JPH0367676B2 - - Google Patents
Info
- Publication number
- JPH0367676B2 JPH0367676B2 JP61180024A JP18002486A JPH0367676B2 JP H0367676 B2 JPH0367676 B2 JP H0367676B2 JP 61180024 A JP61180024 A JP 61180024A JP 18002486 A JP18002486 A JP 18002486A JP H0367676 B2 JPH0367676 B2 JP H0367676B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- carrier
- phospholipase
- phospholipid
- adsorbed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 150000003904 phospholipids Chemical class 0.000 claims description 48
- 238000006243 chemical reaction Methods 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 102000004190 Enzymes Human genes 0.000 claims description 24
- 108090000790 Enzymes Proteins 0.000 claims description 24
- 102000011420 Phospholipase D Human genes 0.000 claims description 24
- 108090000553 Phospholipase D Proteins 0.000 claims description 24
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 20
- 239000002994 raw material Substances 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 10
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 8
- 239000005373 porous glass Substances 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- HXKKHQJGJAFBHI-UHFFFAOYSA-N 1-aminopropan-2-ol Chemical compound CC(O)CN HXKKHQJGJAFBHI-UHFFFAOYSA-N 0.000 claims description 3
- 239000005909 Kieselgur Substances 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 claims description 3
- 235000012239 silicon dioxide Nutrition 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- 229910021536 Zeolite Inorganic materials 0.000 claims description 2
- 239000004927 clay Substances 0.000 claims description 2
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052571 earthenware Inorganic materials 0.000 claims description 2
- 229930182478 glucoside Natural products 0.000 claims description 2
- 150000008131 glucosides Chemical class 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 229910052573 porcelain Inorganic materials 0.000 claims description 2
- 239000010457 zeolite Substances 0.000 claims description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 26
- 229940088598 enzyme Drugs 0.000 description 23
- 238000000034 method Methods 0.000 description 22
- 239000000203 mixture Substances 0.000 description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Natural products CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 239000002904 solvent Substances 0.000 description 12
- 238000001914 filtration Methods 0.000 description 11
- 239000007810 chemical reaction solvent Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 9
- 125000001095 phosphatidyl group Chemical group 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000004809 thin layer chromatography Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000219315 Spinacia Species 0.000 description 6
- 235000009337 Spinacia oleracea Nutrition 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 4
- 240000007124 Brassica oleracea Species 0.000 description 4
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 4
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 4
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 4
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 4
- 239000008351 acetate buffer Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000006276 transfer reaction Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 235000013345 egg yolk Nutrition 0.000 description 3
- 210000002969 egg yolk Anatomy 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 229960001153 serine Drugs 0.000 description 3
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 2
- SFGGITYAMFZATC-UHFFFAOYSA-N ClC(Cl)Cl.CC(C)CC(C)(C)C Chemical compound ClC(Cl)Cl.CC(C)CC(C)(C)C SFGGITYAMFZATC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 2
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 2
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 150000003333 secondary alcohols Chemical class 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 229940083466 soybean lecithin Drugs 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- HOVAGTYPODGVJG-UVSYOFPXSA-N (3s,5r)-2-(hydroxymethyl)-6-methoxyoxane-3,4,5-triol Chemical compound COC1OC(CO)[C@@H](O)C(O)[C@H]1O HOVAGTYPODGVJG-UVSYOFPXSA-N 0.000 description 1
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 1
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- CFWRDBDJAOHXSH-SECBINFHSA-N 2-azaniumylethyl [(2r)-2,3-diacetyloxypropyl] phosphate Chemical compound CC(=O)OC[C@@H](OC(C)=O)COP(O)(=O)OCCN CFWRDBDJAOHXSH-SECBINFHSA-N 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 1
- YGRUUPPVGMDKDK-UHFFFAOYSA-N O.OC.CC(C)=O.CC(O)=O.ClC(Cl)Cl Chemical compound O.OC.CC(C)=O.CC(O)=O.ClC(Cl)Cl YGRUUPPVGMDKDK-UHFFFAOYSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000002473 artificial blood Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012156 elution solvent Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- KDCIHNCMPUBDKT-UHFFFAOYSA-N hexane;propan-2-one Chemical compound CC(C)=O.CCCCCC KDCIHNCMPUBDKT-UHFFFAOYSA-N 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- CXALKVCCFXJRKT-UHFFFAOYSA-N methanol;propan-2-ol;hydrate Chemical compound O.OC.CC(C)O CXALKVCCFXJRKT-UHFFFAOYSA-N 0.000 description 1
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 125000000075 primary alcohol group Chemical group 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、酵素によるリン脂質の製造方法に関
し、特に、塩基構造が変換されたリン脂質を製造
する方法に関する。
（従来の技術）
リン脂質は、単に乳化剤に用い得るのみならず
リポソームの基材として薬剤運搬体、人工血液、
人工細胞等への応用が近年注目されており、ま
た、それ自体生理活性・薬理作用を持つものとし
て、医学・薬学・工学的分野の様々な用途が考え
られている。このような多様な要求に対応するた
めに、各々の用途に応じた構造を有するリン脂質
を効率的に製造する方法を開発することは、産業
上非常に意義あることである。
酵素によるリン脂質の製造方法として、リン脂
質にホスホリパーゼＤを任意の受容体の存在下に
作用させ、ホスフアチジル基転移反応を利用して
目的とする塩基を持つリン脂質を製造する技術は
公知である〔S.F.Yang，et al.，J.Biol.Chem.，
242，(3)477〜484（1967）〕：〔R.M.C.Dawson，
Biochem.J.，102，205〜210（1967）〕。
ホスホリパーゼＤによるホスフアチジル基転移
反応を利用してリン脂質の塩基部分を交換しよう
とする場合、一般に水相と有機溶媒相との二相系
が用いられる。すなわち、主として水溶性である
酵素、受容体、PH緩衝液、無機塩等を含む水溶液
と、主として親油性である原料リン脂質を含む有
機溶媒相とを撹拌・混合する反応系である。前出
の技術をはじめ、その後の研究（K.Bruzik and
M.Tsai，Biochemistry23，(8)1656−1661（1984）
など〕においても広く用いられている。
（発明が解決しようとする問題点）
しかし、従来用いられていたこのような反応系
は、水溶性成分の溶媒としての多量の水の存在が
原因となり、ホスホリパーゼＤが本質的に加水分
解活性を持つているために、副反応として加水分
解が起こり、ホスフアチジン酸（以下PAと略す）
を生成するという欠点を有している。
加水分解によるPAの生成は、反応後の目的リ
ン脂質の分離精製を困難にするばかりでなく、加
水分解反応によつても原料リン脂質が消費される
ため、糖や二級アルコール等の反応性の低い受容
体に対してホスフアチジル基を転移させようとす
る場合、その反応速度が加水分解の反応速度に対
して極端に低いために事実上目的生成物を得るこ
とができなかつた。
このような問題点は、ホスホリパーゼＤ自体が
本来加水分解酵素である以上、水が存在する限り
不可避である。
そこで、本発明者らは、反応系中の水分含量を
酵素が失活しない範囲で極限まで減少させること
によりこの問題を解決すべく、種々検討の結果、
従来水溶液として反応系に添加していた成分を、
担体に吸着あるいは担持せしめて添加する新規な
反応系を見出し、本発明に至つたものである。
（問題点を解決するための手段）
塩基構造が変換されたリン脂質を製造するにあ
たり、原料リン脂質と水酸基を有する受容体と
を、担体に吸着させたホスホリパーゼＤに接触さ
せて、反応系中の水分含量が１重量％以下の状態
で有機溶媒中で反応させることを特徴とする酵素
によるリン脂質の製造方法である。
本発明において用いられる原料リン脂質として
は、ホスホリパーゼＤの基質となり得るものであ
れば、天然から抽出したもの、または抽出後精製
したもの、あるいは合成したものを問わず使用で
きる。また、市販のものあるいは公知の方法で調
製したものを使用しても差し支えない。
例として脱脂大豆レシチン、卵黄レシチン、ホ
スフアチジルコリン（以下PCと略す）、ホスフア
チジルエタノールアミン（以下PEと略す）、ホス
フアチジルセリン（以下PSと略す）、ホスフアチ
ジルグリセロール（以下PGと略す）等またはそ
れらの混合物等があげられる。本発明の効果を最
大に発揮するためには、原料リン脂質として精製
したものないしは組成の単純なものを用いた方が
反応生成物の精製の面で都合が良い。また、原料
コストと入手の容易さ、酵素に対する反応性の面
から特にPC、PEまたはPSが工業的に効果が高
く好ましい。
反応は、原料リン脂質を溶解または懸濁する有
機溶媒の存在下で行うことが好ましく、酵素を失
活させることの少ない溶媒系であればいずれも使
用できる。例として、石油エーテル、ジエチルエ
ーテル、メチルエチルエーテル、ジイソプロピル
エーテル、クロロホルム、ジクロロメタン、四塩
化炭素、ジクロロエタン、ｎ−ヘキサン、シクロ
ヘキサン、ｎ−オクタン、イソオクタン、酢酸エ
チル、ジオキサン、ベンゼン等の溶媒、またはこ
れらの混合溶媒系、またはこれらにアセトン、ア
セトニトリルなどの極性溶媒を配合した混合溶媒
系があげられる。ただしアルコール類は目的反応
の基質となるため、基質として添加する以外に用
いることはあまり好ましくない。
ホスホリパーゼＤとしては、ホスフアチジル基
転移活性を有するものであれば、市販のものある
いは公知の方法で調製したものを問わず使用でき
る。例として、ベーリンガー・マンハイム社
（Boehringer Mannheim GmbH）製のキヤベツ
由来のホスホリパーゼＤ、東洋醸造(株)製の微生物
由来のホスホリパーゼＤ（PLDP）、公知の方法
〔一例としてケーツとサストリイ（M.Kates and
P.S.Sastry）の方法、“Methods in
Enzymology”（J.M.Lowenstein，ed.），vol.14，
pp197−203，Achademic Press，New York
（1969）〕により抽出し精製または部分精製した酵
素標品、または抽出した粗酵素があげられる。
受容体としては、コリン、メタノール、エタノ
ール、エタノールアミン、セリン、グリセロー
ル、グルコース等の従来ホスフアチジル基転移反
応の受容体として知られている化合物のみなら
ず、１−アミノ−２−プロパノール、１−オルソ
メチルグルコシド、トレハロースをはじめとする
従来ホスフアチジル基転移反応の受容体とはなら
ないとされていた糖類を含む一級または二級アル
コール構造を持つ化合物を用いることができる。
ホスホリパーゼＤを吸着あるいは担持させる担
体の材質としては活性炭、活性白土、ケイ酸、シ
リカゲル、ケイ藻土、ゼオライト、アルミナ、多
孔質ガラス、陶器、磁器、樹脂などがあげられ、
形状としては粒径0.02〜0.5mm程度の粒状あるい
はビーズ状が好ましい。またこれらの担体は、原
料リン脂質および受容体を吸着あるいは担持させ
ることもできる。
ホスホリパーゼＤおよび受容体を担体に吸着あ
るいは担持させる方法の例としては、該成分の水
溶液を担体と接触させ、ろ別後過剰の水を凍結乾
燥し、あるいはジエチルエーテルまたはクロロホ
ルム等の酵素を失活させない乾燥有機溶媒を繰り
返しまたは連続的に接触させることにより除去す
る方法、あるいは粉末状にした該成分と担体とを
混合したところへ微量の水を加えて更に混合する
方法などがある。
酵素、受容体その他の成分を吸着または担持さ
せた担体と、原料リン脂質を溶解または懸濁した
有機溶媒とを接触させる方法としては、容器中で
担体を溶媒系中に分散、懸濁し撹拌する方法、ま
たは担体をカラムなどに充填し溶媒系を循環させ
る方法などがある。
原料リン脂質を担体に吸着あるいは担持させる
方法の例としては、低極性有機溶媒に溶解した原
料リン脂質を担体と接触し吸着させた後、濾過に
より溶媒を除去する方法、あるいは原料リン脂質
を溶解した有機溶媒を担体に含浸させた後、溶媒
を溜去する方法などがある。
反応系全体の水分含量は、１％以下に抑えるこ
とにより加水分解反応が従来の反応系に比べて抑
制できるが、更に0.5％以下に抑えることが好ま
しい。特に、水分含量を0.2％以下に制御するこ
とにより従来の反応系では反応し得なかつた受容
体をも反応させることができ、本発明の効果が最
大に発揮される。
反応温度は用いる酵素の至適温度であればよ
く、通常30〜40℃の範囲である。ただし、用いる
溶媒が低沸点のものである場合等はこの限りでは
ない。
反応時間は0.5〜36時間で、好ましくは４〜24
時間である。
このようにして製造した任意の塩基を持つ目的
リン脂質は溶材分画、ケイ酸またはシリカゲルク
ロマトグラフイー、アルミナクロマトグラフイ
ー、DEAE−セルロースクロマトグラフイー等の
公知の手段を適宜用いることにより、容易に精製
することができる。
（発明の効果）
本発明は、反応系中の水分含量を酵素が失活し
ない範囲で極限まで減少させるため、本質的に非
水系の反応系を用いるので、従来の反応系で見ら
れたようなPAの大量の生成は抑制され、反応後
の目的リン脂質の分離精製が容易になり収率が向
上した。
更に、従来の反応系では得ることのできなかつ
た多くの種類の目的リン脂質をも製造することが
可能となつた。
（実施例）
以下、参考例、実施例、および比較例に基づい
て本発明を具体的に説明する。
なお、リン脂質の組成分析、純度検定は薄層ク
ロマトグラフイー（TLC）で行つた。TLC板
（メルク社製 No.5721）に脂質試料20〜100μｇを
直径３〜５mmにスポツトし、クロロホルム−メタ
ノール−水（120：70：５）またはクロロホルム
−アセトン−酢酸−メタノール−水（50：20：
15：10：５）で展開した。検出にはジツトマー試
薬、50％硫酸、ニンヒドリン試薬またはアンスロ
ン試薬を目的に応じて使用した。定量的な測定に
はジツトマー試薬で発色したものを高速薄層クロ
マトスキヤナー（島津製作所製CS−920型）で測
定した。
また、反応生成物の同定は、特に記さない限
り、標準品とのTLC上でのRf値の比較と各種試
薬に対する発色反応で行つた。反応系中の水分は
カールフイツシヤー法により測定した。
参考例 １
ホウレン草のホスホリパーゼＤを前出の公知の
方法の一例、ケーツとサストリイ（M.Kates
and P.S.Sastry）の方法に従つて抽出した。
近在の農家から入手した新鮮なホウレン草（パ
レード種）を水洗後細断し、100ｇに水200mlを加
え、氷冷下で５分間ホモジナイズした。５重にし
たガーゼで濾過した濾液を４℃で2000×ｇ、15分
間遠心分離し、上清210mlを得た。この上清を４
℃で水１に対し３回透析し、４℃で10000×ｇ、
15分間遠心分離した上清195mlをホウレン草粗酵
素液として用いた。なお、この粗酵素液には検出
可能量のリン脂質は含まれていないことを確認し
た。
参考例 ２
大豆PCおよびPEをパルダン（Von H.
Pardun）の方法〔Fette Seifen Anstrichmitte
86，(2)55−62（1984）〕により分離、分画した。
市販脱脂大豆レシチン粉末（PC24％、PE21
％、ホスフアチジルイノシトール14％、PA8％）
20ｇをイソプロパノール−メタノール−水（50：
45：５）100mlに分散し、40℃で加熱撹拌し溶解
した。撹拌しながら20℃まで冷却し、20℃に１時
間保つた。不溶物を20℃に保つたまま遠心分離ま
たはガラスフイルターで減圧濾過した。集めた上
清を減圧下で乾固し、PCおよびPE濃縮物
（PC68％、PE17％、PA7％、PSは含まない）9.7
ｇを得た。
参考例 ３
卵黄レシチンから常法によりPCを精製した。
市販卵黄レシチン（PC67％、PE19％、PA8
％、リゾPC3％）10ｇをシリカゲルカラム（径
3.8cm×60cm）を用いて分画した。溶出溶媒とし
てクロロホルム−メタノール（５：１）1.5、
クロロホルム−メタノール（３：１）３を流
し、PCを多く含む画分を集め、減圧乾固してPC
（PC97％）4.3ｇを得た。
参考例 ４
精製卵黄PCを多孔質ガラスに担持させた。
参考例３で得た精製卵黄PC500mgを100mlのｎ
−ヘキサンに溶かし、直径0.5mm以下に砕いた多
孔質ガラス５ｇに含浸させた後、ｎ−ヘキサンを
減圧乾固して担持させた。
参考例 ５
牛脳からリーズ（M.Lees）の方法〔“Methods
in Enzymology”（S.P.Colowick and N.O.
Kaplan ed.），vol.3，pp328，Achademic
Press，New York（1957）〕により粗セフアリン
を抽出し、DEAE−セルロースカラムクロマトグ
ラフイーで精製した。
近在の屠殺場で入手した新鮮な牛脳の脳膜およ
び血管を取り除いたもの300ｇを1.2のアセトン
中でホモジナイズし、抽出した。濾過残渣をもう
一度1.2のアセトンで抽出する。濾過残渣を1.2
のエタノールで抽出する。濾過残渣を同様にし
て1.2の石油エーテルで２回抽出し、抽出液を
集めて減圧乾固し、粗セフアリン画分3.9ｇを得
た。
このものをクロロホルムに溶解し、酢酸型に調
製したDEAE−セルロースカラム（ワツトマン社
製DE32、径2.5cm×20cm）を用いて分画した。ク
ロロホルム−メタノール（１：４）１でカラム
を洗浄後、酢酸750mlで溶出した画分を集めた。
酢酸溶出画分に等容のクロロホルムを加え、２倍
容の水で４回洗浄した。クロロホルム層を減圧乾
固し、PS（PS98％）0.8ｇを得た。
参考例 ６
市販ケイ藻土を水および溶剤で洗浄し精製し
た。
No.503セライト（ジヨンズ・マンビル・セイル
ズ社製、商品名）100ｇを２の水に懸濁し洗浄
すると同時にデカンテーシヨンにより微粒子を除
去した。同様にして更に水で２回、メタノールで
１回、クロロホルムで１回洗浄し、最後にアセト
ン500mlに懸濁したものを減圧濾過した。風乾後
120℃で５時間乾燥し、精製セライト74ｇを得た。
実施例 １
Ｌ−セリン50ｇ、キヤベツホスホリパーゼＤ
（ベーリンガー・マンハイム社製）0.5ｇ、塩化カ
ルシウム・二水塩0.25ｇを500mlの５ｍＭ酢酸緩
衝液PH5.6に溶かし、特級活性炭（和光純薬(株)製）
40ｇを加え、室温で30分間撹拌した。濾別後、８
時間凍結乾燥し、酵素、受容体その他を吸着させ
た活性炭53.7ｇを得た。
参考例２で得た大豆PCおよびPE濃縮物８ｇを
500mlのジエチルエーテルに溶かし、酵素、受容
体その他を吸着させた活性炭53.5ｇを加え分散さ
せた。密閉容器中で37℃に保温し、17時間
500rpmで撹拌して反応させた。反応系中の水分
は0.1重量％であつた。
反応溶媒を濾過により回収し、活性炭は200ml
のクロロホルムで３回洗つた。反応溶媒と洗液を
合わせて減圧乾固し、リン脂質混合物（PC8％、
PE8％、PA9％、PS72％）6.9ｇを得た。
このものをクロロホルムに溶かしてDEAE−セ
ルロースカラム（ワツトマン社製DE32、径3.8×
27cm）で分画した。カラムをクロロホルム−メタ
ノール（１：４）1.5で洗浄後、酢酸1.2で溶
出した画分から参考例５に示したと同様にして
PS（PS99％）3.4ｇを回収した。
実施例 ２
塩酸でPH5.0に調整した1Mエタノールアミン水
溶液25mlと参考例１で調製したホウレン草粗酵素
液25mlの混合液に参考例６で得た精製セライト
2.5ｇを加え、室温で30分間撹拌した。濾別後少
量のジイソプロピルエーテルに懸濁し、カラム
（径１×3.5cm）に充填した。乾燥したジイソプロ
ピルエーテル2.5を流して過剰の水を吸収、除
去した後、市販卵黄レシチン（PC67％、PE19
％、PA8％、リゾPC3％）３ｇを溶かしたジイソ
プロピルエーテル20mlを定量ポンプを用いて流速
0.3〜0.5ml／分、30℃で６時間循環させ、反応さ
せた。反応系中の水分は0.9重量％であつた。
反応溶媒を回収し、カラムを10mlのクロロホル
ムで３回洗つた。反応溶媒と洗液を合わせて減圧
乾固し、リン脂質混合物（PC1％、PE85％、
PA10％、リゾPC2％）2.7ｇを得た。
このものをクロロホルムに溶かしてシリカゲル
カラム（径2.5×40cm）で分画した。クロロホル
ム−メタノール−水（65：25：２）２で溶出
し、PEを含む画分を集めて減圧乾固し、PE
（PE97％）1.6ｇを得た。
実施例 ３
塩酸でPH5.5に調整した333ｍM1−アミノ−２
−プロパノール水溶液２mlにキヤベツホスホリパ
ーゼＤ（ベーリンガー・マンハイム社製）50mg、
塩化カルシウム・二水塩0.5mgを溶解し、参考例
６で得た精製セライト200mgを加え、15分間よく
振り混ぜた。12000×ｇで15分間遠心分離した沈
殿を６時間凍結乾燥し、酵素、受容体その他を吸
着させたセライト278mgを得た。
10mlのｎ−ヘキサン−アセトン（95：５）に参
考例４で得た精製卵黄PCを担持させた多孔質ガ
ラス600mg、および酵素、受容体その他を吸着さ
せたセライト276mgを懸濁、分散した。37℃で24
時間500rpmで撹拌し、反応させた。反応系中の
水分は0.1重量％であつた。
反応溶媒を濾過により回収し、担体は５mlのク
ロロホルムで３回洗つた。反応溶媒と洗液を合わ
せて減圧乾固し、リン脂質混合物42mgを得た。
このものにはPA4％が含まれるが、PCは検出
されず、残りの94％を占めるものはジツトマー試
薬にもニンヒドリン試薬にも陽性で、TLC上の
Rf値が標準PEときわめて近いことから、１−ア
ミノ−２−プロパノールにホスフアチジル基が導
入された目的リン脂質であると判定した。
実施例 ４
参考例１で得たホウレン草粗酵素液を凍結乾燥
した粉末100mgと活性炭300mgとをよく混ぜ、グリ
セロール（水分３％）60mgを加えてよく練り混ぜ
た。このものを参考例５で得た牛脳PS50mgを溶
かした15mlのクロロホルム中に分散し、35℃で５
時間500rpmで撹拌し、反応させた。反応系中の
水分は0.5重量％であつた。
反応溶媒を濾過により回収し、活性炭は５mlの
クロロホルムで３回洗つた。反応溶媒と洗液を合
わせて減圧乾固し、リン脂質混合物（PS28％、
PA3％、PG67％）44mgを得た。
実施例 ５
１−オルソメチルグルコシド600mg、ホスホリ
パーゼＤ（東洋醸造(株)製PLDP）12μｇを５mlの５
ｍＭ酢酸緩衝液PH5.6に溶かし、活性炭１ｇに実
施例３と同様にして吸着させ、凍結乾燥した。
ジパルミトイルPC45mgを溶かしたクロロホル
ム−イソオクタン（１：１）15mlに酵素、受容体
等を吸着させた活性炭950mgを分散し、38℃で12
時間500rpmで撹拌して反応させた。反応系中の
水分は0.2重量％であつた。
実施例４と同様にしてリン脂質混合物48mgを得
た。
このものをNo.5745分取用TLCプレート（メル
ク社製）を用い、クロロホルム−メタノール−水
（120：70：５）を展開溶媒として分画、分取し、
未同定リン脂質29mgを得た。
このリン脂質をJMS−DX303型質量分析装置
（日本電子(株)製）を用い、下記条件にて分析した
ところ、陽イオン側の親ピークがｍ／e847、陰イ
オン側の親ピークがｍ／e823であつた。
測定条件
イオン化法 ：FAB法
衝撃ガス ：Xe
一次イオン加速電圧 ：6kV
フイラメント電流： 20ｍＡ
検出器 ：コンバーシヨン・ダイノード
押し出し電圧 ：15kV
マトリクス：トリエタノールアミン（陽イオンの
場合塩化ナトリウム添加）
データ処理 ：JMA−DA5000
これらの値は１−オルソメチルグルコシドにホ
スフアチジル基が導入されたと仮定した分子量
（824）の各々ナトリウム塩（分子量824＋23）お
よび陰イオン（分子量824−１）と一致したこと
から、目的リン脂質であると同定した。
実施例 ６
トレハロース１ｇとホスホリパーゼＤ（東洋醸
造(株)製PLDP）10μｇを５mlの水に溶かし、乾燥
した市販のビーズ状スチレン−ジビニルベンゼン
樹脂を加えて４℃で５時間撹拌し、含浸させた。
濾別後18時間凍結乾燥したものをジパルミトイル
PC45mgを溶かした乾燥したジクロロメタン20ml
中に分散させ、37℃で24時間、40rpmで回転浸盪
し、反応させた。反応系中の水は0.1重量％であ
つた。
反応溶媒を濾過により回収し、樹脂を20mlのジ
クロロメタン３回洗つた。反応溶媒と洗液とを合
わせて減圧乾固し、リン脂質混合物43mgを得た。
このものはPC38％、PA5％を含んでいたが、
54％を占めるリン脂質はジツトマー試薬とアンス
ロン試薬に陽性であることから、トレハロースに
ホスフアチジル基が導入された目的リン脂質であ
ると判定した。
更に、実施例５と同様にして一部をTLCで分
取し、質量分析を行つたところ、陽イオン側親ピ
ークｍ／e995、陰イオン側親ピークｍ／e971で、
計算分子量（972）の各々ナトリウム塩（分子量
972＋23）、陰イオン（分子量972−１）と一致し
たことから、目的リン脂質であると同定した。
比較例 １
従来の反応系で、実施例１とできるだけ近い条
件で反応を行つた。
Ｌ−セリン200ｍＭ、塩化カルシウム40ｍＭ、
ホスホリパーゼＤ（ベーリンガー・マンハイム社
製）2.5mgを含むPH5.6の50ｍＭ酢酸緩衝液５ml
と、参考例２で得た大豆PCおよびPEの濃縮物40
mgを溶かしたジエチルエーテル５mlを37℃、
500rpmで撹拌した。
同一のもの５検体を平行して各１、２、４、
８、16時間反応させた。反応後、５mlのジエチル
エーテルで３回脂質を抽出し、リン脂質組成を分
析した。結果を第１表に示す。
【表】
比較例 ２
従来の反応系で、実施例２とできるだけ近い条
件で反応を行つた。
塩酸でPH5.0に調整した1Mエタノールアミン水
溶液0.35mlと参考例１で得たホウレン草粗酵素液
0.375ml、水4.275ml、市販卵黄レシチン45mgを溶
かしたジイソプロピルエーテル５mlを30℃、
500rpmで撹拌した。
同一のもの４検体を平行して各１、２、４、６
時間反応させ、比較例１と同様にして分析した。
結果を第２表に示す。
【表】
比較例 ３
従来の反応系で、実施例５とできるだけ近い条
件で反応を行つた。
１−オルソ−メチルグルコシド600mg、ホスホ
リパーゼＤ（東洋醸造(株)製PLDP）12μｇを溶かし
たPH5.6の50ｍＭ酢酸緩衝液５mlとジパルミトイ
ルPC45mgを溶かしたクロロホルム−イソオクタ
ン（１：１）５mlを38℃、500rpmで撹拌し、反
応させた。
比較例１および２と同様にして経時的に分析し
たが、実施例５で認められた目的リン脂質に相当
するスポツトがTLC上に認められず、８時間反
応した時点で基質が完全に分解されたため、分析
を打ち切つた。
比較例 ４
実施例１に準じた方法でキヤベツホスホリパー
ゼＤのみを活性炭に吸着させて凍結乾燥した。上
記で得た活性炭吸着ホスホリパーゼＤを用いて、
反応系中の水分を10重量％にした以外は比較例１
と同様にして反応させ、同様に分析した。結果を
第３表に示す。
【表】
各比較例の結果から明らかなように、反応系中
に水分が大量に存在すると、時間の経過と共に加
水分解が著しく進行し、PAの量が増大して、目
的物がほとんど得られないことがわかる。 DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing phospholipids using an enzyme, and particularly to a method for producing phospholipids whose base structure has been converted. (Prior art) Phospholipids can be used not only as emulsifiers but also as base materials for liposomes, drug carriers, artificial blood,
Applications to artificial cells have attracted attention in recent years, and as they themselves have physiological activity and pharmacological effects, they are being considered for various uses in the medical, pharmaceutical, and engineering fields. In order to meet such diverse demands, it is of great industrial significance to develop a method for efficiently producing phospholipids having structures suitable for each use. As a method for producing phospholipids using an enzyme, a technique is known in which phospholipase D is applied to phospholipids in the presence of an arbitrary receptor, and a phospholipid having a target base is produced using a phosphatidyl group transfer reaction. [SFYang, et al., J.Biol.Chem.,
242, (3) 477-484 (1967)]: [RMCDawson,
Biochem.J., 102, 205-210 (1967)]. When attempting to exchange the base moiety of a phospholipid using a phosphatidyl group transfer reaction by phospholipase D, a two-phase system of an aqueous phase and an organic solvent phase is generally used. That is, it is a reaction system in which an aqueous solution containing mainly water-soluble enzymes, receptors, PH buffers, inorganic salts, etc., and an organic solvent phase containing mainly lipophilic raw material phospholipids are stirred and mixed. In addition to the above-mentioned technology, subsequent research (K.Bruzik and
M. Tsai, Biochemistry 23 , (8) 1656-1661 (1984)
etc.] is also widely used. (Problems to be Solved by the Invention) However, in such a conventionally used reaction system, the presence of a large amount of water as a solvent for water-soluble components essentially causes phospholipase D to have no hydrolytic activity. As a result, hydrolysis occurs as a side reaction, producing phosphatidic acid (hereinafter abbreviated as PA).
It has the disadvantage of generating . The generation of PA through hydrolysis not only makes it difficult to separate and purify the target phospholipid after the reaction, but also because the raw material phospholipid is consumed in the hydrolysis reaction, which reduces the reactivity of sugars, secondary alcohols, etc. When attempting to transfer a phosphatidyl group to a receptor with a low hydrolysis reaction rate, it has been virtually impossible to obtain the desired product because the reaction rate is extremely low compared to the hydrolysis reaction rate. Such problems are unavoidable as long as water is present, since phospholipase D itself is originally a hydrolytic enzyme. Therefore, the present inventors have conducted various studies in order to solve this problem by reducing the water content in the reaction system to the limit without deactivating the enzyme.
Components that were conventionally added to the reaction system as an aqueous solution,
The present invention was achieved by discovering a new reaction system in which the compound is added by being adsorbed or supported on a carrier. (Means for solving the problem) In producing a phospholipid whose base structure has been changed, a raw material phospholipid and a receptor having a hydroxyl group are brought into contact with phospholipase D adsorbed on a carrier, and the reaction system is This is a method for producing phospholipids using an enzyme, characterized in that the reaction is carried out in an organic solvent with a water content of 1% by weight or less. The raw material phospholipid used in the present invention may be any phospholipid extracted from nature, purified after extraction, or synthesized, as long as it can serve as a substrate for phospholipase D. Moreover, commercially available products or products prepared by known methods may be used. Examples include defatted soybean lecithin, egg yolk lecithin, phosphatidylcholine (hereinafter abbreviated as PC), phosphatidylethanolamine (hereinafter abbreviated as PE), phosphatidylserine (hereinafter abbreviated as PS), phosphatidylglycerol (hereinafter abbreviated as PG). ) or a mixture thereof. In order to maximize the effects of the present invention, it is more convenient to use purified phospholipids or those with a simple composition as raw material phospholipids in terms of purification of the reaction product. In addition, from the viewpoint of raw material cost, ease of availability, and reactivity with enzymes, PC, PE, or PS are particularly preferred because they are highly effective industrially. The reaction is preferably carried out in the presence of an organic solvent that dissolves or suspends the raw material phospholipid, and any solvent system that does not deactivate the enzyme can be used. Examples include solvents such as petroleum ether, diethyl ether, methyl ethyl ether, diisopropyl ether, chloroform, dichloromethane, carbon tetrachloride, dichloroethane, n-hexane, cyclohexane, n-octane, isooctane, ethyl acetate, dioxane, benzene, etc. or a mixed solvent system in which a polar solvent such as acetone or acetonitrile is blended with these solvents. However, since alcohols serve as substrates for the desired reaction, it is not very preferable to use them for purposes other than adding them as substrates. As phospholipase D, any phospholipase D can be used, as long as it has phosphatidyl group transfer activity, regardless of whether it is commercially available or prepared by a known method. Examples include cabbage-derived phospholipase D manufactured by Boehringer Mannheim GmbH, microbial-derived phospholipase D (PLDP) manufactured by Toyo Jozo Co., Ltd., and known methods [for example, M. Kates and
“Methods in
Enzymology” (JMLowenstein, ed.), vol.14,
pp197−203, Academic Press, New York
(1969)] and purified or partially purified enzyme preparations, or extracted crude enzymes. Receptors include not only compounds conventionally known as receptors for phosphatidyl group transfer reactions such as choline, methanol, ethanol, ethanolamine, serine, glycerol, and glucose, but also 1-amino-2-propanol, 1-ortho Compounds having a primary or secondary alcohol structure containing sugars, such as methyl glucoside and trehalose, which were conventionally considered not to be receptors for phosphatidyl group transfer reactions, can be used. Examples of the carrier material that adsorbs or supports phospholipase D include activated carbon, activated clay, silicic acid, silica gel, diatomaceous earth, zeolite, alumina, porous glass, earthenware, porcelain, and resin.
The shape is preferably granular or bead-like with a particle size of about 0.02 to 0.5 mm. These carriers can also adsorb or support raw material phospholipids and receptors. Examples of methods for adsorbing or supporting phospholipase D and receptors on a carrier include contacting an aqueous solution of the components with the carrier, lyophilizing excess water after filtration, or deactivating the enzyme using diethyl ether or chloroform. There are methods for removing the components by repeatedly or continuously bringing them into contact with dry organic solvents that do not cause oxidation, and methods for further mixing by adding a small amount of water to a mixture of the powdered component and the carrier. A method for bringing a carrier adsorbed or loaded with enzymes, receptors, and other components into contact with an organic solvent in which a raw material phospholipid is dissolved or suspended is to disperse or suspend the carrier in a solvent system in a container and stir it. method, or method of filling a column with a carrier and circulating the solvent system. Examples of methods for adsorbing or supporting raw material phospholipids on a carrier include methods in which raw material phospholipids dissolved in a low polar organic solvent are brought into contact with the carrier and adsorbed, and then the solvent is removed by filtration, or methods in which raw material phospholipids are dissolved There is a method in which a carrier is impregnated with an organic solvent and then the solvent is distilled off. By suppressing the water content of the entire reaction system to 1% or less, the hydrolysis reaction can be suppressed compared to conventional reaction systems, but it is preferable to further suppress the water content to 0.5% or less. In particular, by controlling the water content to 0.2% or less, receptors that could not react in conventional reaction systems can be reacted, and the effects of the present invention are maximized. The reaction temperature may be the optimum temperature for the enzyme used, and is usually in the range of 30 to 40°C. However, this does not apply when the solvent used has a low boiling point. The reaction time is 0.5 to 36 hours, preferably 4 to 24 hours.
It's time. The desired phospholipid having an arbitrary base produced in this manner can be easily obtained by appropriately using known means such as solvent fractionation, silicic acid or silica gel chromatography, alumina chromatography, DEAE-cellulose chromatography, etc. It can be refined into (Effects of the Invention) The present invention uses an essentially non-aqueous reaction system in order to reduce the water content in the reaction system to the limit without deactivating the enzyme, so it is different from that seen in conventional reaction systems. The production of a large amount of PA was suppressed, and the separation and purification of the target phospholipid after the reaction was facilitated, resulting in an improved yield. Furthermore, it has become possible to produce many types of target phospholipids that could not be obtained using conventional reaction systems. (Example) Hereinafter, the present invention will be specifically described based on Reference Examples, Examples, and Comparative Examples. The compositional analysis and purity test of phospholipids were performed using thin layer chromatography (TLC). Spot 20 to 100 μg of a lipid sample to a diameter of 3 to 5 mm on a TLC plate (No. 5721, manufactured by Merck & Co., Ltd.), and add chloroform-methanol-water (120:70:5) or chloroform-acetone-acetic acid-methanol-water (50:5). 20:
15:10:5). For detection, Zittmer reagent, 50% sulfuric acid, ninhydrin reagent, or Anthrone reagent was used depending on the purpose. For quantitative measurements, the color developed with the Zittomer reagent was measured using a high-speed thin-layer chromatography scanner (Shimadzu Model CS-920). In addition, unless otherwise specified, reaction products were identified by comparing Rf values with standard products on TLC and by color reaction with various reagents. The water content in the reaction system was measured by the Karl Fischer method. Reference Example 1 An example of the above-mentioned known method for preparing spinach phospholipase D, M. Kates
and PSSastry). Fresh spinach (Parade variety) obtained from a nearby farmer was washed with water and shredded, 200 ml of water was added to 100 g, and homogenized for 5 minutes under ice cooling. The filtrate was filtered through 5 layers of gauze and centrifuged at 2000 xg for 15 minutes at 4°C to obtain 210 ml of supernatant. Add this supernatant to 4
Dialyzed 3 times against 1 part water at ℃, 10000 x g at 4℃,
195 ml of the supernatant obtained by centrifugation for 15 minutes was used as a spinach crude enzyme solution. It was confirmed that this crude enzyme solution did not contain a detectable amount of phospholipid. Reference example 2 Soybean PC and PE were mixed with Paludan (Von H.
Pardun) method [Fette Seifen Anstrichmitte]
86, (2) 55-62 (1984)]. Commercially available defatted soybean lecithin powder (PC24%, PE21
%, phosphatidylinositol 14%, PA 8%)
20g isopropanol-methanol-water (50:
45:5) Dispersed in 100 ml and heated and stirred at 40°C to dissolve. It was cooled to 20°C with stirring and kept at 20°C for 1 hour. The insoluble matter was centrifuged or filtered under reduced pressure using a glass filter while maintaining the temperature at 20°C. The collected supernatant was dried under reduced pressure and the PC and PE concentrate (68% PC, 17% PE, 7% PA, no PS) 9.7
I got g. Reference Example 3 PC was purified from egg yolk lecithin by a conventional method. Commercially available egg yolk lecithin (PC67%, PE19%, PA8
%, LysoPC3%) was added to a silica gel column (diameter
3.8cm x 60cm). Chloroform-methanol (5:1) 1.5 as elution solvent,
Flow chloroform-methanol (3:1), collect fractions containing a lot of PC, and dry under reduced pressure to remove PC.
(PC97%) 4.3g was obtained. Reference Example 4 Purified egg yolk PC was supported on porous glass. 500 mg of purified egg yolk PC obtained in Reference Example 3 was added to 100 ml of n
- After dissolving in hexane and impregnating 5 g of porous glass crushed to a diameter of 0.5 mm or less, n-hexane was dried under reduced pressure to be supported. Reference example 5 M.Lees method from cow brain [“Methods
in Enzymology” (SPColowick and NO.
Kaplan ed.), vol.3, pp328, Academic
Press, New York (1957)] and purified by DEAE-cellulose column chromatography. 300 g of fresh bovine brain membranes and blood vessels removed obtained from a local slaughterhouse were homogenized in 1.2 acetone and extracted. The filter residue is extracted once again with 1.2 parts of acetone. 1.2 filtration residue
Extract with ethanol. The filtration residue was extracted twice with 1.2 petroleum ether in the same manner, and the extracts were collected and dried under reduced pressure to obtain 3.9 g of crude cephalin fraction. This product was dissolved in chloroform and fractionated using a DEAE-cellulose column prepared in an acetic acid form (DE32 manufactured by Watmann, diameter 2.5 cm x 20 cm). After washing the column with chloroform-methanol (1:4), the fractions eluted with 750 ml of acetic acid were collected.
An equal volume of chloroform was added to the acetic acid elution fraction, and the mixture was washed four times with 2 volumes of water. The chloroform layer was dried under reduced pressure to obtain 0.8 g of PS (PS98%). Reference Example 6 Commercially available diatomaceous earth was purified by washing with water and a solvent. 100 g of Celite No. 503 (manufactured by John's Manville Sails, trade name) was suspended in water from step 2, washed, and at the same time fine particles were removed by decantation. In the same manner, it was further washed twice with water, once with methanol, and once with chloroform, and finally, the suspension in 500 ml of acetone was filtered under reduced pressure. After air drying
It was dried at 120°C for 5 hours to obtain 74g of purified Celite. Example 1 L-serine 50g, cabbage phospholipase D
(manufactured by Boehringer Mannheim) and 0.25 g of calcium chloride dihydrate were dissolved in 500 ml of 5mM acetate buffer PH5.6, and special grade activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved.
40 g was added and stirred at room temperature for 30 minutes. After filtering, 8
The product was freeze-dried for hours to obtain 53.7 g of activated carbon adsorbed with enzymes, receptors, etc. 8 g of soybean PC and PE concentrate obtained in Reference Example 2
The mixture was dissolved in 500 ml of diethyl ether, and 53.5 g of activated carbon adsorbed with enzymes, receptors, etc. was added and dispersed. Keep warm at 37℃ in a sealed container for 17 hours.
The reaction was stirred at 500 rpm. The water content in the reaction system was 0.1% by weight. Collect the reaction solvent by filtration, and collect 200 ml of activated carbon.
Washed three times with chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to form a phospholipid mixture (PC8%,
PE8%, PA9%, PS72%) 6.9g was obtained. Dissolve this in chloroform and use a DEAE-cellulose column (Watmann DE32, diameter 3.8 x
27 cm). After washing the column with chloroform-methanol (1:4) 1.5, the fraction eluted with acetic acid 1.2 was purified in the same manner as shown in Reference Example 5.
3.4 g of PS (PS99%) was recovered. Example 2 The purified celite obtained in Reference Example 6 was added to a mixture of 25 ml of 1M ethanolamine aqueous solution adjusted to pH 5.0 with hydrochloric acid and 25 ml of the spinach crude enzyme solution prepared in Reference Example 1.
2.5 g was added and stirred at room temperature for 30 minutes. After filtration, the suspension was suspended in a small amount of diisopropyl ether and packed into a column (diameter 1 x 3.5 cm). After absorbing and removing excess water by running dry diisopropyl ether 2.5, commercially available egg yolk lecithin (PC67%, PE19
%, PA8%, LysoPC3%) dissolved in 20ml of diisopropyl ether using a metering pump to adjust the flow rate.
The mixture was circulated at 0.3 to 0.5 ml/min at 30° C. for 6 hours to react. The water content in the reaction system was 0.9% by weight. The reaction solvent was collected and the column was washed three times with 10 ml of chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to form a phospholipid mixture (PC1%, PE85%,
2.7 g of PA10%, LysoPC2%) was obtained. This product was dissolved in chloroform and fractionated using a silica gel column (diameter 2.5 x 40 cm). Elute with chloroform-methanol-water (65:25:2) 2, collect the fractions containing PE, and dry under reduced pressure.
(PE97%) 1.6g was obtained. Example 3 333mM 1-amino-2 adjusted to PH5.5 with hydrochloric acid
- 50 mg of cabbage phospholipase D (manufactured by Boehringer Mannheim) in 2 ml of propanol aqueous solution,
0.5 mg of calcium chloride dihydrate was dissolved, 200 mg of the purified celite obtained in Reference Example 6 was added, and the mixture was thoroughly shaken for 15 minutes. The precipitate obtained by centrifugation at 12,000×g for 15 minutes was freeze-dried for 6 hours to obtain 278 mg of Celite adsorbed with enzymes, receptors, and the like. In 10 ml of n-hexane-acetone (95:5), 600 mg of the porous glass supporting purified egg yolk PC obtained in Reference Example 4 and 276 mg of Celite adsorbed with enzymes, receptors, etc. were suspended and dispersed. 24 at 37℃
The mixture was stirred at 500 rpm for a reaction time. The water content in the reaction system was 0.1% by weight. The reaction solvent was collected by filtration and the carrier was washed three times with 5 ml of chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to obtain 42 mg of a phospholipid mixture. This contains 4% of PA, but no PC was detected, and the remaining 94% was positive for both Zyttomer and Ninhydrin reagents, and was detected by TLC.
Since the Rf value was very close to that of standard PE, it was determined that the target phospholipid was 1-amino-2-propanol with a phosphatidyl group introduced. Example 4 100 mg of freeze-dried powder of the spinach crude enzyme solution obtained in Reference Example 1 was thoroughly mixed with 300 mg of activated carbon, and 60 mg of glycerol (water content 3%) was added and mixed well. This material was dispersed in 15 ml of chloroform in which 50 mg of bovine brain PS obtained in Reference Example 5 was dissolved, and the mixture was heated to 35°C for 50 minutes.
The mixture was stirred at 500 rpm for a reaction time. The water content in the reaction system was 0.5% by weight. The reaction solvent was collected by filtration and the activated carbon was washed three times with 5 ml of chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to form a phospholipid mixture (PS28%,
44 mg (PA3%, PG67%) was obtained. Example 5 600 mg of 1-orthomethyl glucoside and 12 μg of phospholipase D (PLDP manufactured by Toyo Jozo Co., Ltd.) were added to 5 ml of 5
It was dissolved in mM acetate buffer pH 5.6, adsorbed onto 1 g of activated carbon in the same manner as in Example 3, and freeze-dried. 950 mg of activated carbon adsorbed with enzymes, receptors, etc. was dispersed in 15 ml of chloroform-isooctane (1:1) in which 45 mg of dipalmitoyl PC was dissolved, and the mixture was heated at 38℃ for 12 hours.
The reaction was stirred at 500 rpm for a time. The water content in the reaction system was 0.2% by weight. 48 mg of a phospholipid mixture was obtained in the same manner as in Example 4. This material was fractionated using a No. 5745 preparative TLC plate (manufactured by Merck & Co., Ltd.) using chloroform-methanol-water (120:70:5) as a developing solvent.
29 mg of unidentified phospholipid was obtained. When this phospholipid was analyzed using a JMS-DX303 mass spectrometer (manufactured by JEOL Ltd.) under the following conditions, the parent peak on the cation side was m/e847, and the parent peak on the anion side was m/e847. It was e823. Measurement conditions Ionization method: FAB method Impact gas: Xe Primary ion acceleration voltage: 6kV Filament current: 20mA Detector: Conversion dynode extrusion voltage: 15kV Matrix: Triethanolamine (sodium chloride added for cations) Data processing: JMA -DA5000 These values were consistent with the sodium salt (molecular weight 824 + 23) and anion (molecular weight 824-1) of the molecular weight (824) assuming that a phosphatidyl group was introduced into 1-orthomethylglucoside. It was identified that Example 6 1 g of trehalose and 10 μg of phospholipase D (PLDP manufactured by Toyo Jozo Co., Ltd.) were dissolved in 5 ml of water, and dried commercially available beaded styrene-divinylbenzene resin was added and stirred at 4° C. for 5 hours to impregnate. .
Dipalmitoyl is lyophilized for 18 hours after filtration.
20ml of dry dichloromethane with 45mg of PC dissolved in it
The solution was dispersed in the solution and reacted by rotating and shaking at 40 rpm for 24 hours at 37°C. Water in the reaction system was 0.1% by weight. The reaction solvent was collected by filtration and the resin was washed three times with 20 ml of dichloromethane. The reaction solvent and washing liquid were combined and dried under reduced pressure to obtain 43 mg of a phospholipid mixture. This one contained 38% PC and 5% PA,
Since the phospholipids that accounted for 54% were positive to Zitutomer reagent and Anthrone reagent, it was determined that the target phospholipids were trehalose with a phosphatidyl group introduced. Furthermore, when a portion was fractionated by TLC and subjected to mass spectrometry in the same manner as in Example 5, the parent peak on the cation side was m/e995, the parent peak on the anion side was m/e971,
Calculated molecular weight (972) of each sodium salt (molecular weight
972+23) and an anion (molecular weight 972-1), it was identified as the target phospholipid. Comparative Example 1 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 1. L-serine 200mM, calcium chloride 40mM,
5 ml of 50 mM acetate buffer, pH 5.6, containing 2.5 mg of phospholipase D (manufactured by Boehringer Mannheim)
and soybean PC and PE concentrate 40 obtained in Reference Example 2.
5 ml of diethyl ether dissolved in
Stirred at 500 rpm. 5 identical samples in parallel, 1, 2, 4,
The reaction was allowed to proceed for 8 and 16 hours. After the reaction, lipids were extracted three times with 5 ml of diethyl ether and the phospholipid composition was analyzed. The results are shown in Table 1. [Table] Comparative Example 2 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 2. 0.35ml of 1M ethanolamine aqueous solution adjusted to pH5.0 with hydrochloric acid and spinach crude enzyme solution obtained in Reference Example 1
0.375 ml, water 4.275 ml, and 5 ml of diisopropyl ether in which 45 mg of commercially available egg yolk lecithin were dissolved at 30℃.
Stirred at 500 rpm. 4 identical samples in parallel, 1, 2, 4, 6 each
The mixture was reacted for a period of time and analyzed in the same manner as in Comparative Example 1. The results are shown in Table 2. [Table] Comparative Example 3 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 5. 5 ml of 50 mM acetate buffer (PH 5.6) containing 600 mg of 1-ortho-methyl glucoside and 12 μg of phospholipase D (PLDP manufactured by Toyo Jozo Co., Ltd.) and 5 ml of chloroform-isooctane (1:1) containing 45 mg of dipalmitoyl PC were mixed into 38 The mixture was stirred at 500 rpm and allowed to react. Analysis was performed over time in the same manner as in Comparative Examples 1 and 2, but no spot corresponding to the target phospholipid observed in Example 5 was observed on TLC, indicating that the substrate was completely decomposed after 8 hours of reaction. Therefore, the analysis was discontinued. Comparative Example 4 In the same manner as in Example 1, only cabbage phospholipase D was adsorbed onto activated carbon and freeze-dried. Using the activated carbon-adsorbed phospholipase D obtained above,
Comparative Example 1 except that the water content in the reaction system was 10% by weight
The reaction was carried out in the same manner as above, and the analysis was carried out in the same manner. The results are shown in Table 3. [Table] As is clear from the results of each comparative example, when a large amount of water is present in the reaction system, hydrolysis progresses significantly over time, the amount of PA increases, and most of the target product is not obtained. It turns out that there isn't.
Claims (1)
あたり、原料リン脂質と水酸基を有する受容体と
を、担体に吸着させたホスホリパーゼＤに接触さ
せて、反応系中の水分含量が１重量％以下の状態
で有機溶媒中で反応させることを特徴とする酵素
によるリン脂質の製造方法。 ２ 受容体およびホスホリパーゼＤを吸着させた
担体とを混合し反応させる特許請求の範囲第１項
記載の製造方法。 ３ 原料リン脂質を吸着させた担体と、受容体お
よびホスホリパーゼＤを吸着させた担体とを混合
し反応させる特許請求の範囲第１項記載の製造方
法。 ４ 担体が活性炭、活性白土、ケイ酸、シリカゲ
ル、ケイ藻土、ゼオライト、アルミナ、多孔質ガ
ラス、陶器、磁器、または樹脂である特許請求の
範囲第１、２または３項記載の製造方法。 ５ 受容体が、セリン、エタノールアミン、１−
アミノ−２−プロパノール、１−オルソメチル−
グルコシド、トレハロースのいずれかである特許
請求の範囲第１、２、３または４項記載の製造方
法。[Claims] 1. In producing a phospholipid whose base structure has been converted, a raw material phospholipid and a receptor having a hydroxyl group are brought into contact with phospholipase D adsorbed on a carrier, and the water content in the reaction system is adjusted. 1. A method for producing phospholipids using an enzyme, characterized in that the reaction is carried out in an organic solvent in a state where the amount of phospholipids is 1% by weight or less. 2. The manufacturing method according to claim 1, wherein the receptor and a carrier adsorbed with phospholipase D are mixed and reacted. 3. The manufacturing method according to claim 1, wherein a carrier on which a raw material phospholipid is adsorbed and a carrier on which a receptor and phospholipase D are adsorbed are mixed and reacted. 4. The manufacturing method according to claim 1, 2 or 3, wherein the carrier is activated carbon, activated clay, silicic acid, silica gel, diatomaceous earth, zeolite, alumina, porous glass, earthenware, porcelain, or resin. 5 The receptor is serine, ethanolamine, 1-
Amino-2-propanol, 1-orthomethyl-
5. The manufacturing method according to claim 1, 2, 3 or 4, which is either glucoside or trehalose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18002486A JPS6336791A (en) | 1986-08-01 | 1986-08-01 | Production of phospholipid by enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18002486A JPS6336791A (en) | 1986-08-01 | 1986-08-01 | Production of phospholipid by enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6336791A JPS6336791A (en) | 1988-02-17 |
| JPH0367676B2 true JPH0367676B2 (en) | 1991-10-23 |
Family
ID=16076130
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18002486A Granted JPS6336791A (en) | 1986-08-01 | 1986-08-01 | Production of phospholipid by enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6336791A (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH022381A (en) * | 1988-03-30 | 1990-01-08 | Kanegafuchi Chem Ind Co Ltd | Production of phospholipid |
| JP3791951B2 (en) * | 1995-11-08 | 2006-06-28 | 株式会社ヤクルト本社 | Method for producing oil and fat composition containing polyunsaturated fatty acid-containing phosphatidylserine |
| US5700668A (en) | 1995-12-08 | 1997-12-23 | Italfarmaco Sud S.P.A. | Process for the industrial preparation of phosphatidylserine |
| CA2376604A1 (en) * | 1999-06-15 | 2000-12-21 | Yissum Research Development Company Of The Hebrew University Of Jerusale M | Enzymatic preparation of phospholipids in aqueous media |
| KR100442538B1 (en) * | 2001-05-07 | 2004-07-30 | 주식회사 두산 | Method for producing phosphatidylserine and lysophosphatidylserine in organic solvent |
| KR20030084200A (en) * | 2002-04-25 | 2003-11-01 | 주식회사 두산 | Method for producing phosphatidylethanolamine and lysophosphatidylethanolamine using non-organic solvent system and a water-soluble composition comprising the lysophosphatidylethanolamine produced by the method |
| JPWO2007063886A1 (en) * | 2005-11-30 | 2009-05-07 | ナガセケムテックス株式会社 | Method for producing polyunsaturated fatty acid-containing phospholipid and polyunsaturated fatty acid-containing phospholipid obtained by the production method |
| KR101122388B1 (en) * | 2009-05-23 | 2012-03-23 | 주식회사 고센바이오텍 | The method for biosynthesis of phosphatidylserine by chinese cabbage phospholipase D from egg yolk phospholipid |
| KR101055094B1 (en) * | 2009-05-23 | 2011-08-08 | 주식회사 고센바이오텍 | Biosynthesis Method of Phosphatidylserine from Egg Yolk Phospholipids by Cabbage Phospholipase D |
| CN103074390A (en) * | 2011-11-02 | 2013-05-01 | 江南大学 | Enzyme method for preparing phosphatidyl propanol containing no lysophospholipid or phosphatidic acid |
| CN103074392A (en) * | 2011-11-26 | 2013-05-01 | 江南大学 | Method for improving soybean alcohol-soluble phosphatidylcholine content with enzyme method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59187787A (en) * | 1983-04-11 | 1984-10-24 | Meito Sangyo Kk | Production of sphingolipid derivative using enzymic method |
-
1986
- 1986-08-01 JP JP18002486A patent/JPS6336791A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59187787A (en) * | 1983-04-11 | 1984-10-24 | Meito Sangyo Kk | Production of sphingolipid derivative using enzymic method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6336791A (en) | 1988-02-17 |
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