Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1995-11-09
1998-06-30
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435176, 435177, 435180, 435198, C12P 764, C12N 1114, C12N 1108, C12N 920
Patent
active
057732663
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This application is a 371 of international application PCT/EP94/01308, filed Apr. 22, 1994.
Enzymic conversions of triglycerides, diglycerides, monoglycerides, alkyl esters or free fatty acids, both for the production of triglycerides or other esters and for the hydrolysis of these compounds, in particular for the production of free fatty acids and of free alcohols or glycerol, have been known for many years.
GB 1,577,933, e.g., discloses a process for the preparation of triglycerides by interesterification of a mixture of triglycerides or by reaction of a triglyceride with a free fatty acid. Enzymes that can be used are randomizing enzymes; 1,3-specific enzymes; site-specific enzymes, such as .DELTA..sup.9 -cis specific enzymes etc. The enzymes can be supported on support materials, which enable the application of continuous processes, while simultaneously increasing the activity of the enzymes.
According to U.S. Pat. No. 4,629,742 or EP 322,213, an improvement can be achieved, in particular with respect to enzyme activity, when the enzyme, preferably a lipase, is supported on a hydrophobic support material with a specific pore diameter (i.e. above 50 .mu.m). However, the process for the immobilization of the enzyme requires pre-treatment of the hydrophobic support with a polar organic solvent, if the best results are to be achieved.
According to EP 424,130, a further improvement in enzyme stability can be obtained when the hydrophobic support is pre-treated with alcohol, followed by adsorption of a non-lipase protein, prior to the lipase adsorption.
EP 514,694 discloses an immobilization of lipase on a carrier whereby a surface active agent is added to the lipase-solution to make a micelle around the lipase. The surface active agent can be a non-ionic surface active agent. However no limitations are given for this agent.
We have performed a study in order to find out whether such a solvent pre-treatment could be avoided. Another aim of this study was to find out whether the lipase enzyme could be immobilized by a less complicated process, avoiding any pre-treatment, whereby an in situ immobilization could be achieved in the reaction column per se.
This study has resulted in our invention, which comprises a new process for the immobilization of lipases, resulting in new immobilized lipase enzyme compositions having an enzyme activity that is comparable with that of the prior art products.
SUMMARY OF THE INVENTION
Accordingly, our invention concerns in the first instance a process for the preparation of immobilized lipase enzymes on a dry, porous particulate hydrophobic support material having an average pore size of 0.05-5 .mu.m, wherein the hydrophobic support is contacted with an aqueous solution of the lipase enzyme, either after the support is contacted with a non-ionic surfactant containing at least one fatty acid moiety and having an HLB (=hydrophilic lipophilic balance) value of at least 8, or simultaneously during the contact with the lipase or prior to the contact of the support with the surfactant, whereupon the supported lipase is washed and the washed material is dried.
It is emphasized here that the surfactant can either be completely dissolved in the waterphase (forming a clear solution) or can form a stable dispersion in the waterphase (forming a hazy or an opaque dispersion). This is disclosed in Porter's Handbook of Surfactants 1991, page 42 Ed: Blackie.
From U.S. Pat. No. 4,539,294 it is known that long chain cationic surfactants can be used in the immobilization of lipases. As supports hydrophobic supports, such as Accurel.RTM. are mentioned. However, according to this process a solution of the surfactant in a polar organic solvent must be applied.
According to the process of our invention, the support should be contacted with an aqueous solution of the lipase enzyme while the non-ionic surfactant is to be used at any stage of this contact in order to achieve the required loading and enzyme activity.
As the simplest process is one in which this contact is
REFERENCES:
patent: 4539294 (1985-09-01), Metcalfe et al.
patent: 4629742 (1986-12-01), Brady et al.
patent: 5232843 (1993-08-01), Bosley et al.
Mosbach et al, Methods in Enzymology, vol. 135, Part B, pp. 230-252 (1987).
Chemical Abstracts, vol. 118, No. 25, Abstract No. 250676n (Jun. 21, 1993).
Database WPI, Derwent Publications, Ltd., An 89-051780.
Bosley John Anthony
Moore Stephen Raymond
Loders-Croklaan B.V.
Naff David M.
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