WO1989006278A1 - Immobilized lipase - Google Patents
Immobilized lipase Download PDFInfo
- Publication number
- WO1989006278A1 WO1989006278A1 PCT/DK1988/000219 DK8800219W WO8906278A1 WO 1989006278 A1 WO1989006278 A1 WO 1989006278A1 DK 8800219 W DK8800219 W DK 8800219W WO 8906278 A1 WO8906278 A1 WO 8906278A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lipase
- preparation
- humicola
- immobilized
- dsm
- Prior art date
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 62
- 102000004882 Lipase Human genes 0.000 title claims abstract description 62
- 239000004367 Lipase Substances 0.000 title claims abstract description 62
- 235000019421 lipase Nutrition 0.000 title claims abstract description 62
- 241000223198 Humicola Species 0.000 claims abstract description 23
- 238000009884 interesterification Methods 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims description 15
- 239000011347 resin Substances 0.000 claims description 13
- 229920005989 resin Polymers 0.000 claims description 13
- 150000002148 esters Chemical class 0.000 claims description 10
- 241000223258 Thermomyces lanuginosus Species 0.000 claims description 9
- 241001480714 Humicola insolens Species 0.000 claims description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 5
- 239000003463 adsorbent Substances 0.000 claims description 5
- 241000291718 Hoplocampa brevis Species 0.000 claims description 4
- 239000003957 anion exchange resin Substances 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 238000010931 ester hydrolysis Methods 0.000 claims description 4
- SLGWESQGEUXWJQ-UHFFFAOYSA-N formaldehyde;phenol Chemical compound O=C.OC1=CC=CC=C1 SLGWESQGEUXWJQ-UHFFFAOYSA-N 0.000 claims description 4
- 229920001568 phenolic resin Polymers 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 241000223257 Thermomyces Species 0.000 claims description 3
- 230000000984 immunochemical effect Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 241000228212 Aspergillus Species 0.000 claims description 2
- 240000006439 Aspergillus oryzae Species 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 15
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 238000000034 method Methods 0.000 description 17
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 10
- 239000002904 solvent Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 235000021314 Palmitic acid Nutrition 0.000 description 5
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 5
- 229920002125 Sokalan® Polymers 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 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
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 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
- 241001019663 Acremoniella Species 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241001373560 Humicola sp. Species 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 241001279813 Sepedonium Species 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 244000215068 Acacia senegal Species 0.000 description 1
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 1
- 241001646765 Apini Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 241000223199 Humicola grisea Species 0.000 description 1
- 241000223200 Humicola grisea var. thermoidea Species 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 241001674208 Mycothermus thermophilus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000235403 Rhizomucor miehei Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241001261109 Thermomyces ibadanensis Species 0.000 description 1
- 241001285933 Thermomyces sp. Species 0.000 description 1
- 241000123975 Trichoderma polysporum Species 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000006136 alcoholysis reaction 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
- 238000004458 analytical method Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000012512 characterization method 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
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001030 gas--liquid chromatography Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 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
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
-
- 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
- C12P7/6454—Glycerides by esterification
-
- 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
- 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
-
- 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
Definitions
- This invention relates to an immobilized, 1,3- specific lipase preparation and its use in ester hydrolysis, ester synthesis and interesterification.
- EP 140,542 (Novo) discloses immobilized lipase from Mucor miehei and its use in continuous interesterification.
- 1,3-specific lipases are known from many sources.
- One example is lipase from Humicola lanuginosa disclosed in JP 53-45, 394B and shown to be specific in A.R. Macrae et al., Biotechnology and Genetic Engineering Reviews, Vol. 3, pp. 193,217
- the invention provides an immobilized, positionally specific lipase preparation, characterized in that said lipase is produced by cultivation of a microorganism containing a gene encoding for and expressing a lipase derived from a strain of Humicola (incl. Thermomyces).
- the invention further provides an immobilized, 1,3-specific lipase preparation, characterized in that said lipase shows immunochemical identity to a lipase derived from a strain of Humicola (incl. Thermomyces).
- the invention also provides use of said immobilized lipase in ester hydrolysis, ester synthesis and interesterification.
- Preferred lipases are those derived from strains of thermophilic Humicola sp., including thermophilic Thermomyces sp., such as H. lanuginosa (Griffon and Maublanc) Bunce, H. stellata Bunce, H. grisea var. thermoidea, Cooney & Emerson, H. insolens, Cooney & Emerson, Thermomyces ibadanensis, Apinis & Eggins, H. hyalothermoph.ila Moubasher, Mazen and Abdel-Hafez, H. grisea var. indica Subrahmanyam, H. brevis var. thermoidea Subrahmanyam and Thirumalachar and H.
- thermophilic Humicola sp. including thermophilic Thermomyces sp., such as H. lanuginosa (Griffon and Maublanc) Bunce, H. stellata Bunce, H. grisea
- the lipase is derived from H. lanuginosa (Griffon and Maublanc) Bunce, H. brevispora Subrahmanyam and Thirumalachar, H. brevis var. thermoidea Subrahmanyam and Thirumalachar or H. insolens Cooney & Emerson.
- H. lanuginosa has also been described under the synonyms Thermomyces lanuginosus Tsiklinsky, Sepedonium lanuginosum Griffon and Maublanc, Sepedonium thermaphilum cyclosporum and S. thermaphilum ovosporum Velich, Acremoniella sp. Rege, Acremoniella thermophila Curzi and Monotospora lanuginosa (Griffon and Maublanc) Mason.
- the species Scytalidium thermophilum (Cooney & Emerson) Austwick was by Hedger (1975, The ecology of thermophilic fungi in Indonesia. In biodegradation et Humification. Rapport due ler Colloque International - Nancy 1974 (ed. G. Kilbertius, O. Reisinger, A. Mourey & J. A. Cancela Da Fonseca), Sarreguemines : Pierron Editeur - 57206) considered as belonging to Humicola insolens.
- the lipase is derived from one of the following strains:
- DSM indicates Deutsche Sammlung von Mikroorganismen. The strains have been deposited under the terms of the Budapest Treaty.
- Humicola lipase according to JP 53-45, 394B and JP 62-79, 782A (Unitika) can also be used. Lipase production
- Lipase for use in the invention may be produced by aerobic cultivation of one of the above strains according to principles known in the art, e.g. according to DK 4499/87 (Novo), JP 53-45, 394B or JP 62-79, 782A.
- Lipase may also be produced by cultivation of a transformed host organism which contains a gene encoding for a Humicola lipase, and where the lipase can be expressed and secreted extracellularly.
- the transformation can be done according to methods known in the art, e.g. EP 238,023 (Novo).
- Preferred host organisms are Humicola, Aspergillus (especially A. oryzae) and yeasts.
- the preferred lipases of the invention crossreact immunologically with (are antigenically identical or partially antigenically identical to) a lipase from Humicola sp., more particularly with the lipase from one of the above-mentioned species, particularly H. lanuginosa and especially from one of the above-mentioned strains, notably DSM 3819 and DSM 4109.
- the identity (cross-reaction) tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectrophoresis according to N.H. Axelsen: Handbook of Immunoprecipitation-in-Gel Techniques (Blackwell Scientific Publication, 1983), Chapters 5 and 14.
- the terms "antigenic identity” and "partial antigenic identity” are described in the same book, Chapters 5, 19 and 20.
- monospecific rabbit antiserum raised against purified lipase from DSM 4109 we found that the lipases from strains DSM 3819, DSM 4109, DSM 4110 and DSM 4111 are all antigenically identical by both of the above-mentioned methods. Production of antiserum is described in N.H.
- lipase may be immobilized by any method known in the art, e.g. in K. Mosbach (ed.): Methods in Enzymology, 44, "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 orother 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 crosslinking agent known in the art.
- the resin is preferably an adsorbent resin or a weakly basic anion exchange resin.
- the matrix of the resin is preferably of the phenolformaldehyde or the acrylic type. Immobilization on weakly basic anion exchange resin is preferably done according to EP 140,542, incorporated herein by reference.
- Immobilization on an adsorbent resin of the phenol-formaldehyde type is preferably done according to DK 85/878 (Novo), 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 described in K. Mosbach (ed.): Methods in Enzymology, 44, "Immobilized Enzymes” (Academic Press, 1976).
- the lipases of the invention may be used in any of the following processes (reactants indicated in parenthesis):
- the alcohol may be a mono- or polyvalent primary 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.
- the immobilized lipase of the invention is particularly suited for continuous interesterification where the enzyme is in fixed-bed columns.
- the lipase should be partly hydrated, and the reaction mixture should be partly saturated with water to activate the enzyme.
- a suitable temperature is around 60°C.
- a solvent such as hexane or other hydrocarbon can be used, but is usually not necessary.
- An example of this process is acidolysis of triglyceride and fatty acid to produce triglyceride with mainly unsaturated acyl in the 2-position and mainly saturated acyl groups in the 1- and 3-positions, for confectionery use.
- the method is based on hydrolysis of tributyrine 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. Acidolysis activity of immobilized lipase (BIU)
- the activity is determined by reacting palmitic acid with triolein with or without solvent. Total incorporation of palmitic acid is measured by FAME-GLC of triglyceride.
- FAME-GLC Fatty acid methyl ester - gas-liquid chromatography
- AOCS American Oil Chemists' Society
- the reaction mixture consists of 0.6 g of triolein, 0.174 g of palmitic acid and 8.083 g of petroleum ether.
- the reaction without solvent 3.0 g of triolein and 0.87 g of palmitic acid is used.
- a suitable amount of enzyme is hydrated, incubated with the above reaction mixture at a given temperature for 1-4 hours, and then filtering to stop the reaction.
- the filtrate is purified on an alumina column, and the triglycerides are analyzed by FAME-GLC.
- One BIU (Batch Interesterification Unit) is the amount of immobilized lipase that incorporates palmitic acid at an initial rate of 1 ⁇ mole/minute at the given temperature with or without solvent.
- This example shows the effect of pH during immobilization of Humicola lipase on Duolite ® ES 562 macroporous, phenolic anionic resin.
- Immobilizations were made by adjusting 4.25 g D.S. of Duolite ® ES 562 (Rohm & Haas) to the appropriate pH in aqueous suspension and adding 1.0 g of Humicola lipase of 91,700 LU/g (prepared according to Example 1 of DK 4117/86) in 12.5 ml water to the recovered, wet resin. After rotation for 8 hours at room temperature the preparations were filtered, washed with 25 ml water and dried in vacuum at room temperature.
- BIU was measured at 40°C with solvent.
- Humicola lipase in a somewhat larger scale, but otherwise similar to the procedure in Example 1.
- BIU was measured at 40°C with solvent.
- This example shows the immobilization of Humicola lipase on a macroporous, acrylic non-ionic resin.
- BIU was measured at 40°C with solvent and at 60°C without solvent.
- This example shows that very high interesterification activities can be obtained with the combination of Humicola lipase and non-ionic acrylic carrier.
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- Bioinformatics & Cheminformatics (AREA)
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- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Immobilization of Humicola lipase leads to a product with higher interesterification activity than those previously known. The high activity in immobilized form can be obtained from the same activity of native enzyme as the prior art. Immobilized Humicola has good thermostability and can be used for continuous interesterification in column at 60°C, whereby a smaller column size is needed than in the prior art.
Description
IMMOBILIZED LIPASE
This invention relates to an immobilized, 1,3- specific lipase preparation and its use in ester hydrolysis, ester synthesis and interesterification.
BACKGROUND ART
Immobilized, 1,3-specific lipases are known.
Thus, EP 140,542 (Novo) discloses immobilized lipase from Mucor miehei and its use in continuous interesterification.
Native (i.e. not immobilized), 1,3-specific lipases are known from many sources. One example is lipase from Humicola lanuginosa disclosed in JP 53-45, 394B and shown to be specific in A.R. Macrae et al., Biotechnology and Genetic Engineering Reviews, Vol. 3, pp. 193,217
(1985).
STATEMENT OF THE INVENTION
We have surprisingly found that immobilization of Humicola lipase leads to a product with higher interesterification activity than those previously known. The high activity in immobilized form can be obtained from the same activity of native enzyme as the prior art. Immobilized Humicola has good thermostability and can be used for continuous interesterification in column at 60°C, whereby a smaller column size is needed than in the prior art.
Accordingly, the invention provides an immobilized, positionally specific lipase preparation, characterized in that said lipase is produced by cultivation of a microorganism containing a gene encoding for and expressing a lipase derived from a strain of Humicola (incl. Thermomyces).
The invention further provides an immobilized, 1,3-specific lipase preparation, characterized in that said lipase shows immunochemical identity to a lipase derived from a strain of Humicola (incl. Thermomyces).
The invention also provides use of said immobilized lipase in ester hydrolysis, ester synthesis and interesterification.
DETAILED DESCRIPTION OF THE INVENTION
Humicola lipase
Preferred lipases are those derived from strains of thermophilic Humicola sp., including thermophilic Thermomyces sp., such as H. lanuginosa (Griffon and Maublanc) Bunce, H. stellata Bunce, H. grisea var. thermoidea, Cooney & Emerson, H. insolens, Cooney & Emerson, Thermomyces ibadanensis, Apinis & Eggins, H. hyalothermoph.ila Moubasher, Mazen and Abdel-Hafez, H. grisea var. indica Subrahmanyam, H. brevis var. thermoidea Subrahmanyam and Thirumalachar and H. brevispora Subrahmanyam and Thirumalachar. In a specially preferred embodiment, the lipase is derived from H. lanuginosa (Griffon and Maublanc) Bunce, H. brevispora Subrahmanyam and Thirumalachar, H. brevis var. thermoidea Subrahmanyam and Thirumalachar or H. insolens Cooney & Emerson.
H. lanuginosa has also been described under the synonyms Thermomyces lanuginosus Tsiklinsky, Sepedonium lanuginosum Griffon and Maublanc, Sepedonium thermaphilum cyclosporum and S. thermaphilum ovosporum Velich, Acremoniella sp. Rege, Acremoniella thermophila Curzi and Monotospora lanuginosa (Griffon and Maublanc) Mason.
Moreover, the species Scytalidium thermophilum (Cooney & Emerson) Austwick was by Hedger (1975, The ecology of thermophilic fungi in Indonesia. In biodegradation et Humification. Rapport due ler Colloque International - Nancy 1974 (ed. G. Kilbertius, O. Reisinger, A. Mourey & J. A. Cancela Da Fonseca), Sarreguemines : Pierron Editeur - 57206) considered as belonging to Humicola insolens. In a particularly preferred embodiment the lipase is derived from one of the following strains:
taxonomic internal No. deposit No. deposit date designation
H. lanuginosa A 1231 DSM 3819 13 Aug 1986
H. lanuginosa H 126 DSM 4109 4 May 1987
H. brevispora A 2121 DSM 4110 4 May 1987
H. brevis var. thermoidea A 2106 DSM 4111 4 May 1987
H. insolens C 579 DSM 1800 1 Oct 1981
DSM indicates Deutsche Sammlung von Mikroorganismen. The strains have been deposited under the terms of the Budapest Treaty.
Humicola lipase according to JP 53-45, 394B and JP 62-79, 782A (Unitika) can also be used.
Lipase production
Lipase for use in the invention may be produced by aerobic cultivation of one of the above strains according to principles known in the art, e.g. according to DK 4499/87 (Novo), JP 53-45, 394B or JP 62-79, 782A.
Lipase may also be produced by cultivation of a transformed host organism which contains a gene encoding for a Humicola lipase, and where the lipase can be expressed and secreted extracellularly. The transformation can be done according to methods known in the art, e.g. EP 238,023 (Novo). Preferred host organisms are Humicola, Aspergillus (especially A. oryzae) and yeasts.
Immunochemical characterization of lipases
The preferred lipases of the invention crossreact immunologically with (are antigenically identical or partially antigenically identical to) a lipase from Humicola sp., more particularly with the lipase from one of the above-mentioned species, particularly H. lanuginosa and especially from one of the above-mentioned strains, notably DSM 3819 and DSM 4109.
The identity (cross-reaction) tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectrophoresis according to N.H. Axelsen: Handbook of Immunoprecipitation-in-Gel Techniques (Blackwell Scientific Publication, 1983), Chapters 5 and 14. The terms "antigenic identity" and "partial antigenic identity" are described in the same book, Chapters 5, 19 and 20.
Using monospecific rabbit antiserum raised against purified lipase from DSM 4109, we found that the lipases from strains DSM 3819, DSM 4109, DSM 4110 and DSM 4111 are all antigenically identical by both of the above-mentioned methods. Production of antiserum is described in N.H. Axelsen's book, Chapter 41. Purification of Humicola lipase is described in W-H Liu, Agr. Biol. Chem., 37(1), 157-163 (1973); however, we found that the column chromatography may be more conveniently performed by use of: DEAE-sepharose ( anion exchange chromatography), phenyl sepharose (hydrophobic interaction chromatography), followed by gel filtration on TSK G3000SW.
Immobilized lipase
For the practice of this invention, lipase may be immobilized by any method known in the art, e.g. in K. Mosbach (ed.): Methods in Enzymology, 44, "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 orother adsorbent materials. Also, 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 crosslinking agent known in the art. The resin is preferably an adsorbent resin or a weakly basic anion exchange resin. The matrix of the resin is preferably of the phenolformaldehyde or the acrylic type.
Immobilization on weakly basic anion exchange resin is preferably done according to EP 140,542, incorporated herein by reference.
Immobilization on an adsorbent resin of the phenol-formaldehyde type is preferably done according to DK 85/878 (Novo), incorporated herein by reference.
Examples of commerically available resins are:
- Lewatit® 1999/85 (Bayer, West Germany), and Duolite® ES-568 (Rohm & Haas, USA), weakly basic anion exchange resins of the acrylic and phenol-formaldehyde type, respectively.
- Lewatit® E2001/85 (Bayer), an adsorbent resin of acrylic type.
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 described in K. Mosbach (ed.): Methods in Enzymology, 44, "Immobilized Enzymes" (Academic Press, 1976).
Lipase-catalyzed processes
The lipases of the invention may be used in any of the following processes (reactants indicated in parenthesis):
- Ester hydrolysis (ester + water) - Ester synthesis (acid + alcohol)
- Interesterification, including
- Acidolysis (ester + acid)
- Alcoholysis (ester + alcohol)
- Ester interchange or transesterification (ester + ester)
The alcohol may be a mono- or polyvalent primary 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.
The immobilized lipase of the invention is particularly suited for continuous interesterification where the enzyme is in fixed-bed columns. The lipase should be partly hydrated, and the reaction mixture should be partly saturated with water to activate the enzyme. A suitable temperature is around 60°C. A solvent such as hexane or other hydrocarbon can be used, but is usually not necessary.
An example of this process is acidolysis of triglyceride and fatty acid to produce triglyceride with mainly unsaturated acyl in the 2-position and mainly saturated acyl groups in the 1- and 3-positions, for confectionery use.
EXEMPLARY PRACTICE OF THE INVENTION
Assays for activity of soluble lipase (LU)
The method is based on hydrolysis of tributyrine in a pH-stat. 1 LU (Lipase Unit) is the amount of enzyme which liberates 1 μmol 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.
Acidolysis activity of immobilized lipase (BIU)
The activity is determined by reacting palmitic acid with triolein with or without solvent. Total incorporation of palmitic acid is measured by FAME-GLC of triglyceride.
FAME-GLC (Fatty acid methyl ester - gas-liquid chromatography) may be done according to methods Ce 2-66 and Ce 1-62 published by the American Oil Chemists' Society (AOCS).
In case of reaction with solvent, the reaction mixture consists of 0.6 g of triolein, 0.174 g of palmitic acid and 8.083 g of petroleum ether. For reaction without solvent, 3.0 g of triolein and 0.87 g of palmitic acid is used.
In either case, a suitable amount of enzyme is hydrated, incubated with the above reaction mixture at a given temperature for 1-4 hours, and then filtering to stop the reaction. The filtrate is purified on an alumina column, and the triglycerides are analyzed by FAME-GLC.
One BIU (Batch Interesterification Unit) is the amount of immobilized lipase that incorporates palmitic acid at an initial rate of 1 μmole/minute at the given temperature with or without solvent.
EXAMPLE 1
This example shows the effect of pH during immobilization of Humicola lipase on Duolite® ES 562 macroporous, phenolic anionic resin.
Immobilizations were made by adjusting 4.25 g D.S. of Duolite® ES 562 (Rohm & Haas) to the appropriate pH in aqueous suspension and adding 1.0 g of Humicola lipase of 91,700 LU/g (prepared according to Example 1 of DK 4117/86) in 12.5 ml water to the recovered, wet resin.
After rotation for 8 hours at room temperature the preparations were filtered, washed with 25 ml water and dried in vacuum at room temperature.
Results appear from the table below:
Immobilization | Load | Activity pH | Yield (%) | LU/mg D.S. | BlU/g | | |
5.0 | 87 | 18 | 37
5.9 | 85 | 18 | 34
6.8 | 90 | 19 | 30
BIU was measured at 40°C with solvent.
These results indicate (1) that optimal initial activity is obtained around pH 5 (2) that the lipase is immobilized in high yields and (3) that the loaded hydrolytic activity is more efficiently expressed in terms of interesterification activity than usually seen with Mucor lipase (30 LU/mg and 30 BlU/g).
EXAMPLE 2
This example shows the immobilization of
Humicola lipase in a somewhat larger scale, but otherwise similar to the procedure in Example 1.
98 g D.S. Duolite® ES 568N (Rohm & Haas) was adjusted to pH 5 and mixed with 20 g Humicola lipase of 146,100 LU/g dissolved in water to 250 g enzyme solution. After readjustment to pH 5 and stirring with a propeller for 5 hours at room temperature the preparation was filtered and washed with 400 ml of water. The product was dried in vacuum at room temperature. Results appear below:
Immobilization | Load | Activity pH | Yield (%) | LU/mg D.S. | BlU/g | | |
5.3 | 98 | 29 | 35
BIU was measured at 40°C with solvent.
EXAMPLE 3
This example shows the immobilization of Humicola lipase on a macroporous, acrylic non-ionic resin.
4.25 g D.S. of Lewatit® E2001/85 (Bayer) was mixed with 0.71 g of Humicola lipase of 211,100 LU/g dissolved in water to 12.5 g enzyme solution. After readjustment to pH 5 and rotation for 6 hours at room temperature the preparation was filtered and washed with 50 ml water. The product was dried in vacuum at room temperature. Results appear below:
Immobilization | Load | Activity pH | Yield (%) | LU/mg D.S. | BlU/g (40°C) | BIU/g (60°C)
| | | | 5.6 | 93 | 33 | 186 | 226
BIU was measured at 40°C with solvent and at 60°C without solvent.
Claims
1. An immobilized, positionally specific lipase preparation, characterized in that said lipase is produced by cultivation of a microorganism containing a gene encoding for and expressing a lipase derived from a strain of Humicola (incl. Thermomyces).
2. The preparation of Claim 1, characterized in that the Humicola strain belongs to H. lanuginosa, H. brevispora, H. brevis var. thermoidea or H. insolens
3. The preparation of Claim 2, characterized in that the strain is DSM 3819, DSM 4109, DSM 4110, DSM 4111 or DSM 1800.
4. The preparation of Claims 1 - 3, characterized in that the lipase is produced by cultivation of the Humicola strain or a mutant or variant thereof.
5. The preparation of Claims 1 - 3, characterized in that the lipase is produced by cultivation of a transformed host organism.
6. The preparation of Claim 5, characterized in that the host organism is a Humicola strain, an Aspergillus strain or a yeast, preferably A. oryzae.
7. An immobilized, 1,3-specific lipase preparation, characterized in that said lipase shows immunochemical identity to a lipase derived from a Humicola strain as defined in Claims 1 - 3.
8. The preparation of Claims 1 - 7, characterized by being immobilized on a particulate, macroporous resin, preferably an adsorbent or a weakly basic anion exchange resin, and preferably of the phenol-formaldehyde or acrylic type.
9. Use of the immobilized lipase preparation of Claims 1 - 8 in ester hydrolysis, ester synthesis or interesterification.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK687387A DK687387D0 (en) | 1987-12-28 | 1987-12-28 | IMMOBILIZED LIPASE |
DK6873/87 | 1987-12-28 |
Publications (1)
Publication Number | Publication Date |
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WO1989006278A1 true WO1989006278A1 (en) | 1989-07-13 |
Family
ID=8149135
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/DK1988/000219 WO1989006278A1 (en) | 1987-12-28 | 1988-12-22 | Immobilized lipase |
Country Status (2)
Country | Link |
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DK (1) | DK687387D0 (en) |
WO (1) | WO1989006278A1 (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0424130A1 (en) * | 1989-10-20 | 1991-04-24 | Unichema Chemie B.V. | Supported enzyme |
WO1991014784A1 (en) * | 1990-03-23 | 1991-10-03 | Novo Nordisk A/S | A process for increasing the amount of triglyceride of a fat or oil |
US5232843A (en) * | 1989-10-20 | 1993-08-03 | Unilever Patent Holdings Bv | Preparation of immobilized lipase by adsorption of lipase and a non-lipase protein on a support |
US5914306A (en) * | 1991-05-01 | 1999-06-22 | Novo Nordisk A/S | Stabilized enzymes |
US6110508A (en) * | 1992-08-21 | 2000-08-29 | Novo Nordisk A/S | Use of lipase in baking |
US10563094B2 (en) | 2011-09-09 | 2020-02-18 | Toyota Motor Engineering & Manufacturing North America, Inc. | Coatings containing polymer modified enzyme for stable self-cleaning of organic stains |
US10767141B2 (en) | 2010-06-21 | 2020-09-08 | Toyota Motor Engineering & Manufacturing North America, Inc. | Thermolysin for easy-cleaning of insect body stains |
US10781438B2 (en) | 2006-11-22 | 2020-09-22 | Toyota Motor Engineering & Manufacturing North America, Inc. | Biofunctional materials |
US10988714B2 (en) | 2010-06-21 | 2021-04-27 | Regents Of The University Of Minnesota | Methods of facilitating removal of a fingerprint from a substrate or a coating |
US11015149B2 (en) | 2010-06-21 | 2021-05-25 | Toyota Motor Corporation | Methods of facilitating removal of a fingerprint |
US11624044B2 (en) | 2010-06-21 | 2023-04-11 | Toyota Motor Corporation | Compositions for facilitating biological stain removal |
Citations (1)
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EP0140542A1 (en) * | 1983-09-05 | 1985-05-08 | Novo Nordisk A/S | An immoblized lipase preparation and use thereof |
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1987
- 1987-12-28 DK DK687387A patent/DK687387D0/en not_active Application Discontinuation
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EP0140542A1 (en) * | 1983-09-05 | 1985-05-08 | Novo Nordisk A/S | An immoblized lipase preparation and use thereof |
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CHEMICAL ABSTRACTS, Vol. 105, (1986), abstract No. 222345s, MORINAGA, TSUTOMU et al., J. FERMENT. TECHNOL. 1986, 64(5), 451-3 (Eng). * |
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Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0424130A1 (en) * | 1989-10-20 | 1991-04-24 | Unichema Chemie B.V. | Supported enzyme |
US5232843A (en) * | 1989-10-20 | 1993-08-03 | Unilever Patent Holdings Bv | Preparation of immobilized lipase by adsorption of lipase and a non-lipase protein on a support |
WO1991014784A1 (en) * | 1990-03-23 | 1991-10-03 | Novo Nordisk A/S | A process for increasing the amount of triglyceride of a fat or oil |
US5914306A (en) * | 1991-05-01 | 1999-06-22 | Novo Nordisk A/S | Stabilized enzymes |
US6110508A (en) * | 1992-08-21 | 2000-08-29 | Novo Nordisk A/S | Use of lipase in baking |
US11236323B2 (en) | 2006-11-22 | 2022-02-01 | Toyota Motor Corporation | Biofunctional materials |
US11225654B2 (en) | 2006-11-22 | 2022-01-18 | Toyota Motor Corporation | Biofunctional materials |
US10781438B2 (en) | 2006-11-22 | 2020-09-22 | Toyota Motor Engineering & Manufacturing North America, Inc. | Biofunctional materials |
US10988714B2 (en) | 2010-06-21 | 2021-04-27 | Regents Of The University Of Minnesota | Methods of facilitating removal of a fingerprint from a substrate or a coating |
US11015149B2 (en) | 2010-06-21 | 2021-05-25 | Toyota Motor Corporation | Methods of facilitating removal of a fingerprint |
US10767141B2 (en) | 2010-06-21 | 2020-09-08 | Toyota Motor Engineering & Manufacturing North America, Inc. | Thermolysin for easy-cleaning of insect body stains |
US11254898B2 (en) | 2010-06-21 | 2022-02-22 | Toyota Motor Corporation | Bioactive protein-polymer compositions |
US11624044B2 (en) | 2010-06-21 | 2023-04-11 | Toyota Motor Corporation | Compositions for facilitating biological stain removal |
US11692156B2 (en) | 2010-06-21 | 2023-07-04 | Toyota Motor Corporation | Bioactive protein-polymer compositions for stain removal |
US10563094B2 (en) | 2011-09-09 | 2020-02-18 | Toyota Motor Engineering & Manufacturing North America, Inc. | Coatings containing polymer modified enzyme for stable self-cleaning of organic stains |
US11535773B2 (en) | 2011-09-09 | 2022-12-27 | Toyota Motor Corporation | Coatings containing polymer modified enzyme for stable self-cleaning of organic stains |
US11542410B2 (en) | 2011-09-09 | 2023-01-03 | Toyota Motor Corporation | Coatings containing enzyme for stable self-cleaning of organic stains |
US11566149B2 (en) | 2011-09-09 | 2023-01-31 | Toyota Motor Corporation | Coatings containing polymer modified enzyme for stable self-cleaning of organic stains |
US11597853B2 (en) | 2011-09-09 | 2023-03-07 | Toyota Motor Corporation | Coatings containing polymer modified enzyme for stable self-cleaning of organic stains |
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