Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1990-03-28
1992-10-20
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435134, 435180, 435874, 435921, 435931, C12P 762, C12P 764, C12N 1108
Patent
active
051569639
DESCRIPTION:
BRIEF SUMMARY
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. Biotechnol. (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 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.
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) crosslinked with divinyl benzene. They are macroporous and typically have average pore radius about 100-200 .ANG. and a total surface area of 25-150 m.sup.2 /g (by N.sub.2 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 .mu.m, e.g. 100-500 .mu.m.
Examples of resins are Lewatit.RTM. E 2001/85 (Bayer, West Germany), Amberlite.RTM. 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: (Novo) Lipase OF) and C. antarctica, see PCT/DK87/00127 (Novo). 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 acidolysis (reaction of ester+acid), alcoholysis (ester+alcohol) and transesterification (ester+ester). Besides triglycerides, 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.
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 .mu.mol titratable butyric acid per minute at 30.degree. C, pH 7.0 with
REFERENCES:
patent: 4338398 (1982-07-01), Yoneyama
patent: 4472503 (1984-09-01), Matsuo et al.
patent: 4798793 (1989-01-01), Eigtved
patent: 4818695 (1989-04-01), Eigtved
patent: 4898822 (1990-02-01), Asada et al.
Lambiris Elias J.
Naff David M.
Novo Nordisk A S
Zelson Steve T.
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