VR1) channels expressed in Xenopus oocytes, Mol Cells, 2001, the 12:342-346 induced by capsicine and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, Antinociceptive effects ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model, Planta Med, 2003,69:1001-1004) and extend (Can ginsenosides protect human erythrocytes against free-radical-induced hemolysis retardation time that haemolysis occurs, Biochim Biophys Acta, 2002,1572 (1): 58-66), Rd has the neural stem cell of promotion differentiation (Ginsenoside-Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells, Life Sci, 2005, 76 (9): 983-995), the lethality excitotoxicity that the acid of selectivity antagonism cacaine causes (Effects of ginsenoside Rd and decursinol on the neurotoxic responses induced by kainic acid in mice, Planta Med, 2003, 69 (3): 230-234), weaken pain (Effects of ginsenosides on vanilloid receptor (VR1) the channels expressed in Xenopus oocytes induced by capsicine and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, Mol Cells, 2001, 12:342-346, Antinociceptive effects ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model, Planta Med, 2003,69:1001-1004) and anti-ageing (Ginsenoside-Rd attenuates oxidative damage related to aging in senescence-accelerated mice, J Pharm Pharmacol, 2004,56 (1): the effect such as 107-113), Rg
3there is significant effect (ginsenoside Rg at inducing apoptosis of tumour cell, inhibition tumor cell propagation, inhibition tumor cell invasion and attack and the aspects such as transfer and enhancing body immunizing power
3advances in studies on antitumor activity, modern tumour medical science, 2008,16 (4): 648-652), Rh
2kinds of tumors is all had to certain restraining effect, there is stronger antitumor synergism (Rh
2synergistically enhances paclitaxel or mitoxantrone in prostate cancer models, J Urol, 2006,175 (5): 1926-1931), in addition, also there is hypoglycemic effect (Mediation of β-endorphin by ginsenoside Rh2to lower plasma glucose in streptozotocin-induced diabetic rats, Planta Med, 2006,72 (1): 9-13).In above-mentioned saponin(e, Rb
1, Rc and other three kinds of saponin monomer Rb
2, Re and Rg
1content in ginseng surpasses 80%, and Rd, Rg
3and Rh
2content is but extremely low, but they have stronger pharmacologically active (Microbial conversion of major ginsenoside Rb
1to pharmaceutically active minor ginsenoside Rd, Journal of Microbiology, 2005,43 (5): 456-462).In addition, natural high-content methods of glycosides is difficult to absorb at enteron aisle, and molecular structure is not the optimum activity state, is difficult to directly bring into play drug effect, and therefore, rare saponin(e becomes main research and development object both at home and abroad.
The mechanism of ginsenoside in the ginseng metabolic process is also indefinite, can't in process of growth, be controlled with orientation accumulation rare ginsenoside.The investigator often passes through the more much higher glycosyl ginsenoside of content as Rb
1and Rc is hydrolyzed to obtain Rd, the Rg of low glycosyl
3or Rh
2etc. rare saponin(e.The method of ginsenoside glycosyl transformation has chemical method, enzyme process and microbe transformation method; wherein enzyme process and microbe transformation method are simple to operate; can protect to greatest extent chemical composition of Chinese materia medica to exempt from destruction; be conducive to environment protection simultaneously; reducing production costs, is to take the desirable mode of production that Radix Ginseng total saponins produces rare saponin(e as feedstock conversion.At present, having and utilizing the glycosyl hydrolase of institute's production capacity hydrolysis ginsenoside glycosidic link in microorganism or animals and plants metabolic process is catalyzer, the directed technological method that generates rare saponin(e that transforms.The microorganism type related in research is mainly bacterium and mould (screening of high yield ginsenoside beta-glucosidase bacterial classification, Journal of Dalian Institute of Light, 2000,19 (3): 195-198; Find out ginsenosidases from a kind of bacterium of new screening, Journal of Dalian Institute of Light, 2003,22 (3): 164-166; Microbial conversion of major ginsenoside Rb
1to pharmaceutically active minor ginsenoside Rd, Journal of Microbiology, 2005,43 (5): 456-462; The panoxadiol saponins is converted into to Rg
3the bacteria screening that generates of extraordinary ginsenoside Glycosylase, Dalian Polytechnic University's journal, 2008,27 (2): 97-101), report (the Purification and characterization of ginsenoside-β-glucosidase from ginseng that isolates the ginsenoside lytic enzyme and use the animal digestion enzyme to be studied from ginseng is also arranged, Chem.Pharm.Bull, 2001,49 (7): 795-798; Purification and characterization of ginsenoside-α-arabinofuranase hydrolyzing ginsenoside Rc into Rd from the fresh root of Panax ginseng, Process Biochemistry, 2002,37:793-798; A kind of ginsenoside Rb in helicase
1the separation and purification of enzyme, the biotechnology journal, 2005,11:929-933), in addition, (fungi is to ginseng soap former times Rb to use in addition fungi
1and the metabolism of panaxadiol saponins, Acta Pharmaceutica Sinica, 2001,3 (18): 603-605) or large-scale medicinal fungi adopt solid fermentation to carry out the research report (research of Microbiological Transformation of Ginsenoside of microbial transformation, Jilin: Jilin Agriculture University, 2004).Obtaining more enzyme in research is ginsenoside Rb
1lytic enzyme, its hydrolysate is mainly Rd, in addition in addition can be by Rg
3hydrolysis generates Rh
2the ginsenoside Glycosylase, and separation and purification goes out the ginsenoside-α that Rc hydrolysis can be generated to Rd-arabinofuranosidase/xylosidase from ginseng.
In these method for transformation, mostly use the inclined-plane seed activation, prepare liquid seeds and carry out microbial transformation, when carrying out bio-transformation, except the ginsenoside raw material, also add other auxiliary material as glucose simultaneously, inorganic salt etc., and transformation efficiency is low, mostly is about 15%～20%.Also utilize active dry yeast to transform ginsenoside Rb
1report, transformation efficiency is only 30%～36%.
Summary of the invention
The purpose of this invention is to provide a kind of conversion ginsenoside Rb
1generate the yeast of Rd.
In order to realize above purpose, the technical solution adopted in the present invention is to provide a kind of conversion ginsenoside Rb
1generate the yeast of Rd, described yeast is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GWYS01, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preserving number: CCTCC NO:M2013038, preservation date: on January 22nd, 2013.
Yeast is a kind of unicellular fungi, there is the enzyme system that various active is powerful, as amylase, saccharifying enzyme, Glycosylase, lipase, proteolytic enzyme, cellulase etc., particularly glycosidase activity is strong, and ginsenoside is comprised of aglycon and sugar chain, utilize Glycosylase that yeast produces, the glycosyl on degraded ginsenoside molecule, make it be converted into the saponin(e aglycon, can improve the bioavailability of ginsenoside.
In metabolism of yeasts product of the present invention, containing can intense hydrolysis ginsenoside Rb
1the ginsenoside Glycosylase, 24 hours Rb
1transformation efficiency reach 97.5%, the ratio that transform to form Rd is 62%.
The present invention also aims to provide a Yeasts transforming ginsenoside Rb
1generate the application of Rd aspect.
The technical solution adopted in the present invention also is to provide a Yeasts transforming ginsenoside Rb
1generate the application of Rd aspect.
The present invention also aims to provide a kind of conversion of saccharomycetes ginsenoside Rb
1generate the method for Rd.
The technical solution adopted in the present invention also is to provide a kind of conversion of saccharomycetes ginsenoside Rb
1generate the method for Rd, comprise the following steps:
1) preparation of enzyme liquid:
Saccharomycetes to make fermentation is cultivated, then by filtering fermentation liquor, centrifugal for the first time, remove bacterial sediment, collect supernatant liquor; Under 20-30 ℃ of condition, add ammonium sulfate to the 60-80% saturation ratio, 4-6 ℃ of standing over night; Then centrifugal for the second time, abandon supernatant liquor, collect protein precipitation; By the dialysis tubing of packing into after the sodium-acetate buffer 6-10mL of 0.02mol/L, pH4.5-5.5 for protein precipitation dissolving, with identical sodium-acetate buffer dialysis 22-26h; After dialysis, centrifugal for the third time, supernatant liquor is enzyme liquid;
2) conversion reaction of enzyme liquid:
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5-5.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with enzyme liquid substrate reactions liquid according to volume ratio 1:5-9, under 35-45 ℃ of condition, react, and obtains the Ginsenoside Rd.
The concrete grammar that described saccharomycetes to make fermentation is cultivated is: yeast is inoculated on the PDA substratum, cultivate 35-45h under 28-32 ℃ of condition, then yeast is inoculated in the YPD substratum, under 34-38 ℃ of condition, shaking culture 45-51h, obtain saccharomycetes to make fermentation liquid.
Described PDA substratum comprises following component: potato 200g, and glucose 20g, agar 15-20g, water is settled to 1000mL, natural pH.
Described YPD substratum comprises the component of following massfraction: 1% yeast extract, 2% peptone, 2% glucose.
Described centrifugal condition for the first time is: centrifuging temperature 15-17 ℃, rotating speed 10000-12000r/min, centrifugation time 15-20min.
Described centrifugal condition for the second time is: centrifuging temperature 4-6 ℃, rotating speed 12000-14000r/min, centrifugation time 15-20min.
Described centrifugal condition for the third time is: centrifuging temperature 15-17 ℃, rotating speed 12000-14000r/min, centrifugation time 8-12min.
The present invention utilizes metabolism of yeasts product Glycosylase by ginsenoside Rb
1be converted into Rd, improved the bioavailability of effective constituent in the ginseng.Cost of the present invention is lower, simple to operate, and the transformation period is short, has promoted the development of the modernization of Chinese medicine ginsenoside and industrialization.
Embodiment
The preparation of substratum:
The PDA substratum: potato 200g, glucose 20g, agar 15-20g, water is settled to 1000mL, natural pH, 115 ℃, 0.1MPa autoclaving 20min.
YPD substratum: 1% yeast extract, 2% peptone, 2% glucose, 115 ℃, 0.1MPa autoclaving 20min.The YPD Agar substratum i.e. 2% agar in the YPD substratum.
Malt juice liquid medium: kilned malt is pulverized, and a Fructus Hordei Germinatus adds four parts of water, saccharification 3-4h in 65 ℃ of water-baths, by saccharified liquid boil rear by filtered through gauze to clarification, be diluted to the 5-6 degree Beaume, pH6.4,121 ℃, 0.1MPa, autoclaving 20min.
Glucose fermentation substratum: soybean sprout 125g, yeast extract paste 6g, glucose 20g, water 1000mL, 115 ℃ of sterilizings, 20min.
Nitrogenous source assimilation substratum: glucose 20g, KH
2pO
41g, MgSO
47H
2o0.5g, yeast extract paste 0.2g, agar 20g, water 1000mL, packing test tube after filtering, 115 ℃ of sterilizing 20min.
Embodiment 1,
1, saccharomycetic screening
Brewery is stored to mud to be inoculated in malt juice liquid medium and to be increased bacterium and cultivate, then adopt the YPD substratum, with the plate dilution method isolated strains, cultivate 2d respectively under 25 ℃ and 37 ℃ of two temperature condition, take the bacterium colony mode of appearance as main, coordinate microscopy to observe thalli morphology, it is some that acquisition meets the bacterial strain of yeast colony characteristics, then carry out repeatedly purifying and obtain 5 bacterial strains, access respectively the YPD Agar medium slant, be placed in 4 ℃, refrigerator and save backup.5 bacterial strains that separation and purification obtains are called after GWYS01, GWYS02, GWYS03, GWYS04, GWYS05 respectively.
2, the preparation of enzyme liquid
The corresponding enzyme liquid of 5 bacterial strains that Preparation Example 1 is screened respectively, concrete steps are as follows:
1) cultivation of bacterial strain and fermentation
By the inoculation of embodiment 1 separation and purification in the PDA substratum, 30 ℃ cultivate 40h after, then proceed to the fermentation of YPD substratum according to the ratio of volume fraction 5%, under 36 ℃, 160r/min condition, shaking culture 48h, obtain fermented liquid;
2) preparation of enzyme liquid:
By filtering fermentation liquor, under 15 ℃, 10000r/min condition, centrifugal 20min, remove bacterial sediment, collects supernatant liquor; Under 25 ℃ of conditions, add ammonium sulfate to 70% saturation ratio, 4 ℃ of standing over night; Then under 4 ℃, 13000r/min condition, centrifugal 20min, abandon supernatant liquor, collects protein precipitation; By the dialysis tubing of packing into after the sodium-acetate buffer 8mL of 0.02mol/L, pH5.0 for protein precipitation dissolving, with identical sodium-acetate buffer dialysis 24h; After dialysis, at 15 ℃, under the 13000r/min condition, centrifugal 10min, supernatant liquor is enzyme liquid.
3, conversion reaction
By 5 corresponding enzyme liquid of bacterial strain under the same conditions with ginsenoside Rb
1reaction, screening obtains 1 and can effectively transform ginsenoside Rb
1generating the bacterial strain of Rd, is GWYS01.
The GWYS01 streak inoculation, in the YPD substratum, is cultivated to 2d under 37 ℃, its colony morphology characteristic of visual inspection, microscopy observation of cell form.Select the glucose fermentation substratum, inoculate rear 37 ℃ of cultivations, every 12h, observe the fermentation situation, select nitrogenous source assimilation substratum simultaneously, inoculate latter 37 ℃ and cultivate the 2d observation.Observe colonial morphology, the cellular form of bacterial strain GWYS01, and the Physiology and biochemistry cultural characters, as shown in table 1.
Table 1 morphologic observation and physio-biochemical characteristics test result
18S rDNA fragment amplification and order-checking:
Extract its genome, adopt EF3, EF4 universal primer to carry out 18S rDNA fragment amplification and order-checking, wherein,
EF3：5'-TCCTCTAAATGACCAAGTTTG-3'（SEQ?ID?NO:1）
EF4：5'-GGAAGGGRTGTATTTATTAG-3'（SEQ?ID?NO:2）
Wherein, R represents A or G.
18S rDNA amplified fragments sequencing result:
gatggagttg?cccccttctc?taagcagatc?ctgaggcctc?actaagccat?tcaatcggta?ctagcgacgg?gcggtgtgta
caaagggcag?ggacgtaatc?aacgcaagct?gatgacttgc?gcttactagg?aattcctcgt?tgaagagcaa?taattacaat?gctctatccc
cagcacgacg?gagtttcaca?agattaccaa?gacctctcgg?ccaaggttag?actcgctggc?tccgtcagtg?tagcgcgcgt
gcggcccaga?acgtctaagg?gcatcacaga?cctgttattg?cctcaaactt?ccatcggctt?gaaaccgata?gtccctctaa?gaagtggata
accagcaaat?gctagcacca?ctatttagta?ggttaaggtc?tcgttcgtta?tcgcaattaa?gcagacaaat?cactccacca?actaagaacg
gccatgcacc?accacccaca?aaatcaagaa?agagctctca?atctgtcaat?ccttattgtg?tctggacctg?gtgagtttcc?ccgtgttgag
tcaaattaag?ccgcaggctc?cactcctggt?ggtgcccttc?cgtcaattcc?tttaagtttc?agccttgcga?ccatactccc?cccagaaccc
aaagactttg?atttctcgta?aggtgccgag?tgggtcatta?aaaaaacacc?acccgatccc?tagtcggcat?agtttatggt?taagactacg
acggtatctg?atcatcttcg?atcccctaac?tttcgttctt?gattaatgaa?aacgtccttg?gcaaatgctt?tcgcagtagt?tagtcttcaa
taaatccaag?aatttcacct?ctgacaattg?aatactgatg?cccccgaccg?tccctattaa?tcattacgat?ggtcctagaa?accaacaaaa
tagaaccaaa?cgtcctattc?tattattcca?tgctaatata?ttcgagcaat?acgcctgctt?tgaacactct?aattttttca?aagtaaaagt
cctggttcgc?caagagccac?aaggactcaa?ggttagccag?aaggaaaggc?cccgttggaa?atccagtaca?cgaaaaaatc
ggaccggcca?accgggccca?aagttcaact?acgagctttt?taactgcaac?aactttaata?tacgctattg?gagctggaat?taccgcggct
gctggcacca?gacttgccct?ccaattgttc?ctcgttaagg?tatttacatt?gtactcattc?caattacaag?acccgaatgg?gccctgtatc
gttatttatt?gtcactacct?ccctgaatta?ggattgggta?atttgcgcgc?ctgctgcctt?ccttggatgt?ggtagccgtt?tctcaggctc
cctctccgga?atcgaaccct?tattccccgt?tacccgttga?aaccatggta?ggccactatc?ctaccatcga?aagttgatag?ggcagaaatt
tgaatgaacc?atcgccagca?caaggccatg?cgattcgaaa?agttattatg?aatcatcaaa?gagtccga
Sequencing result carries out the homology sequence compare of analysis in the GenBank database, and result shows that GWYS01 and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genes involved fragment have 100% similar value on sequence.Combining form observation, physio-biochemical characteristics and Molecular Identification, identify that GWYS01 is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
Embodiment 2
1) saccharomycetic cultivation and fermentation
GWYS01 is inoculated in the PDA substratum, 32 ℃ cultivate 35h after, then proceed to the fermentation of YPD substratum according to the ratio of volume fraction 5%, under 38 ℃, 160r/min condition, shaking culture 45h, obtain fermented liquid;
2) preparation of enzyme liquid:
By filtering fermentation liquor, under 16 ℃, 12000r/min condition, centrifugal 17min, remove bacterial sediment, collects supernatant liquor; Under 30 ℃ of conditions, add ammonium sulfate to 80% saturation ratio, 5 ℃ of standing over night; Then under 5 ℃, 12000r/min condition, centrifugal 15min, abandon supernatant liquor, collects protein precipitation; By the dialysis tubing of packing into after the sodium-acetate buffer 6mL of 0.02mol/L, pH5.5 for protein precipitation dissolving, with identical sodium-acetate buffer dialysis 22h; After dialysis, at 16 ℃, under the 12000r/min condition, centrifugal 12min, supernatant liquor is enzyme liquid.
Embodiment 3
1) saccharomycetic cultivation and fermentation
GWYS01 is inoculated in the PDA substratum, 28 ℃ cultivate 45h after, then proceed to the fermentation of YPD substratum according to the ratio of volume fraction 5%, under 34 ℃, 160r/min condition, shaking culture 51h, obtain fermented liquid;
2) preparation of enzyme liquid:
By filtering fermentation liquor, under 17 ℃, 11000r/min condition, centrifugal 15min, remove bacterial sediment, collects supernatant liquor; Under 20 ℃ of conditions, add ammonium sulfate to 60% saturation ratio, 6 ℃ of standing over night; Then under 6 ℃, 14000r/min condition, centrifugal 17min, abandon supernatant liquor, collects protein precipitation; By the dialysis tubing of packing into after the sodium-acetate buffer 10mL of 0.02mol/L, pH4.5 for protein precipitation dissolving, with identical sodium-acetate buffer dialysis 26h; After dialysis, at 17 ℃, under the 14000r/min condition, centrifugal 8min, supernatant liquor is enzyme liquid.
Embodiment 4
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 1 makes substrate reactions liquid according to volume ratio 1:5, under 45 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 5
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 1 makes substrate reactions liquid according to volume ratio 7:50, under 40 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 6
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 1 makes substrate reactions liquid according to volume ratio 1:9, under 35 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 7
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.0, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 1 makes substrate reactions liquid according to volume ratio 7:50, under 40 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 8
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.0, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 2 makes substrate reactions liquid according to volume ratio 1:5, under 35 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 9
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.0, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 2 makes substrate reactions liquid according to volume ratio 1:9, under 45 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 10
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 3 makes substrate reactions liquid according to volume ratio 1:5, under 35 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 11
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 3 makes substrate reactions liquid according to volume ratio 7:50, under 45 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Embodiment 12
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with the enzyme liquid that GWYS01 in embodiment 3 makes substrate reactions liquid according to volume ratio 1:9, under 40 ℃ of conditions, react 24h, then in 100 ℃ of water-baths, places 15min, obtains the Ginsenoside Rd.
Experimental example, conversion of saccharomycetes ginsenoside Rb
1generate the transformation efficiency analysis of Rd
By the enzyme reaction solution of embodiment 4-12 with do not add the substrate reactions liquid of enzyme liquid to dry under 60 ℃, then be dissolved in respectively in 95% methyl alcohol 700 μ L the centrifugal 10min of 8000rmp.Draw supernatant liquor with syringe, be stored in sample bottle to be measured after 0.45 μ m pin type filter filters.
Enzyme reaction solution detects analysis by HPLC, the HPLC chromatographic condition: detector is UV/VIS, detects wavelength 203nm; Chromatographic column: ODS C18(150mm * 4.6mm, 5 μ m); Sample size 10 μ L, 35 ℃ of column temperatures, flow velocity 1mL/min, moving phase: 0-23min, acetonitrile: 0.05% phosphoric acid water=32:68; 23-80min, acetonitrile: 0.05% phosphoric acid water=47:53.Record the content of the various compositions in reaction front and back according to typical curve, according to reaction substrate and product molar ratio relation, calculate the ginsenoside transformation efficiency, result is as shown in table 2.
Table 2 conversion of saccharomycetes ginsenoside Rb
1generate the transformation efficiency analysis of Rd
| ? | Ginsenoside Rb 1Transformation efficiency % | Transform the ratio % that forms the Ginsenoside Rd |
| Embodiment 4 | 94 | 51 |
| Embodiment 5 | 95 | 54 |
| Embodiment 6 | 97 | 55 |
| Embodiment 7 | 97.5 | 62 |
| Embodiment 8 | 97 | 60 |
| Embodiment 9 | 98 | 57 |
| Embodiment 10 | 92 | 55 |
| Embodiment 11 | 98 | 58 |
| Embodiment 12 | 96 | 53 |
Claims (9)
1. one kind transforms ginsenoside Rb
1generate the yeast of Rd, it is characterized in that, described yeast is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GWYS01, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preserving number: CCTCCNO:M2013038, preservation date: on January 22nd, 2013.
2. a yeast as claimed in claim 1 is transforming ginsenoside Rb
1generate the application of Rd aspect.
3. a conversion of saccharomycetes ginsenoside Rb as claimed in claim 1
1generate the method for Rd, it is characterized in that, comprise the following steps:
1) preparation of enzyme liquid:
Saccharomycetes to make fermentation is cultivated, then by filtering fermentation liquor, centrifugal for the first time, remove bacterial sediment, collect supernatant liquor; Under 20-30 ℃ of condition, add ammonium sulfate to the 60-80% saturation ratio, 4-6 ℃ of standing over night; Then centrifugal for the second time, abandon supernatant liquor, collect protein precipitation; By the dialysis tubing of packing into after the sodium-acetate buffer 6-10mL of 0.02mol/L, pH4.5-5.5 for protein precipitation dissolving, with identical sodium-acetate buffer dialysis 22-26h; After dialysis, centrifugal for the third time, supernatant liquor is enzyme liquid;
2) conversion reaction of enzyme liquid:
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5-5.5, be mixed with ginsenoside Rb
1the substrate reactions liquid that concentration is 1.70mg/mL, then mix with enzyme liquid substrate reactions liquid according to volume ratio 1:5-9, under 35-45 ℃ of condition, react, and obtains the Ginsenoside Rd.
4. conversion of saccharomycetes ginsenoside Rb according to claim 3
1generate the method for Rd, it is characterized in that, the concrete grammar that described saccharomycetes to make fermentation is cultivated is: yeast is inoculated on the PDA substratum, cultivate 35-45h under 28-32 ℃ of condition, then yeast is inoculated in the YPD substratum, under 34-38 ℃ of condition, shaking culture 45-51h, obtain saccharomycetes to make fermentation liquid.
5. conversion of saccharomycetes ginsenoside Rb according to claim 4
1generate the method for Rd, it is characterized in that, described PDA substratum comprises following component: potato 200g, and glucose 20g, agar 15-20g, water is settled to 1000mL, natural pH.
6. conversion of saccharomycetes ginsenoside Rb according to claim 4
1generate the method for Rd, it is characterized in that, described YPD substratum comprises the component of following massfraction: 1% yeast extract, 2% peptone, 2% glucose.
7. conversion of saccharomycetes ginsenoside Rb according to claim 3
1generate the method for Rd, it is characterized in that, described centrifugal condition for the first time is: centrifuging temperature 15-17 ℃, rotating speed 10000-12000r/min, centrifugation time 15-20min.
8. conversion of saccharomycetes ginsenoside Rb according to claim 3
1generate the method for Rd, it is characterized in that, described centrifugal condition for the second time is: centrifuging temperature 4-6 ℃, rotating speed 12000-14000r/min, centrifugation time 15-20min.
9. conversion of saccharomycetes ginsenoside Rb1 according to claim 3 generates the method for Rd, it is characterized in that, described centrifugal condition for the third time is: centrifuging temperature 15-17 ℃, rotating speed 12000-14000r/min, centrifugation time 8-12min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310038798.4A CN103146592B (en) | 2013-01-31 | 2013-01-31 | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310038798.4A CN103146592B (en) | 2013-01-31 | 2013-01-31 | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103146592A true CN103146592A (en) | 2013-06-12 |
| CN103146592B CN103146592B (en) | 2014-06-25 |
Family
ID=48544916
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310038798.4A Active CN103146592B (en) | 2013-01-31 | 2013-01-31 | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103146592B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104435032A (en) * | 2013-09-18 | 2015-03-25 | 长春三德天晟科技有限公司 | Method for preparing health-care products from fresh ginseng roots, fruits, stems and leaves |
| CN105533707A (en) * | 2015-12-28 | 2016-05-04 | 江瀚生物科技（上海）有限公司 | Ginseng small molecule nutritional ingredient preparation and preparation method thereof |
| CN106520891A (en) * | 2016-10-27 | 2017-03-22 | 辽宁中医药大学 | Method for preparing ginsenoside-Rd through immobilization of cellulase and enzymolysis of ginsenoside-Rb1 by virtue of covalent cross-linking process |
| CN108138210A (en) * | 2015-10-22 | 2018-06-08 | 株式会社爱茉莉太平洋 | The method for selectively preparing ginsenoside Rd from the saponin of ginseng using enzyme process |
| CN113249235A (en) * | 2021-04-26 | 2021-08-13 | 安琪纽特股份有限公司 | Pichia fischeri for producing beta-D-glucosidase and application thereof |
| CN114134060A (en) * | 2021-12-09 | 2022-03-04 | 北京理工大学 | 4 engineering microzyme capable of efficiently synthesizing ginsenoside Ro synthetic pathway intermediate product or final product and method |
| CN116396876A (en) * | 2022-11-17 | 2023-07-07 | 云南农业大学 | Saccharomyces cerevisiae engineering bacteria for producing ginsenoside Rd and construction method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1479787A (en) * | 2000-12-29 | 2004-03-03 | Ginsenoside glycosidase of hydrolyze ginseng saponin glycosyl and its application | |
| CN102762738A (en) * | 2009-09-18 | 2012-10-31 | 韩国生命工学研究院 | Rhodanobacter ginsenosidimutans kctc22231t-derived ginsenoside glycosidase, and use thereof |
| CN102782125A (en) * | 2009-10-23 | 2012-11-14 | 韩国生命工学研究院 | Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof |
-
2013
- 2013-01-31 CN CN201310038798.4A patent/CN103146592B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1479787A (en) * | 2000-12-29 | 2004-03-03 | Ginsenoside glycosidase of hydrolyze ginseng saponin glycosyl and its application | |
| CN102762738A (en) * | 2009-09-18 | 2012-10-31 | 韩国生命工学研究院 | Rhodanobacter ginsenosidimutans kctc22231t-derived ginsenoside glycosidase, and use thereof |
| CN102782125A (en) * | 2009-10-23 | 2012-11-14 | 韩国生命工学研究院 | Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| 《Molecules》 20090608 Chang-Chun Ruan et al. Biotransformation of Ginsenoside Rf to Rh1 by Recombinant beta-Glucosidase 2043-2048 1-9 第14卷, 第6期 * |
| CHANG-CHUN RUAN ET AL.: "Biotransformation of Ginsenoside Rf to Rh1 by Recombinant β-Glucosidase", 《MOLECULES》 * |
| CHANG-SU PARK ET AL.: "Biotransformation of ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104435032A (en) * | 2013-09-18 | 2015-03-25 | 长春三德天晟科技有限公司 | Method for preparing health-care products from fresh ginseng roots, fruits, stems and leaves |
| CN108138210A (en) * | 2015-10-22 | 2018-06-08 | 株式会社爱茉莉太平洋 | The method for selectively preparing ginsenoside Rd from the saponin of ginseng using enzyme process |
| CN105533707A (en) * | 2015-12-28 | 2016-05-04 | 江瀚生物科技（上海）有限公司 | Ginseng small molecule nutritional ingredient preparation and preparation method thereof |
| CN105533707B (en) * | 2015-12-28 | 2018-06-19 | 江瀚生物科技（上海）有限公司 | A kind of ginseng small molecule nutritional ingredient preparation and preparation method thereof |
| CN106520891A (en) * | 2016-10-27 | 2017-03-22 | 辽宁中医药大学 | Method for preparing ginsenoside-Rd through immobilization of cellulase and enzymolysis of ginsenoside-Rb1 by virtue of covalent cross-linking process |
| CN106520891B (en) * | 2016-10-27 | 2019-06-04 | 辽宁中医药大学 | A kind of covalent cross-linking method anchoring fiber element enzyme enzymatic hydrolysis ginsenoside-Rb1The method for preparing ginsenoside-Rd |
| CN113249235A (en) * | 2021-04-26 | 2021-08-13 | 安琪纽特股份有限公司 | Pichia fischeri for producing beta-D-glucosidase and application thereof |
| CN113249235B (en) * | 2021-04-26 | 2023-03-10 | 安琪纽特股份有限公司 | Pichia pastoris for producing beta-D-glucosidase and application thereof |
| CN114134060A (en) * | 2021-12-09 | 2022-03-04 | 北京理工大学 | 4 engineering microzyme capable of efficiently synthesizing ginsenoside Ro synthetic pathway intermediate product or final product and method |
| CN114134060B (en) * | 2021-12-09 | 2023-09-19 | 北京理工大学 | Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method |
| CN116396876A (en) * | 2022-11-17 | 2023-07-07 | 云南农业大学 | Saccharomyces cerevisiae engineering bacteria for producing ginsenoside Rd and construction method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103146592B (en) | 2014-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103146592B (en) | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof | |
| CN1479787B (en) | Ginsenoside glycosidase of hydrolyze ginseng saponin glycosyl and its application | |
| CN104013657A (en) | Post-fermentation extracting method of extracting saponin from American ginseng medicinal material | |
| CN103966105B (en) | One kind conversion ginsenoside Rg3Produce Rh2Aspergillus oryzae, production method and application | |
| CN102559828B (en) | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms | |
| CN102676627B (en) | Microbial fermentation extraction method of total arasaponin | |
| CN111321183A (en) | Polysaccharide fermentation composition with anticancer, antiviral, anti-inflammation, osteoblast proliferation promoting and intestinal stem cell proliferation promoting effects and preparation method thereof | |
| CN104357332B (en) | Aspergillus niger JH-2 and application to biotransformation and synthesis of asiatic acid | |
| CN106086147A (en) | Fungal elicitor is utilized to induce the method extracting betulic acid from Inonqqus obliquus | |
| CN117089465B (en) | Aspergillus wart and application thereof | |
| CN117247842A (en) | Metascus fungus X-Z-5 for converting ginsenoside Rb1 and application thereof | |
| CN103333872B (en) | Method for preparing Beta-glucuronidase crude enzyme preparation | |
| CN111996223A (en) | Method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing bidirectional fermentation technology | |
| CN113717860B (en) | Application of Huang Lanzhuang bacteria in conversion of total saponins of Notoginseng radix into small-polarity ginsenoside | |
| CN113502230B (en) | Hericium erinaceus strain and culture method thereof, hericium erinaceus-ginseng bidirectional solid fermentation method and method for efficiently converting rare ginsenoside | |
| CN104278070A (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| CN111485012A (en) | Method for preparing glycyrrhetinic acid monoglucuronide by liquorice fermentation | |
| CN113249235B (en) | Pichia pastoris for producing beta-D-glucosidase and application thereof | |
| CN116103161A (en) | Plant endophytic fungus for producing eupatorium and application thereof | |
| Chen et al. | Biotransformation of saponins to astragaloside IV from Radix Astragali by immobilized Aspergillus niger | |
| CN111763705B (en) | Preparation method and application of ginsenoside composition | |
| TU et al. | Extraction of Polysaccharide from Hericium corallinum and Analysis on Its in vitro Antioxidant Activity | |
| CN103865804B (en) | Beta-glucosidase Producing Strain and the application in resveratrol is prepared in conversion thereof | |
| CN102286565A (en) | Preparation method of theaflavin monomer | |
| CN105199966B (en) | A kind of conversion ginsenoside Rb1 produces aspergillus and the application of Rd |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240709 Address after: No. 6 Yingjie Avenue, Houhe Professional Park, Houhe Town, Weihui City, Xinxiang City, Henan Province, China 453100 Patentee after: Henan Yingjie Food Co.,Ltd. Country or region after: China Address before: 453003. Eastern section of Hua Hua Avenue, Henan, Xinxiang Patentee before: Henan Institute of Science and Technology Country or region before: China |
|
| TR01 | Transfer of patent right |