WO1989006278A1 - Immobilized lipase - Google Patents

Immobilized lipase Download PDF

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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
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Prior art keywords
lipase
preparation
humicola
immobilized
dsm
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PCT/DK1988/000219
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French (fr)
Inventor
Peter Eigtved
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Novo Industri A/S
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    • 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.

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.
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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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.
PCT/DK1988/000219 1987-12-28 1988-12-22 Immobilized lipase WO1989006278A1 (en)

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

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Cited By (11)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0140542A1 (en) * 1983-09-05 1985-05-08 Novo Nordisk A/S An immoblized lipase preparation and use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0140542A1 (en) * 1983-09-05 1985-05-08 Novo Nordisk A/S An immoblized lipase preparation and use thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 105, (1986), abstract No. 222345s, MORINAGA, TSUTOMU et al., J. FERMENT. TECHNOL. 1986, 64(5), 451-3 (Eng). *
CHEMICAL ABSTRACTS, Vol. 107, (1987), abstract No. 214029t, OMAR, IBRAHIM CHE et al., AGRIC. BIOL. CHEM. 1987, 51(8), 2153-9 (Eng). *
CHEMICAL ABSTRACTS, Vol. 108, (1988), abstract No. 146248g, OMAR, IBRAHIM CHE et al., AGRIC. BIOL. CHEM. 1988, 52(1), 99-105 (Eng). *
CHEMICAL ABSTRACTS, Vol. 110, (1989), abstract No, 55933b, OMAR, IBRAHIM CHE et al., BIOTECHNOL. LETT. 1988, 10(11), 799-804 (Eng). *
CHEMICAL ABSTRACTS, Vol. 110, (1989), abstract No. 53732m, OMAR, IBRAHIM CHE et al., AGRIC. BIOL. CHEM. 1988, 52(11), 2923-5 (Eng). *
CHEMICAL ABSTRACTS, Vol. 80, (1974), abstract No. 69149z; & JP,A,48 062 990, ARIMA, KEI et al. *

Cited By (19)

* Cited by examiner, † Cited by third party
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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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