WO1991003565A1 - Hydrolysis of phospholipids using immobilized lipase - Google Patents
Hydrolysis of phospholipids using immobilized lipase Download PDFInfo
- Publication number
- WO1991003565A1 WO1991003565A1 PCT/DK1990/000224 DK9000224W WO9103565A1 WO 1991003565 A1 WO1991003565 A1 WO 1991003565A1 DK 9000224 W DK9000224 W DK 9000224W WO 9103565 A1 WO9103565 A1 WO 9103565A1
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- WO
- WIPO (PCT)
- Prior art keywords
- process according
- lipase
- immobilized
- enzyme
- phospholipid
- Prior art date
Links
- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 24
- 108090001060 Lipase Proteins 0.000 title claims description 33
- 102000004882 Lipase Human genes 0.000 title claims description 33
- 239000004367 Lipase Substances 0.000 title claims description 33
- 235000019421 lipase Nutrition 0.000 title claims description 33
- 230000007062 hydrolysis Effects 0.000 title description 8
- 238000006460 hydrolysis reaction Methods 0.000 title description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 125000002252 acyl group Chemical group 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 25
- 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 claims description 11
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 241000235403 Rhizomucor miehei Species 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 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 claims description 5
- 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 claims description 5
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 5
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims description 5
- 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 claims description 5
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 5
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- -1 phospho Chemical class 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 241000589513 Burkholderia cepacia Species 0.000 claims description 2
- 241000223198 Humicola Species 0.000 claims description 2
- 241000235402 Rhizomucor Species 0.000 claims description 2
- 239000003463 adsorbent Substances 0.000 claims description 2
- 239000003957 anion exchange resin Substances 0.000 claims description 2
- 150000008280 chlorinated hydrocarbons Chemical class 0.000 claims description 2
- 238000010924 continuous production Methods 0.000 claims description 2
- FRKBLBQTSTUKOV-UHFFFAOYSA-N diphosphatidyl glycerol Natural products OP(O)(=O)OCC(OP(O)(O)=O)COP(O)(O)=O FRKBLBQTSTUKOV-UHFFFAOYSA-N 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 2
- 239000012454 non-polar solvent Substances 0.000 claims description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 claims description 2
- 239000011541 reaction mixture Substances 0.000 claims description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims 1
- 241000047214 Cyclocybe cylindracea Species 0.000 claims 1
- 241000055915 Heterocoma lanuginosa Species 0.000 claims 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 claims 1
- 241000589516 Pseudomonas Species 0.000 claims 1
- 238000010923 batch production Methods 0.000 claims 1
- 235000014113 dietary fatty acids Nutrition 0.000 claims 1
- 239000000194 fatty acid Substances 0.000 claims 1
- 229930195729 fatty acid Natural products 0.000 claims 1
- 150000004665 fatty acids Chemical class 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 claims 1
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 11
- 239000008777 Glycerylphosphorylcholine Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 7
- SUHOQUVVVLNYQR-MRVPVSSYSA-N choline alfoscerate Chemical compound C[N+](C)(C)CCOP([O-])(=O)OC[C@H](O)CO SUHOQUVVVLNYQR-MRVPVSSYSA-N 0.000 description 7
- 229960004956 glycerylphosphorylcholine Drugs 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- 241000223258 Thermomyces lanuginosus Species 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000015439 Phospholipases Human genes 0.000 description 3
- 108010064785 Phospholipases Proteins 0.000 description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 3
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 240000006439 Aspergillus oryzae Species 0.000 description 2
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 229920002125 Sokalan® Polymers 0.000 description 2
- 241000179532 [Candida] cylindracea Species 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- SLGWESQGEUXWJQ-UHFFFAOYSA-N formaldehyde;phenol Chemical compound O=C.OC1=CC=CC=C1 SLGWESQGEUXWJQ-UHFFFAOYSA-N 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 229920001568 phenolic resin Polymers 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 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 1
- 240000008791 Antiaris toxicaria Species 0.000 description 1
- 241001637800 Caulerpa cylindracea Species 0.000 description 1
- 241000222175 Diutina rugosa Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 101000968489 Rhizomucor miehei Lipase Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 150000002190 fatty acyls Chemical group 0.000 description 1
- 125000001924 fatty-acyl group Chemical group 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 150000002327 glycerophospholipids Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002440 hydroxy compounds Chemical class 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000009884 interesterification Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 239000000725 suspension Substances 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
- 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
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
-
- 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
- C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
Definitions
- This invention relates to an enzymatic process for hydrolyzing one or both acyl groups in a phospholipid.
- Phospholipids such as phosphatidyl choline, consist of glycerol esterified with 2 fatty acyl groups and one phosphate or esterified phosphate group.
- hydrolysis of phospholipid can be catalyzed by lipase (EP 260,573 and JP-A 63-42,691) and by chemically modified (derivatized) lipase (JP-A 63-105,685). It is the object of this invention to provide an enzymatic hydrolysis process whereby the enzyme can be easily separated from the reaction mixture and be re-used, without the need for a large excess of water.
- the invention provides an enzymatic process for hydrolyzing one or both acyl groups in a phospholipid, characterized in that the enzyme is immobil ⁇ ized on a particulate macroporous carrier.
- the enzyme used in the invention is immobilized on a particulate macroporous carrier.
- the enzyme may be simply adsorbed on the carrier, or it may be attached to the carrier by cross-linking with glutaraldehyde or other cross- linking agent known in the art.
- a preferred carrier type is weakly basic anion exchange resin, e.g. of acrylic, polystyrene or phenolformaldehyde type.
- weakly basic anion exchange resin e.g. of acrylic, polystyrene or phenolformaldehyde type.
- Examples of commercial products are Lewatit ® E 1999/85 (product of Bayer, West Germany) and Duolite ® ES-568 (Rohm & Haas).
- Another preferred carrier type is an adsorbent (non-ionic) carrier, e.g. of the phenol-formaldehyde type, acrylic type or polypropylene type.
- adsorbent non-ionic carrier
- examples of commercial products are Lewatit E2001/85 (acrylic, product of Bayer) and Accurel EP-100 (polypropylene, product of AKZO).
- Another preferred immobilization method uses an inorganic support material, and the enzyme is preferably attached to the support by adsorption or covalent coupling.
- Such support materials and immobilization techniques are described in K. Mosbach (ed.): Methods in Enzymology, 44, "Immobilized
- a preferred inorganic support material is macroporous silica or silicate carriers e.g. macroporous silica carriers from Grace Chemicals described in Biocatalyst Supports SG BC 1E/June 1987 in which more than 90% of the particles have particle sizes between 100 and 1000 p, wherein more than 80% of the pores in the particles exhibit a diameter between 5 and 45 times the diameter of the enzyme globules.
- macroporous silica or silicate carriers e.g. macroporous silica carriers from Grace Chemicals described in Biocatalyst Supports SG BC 1E/June 1987 in which more than 90% of the particles have particle sizes between 100 and 1000 p, wherein more than 80% of the pores in the particles exhibit a diameter between 5 and 45 times the diameter of the enzyme globules.
- the immobilized enzymes useful for interesterification of phospholipids typically are loaded with 20,000 - 200,000 LU per g (dry weight) of catalyst (LU, Lipase Unit is defined in US 4,810,414).
- the enzyme to be used may be a lipase of animal, plant or microbial origin.
- a microbially produced lipase is preferred, e.g. a bacterial or fungal lipase.
- suitable enzymes are lipases derived from the following organisms:
- Rhizomucor also designated M ⁇ co ⁇ , especially R. miehei ⁇ M. miehei
- LipozymeTM Novo Nordisk a/s
- Candida rugosa also termed C. cylindraceae, the lipase being available as Lipase OF (Meito Sangyo)).
- the process is preferably carried out in a non-polar solvent like hexane, heptane, petroleum ether, or chlorinated hydrocarbons with a humidified immobilized enzyme.
- a non-polar solvent like hexane, heptane, petroleum ether, or chlorinated hydrocarbons with a humidified immobilized enzyme.
- the necessary amount of water is generally in the range 0.5-5% by weight and may be provided simply by humidifying the immobilized enzyme, e.g. to a water content of 5-50% (w/w), especially 5-25%.
- the solvent may also be saturated with water, e.g. in a continuous process.
- the process temperature should be chosen after considering thermostability of the immobilized enzyme. In many cases 20-60°C will be suitable.
- thermostable enzymes temperatures as high as 80°C may be used.
- the process may be carried out as a batch reaction, where the ingredients are stirred gently throughout the reaction period.
- the amount of immobilized enzyme will typically be 1-10% (w/w), and the reaction time will generally be 10 minutes - 24 hours. After the reaction the reaction products can be separated from the immobilized enzyme simply by decanting or filtration.
- the process may be carried out continuously by letting the phospholipid in solvent pass through a fixed bed column of immobilized enzyme.
- the residence time will typically be 1 - 12 hours.
- Phospholipid The process of the invention may be applied to any desired kind of phospholipid containing fatty acyl ester groups and one or two phosphate groups which may be esterified.
- Examples of such naturally occurring phospholipids are phosphatidic acid, phosphatidyl chol ⁇ ne, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine and diphosphatidyl glycerol.
- Synthetic phospholipids with various hydroxy compounds esterified to the phosphate group may also be processed.
- Rhizomucor miehei lipase produced by cultivating a transformed
- Aspergillus oryzae was immobilized on Duolite ® ES-568 N.
- the load was 99,200 LU per g (dry weight) of catalyst.
- Humicola lanuginosa lipase produced by cultivating a transformed Aspergillus oryzae was immobilized on a macroporous silica carrier (Grace 6, product of Grace Chemicals). The load was 166,500 LU per g (dry weight) of catalyst.
- Candida cylindracea lipase (Lipase-OF from Meito Sangyo) was immobilized on Accurel EP-100. The load was 34,200 LU per g (dry weight) of catalyst.
- Epikuron 200 As phospholipid was used the commercial product Epikuron 200 from Lucas Meyer GmbH. This is a fractionated soybean lecithin claimed to contain min. 95% phosphatidyl choline (PC), max. 4% lysophosphatidyl choline (LPC) and a moisture and oil content of max. 3%.
- 1.0 g of Epikuron 200 was mixed with 20 ml petroleum ether (b.p. 80-100°C).
- Each of the above immobilized R. miehei and H. lanuginosa lipases corresponding to a dry weight of 125 mg was weighed into a vial. The lipases were humidified overnight to a water content of 25% (w/w). 1.5 ml of the above mixture was added to the immobilized lipase.
- PC was separated from LPC and glycerophosphorylcholine (GPC) by thin layer chromatography on Silica gel 60 plates (Merck art. 5721) using CHCI3 : CH 3 OH : H 0 (65:25:4, v/v/v) as solvent. After elution the plates were dried, and spots visualized by iodine vapors. The area corresponding to phosphatidyl choline and to LPC + GPC were scraped off.
- GPC glycerophosphorylcholine
- PC and LPC + GPC were extracted from the scraped off silica gel using in turn the following media 1.5 ml CHCI 3 :CH 3 OH:H 2 0 (65:25:4, v/v/v) 1.0 ml CHCI 3 :CH 3 OH:H 2 0 (65:25:4, v/v/v) 1.0 ml CH 3 OH 1.0 ml CH 3 OH:CH 3 COOH:H 2 0 (94:1:5)
- the extracts were pooled and adjusted to 5 ml by CHCI 3 :CH 3 OH:H 2 0 (65:25:4, v/v/v).
- PC and LPC + GPC were quantitated by taking a 1 ml sample of the pooled extracts, evaporating the solvents and determining phosphorus in the residue according to Bruce N. Ames, Methods Enzymol. (8), 115-117, (1966). The degree of PC hydrolysis (DPCH) was calculated as amount of P in LPC + GPC
- phosphatidic acid PA
- PG phosphatidyl glycerol
- PE phosphatidyl ethanolamine
- PS phosphatidyl serine
- LPC lysophosphatidyl choline
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- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
One or both acyl groups in a phospholipid are efficiently hydrolyzed by using an enzyme immobilized on a particulate macroporous carrier. Only a slight excess of water is required above the stoichiometric amount.
Description
HYDROLYSIS OF PHOSPHOLIPIDS USING IMMOBILIZED LIPASE
TECHNICAL FIELD
This invention relates to an enzymatic process for hydrolyzing one or both acyl groups in a phospholipid.
BACKGROUND ART
Phospholipids (glycerophospholipids), such as phosphatidyl choline, consist of glycerol esterified with 2 fatty acyl groups and one phosphate or esterified phosphate group. For some applications of the phospholipid, it is desirable to hydrolyze one or both acyl groups, e.g. in order to modify the emul- sification properties or create glycerophosphoryl compounds which are building blocks for synthetic phospholipids.
Use of phospholipase A-j and A2 for this hydrolysis is well known. In many cases, it is desirable to remove the residual enzyme activity, and due to the stability of some phospholipases like porcine pancreatic phospholipase A2 this may require special measures such as extensive heating or treatment with protease. See JP-A 63-233,750.
It is also known that hydrolysis of phospholipid can be catalyzed by lipase (EP 260,573 and JP-A 63-42,691) and by chemically modified (derivatized) lipase (JP-A 63-105,685). It is the object of this invention to provide an enzymatic hydrolysis process whereby the enzyme can be easily separated from the reaction mixture and be re-used, without the need for a large excess of water.
STATEMENT OF THE INVENTION
Surprisingly, we have found that phospholipids can be efficiently hydrolyzed by immobilized enzymes with a slight stoichiometric excess of water.
Accordingly, the invention provides an enzymatic process for hydrolyzing one or both acyl groups in a phospholipid, characterized in that the enzyme is immobil¬ ized on a particulate macroporous carrier.
Immobilization The enzyme used in the invention is immobilized on a particulate macroporous carrier. The enzyme may be simply adsorbed on the carrier, or it may be attached to the carrier by cross-linking with glutaraldehyde or other cross- linking agent known in the art.
A preferred carrier type is weakly basic anion exchange resin, e.g. of acrylic, polystyrene or phenolformaldehyde type. .Examples of commercial products are Lewatit® E 1999/85 (product of Bayer, West Germany) and Duolite® ES-568 (Rohm & Haas).
Another preferred carrier type is an adsorbent (non-ionic) carrier, e.g. of the phenol-formaldehyde type, acrylic type or polypropylene type. Examples of commercial products are Lewatit E2001/85 (acrylic, product of Bayer) and Accurel EP-100 (polypropylene, product of AKZO).
Another preferred immobilization method uses an inorganic support material, and the enzyme is preferably attached to the support by adsorption or covalent coupling. Such support materials and immobilization techniques are described in K. Mosbach (ed.): Methods in Enzymology, 44, "Immobilized
Enzymes" (Academic Press, 1976).
A preferred inorganic support material is macroporous silica or silicate carriers e.g. macroporous silica carriers from Grace Chemicals described in Biocatalyst Supports SG BC 1E/June 1987 in which more than 90% of the particles have particle sizes between 100 and 1000 p, wherein more than 80% of the pores in the particles exhibit a diameter between 5 and 45 times the diameter of the enzyme globules.
The immobilized enzymes useful for interesterification of phospholipids typically are loaded with 20,000 - 200,000 LU per g (dry weight) of catalyst (LU, Lipase Unit is defined in US 4,810,414).
Enzyme
The enzyme to be used may be a lipase of animal, plant or microbial origin. For reasons of economy, a microbially produced lipase is preferred, e.g. a bacterial or fungal lipase. Some examples of suitable enzymes are lipases derived from the following organisms:
- Positionally specific lipase from Rhizomucor (also designated Mυcoή, especially R. miehei {M. miehei), commercially available as Lipozyme™ (Novo Nordisk a/s). - Positionally specific lipase from Humicola, especially H. lanuginosa
(also designated Thermomyces lanuginosus), see US 4,810,414, EP 305,216.
- Positionally non-specific lipase from Candida rugosa (also termed C. cylindraceae, the lipase being available as Lipase OF (Meito Sangyo)).
- Positionally non-specific lipase from Pseudomonas cepacia (WO 89/01032).
Other enzymes are those indicated in JP-A 63-42,691, incorporated herein by reference, at col. 6-7.
Process conditions
The process is preferably carried out in a non-polar solvent like hexane, heptane, petroleum ether, or chlorinated hydrocarbons with a humidified immobilized enzyme.
In the process there has to be a sufficient amount of water surrounding the enzyme molecules in order to keep the enzyme molecules catalytically active. Secondly there has to be a sufficient amount of water to hydrolyze the ester bonds in the phospholipids. The necessary amount of water is generally in the range 0.5-5% by weight and may be provided simply by humidifying the immobilized enzyme, e.g. to a water content of 5-50% (w/w), especially 5-25%. The solvent may also be saturated with water, e.g. in a continuous process. The process temperature should be chosen after considering thermostability of the immobilized enzyme. In many cases 20-60°C will be suitable.
For very thermostable enzymes temperatures as high as 80°C may be used.
The process may be carried out as a batch reaction, where the ingredients are stirred gently throughout the reaction period. The amount of immobilized enzyme will typically be 1-10% (w/w), and the reaction time will generally be 10 minutes - 24 hours. After the reaction the reaction products can be separated from the immobilized enzyme simply by decanting or filtration.
Alternatively, the process may be carried out continuously by letting the phospholipid in solvent pass through a fixed bed column of immobilized enzyme. The residence time will typically be 1 - 12 hours.
Phospholipid The process of the invention may be applied to any desired kind of phospholipid containing fatty acyl ester groups and one or two phosphate groups which may be esterified. Examples of such naturally occurring phospholipids are phosphatidic acid, phosphatidyl cholϊne, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine and diphosphatidyl glycerol. Synthetic phospholipids with various hydroxy compounds esterified to the phosphate group may also be processed.
EXAMPLES
The following immobilized upases were prepared for use in the examples: Rhizomucor miehei lipase produced by cultivating a transformed
Aspergillus oryzae was immobilized on Duolite® ES-568 N. The load was 99,200 LU per g (dry weight) of catalyst.
Humicola lanuginosa lipase produced by cultivating a transformed Aspergillus oryzae was immobilized on a macroporous silica carrier (Grace 6, product of Grace Chemicals). The load was 166,500 LU per g (dry weight) of catalyst.
Candida cylindracea lipase (Lipase-OF from Meito Sangyo) was immobilized on Accurel EP-100. The load was 34,200 LU per g (dry weight) of catalyst.
EXAMPLE 1
As phospholipid was used the commercial product Epikuron 200 from Lucas Meyer GmbH. This is a fractionated soybean lecithin claimed to contain min. 95% phosphatidyl choline (PC), max. 4% lysophosphatidyl choline (LPC) and a moisture and oil content of max. 3%. 1.0 g of Epikuron 200 was mixed with 20 ml petroleum ether (b.p. 80-100°C). Each of the above immobilized R. miehei and H. lanuginosa lipases corresponding to a dry weight of 125 mg was weighed into a vial. The lipases were humidified overnight to a water content of 25% (w/w). 1.5 ml of the above mixture was added to the immobilized lipase.
Gentle stirring was then carried out at 40°C for 24 hours. Then the substrate was separated from the enzyme catalyst.
To estimate the degree of hydrolysis the following was performed: PC was separated from LPC and glycerophosphorylcholine (GPC) by thin layer chromatography on Silica gel 60 plates (Merck art. 5721) using CHCI3 : CH3OH : H 0 (65:25:4, v/v/v) as solvent. After elution the plates were dried, and spots visualized by iodine vapors. The area corresponding to phosphatidyl choline and to LPC + GPC were scraped off. PC and LPC + GPC were extracted from the scraped off silica gel using in turn the following media 1.5 ml CHCI3:CH3OH:H20 (65:25:4, v/v/v) 1.0 ml CHCI3:CH3OH:H20 (65:25:4, v/v/v) 1.0 ml CH3OH 1.0 ml CH3OH:CH3COOH:H20 (94:1:5)
The extracts were pooled and adjusted to 5 ml by CHCI3:CH3OH:H20 (65:25:4, v/v/v).
PC and LPC + GPC were quantitated by taking a 1 ml sample of the pooled extracts, evaporating the solvents and determining phosphorus in the residue according to Bruce N. Ames, Methods Enzymol. (8), 115-117, (1966). The degree of PC hydrolysis (DPCH) was calculated as
amount of P in LPC + GPC
DPCH = x 100% amount of P in PC + LPC + GPC
The results were:
Substrate DPCH
Untreated 5%
Treated with R. miehei lipase 85%
Treated with H. lanuginosa lipase 93%
EXAMPLE 2
Reaction was carried out as in Example 1 , but using the immobilized
C. cylindracea lipase.
After 24 hours essentially all of the phosphatidyl choline had become hydrolyzed.
EXAMPLE 3
10 mg/ml solutions or suspensions of phosphatidic acid (PA), phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and lysophosphatidyl choline (LPC) in petroleum ether (b.p. 80-100°C) were made.
Samples of immobilized R. miehei lipase corresponding to a dry weight of 125 mg were weighed into vials and humidified overnight to water contents as shown in the table below. 1.5 ml of above substrates were added (at time t = 0). Gentle stirring was carried out at 40°C. 50 μ\ samples were taken at times t = 0, 2, 4, 6, and 24 hours and applied to Silica gel 60 plates. The plates were eluted with CHCI3:CH3OH:H20 (65:25:4, v/v/v) and spots visualized by iodine vapors.
In all cases spots corresponding to PA, PG, PE, PS, and LPC disappeared within 24 hours. In the table below is shown the hydrolysis time T in which the phospholipid in question could no longer be discerned by TLC.
Water content (% w/w) Hydrolysis time Phos holi id of immobilized li ase T h r
Claims
1. An enzymatic hydrolysis process for removing one or both acyl groups from a phospholipid, characterized in that the enzyme is immobilized on a particulate macroporous carrier.
2. A process according to Claim 1, wherein the macroporous carrier is a weakly basic anion exchange resin, an adsorbent carrier, silica or a silicate carrier.
3. A process according to a preceding claim, wherein the amount of water in the reaction system is 0.5-5% by weight.
4. A process according to Claims 1 - 3, wherein the immobilized enzyme is humidified to a water content in the range 5-50%, preferably 5-25% by weight prior to contact with the phospholipid.
5. A process according to any preceding claim, wherein the enzyme is a lipase, preferably a microbially produced lipase.
6. A process according to Claim 5, wherein the lipase is a positionally specific lipase preferably derived from Humicola (especially H. lanuginosa) or Rhizomucor (especially R. miehei).
7. A process according to Claim 5, wherein the lipase is a positionally nonspecific lipase preferably derived from Candida (especially C. cylindracea), or Pseudomonas (especially P. cepacia).
8. A process according to any preceding claim, wherein the phospho¬ lipid is phosphatidyl choline, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidic acid, diphosphatidyl glycerol, synthetic phospholipids containing esterified fatty acids, or a mixture of two or more of these.
9. A process according to any preceding claim, wherein the temperature is 20 - 80°C.
10. A process according to any preceding claim, carried out in the presence of a non-polar solvent, preferably hexane, heptane, petroleum ether or a chlorinated hydrocarbon.
11. A continuous process according to any of Claims 1-10, wherein phospholipid, and optionally water, suspended in a solvent is passed through a fixed bed of immobilized lipase with a residence time of 1-12 hours.
12. A batch process according to any of Claims 1 - 10, wherein a mixture of immobilized lipase, phospholipid, solvent, and optionally water, is stirred for 10 minutes - 24 hours, and the immobilized lipase is later separated from the mixture.
13. A process according to Claim 12, wherein the amount of the immobilized enzyme in the reaction mixture is 1-10% w/w.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK4259/89 | 1989-08-30 | ||
| DK425989A DK425989D0 (en) | 1989-08-30 | 1989-08-30 | HYDROLYSE OF PHOSPHOLIPIDES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1991003565A1 true WO1991003565A1 (en) | 1991-03-21 |
Family
ID=8131827
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DK1990/000224 WO1991003565A1 (en) | 1989-08-30 | 1990-08-29 | Hydrolysis of phospholipids using immobilized lipase |
Country Status (2)
| Country | Link |
|---|---|
| DK (1) | DK425989D0 (en) |
| WO (1) | WO1991003565A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0579928A1 (en) * | 1992-05-25 | 1994-01-26 | The Nisshin Oil Mills, Ltd. | Immobilized lipase, process for producing the same and process for transesterifying oil and fat with the same |
| FR2753200A1 (en) * | 1996-09-06 | 1998-03-13 | Fabre Pierre Dermo Cosmetique | New dermatological and cosmetic products for dry skin |
| EP1073339A1 (en) | 1998-04-20 | 2001-02-07 | Novozymes A/S | Preparation of dough and baked products |
| WO2020153902A1 (en) * | 2019-01-25 | 2020-07-30 | Wilmar International Limited | A process for hydrolyzing oil with high melting point by lipase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4798793A (en) * | 1983-09-05 | 1989-01-17 | Novo Industri A/S | Immobilized Mucor miehei lipase for transesterification |
| WO1989002748A1 (en) * | 1987-09-25 | 1989-04-06 | Massachusetts Institute Of Technology | Reduction of low density lipoproteins in biological fluids |
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1989
- 1989-08-30 DK DK425989A patent/DK425989D0/en unknown
-
1990
- 1990-08-29 WO PCT/DK1990/000224 patent/WO1991003565A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4798793A (en) * | 1983-09-05 | 1989-01-17 | Novo Industri A/S | Immobilized Mucor miehei lipase for transesterification |
| US4818695A (en) * | 1983-09-05 | 1989-04-04 | Novo Industri A/S | Immobilized Mucor miehe lipase for transesterification |
| WO1989002748A1 (en) * | 1987-09-25 | 1989-04-06 | Massachusetts Institute Of Technology | Reduction of low density lipoproteins in biological fluids |
Non-Patent Citations (4)
| Title |
|---|
| CHEMICAL ABSTRACTS, Volume 90, No. 13, abstract 99580G, 26 March 1979, (Columbus, Ohio, US), MARMER, WILLIAM N. et al., "Rapid Enzyme-Induced Hydrolysis of Microgram Amounts of Phosphatidylcholine on Phospholiphase A2/Celite Columns", see pages 229; & LIPIDS 1978, 13(12), 840-843. * |
| DIALOG INFORMATION SERVICES, File 351, World Patents Index, Latest 1981 + Dialog acc. no. 4799201, (SHOWA SANGYO KK), "Modification of Phospholipid to Lyso-Type Using Lipase - where Lipase is Produced from Aspergiluis or Pseudomonas Microorganism"; & JP,A,63 042 691, 23-02-88. * |
| PATENT ABSTRACTS OF JAPAN, Vol. 10, No. 258, C370; & JP,A,61 085 195, 30-04-1986, AGENCY OF IND SCIENCE & TECHNOL. * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0579928A1 (en) * | 1992-05-25 | 1994-01-26 | The Nisshin Oil Mills, Ltd. | Immobilized lipase, process for producing the same and process for transesterifying oil and fat with the same |
| FR2753200A1 (en) * | 1996-09-06 | 1998-03-13 | Fabre Pierre Dermo Cosmetique | New dermatological and cosmetic products for dry skin |
| EP1073339A1 (en) | 1998-04-20 | 2001-02-07 | Novozymes A/S | Preparation of dough and baked products |
| WO2020153902A1 (en) * | 2019-01-25 | 2020-07-30 | Wilmar International Limited | A process for hydrolyzing oil with high melting point by lipase |
| CN111485007A (en) * | 2019-01-25 | 2020-08-04 | 丰益国际有限公司 | Method for hydrolyzing oil with high melting point by lipase |
| CN111485007B (en) * | 2019-01-25 | 2024-04-26 | 丰益国际有限公司 | Method for hydrolyzing oils with high melting point by lipase |
| US12129508B2 (en) | 2019-01-25 | 2024-10-29 | Wilmar International Limited | Process for hydrolyzing oil with high melting point by lipase |
Also Published As
| Publication number | Publication date |
|---|---|
| DK425989D0 (en) | 1989-08-30 |
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