CN108823275A - A method of phytosterol bio-conversion prepares androstenedione in system containing quaternary ammonium salt ionic liquid - Google Patents
A method of phytosterol bio-conversion prepares androstenedione in system containing quaternary ammonium salt ionic liquid Download PDFInfo
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- CN108823275A CN108823275A CN201810622695.5A CN201810622695A CN108823275A CN 108823275 A CN108823275 A CN 108823275A CN 201810622695 A CN201810622695 A CN 201810622695A CN 108823275 A CN108823275 A CN 108823275A
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- Prior art keywords
- phytosterol
- ionic liquid
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- bioconversion
- whole cell
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- 239000002608 ionic liquid Substances 0.000 title claims abstract description 57
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 33
- 150000003242 quaternary ammonium salts Chemical class 0.000 title claims abstract description 10
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 title claims abstract 16
- 229960005471 androstenedione Drugs 0.000 title claims abstract 16
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 title claims abstract 16
- 239000006184 cosolvent Substances 0.000 claims abstract description 31
- 230000000284 resting effect Effects 0.000 claims abstract description 24
- 239000000872 buffer Substances 0.000 claims abstract description 19
- 239000008103 glucose Substances 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 13
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 11
- -1 carboxylic acid quaternary ammonium salt Chemical class 0.000 claims abstract description 11
- 241000187488 Mycobacterium sp. Species 0.000 claims abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 8
- 230000004151 fermentation Effects 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 7
- 239000004310 lactic acid Substances 0.000 claims abstract description 6
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 6
- 230000010148 water-pollination Effects 0.000 claims abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 6
- DZLFLBLQUQXARW-UHFFFAOYSA-N tetrabutylammonium Chemical compound CCCC[N+](CCCC)(CCCC)CCCC DZLFLBLQUQXARW-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 229920000136 polysorbate Polymers 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims description 3
- 229960001231 choline Drugs 0.000 claims description 3
- 239000006185 dispersion Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims description 2
- 239000011324 bead Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000004090 dissolution Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 230000020411 cell activation Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 47
- 239000007853 buffer solution Substances 0.000 description 12
- 238000001228 spectrum Methods 0.000 description 12
- 230000003197 catalytic effect Effects 0.000 description 10
- 238000006555 catalytic reaction Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 229910052739 hydrogen Inorganic materials 0.000 description 8
- 239000001257 hydrogen Substances 0.000 description 8
- 229930182558 Sterol Natural products 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 150000003432 sterols Chemical class 0.000 description 7
- 235000003702 sterols Nutrition 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 239000011942 biocatalyst Substances 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006356 dehydrogenation reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 150000003431 steroids Chemical class 0.000 description 4
- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 description 4
- IQQRAVYLUAZUGX-UHFFFAOYSA-N 1-butyl-3-methylimidazolium Chemical compound CCCCN1C=C[N+](C)=C1 IQQRAVYLUAZUGX-UHFFFAOYSA-N 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000203720 Pimelobacter simplex Species 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 3
- 150000001735 carboxylic acids Chemical class 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical group NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical group C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- NXQOQNROJJFYCJ-FZFXZXLVSA-N androst-16-ene Chemical compound C1CCC[C@]2(C)[C@H]3CC[C@](C)(C=CC4)[C@@H]4[C@@H]3CCC21 NXQOQNROJJFYCJ-FZFXZXLVSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-M octanoate Chemical compound CCCCCCCC([O-])=O WWZKQHOCKIZLMA-UHFFFAOYSA-M 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N phosphine group Chemical group P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- 235000002378 plant sterols Nutrition 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 125000002328 sterol group Chemical group 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- NJMWOUFKYKNWDW-UHFFFAOYSA-N 1-ethyl-3-methylimidazolium Chemical compound CCN1C=C[N+](C)=C1 NJMWOUFKYKNWDW-UHFFFAOYSA-N 0.000 description 1
- VSGTWMOZGFNRPM-RSWUNGFYSA-N 11beta-17-Dihydroxy-6alpha-methylpregn-4-ene-3,20-dione Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(C)=O)CC[C@H]21 VSGTWMOZGFNRPM-RSWUNGFYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- OILXMJHPFNGGTO-NRHJOKMGSA-N Brassicasterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@](C)([C@H]([C@@H](/C=C/[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 OILXMJHPFNGGTO-NRHJOKMGSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Chemical group CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- OILXMJHPFNGGTO-ZRUUVFCLSA-N UNPD197407 Natural products C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)C=C[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZRUUVFCLSA-N 0.000 description 1
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- OILXMJHPFNGGTO-ZAUYPBDWSA-N brassicasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZAUYPBDWSA-N 0.000 description 1
- 235000004420 brassicasterol Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000002153 concerted effect Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Chemical group CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 231100000584 environmental toxicity Toxicity 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229960002413 ferric citrate Drugs 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 1
- 150000003053 piperidines Chemical group 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000003235 pyrrolidines Chemical group 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000010499 rapseed oil Substances 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011829 room temperature ionic liquid solvent Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- 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
- C12P33/00—Preparation of steroids
-
- 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
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the methods that phytosterol Whole Cell Bioconversion in a kind of system containing quaternary ammonium salt ionic liquid prepares androstenedione (AD).Mycobacteria Mycobacterium sp.NRRL B-3683 strain is subjected to seed liquor culture, seed liquor is inoculated into fermentation medium again and is cultivated to prepare resting cell, gained resting cell activation culture 7h in the Tris-HCl buffer containing glucose, then hydrophily lactic acid/carboxylic acid quaternary ammonium salt ionic liquid of substrate phytosterol and 0.1~1vol.% are put into as cosolvent, bioconversion reaction is carried out, AD is prepared.Quaternary ammonium salt ionic liquid used in the present invention is up to 8~565g L to the solubility of substrate phytosterol‑1, it is much higher than common commercial ionic liquid, substrate and product dissolution can be effectively facilitated, improve process transformation efficiency, and ionic liquid materials are few, is conducive to save production cost.
Description
Technical field
The present invention relates to phytosterol Whole Cell Bioconversions in a kind of system containing quaternary ammonium salt ionic liquid to prepare androstene two
The method of ketone.
Background technique
4-AD (AD) is important Steroid medicine intermediates, can be used for synthesizing hundreds of steroidal and swashs
Plain class drug.Using phytosterol abundant in nature as substrate, before preparing AD with wide application by biotransformation method
Scape.However, mycobacteria degrading plant sterol side chain obtain AD need to the 16 steps reaction by 11 kinds of function enzyme system concerted catalysis go through
Journey can only be completed in full cell so far.There is two main problems：(1) low aqueous solubility of sterol substrates；(2) it dredges
Aqueous product AD is after the inhibition intracellular to cell activity even toxicity, and precipitation to the inclusion of substrate particle and cell.It is logical
Often, based on solvent engineering strategy addition cosolvent (cosolvent), substrate and product can be promoted to dissolve, improves process conversion effect
Rate.
As the requirement that people develop green processes is more more and more urgent, a kind of novel green solvent --- ionic liquid
The visual field of people is progressed into its unique physicochemical property.Ionic liquid (ionic liquid, IL) is by cation and yin
The organic salt being in a liquid state under critical-temperature (generally 100 DEG C) that ion is constituted.Currently used ionic liquid is mainly by 9
Cationoid (alkyl imidazole, alkyl pyridine class, quaternary ammonium, quaternary phosphine, pyrrolidines, piperidines, morpholine, guanidine etc.) and 28 anionoids
(halogen, hexafluorophosphoric acid type, tetrafluoro boric acid type, nitric acid type, sulfonic acid type, carboxylic acid type, imine etc.) is constituted, and is mainly used for organic
The fields such as synthesis and catalysis, extraction and separation, electrochemistry, functional material, the ionic liquid of poor biocompatibility is directly used as giving birth to
The problems such as object catalysis reaction medium can bring low catalytic efficiency, the wasting of resources, eco-toxicity.Therefore, for biocatalytic reaction
The ionic liquid of process screening and synthesis specific structure is to improve one of process efficiency, the approach for realizing green catalysis.
History of the ionic liquid for Whole Cell Biocatalysis process can trace back to (Biotechnology in 2000
and Bioengineering.2000,69:227-233).But application of the ionic liquid in steroid drugs bioconversion is also
It is less.(the Biotechnology and Bioprocess Engineering.2011,16 such as Wang in 2011:852-857) will
2% hydrophily lactate ions liquid [BMIM] [Lac] is added in toluene/buffer two-phase system as cosolvent, uses
In the dehydrogenation reaction of 11 beta-hydroxy Medroxyprogesterone of Arthrobacter simplex whole-cell catalytic, so that conversion ratio is from original
56.4% is increased to 83.2%.2012, author (Applied Biochemistry and Biotechnology.2012,
167:It 2131-2143) is catalyzed -16 Beta-methyl of 17 Alpha-hydroxy-pregnant steroid -4 in Arthrobacter simplex immobilized cell again,
9 (11)-diene -3,20- diketone C1The hydrophilic ionic-liquid [EMIM] [Lac] of addition 0.3% in the dehydrogenation reaction of position, will turn
Rate is increased to 93.4%.Recently, (the Journal of Chemical Technology and such as Shen
Biotechnology.2018,93:426-431) also reporting can in Arthrobacter simplex whole-cell catalytic acetic acid
Loose C1[PrMIM] [BF of addition 0.5% in the dehydrogenation reaction of position4]、[PrMIM][PF6] plasma liquid as cosolvent, and
Various ionic liquids are had studied in detail to growth of microbial cells, cell activity, permeability of cell membrane, cell composed structure and are urged
The influence for changing transformation efficiency etc., has understanding more profound to whole-cell catalytic conversion process in the presence of ionic liquid.But this
A little researchs all concentrate on the C of steroid drugs1In the dehydrogenation reaction of position, it yet there are no and be used successfully to sterol for ionic liquid as cosolvent
The report of Side chain cleavage reaction.In view of commercialization ionic liquid to sterol dissolubility difference and to cell biological compatibility still not
Enough ideal situation (ACS Sustainable Chemistry&Engineering.2017,5:10702-10709), of the invention
The acid and full cell of the hydrophobicity and hydrogen bond of marisone is as this two major features of catalyst, with good biocompatibility, hydrogen bond alkali
Property strong biomolecule lactic acid/carboxylic acid be anionic donor, biocompatibility and the preferable quaternary ammonium salt of solubility property be sun from
Sub- donor synthesizes ionic liquid, as the cosolvent for promoting substrate and product dissolution, to improve phytosterol whole-cell biological
Conversion process efficiency.
Summary of the invention
The object of the present invention is to provide phytosterol Whole Cell Bioconversion systems in a kind of system containing quaternary ammonium salt ionic liquid
The method of standby AD.
Provide a kind of side that AD is prepared containing mycobacteria resting cell degrading plant sterol side chain in ion liquid system
Method includes the following steps：
(a) it with mycobacteria Mycobacterium sp.NRRL B-3683 (bacterium numbering ATCC 29472) for strain, carries out
Seed liquor culture；Wherein strain is commercially available from https://www.atcc.org/Search_Results.aspx？DsNav=
Ntk:PrimarySearch%7c29472%7c3%7c, Ny:True,Ro:0,N:1000552&searchTerms=
29472&redir=1.The strain is also disclosed report in CN1507489A.
(b) seed liquor obtained by step (a) is inoculated into fermentation medium and is cultivated, prepare resting cell, freezen protective is spare；
(c) it by resting cell 2~12h of activation culture in the Tris-HCl buffer containing glucose obtained by step (b), then throws
Enter 1~30g L-1The hydrophily of substrate phytosterol and 0.1~1vol.% lactic acid/carboxylic acid quaternary ammonium salt ionic liquid are as molten altogether
Agent carries out bioconversion reaction, prepares AD.
Preferably, bead is added in the seed culture medium with cell dispersion, seed liquor cultivation temperature is 28 DEG C, shaking table
Revolving speed is 180rpm, and incubation time is 24~48h.
Preferably, the fermented and cultured is in the 500mL conical flask that bottom is machined with 2 sizes about 15 × 8 × 2mm baffle
It carries out, 1~2g L is added in fermentation medium-1The phytosterol for using 80 aqueous solution of tween pre-dispersed is inoculated with as inducer
Amount is 8~12vol.%, and fermented and cultured temperature is 30 DEG C, shaking speed 200rpm, and incubation time is 48~60h.Fermentation training
After the completion of supporting, thalline were collected by centrifugation, is washed with the phosphate buffer of pH 7.0 to get the resting cell.
Preferably, the resting cell concentration is 50g L-1, concentration of glucose is 2g L-1, Tris-HCl buffer is pH
7.5,0.1M, the phytosterol is white powder, and main component is cupreol (45%), stigmasterol (26.2%), rape oil
Sterol (23.5%) and brassicasterol (3.2%), purity >=95%.
Preferably, the cation of the hydrophilic quaternary ammonium salt ionic liquid is tetrabutylammonium (N4,4,4,4 +) or choline (Ch+),
Anion is lactate or the carboxylate radical containing 8 carbon.Synthesis step is：By tetrabutylammonium hydroxide or choline and equimolar amounts
Lactic acid/carboxylic acid mixing is stirred to react at 45 DEG C to complete neutralization, then by heating stirring and with air in round-bottomed flask
Purging is until constant weight obtains ionic liquid to remove solvent.
Preferably, the bioconversion reaction carries out in the conical flask that bottom is machined with 2 baffles, reaction temperature 30
DEG C, shaking speed 200rpm.
Advantage of the invention is that：Hydrophily lactic acid/carboxylic acid quaternary ammonium salt ionic liquid as cosolvent is to substrate plant
The solubility of sterol is up to 8~565g L-1, it is much higher than common commercial ionic liquid；Its low concentration aqueous solution is to substrate and production
The solvability of object is also superior to the system that organic solvent participates in, and viscosity is no more than 1.6cP, and cell and substrate dispersion situation are good
Good, cell membrane integrity is hardly influenced by ionic liquid.Therefore, these ionic liquids can effectively facilitate bottom as cosolvent
Object and product dissolution, improve process transformation efficiency, and ionic liquid materials are few, are conducive to save production cost.
Detailed description of the invention
Fig. 1 is ionic liquid tetrabutylammonium the caprylate ([N of synthesis4,4,4,4][C7H15COO])1H NMR spectra；
Fig. 2 is ionic liquid tetrabutylammonium the caprylate ([N of synthesis4,4,4,4][C7H15COO])13C NMR(500MHz,
DMSO-d6) spectrogram.
Specific embodiment
Specific embodiment below expands detailed description to the present invention, but the present invention is not restricted to following implementation
Example.
Embodiment 1：Tetrabutylammonium octanoic acid ionic liquid ([N4,4,4,4][C7H15COO]) in-buffer cosolvent system
Phytosterol bio-conversion prepares AD, specific as follows：
Step 1：Prepare ionic liquid [N4,4,4,4][C7H15COO].Specifically, by tetrabutylammonium hydroxide ([N4,4,4,4]
[OH]) the octanoic acid of 25wt.% aqueous solution and equimolar amounts mix, 12h is stirred to react at 45 DEG C in round-bottomed flask to complete
It neutralizes, then by heating stirring and with air purging until constant weight, to remove solvent, obtains transparent liquid at room temperature
Ionic liquid [N4,4,4,4][C7H15COO]。
Use nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13[the N of C NMR characterization synthesis4,4,4,4][C7H15COO]。1H NMRδ/ppm
(500MHz,DMSO-d6):0.84-0.87(3H,t；COO-C6-CH3),0.92-0.95(12H,t；N-C3-CH3),1.14-1.37
(18H,m；N-C2-CH2and COO-C-(CH2)5),1.54-1.60(8H,m；N-C-CH2),1.70-1.73(2H,t；COO-
CH2),3.16-3.19(8H,m；N-CH2).13C NMRδ/ppm(500MHz,DMSO-d6):13.46(s；N-C4),13.95(s；
COO-C8),19.18(s；N-C3),22.13(s；COO-C7),23.06(s；N-C2),26.80(s；COO-C6),28.90(s；
COO-C5),29.58(s；COO-C4),31.41(s；COO-C3),39.17(s；COO-C2),57.45-57.50(t；N-C1),
174.83(s；COO-C1).Spectrogram is as illustrated in fig. 1 and 2.Analysis shows the ionic liquid structure of synthesis is errorless and with higher
Purity.
Step 2：Seed liquor culture.Aseptically, by test tube slant mycobacteria Mycobacterium
Sp.NRRL B-3683 strain inoculated is into the 250mL conical flask equipped with 50mL seed culture medium and 15 beades, in constant temperature
28 DEG C in shaken cultivation case, 48h is cultivated under the conditions of 180rpm.
The seed culture based formulas is：Glucose 5g L-1, beef extract 3g L-1, tryptone 5g L-1, NaCl 15g
L-1, glycerol 1.5vol.%, pH 7.0；High pressure steam sterilization 30min at 115 DEG C.
Step 3：Fermented and cultured prepares resting cell.The seed liquor cultivated 2 days is transferred to the inoculum concentration of 12vol.%
In 500mL tool baffle conical flask equipped with 100mL fermentation medium.Wherein, fermentation medium adds 1g L before sterilization-1With
The pre-dispersed phytosterol of 80 aqueous solution of tween is to induce mycobacteria producing enzyme (to control 80 concentration of tween in final culture medium
0.05%), to be placed in constant-temperature shaking incubator, at 30 DEG C, cultivate under the conditions of 200rpm to mycobacterial cells logarithmic growth
In latter stage (60h), cell concentration and active enzyme amount higher and intracellular are abundant at this time.It is centrifuged (4 DEG C, 8000rpm, 2min) collection bacterium
Body, and washed 3 times with phosphate buffer, wet cell (i.e. resting cell, every gram of wet cell is containing about stem cell weight 0.2g) is obtained,
It is spare in -20 DEG C of stored frozens.
The fermentative medium formula is：Glucose 8g L-1, citric acid 2g L-1, ferric citrate 0.05g L-1, MgSO4
0.5g L-1, K2HPO4 0.5g L-1, (NH4)2HPO4 6g L-1, pH 7.0；High pressure steam sterilization 30min at 115 DEG C.
Step 4：Phytosterol bio-conversion prepares AD in ionic liquid-buffer cosolvent system.Point of aerobic environment
Branch bacillus resting cell catalysis phytosterol Side chain cleavage process carries out in 100mL tool baffle conical flask.Take out culture in advance
And in the mycobacteria Mycobacterium sp.NRRL B-3683 resting cell of -20 DEG C of stored frozens as biocatalysis
Agent.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added to pH
In 7.5 Tris-HCl buffer, 7h is activated under the conditions of 30 DEG C and 200rpm.3g L is added into system-1Phytosterol and
Ionic liquid [the N of 0.1vol.%4,4,4,4][C7H15COO] it is used as cosolvent, bioconversion reaction is carried out, not add ion
The buffer solution system of liquid is as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time yield (conversion speed
Rate) it is 116mg L-1h-1, much higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 2：Tetrabutylammonium octanoic acid ionic liquid ([N4,4,4,4][C7H15COO]) in-buffer cosolvent system
Phytosterol bio-conversion prepares AD, specific as follows：Ionic liquid [N is prepared as described in Example 14,4,4,4][C7H15COO],
And carry out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13The characterization of C NMR.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL
Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens
Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g
L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system
Add 3g L-1Ionic liquid [the N of phytosterol and 0.4vol.%4,4,4,4][C7H15COO] it is used as cosolvent, carry out bioconversion
Reaction, not add the buffer solution system of ionic liquid as compareing.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD
Space-time yield (conversion rate) is 65mg L-1h-1, higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 3：Tetrabutylammonium lactate ions liquid ([N4,4,4,4] [Lac]) plant in-buffer cosolvent system
Sterol bioconversion prepares AD, specific as follows：
Ionic liquid [N is prepared as described in Example 14,4,4,4] [Lac], and carry out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C
The characterization of NMR.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL
Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens
Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g
L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system
Add 3g L-1Ionic liquid [the N of phytosterol and 0.4vol.%4,4,4,4] [Lac] be used as cosolvent, carry out bioconversion reaction,
Not add the buffer solution system of ionic liquid as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time
Yield (conversion rate) is 72mg L-1h-1, higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 4：Tetrabutylammonium lactate ions liquid ([N4,4,4,4] [Lac]) plant in-buffer cosolvent system
Sterol bioconversion prepares AD, specific as follows：
Ionic liquid [N is prepared as described in Example 14,4,4,4] [Lac], and carry out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C
The characterization of NMR.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL
Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens
Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g
L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system
Add 3g L-1Ionic liquid [the N of phytosterol and 1vol.%4,4,4,4] [Lac] be used as cosolvent, carry out bioconversion reaction, with
The buffer solution system of ionic liquid is not added as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate the production of AD space-time
Rate (conversion rate) is 86mg L-1h-1, much higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 5：Phytosterol bio in choline lactate ions liquid ([Ch] [Lac])-buffer cosolvent system
Conversion prepares AD, specific as follows：
Ionic liquid [Ch] [Lac] is prepared as described in Example 1, and carries out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C NMR
Characterization.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL
Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens
Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g
L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system
Add 3g L-1The ionic liquid [Ch] [Lac] of phytosterol and 0.1vol.% are used as cosolvent, bioconversion reaction are carried out, with not
The buffer solution system of ionic liquid is added as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time yield
(conversion rate) is 109mg L-1h-1, much higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 6：Phytosterol bio in choline lactate ions liquid ([Ch] [Lac])-buffer cosolvent system
Conversion prepares AD, specific as follows：
Ionic liquid [Ch] [Lac] is prepared as described in Example 1, and carries out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C NMR
Characterization.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL
Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens
Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g
L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system
Add 3g L-1The ionic liquid [Ch] [Lac] of phytosterol and 0.4vol.% are used as cosolvent, bioconversion reaction are carried out, with not
The buffer solution system of ionic liquid is added as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time yield
(conversion rate) is 91mg L-1h-1, higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Claims (10)
1. a kind of method that phytosterol Whole Cell Bioconversion prepares androstenedione (AD) in system containing quaternary ammonium salt ionic liquid,
Characterized by the following steps：
(a) it with mycobacteria Mycobacterium sp.NRRL B-3683 (bacterium numbering ATCC 29472) for strain, carries out
Seed liquor culture；
(b) seed liquor obtained by step (a) is inoculated into fermentation medium and is cultivated, prepare resting cell, freezen protective is spare；
(c) it by resting cell 2~12h of activation culture in the Tris-HCl buffer containing glucose obtained by step (b), then throws
Enter 1~30g L-1Substrate phytosterol uses hydrophily lactic acid/carboxylic acid quaternary ammonium salt ionic liquid of 0.1~1vol.% as altogether
Solvent carries out bioconversion reaction, prepares AD.
2. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
Bead is added in seed culture medium in step (a) with cell dispersion.
3. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
Seed liquor cultivation temperature is 28 DEG C, shaking speed 180rpm in step (a), and incubation time is 24~48h.
4. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
1~2g L is added in fermentation medium in step (b)-1The phytosterol for using 80 aqueous solution of tween pre-dispersed as induction
Agent.
5. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
Bioconversion reaction is machined with 2 size 15 × 8 × 2mm baffles in bottom in fermented and cultured and step (c) in step (b)
500mL conical flask in carry out.
6. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
Seed liquor inoculum concentration is 8~12vol.% in step (b), and fermented and cultured temperature is 30 DEG C, shaking speed 200rpm, culture
Time is 48~60h.
7. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
In step (b) after the completion of fermented and cultured, thalline were collected by centrifugation, is washed with the phosphate buffer of pH 7.0 to get described quiet
Cease cell.
8. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
Resting cell concentration is 50g L in step (c)-1, concentration of glucose is 2g L-1, Tris-HCl buffer be pH 7.5,
0.1M。
9. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
The cation of hydrophilic quaternary ammonium salt ionic liquid is tetrabutylammonium or choline in step (c), and anion is for lactate or containing 8
The carboxylate radical of carbon.
10. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist
Bioconversion reaction temperature is 30 DEG C in step (c), shaking speed 200rpm.
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