EP0382738A1 - Immobilisierte, nichtpositionsspezifische lipase, ihre herstellung und verwendung - Google Patents
Immobilisierte, nichtpositionsspezifische lipase, ihre herstellung und verwendungInfo
- Publication number
- EP0382738A1 EP0382738A1 EP88907259A EP88907259A EP0382738A1 EP 0382738 A1 EP0382738 A1 EP 0382738A1 EP 88907259 A EP88907259 A EP 88907259A EP 88907259 A EP88907259 A EP 88907259A EP 0382738 A1 EP0382738 A1 EP 0382738A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- lipase
- use according
- immobilized
- preparation
- specific
- 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.)
- Ceased
Links
- 108090001060 Lipase Proteins 0.000 title claims abstract description 104
- 239000004367 Lipase Substances 0.000 title claims abstract description 104
- 102000004882 Lipase Human genes 0.000 title claims abstract description 104
- 235000019421 lipase Nutrition 0.000 title claims abstract description 104
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 238000000034 method Methods 0.000 claims abstract description 37
- 150000002148 esters Chemical class 0.000 claims abstract description 24
- 241000589513 Burkholderia cepacia Species 0.000 claims abstract description 21
- 230000008569 process Effects 0.000 claims abstract description 20
- 238000009884 interesterification Methods 0.000 claims abstract description 17
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 9
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 8
- 238000010931 ester hydrolysis Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000002360 preparation method Methods 0.000 claims description 22
- 239000011347 resin Substances 0.000 claims description 15
- 229920005989 resin Polymers 0.000 claims description 15
- 239000000376 reactant Substances 0.000 claims description 13
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 10
- 239000000194 fatty acid Substances 0.000 claims description 10
- 229930195729 fatty acid Natural products 0.000 claims description 10
- 150000004665 fatty acids Chemical class 0.000 claims description 10
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 8
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 150000001733 carboxylic acid esters Chemical class 0.000 claims description 4
- 238000004132 cross linking Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000001179 sorption measurement Methods 0.000 claims description 4
- 239000003463 adsorbent Substances 0.000 claims description 3
- 238000006136 alcoholysis reaction Methods 0.000 claims description 3
- 239000003957 anion exchange resin Substances 0.000 claims description 3
- 238000010923 batch production Methods 0.000 claims description 3
- 238000010924 continuous production Methods 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 150000001840 cholesterol esters Chemical class 0.000 claims description 2
- 125000004494 ethyl ester group Chemical group 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims description 2
- 230000004151 fermentation Effects 0.000 claims description 2
- SLGWESQGEUXWJQ-UHFFFAOYSA-N formaldehyde;phenol Chemical compound O=C.OC1=CC=CC=C1 SLGWESQGEUXWJQ-UHFFFAOYSA-N 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 229920001568 phenolic resin Polymers 0.000 claims description 2
- 238000011160 research Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims 2
- 230000003100 immobilizing effect Effects 0.000 claims 1
- 230000000984 immunochemical effect Effects 0.000 claims 1
- 125000003198 secondary alcohol group Chemical group 0.000 claims 1
- 229940040461 lipase Drugs 0.000 description 80
- 239000003925 fat Substances 0.000 description 21
- 235000019197 fats Nutrition 0.000 description 21
- IPCSVZSSVZVIGE-UHFFFAOYSA-N palmitic acid group Chemical group C(CCCCCCCCCCCCCCC)(=O)O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 18
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 150000003626 triacylglycerols Chemical class 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 235000021314 Palmitic acid Nutrition 0.000 description 9
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 9
- 239000005639 Lauric acid Substances 0.000 description 8
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 7
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 7
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 7
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 7
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 7
- 239000005642 Oleic acid Substances 0.000 description 6
- 238000002844 melting Methods 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 238000010348 incorporation Methods 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 229920006395 saturated elastomer Polymers 0.000 description 5
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 4
- DCXXMTOCNZCJGO-UHFFFAOYSA-N Glycerol trioctadecanoate Natural products CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 4
- 235000021355 Stearic acid Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- VKOBVWXKNCXXDE-UHFFFAOYSA-N icosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(O)=O VKOBVWXKNCXXDE-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical group CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 4
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Chemical group CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 239000008117 stearic acid Chemical group 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 description 3
- BAECOWNUKCLBPZ-HIUWNOOHSA-N Triolein Natural products O([C@H](OCC(=O)CCCCCCC/C=C\CCCCCCCC)COC(=O)CCCCCCC/C=C\CCCCCCCC)C(=O)CCCCCCC/C=C\CCCCCCCC BAECOWNUKCLBPZ-HIUWNOOHSA-N 0.000 description 3
- PHYFQTYBJUILEZ-UHFFFAOYSA-N Trioleoylglycerol Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCCCCCCCC)COC(=O)CCCCCCCC=CCCCCCCCC PHYFQTYBJUILEZ-UHFFFAOYSA-N 0.000 description 3
- 241000179532 [Candida] cylindracea Species 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 3
- 229940110456 cocoa butter Drugs 0.000 description 3
- 235000019868 cocoa butter Nutrition 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 150000003333 secondary alcohols Chemical class 0.000 description 3
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 3
- 229940117972 triolein Drugs 0.000 description 3
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 241000186427 Cutibacterium acnes Species 0.000 description 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 2
- 108010048733 Lipozyme Proteins 0.000 description 2
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 2
- 125000001924 fatty-acyl group Chemical group 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 235000020778 linoleic acid Nutrition 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 235000019626 lipase activity Nutrition 0.000 description 2
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 2
- 235000013310 margarine Nutrition 0.000 description 2
- 239000003264 margarine Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000005809 transesterification reaction Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- UOCLXMDMGBRAIB-UHFFFAOYSA-N 1,1,1-trichloroethane Chemical compound CC(Cl)(Cl)Cl UOCLXMDMGBRAIB-UHFFFAOYSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 244000300657 Alchornea rugosa Species 0.000 description 1
- 208000016444 Benign adult familial myoclonic epilepsy Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 244000168141 Geotrichum candidum Species 0.000 description 1
- 235000017388 Geotrichum candidum Nutrition 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 241001507683 Penicillium aurantiogriseum Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000235403 Rhizomucor miehei Species 0.000 description 1
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003518 caustics Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 125000004177 diethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000009886 enzymatic interesterification Methods 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- ZKQFHRVKCYFVCN-UHFFFAOYSA-N ethoxyethane;hexane Chemical compound CCOCC.CCCCCC ZKQFHRVKCYFVCN-UHFFFAOYSA-N 0.000 description 1
- 208000016427 familial adult myoclonic epilepsy Diseases 0.000 description 1
- 125000005313 fatty acid group Chemical group 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 239000012442 inert solvent Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 150000002889 oleic acids Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940055019 propionibacterium acne Drugs 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 108010079522 solysime Proteins 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- WBWWGRHZICKQGZ-HZAMXZRMSA-N taurocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005292 vacuum distillation Methods 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/091—Phenol resins; Amino resins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
Definitions
- Lipase is taken to mean an enzyme that catalyzes reactions involving ester bonds (such as hydrolysis, synthesis and exhange of ester bonds) in water-insoluble carboxylic acid esters e.g. at an interface between aqueous and an organic phase.
- Lipase may be in soluble, derivatized or immobilized form.
- Immobilized lipase denotes lipase in the form of immobilized enzyme or immobilized cells, as defined in "Guidelines for the characterization of immobilized biocatalysts” (1983), Enzyme Microb. Technol., 5_, 304-307.
- Derivatized lipase denotes lipase that has been chemically modified without immobiliz.ing " it T__5oluble lipase denotes un-modified lipase. T *-- ⁇ ⁇ " "y ⁇ - - ⁇ - ⁇ ⁇ ⁇
- Lipases may be divided according to their positional specificity. As used in this specification, a positionally specific lipase (or specific lipase for short) is one that reacts only with the fatty acyl groups in the 1- and 3- positions of a triglyceride molecule, and a positionally non-specific lipase (or non-specific lipase for short) is one that reacts with all three fatty acyl groups of a triglyceride.
- Non-specific lipase activity can be determined in an interesteri-fication reaction with a triglyceride by measuring the reaction rate in the middle position, e.g. by reacting pure triolein or cocoa butter stearin with a pure fatty acid.
- Lipase-catalyzed processes are ester hydrolysis, ester synthesis and interesterification.
- the esters involved may be triglycerides or other carboxylic acid esters.
- Random interesterification of fats refers to interesterification reactions in which all three acyl groups in the triglycerides react, whereby a nearly random distribution of fatty acids in all three positions can be obtained. This may be achieved by use of a chemical catalyst or a non-specific lipase.
- This,-invention relates to an immobilized, non ⁇ specific lipase preparation in particulate form, to a method for producing it, and to its use in a lipase- catalyzed process, i.e. interesterification, ester synthesis and ester hydrolysis.
- a lipase- catalyzed process i.e. interesterification, ester synthesis and ester hydrolysis.
- it relates to such an immobilized lipase with improved heat
- the lipase is derived from Candida cylindracea, and the data in- the article show that the immobilized lipase has optimum temperature about 40°C, and that there is significant deactivation at 50°C.
- thermostable, immobilized, non-specific lipase for
- the object of the invention to provide immobilized, non-specific lipase that is thermostable enough for long-term use at 60°C or higher.
- the lipase should be microbial, as these can be produced
- thermostability data have been published for the following: Staphylococcus aureus (Vadehra, D.V. (1974). Lipids, 9_, 158), Penicillium cyclopium (Okumura, S., et al. (1976). Agricultural and Biological Chemistry, 40, 655 and Renshaw E.C. and San Clemente CL. (1966) Developments in Industrial Microbiology, , 214), Corynebacterium acnes (Hassing, G.S. (1971). Biochimica et Biophysica Acta, 242, 381 and Pablo
- positionally non-specific lipase can be obtained from Pseudomonas cepacia. And it has surprisingly been found that this lipase in immobilized form is heatstable enough for long- term use at 60°C, and is thus more heat-stable than previously known immobilized, non-specific lipases.
- the immobilized lipase -i-s " -particularly suited for randomization of fat at temperatures as high as 60-80°C. Most fats are liquid at such temperatures and can be randomized without the presence of a solvent.
- Lipases are known from Pseudomonas cepacia and from other species of Pseudomonas, but none of these are known to be positionally non-specific towards triglycerides.
- P. Eigtved et al. in a paper presented at the AOCS/JOCS Meeting in Honolulu on 1986-05-17 describe a Pseudomonas cepacia lipase that can esterify secondary alcohols (in contrast to most specific lipases), but is positionally specific ir its action on triglycerides.
- the first aspect of the invention provides an immobilized, positionally non-specific lipase preparation in particulate form, characterized in that the non-specific lipase is producible from Pseudomonas cepacia and/ or that it has immunological properties identical to those of the non-specific lipase from the P. cepacia strain DSM 3959.
- This preparation has a half-life at 60°C above 1,000 hours when measured in continuous, fixed-bed interesterification.
- Another aspect of the invention provides use of the above-mentioned immobilized preparation in a lipase- catalyzed process.
- non-specific lipases that can be used in the practice of the invention are those that are producible by a strain of Pseudomonas cepacia and/or have immunological properties identical to those of the non ⁇ specific lipase from P. cepacia strain DSM 3959. Immunological identity may be determined according to N.H. Axelsen et al. (ed.): Quantitative Immunoelectrophoresis, -(Blackwell Scientific Publications, 1973), especially • . __ chapter 10 and to Ivan Roitt: Essential Immunology, 5th ed. (Blackwell Scientific Publications, 1984), especially Chapter 6.
- Non-specific lipase for use in the invention may contain specific lipase as well, e.g.
- Non-specific lipase for use in the invention may be produced by cultivating the strain DSM 3959 under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen together with other essential nutrients, the medium being composed in accordance with principles known in the art.
- the strain DSM 3959 was deposited at Deutsche Sammlung von Mikroorganismen (DSM) in West Germany on Jan. 30, 1987 under the terms of the Budapest Treaty. It has been identified as Pseudomonas cepacia. The strain also produces specific lipase.
- Non-specific lipase for use in the invention may also be obtained according to Japanese published application 57-63,087 from the P. cepacia strain with deposit number 5494 at the Fermentation Research Institute, Japan. Specificity of the lipase preparation -..isclosed in said application has not previously been described, but we have found that the preparation contains non-specific lipase.
- lipase may be immobilized by any method known in the art, e.g. in . Mosbach (ed. ): Methods in Enzymology, _4_4, "Immobilized Enzymes", (Academic Press, New York, 1976).
- Available methods for enzyme immobilization include: cross-linking of cell homogenates, covalent coupling to insoluble inorganic or organic carriers, entrapment in gels and adsorption on ion-exchange resins or other adsorbent materials.
- coating on a particulate support may be used, as described in Macrae A.R. and Hammond R.C. (1985), Biotechnology and Genetic Engineering Reviews, 3, 193.
- a preferred immobilization method uses a particulate, macroporous resin.
- the lipase may be simply adsorbed on the resin, or it may be attached to the resin by cross-linking with glutaraldehyde or other cross-linking agent known in the art.
- a preferred resin type is weakly basic anion exchange resin, e.g. of acrylic, polystyrene or phenolic type.
- An example of a commercial product is Lewatit® E 1999/85 (product of Bayer, West Germany).
- the immobilization on this type of resin is preferably according to EP 0 140 542, incorporated herein by reference.
- Another preferred resin type is an adsorbent resin of the phenol-formaldehyde type.
- the immobilization on this resin is preferably done according to DK 85/878, incorporated herein by reference.
- Another preferred immobilization method uses an inorganic support material, and the lipase is preferably attached to the support by adsorption or covalent coupling.
- Such support materials and immobilization techniques are describe ' d in K. Mosbach (ed.): Methods in Enzymol ⁇ gy, _44, "Immobilized Enzymes” (Academic Press, 1976).
- the lipase-catalyzed process of this invention may be any of the following types. In each case reactant types are listed in parentheses:
- the alcohol may be any mono- or polyvalent primary and/or secondary alcohol or a mixture of these.
- the acid may be any carboxylic acid or a mixture of these.
- the ester may be any ester derived from the mentioned alcohol and acid, or a mixture of these. Use of the immobilized lipase of the invention is particularly advantageous with triglycerides, where reaction in all three positions is desired.
- the temperature in the process of this invention is preferably above 60°C, where most substrates and products of interest are liquid. Higher temperatures are generally preferred as reaction rate increases, and the diffusion resistance to mass transfer into and out of the immobilized lipase decreases. Also, in the case of column operation higher temperatures may be preferred to reduce the pressure drop over the column. On the other hand, in many cases the substrates and products will be degraded at the higher temperatures. Thus, a preferred range is 60- 90°C, more preferably 60-80°C.
- Preferred embodiments of this process are fat splitting and hydrolysis of cholesterol esters. This may be performed either batch-wise or continuously.
- the fat and water are mixed mechanically together with the necessary amount of immobilized lipase.
- the water content will usually be kept below 40% w/w.
- the temperature should be above the melting point of the fat, and may be as high as 80°C. Reaction time depends on enzyme dosage and desired conversion, but may be up to several days.
- the immobilized lipase may be recovered and reused, thereby improving process economy.
- fat above its melting point is passed through a reactor in which the immobilized lipase is retained.
- Water may be added to the system in several ways, e.g. by dispersing water in the fat or by intermittently absorbing water in the immobilized lipase.
- the process of this invention is particularly advantageous for the synthesis of esters of secondary alcohols that are otherwise difficult to produce, including those where the acid or alcohol is high-melting.
- the process may be performed batch-wise or continuously.
- the immobilized lipase may be recovered and reused to improve economy.
- water is removed during reaction, e.g. by vacuum distillation or by absorption on molecular sieves.
- the temperature should be such that the reaction mixture is liquid, preferably 60-90°C, preferably 60-80°C.
- the reactants comprise a triglyceride fat and a fatty acid.
- a preferred embodiment of this process is random interesterification of fat, where the reactant mixture comprises triglyceride fat, and reaction occurs by exchange of acyl groups between triglyceride molecules.
- the reactant mixture may consist of a single fat fraction, whereby exchange between acyl groups in the three different positions occurs.
- the reactant mixture may also consist of two or more types of fat, especially one being liquid at ambient temperature and one being a high-melting fat. The latter may be obtained by fractionation from natural sources or by hydrogenation. The product obtained by randomization of such mixtures is useful in margarine production.
- the reactants comprise a triglyceride fat and a carboxyl acid ester, especially a methyl or ethyl ester.
- the reactant mixture may also include a small amount of water, in order to maintain the activity of the enzyme. Water content up to saturation may be used, but a high water content leads to an undesired high degree of by-product formation by hydrolysis.
- purification may be needed prior to carrying out the reaction in order to achieve the highest productivity of the immobilized lipase.
- Conventional purification methods may be used, such as treatment with bleaching-earth or activated carbon.
- reaction temperature may be as high as 80°C.
- the lower limit for reaction temperature is determined by the melting point and viscosity of the reactant mixture. Preferred temperatures are from 60 to 90°C, most preferably from 60 to 80°C.
- the reaction may be performed batch-wise or con- tinuously.
- the substrate and if convenient solvent is mixed in a batch reactor whi>_-h is heated to the preferred temperature together with the immobilized lipase.
- the substrate can be partly or fully saturated with water.
- the enzyme dosage can be up to 10% depending on the desired conversion and reaction time.
- the reaction time can be from a few hours to several days. After reaction the enzyme can be filtered off and reused, if convenient after a solvent wash.
- the substrate is passed through a column containing the immobilized lipase.
- the substrate can if convenient be dissolved in hexane or similar inert solvents.
- the substrate can be partly or fu ' i y saturated with water before entering the enzyme column. This can e.g. be done by a precolumn containing water saturated resin or by saturating the substrate in the substrate container.
- the desired conversion can be achieved by adjusting the flow rate through the column, i.e. changing the contact time.
- the operation time in such a system can be up to several thousand hours.
- the slow loss of activity occuring can be compensated for by decreasing the flow rate, i.e. increasing the residence time of the reactant mixture.
- a typical initial residence time will depend. on desired conversion and can be from 5 min up to 2 hours.
- the products may be further processed. ⁇ By-products such as free fatty acids may be removed afterwards by conventional methods such as caustic refining.
- the product itself can be fractionated, blended with other oils or similar, depending on the specific application.
- the method is based on hydrolysis of tributyrin in a pH-stat.
- 1 LU Lipase Unit
- 1 LU is the amount of enzyme which liberates 1 ⁇ mol titratable butyric acid per minute at 30°C, pH 7.0 with gum arabic as an emulsifier. Further details are given in Novo Analytical Method AF 95/5, available on request.
- FAME fatty acid methyl esters
- LPC gas chromatography
- the ⁇ decrease-in oleic acid content expresses the non ⁇ specific lipase activity, and conveniently a Non- Specificity Index (NSI) may be calculated from the decrease of oleic acid and the total incorporation of lauric acid.
- NBI Non- Specificity Index
- the immobilized lipase (generally 250 mg as dry matter) is hydrated as required for activation usually to about 10% of water.
- the following mixture is used: - 345 mg of cocoa butter stearin, (supplied by Aarhus Olie A/S, Denmark, and containing about 95% of SOS, POS and
- NSI 3 x 33,3 % ° % La where % 0 is mole % oleic acid, and % La is mole % lauric acid
- a culture of Pseudomonas cepacia strain DSM 3959 was transferred to a 500 ml shakeflask with 100 ml PSC-3 medium and shaken at 30°C for 1 day.
- the composition of PSC-3 was as follows:
- the resulting broth was used as seed culture for a 5
- the medium was inoculated with 30 ml seed culture and fermented 5 days with good agitation and aeration at 30 C with a continuous feed of 1.5 ml/hour soybea oil and 4.2 ml/hour 18.6% (NH 4 )_S0 4 .
- the pH was controlled at 6.3 with carbonate.
- the culture broth was concentrated to 800 ml by an Amicon Diaflow hollow fibre cartridge. One volume 96% ethanol was added to the concentrate, the mixture was stirred for 60 minutes at 4°C and centrifugated at 4200 g. The supernatant was freezedried, .-.',> ⁇ the powder was dissolved in water to giv 8000 LU/ml.
- lipase preparation is non ⁇ specific.
- Cocoa " Butter Stearin (CBS) (27.2 mole-% palmitic acid, 39.6 mole-% stearic acid, 31.3 mole-% oleic acid, 0.9 mole-% linoleic acid and 1.1 mole-% arachidic acid) was used as test substrate as it has the unsaturated fatty acid (oleic and linoleic) concentrated in the 2-position of the triglycerides.
- CBS Butter Stearin
- the chemical randomization was carried out as follows: 3.0 g CBS was dried at 95°C for 30 minutes under vacuum by a rotary evaporator. Before addition of 30 mg sodium methoxylate (NaOCH 3 ) the temperature was reduced to 85°C and the vacuum was replaced with nitrogen at atmospheric pressure. The reaction was carried out for 1 1/2 hour under rotation before it was stopped by addition of 0.6 ml 1M HCl and washed 3 times with 5 ml deionized water at 60°C. The sample was dried at 95°C for 1 hour in the rotary evaporator before analysis. The enzymatic interesterifications were carried out with immobilized P. cepacia lipase (prepared similarly to
- Example 2 but with a lipase load of 32,000 LU/g resin) and with Lipozyme TM IM20 (product of Novo Industri A/S), an immobilized, positionally specific lipase from Mucor miehei prepared according to EP 0 140 542. Both by the following procedure: 250 mg dry weight immobilized lipase was hydrated to 10% and 1.7 g CBS was added. The mixture was placed in a
- the fatty acid composition in the 2-position of the triglycerides was analysed as follows: 100 mg CBS or interest -xified CBS, 3 ml pancreatic lipase solution (250 mg porcine pancreas lipase grade II from Sigma cat. no. L3126 dissolved in 10 ml 1M trisbuffer pH 8), 300 ⁇ l 2M CaCl 2 , and 0.75 ml 0.2% w/v taurocholate were mixed.
- the emulsion was heated in a water bath at 40°C for 2 minutes and mixed on a Whirley mixer for 1 1/2 minute before the reaction was stoppe by addition of 4 ml 96% ethanol.
- the sample was transferred t a separation funnel and extracted with 4 x 20 ml diethyl
- the monoglyceride band was identified by iodine vapour, scraped off and extracted by 3 times 10 ml diethyl ether.
- the ether fase was filtered, evaporated and the sample was methylated and analysed on GLC (procedure as described in AF 206/2 available from Novo Industri). The results are presented below:
- Unsaturated fatty acid oleic and linoleic acid
- Example 2 4.5 g of the immobilized lipase of Example 2 was filled into a water jacketed column, having an internal diameter of 1.5 cm.
- the column was heated by use of hot circulating water, i.e. 60°C.
- a precolumn containing water-saturated resin, (Duolite® ES561) was placed before the enzyme column ⁇ d also heated to 60 C.
- a substrate consisting of 71% highly refined bleached and deoinized soy bean oil with a peroxide value less than 3 and 29% analytical grade lauric acid was pumped through the columns.
- At the outlet from the enzyme column samples were taken for analysis, and the incorporation of lauric acid measured by GLC. An incorporation of 14% w/w lauric acid was attempted and the flow rate was adjusted in order to keep the conversion at that value. Whenever the precolumn was dry it was replaced by a fresh one.
- the samples were analysed by removing the free fatty acid and mono- and diglyceride by Al 2 0 3 -column chromatography thereafter methylation of the triglyceride by NaOCH, and finally analysis of the methylester on a GLC.
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Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DK3993/87 | 1987-07-31 | ||
DK399387A DK399387D0 (da) | 1987-07-31 | 1987-07-31 | Immobiliseret lipase og dennes anvendelse |
Publications (1)
Publication Number | Publication Date |
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EP0382738A1 true EP0382738A1 (de) | 1990-08-22 |
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ID=8129070
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP88907259A Ceased EP0382738A1 (de) | 1987-07-31 | 1988-07-22 | Immobilisierte, nichtpositionsspezifische lipase, ihre herstellung und verwendung |
Country Status (4)
Country | Link |
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EP (1) | EP0382738A1 (de) |
JP (1) | JPH02504342A (de) |
DK (1) | DK399387D0 (de) |
WO (1) | WO1989001032A1 (de) |
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1987
- 1987-07-31 DK DK399387A patent/DK399387D0/da not_active Application Discontinuation
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1988
- 1988-07-22 JP JP63506782A patent/JPH02504342A/ja active Pending
- 1988-07-22 EP EP88907259A patent/EP0382738A1/de not_active Ceased
- 1988-07-22 WO PCT/DK1988/000125 patent/WO1989001032A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
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See references of WO8901032A1 * |
Also Published As
Publication number | Publication date |
---|---|
DK399387D0 (da) | 1987-07-31 |
WO1989001032A1 (en) | 1989-02-09 |
JPH02504342A (ja) | 1990-12-13 |
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