METHOD FOR IMMOBILIZING LIPASE

Abstract: Immobilization of lipase on a macroporous adsorbent resin of the acrylic type leads to a product with higher interesterification activity than prior-art methods, even when the same amount of native lipase is used in the two methods. This immobilization method is applicable to a wide variety of microbial lipases from bacteria, yeasts or fungi, including both 1,3-specific and positionally non-specific lipases. All these lipases immobilized by said method have good interesterification activity and enhanced thermostability compared to the native lipase.

Description: METHOD FOR IMMOBILIZING LIPASE This invention relates to a method for immobilizing lipase, to an immobilized lipase preparation and to use of the immobilized lipase. BACKGROUND ART Various immobilization methods for lipase are known. Thus, EP 140,542 (Novo) discloses immobilization of Mucor lipase by adsorption on a weakly basic anion exchange resin, and DK 85/878 (Novo) discloses immobilization of the same lipase on adsorbent resin of phenol-formaldehyde type. Kimura et . al♦ , Eur.J.Appl. Microbiol. Biotechno] . (1983) 17:107-112 discloses Candida lipase immobilized by adsorption on phenol-formaldehyde resins of various functionality. STATEMENT OF THE INVENTION We have surprisingly found that immobilization of lipase on a macroporous adsorbent resin of the acrylic type leads to a product with higher interesterification activity than prior-art methods, even when the same amount of native lipase is used in the two methods. This immobilization method is applicable to a wide variety of microbial lipases fror bacteria, yeasts or f ngi, including both 1,3-specific and positionally non-specific lipases. All these lipases immobilized by said method have good interesterification activity and enchanced thermostability compared to the native lipase . Accordingly, the invention provides a method for immobilizing lipase, characterized by adsorption of the lipase on a particulate, macroporous adsorbent resin of the acrylic type. The invention also provides an immobilized lipase preparation, characterized in that the lipase is adsorbed on a particulate, macroporous resin of the acrylic type. Further, the invention provides use of said immobilized lipase in interesterification, ester synthesis and ester hydrolysis. DETAILED DESCRIPTION OF THE INVENTION Resin Typical resins for use in the invention consist of poly- (meth)acrylic acid esters, (e.g. poly-methyl methacrylate) croεslinked with divinyl benzene. They are macroporous and typically have average pore radius about 2 100-200 A and a total surface area of 25-150 m /g (by K ^ adsorption method) . For use in continuous interesterification in a fixed-bed column the particles should preferably be spherical and of uniform size, preferably 100-1000 μ , e.g. 100 - 500 μm. Examples of resins are Lewatit® E 2001/85 (Bayer, West Germany), Amberlite® XAD-8 (Rohm & Haas, USA). Immobilization process The immobilization is conveniently carried out simply by contacting an aqueous solution of the lipase with the resin, thereafter separating the thus formed immobilized lipase from the aqueous phase followed by washing and drying of the separated immobilized lipase. Suitable temperature and pH will depend on the characteristics of the lipase, but in many cases ambient temperature and pH near neutral may conveniently be used. Contact time will usually be chosen as needed for essentially complete adsorption. This will typically be from 1-2 hours up to 24 hours. Microbial lipase The lipase to be immobilized is preferably microbial. Some preferred lipases are derived from the following organisms: - Mucor sp . , especially M. miehei, commercially available as Lipozyme® (Novo) ~ Candida sp. , especially C. rugosa (available fr Meito Sangyo, Japan, as Lipase OF) and C. antarctica, see PCT/DK87/00127 (Novo). - Pseudomonas sp., especially Ps. cepacia, see DK 87/3993 (Novo). - Humicola sp. , especially H. lanuginosa, see JP 53-45, 394B (Arima), DK 4499/87 (Novo) and JP 62-79, 782A (Unitika). Lipase-catalyzed processes The immobilized lipases of the invention can be used in ester hydrolysis, ester synthesis and interesterification. The latter term includes acidolyεiε (reaction of ester + acid), alcoholysis (ester + alcohol) and transesterification (ester + ester). Besides triglycerideε, other esters may be used depending on substrate specificity of the lipase. The immobilized lipases of the invention are particularly useful for continuous interesterification in a fixed-bed column. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the results of continuous acidolysis. Details are given in Example 2. EXEMPIARY 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 arable as an emulsifier. Further details are given in Novo Analytical Method AF 95/5, available on reques . 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 Immobilization of lipases on Lewatit® E 2001/85 In general an aqueous solution of a given lipase was mixed by rotation with the resin (in this case Lewatit € E2001/85, product of Bayer) at fixed pH, at room temperature. Then the resin with immobilized lipase was collected by filtration, followed by washing with water and drying in vacuum. The removal of adsorbed lipase was calculated frorr. the hydrolytic lipase activity left in the filtrateε (LU- assay) . The interesterification activity of the immobilized preparations was measured by the BlU-aεsay, at 60°C without solvent. Data and results are summarized in the following table. incorporation of lauric acid measured by GLC. An incorporation of 14% w/w lauric acid was attempted and the flow rate was adjusted in order to keep the conversion at that value. Measurements of flow rate were taken when the actual conversion waε 14 ± 1%. Whenever the precolumn was dry it was replaced by a fresh one. The samples were analysed by removing the free fatty acid and mono- and diglyceride by Al^O,-column chromatography, thereafter methylation of the triglyceride by NaOCH, and finally analysis of the methylester on a GLC. The resultε, shown in fig. 1 aε the natural logarithm of flow rate (g triglyceride/hour/g immobilized enzyme) versuε time (hours), indicate a half-life of about 1,800 hours at 60°C. EXAMPLE 3 Immobilization of lipases on Amberlite® XAD-8 Immobilizations were carried out baεically aε described in Example 1. Data and results are summarized in the following table. Amberlite® XAD-8 This resin is a macroporous, non-ionic adsorbent resin of the polyacrylate type. The average pore radius is 2 arund 120 A and total surface area around 140 m /g.

Claims: CLAIMS 1. A method for immobilizing lipase, characterized by comprising adsorption of the lipase on a particulate, macroporous adsorbent resin of the acrylic type. 2. The method of Claim 1, characterized by comprising contacting an aqueous solution of the lipase with the reεin, thereafter separating the thus-formed immobilized lipase from the aqueous phase followed by washing and drying of the separated immobilized lipase. 3. The method of Claim 1 or 2, characterized in that the lipase is derived from a strain of Mucor, Candida, Pseudomonaε or Humicola, preferably from M. miehei, C . antarctica, Ps. cepacia or H. lanuginosa. 4. Immobilized lipase produced by the method of any of Claims 1 - 3. 5. Use , of immobilized lipase according to Claim 4 in interesterification, ester synthesis or ester hydrolyεiε. 6. • Use according to Claim 5 in continuous interesterifica-tion, whereby the immobilized lipase is kept in a fixed bed.

Inventors: EIGTVED PETER, EIGTVED, PETER
Applicants: NOVO INDUSTRI AS, NOVO INDUSTRI A/S
Application year: 1988
Main classification: C12N9
IPC codes: C12N9, C12N11, C12P7