Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-12-02
2004-04-06
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S135000, C435S180000
Reexamination Certificate
active
06716610
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for preparing an immobilized enzyme showing a high activity with a less loss in the enzyme activity, which is used for hydrolysis of fats and oils, ester-exchange of fats and oils, and esterification of aliphatic acids and alcohols. The term of “fats and oils” means an inclusion of a fat, an oil, a lard, a grease and so on.
BACKGROUND ART
In hydrolyzing fats and oils by a lipolytic (or fat and/or oil-decomposing) enzyme, an immobilized enzyme prepared by immobilizing a lipolytic enzyme onto an inorganic or organic carrier is used for efficient use of the enzyme. To raise the absorptivity of the enzyme onto a carrier and to improve an enzyme activity, various studies have been made, and for example, JP-A 9-257 discloses a process for producing an immobilized enzyme carrier prepared by immobilizing a lipase onto an inorganic carrier treated with a silane coupling agent having a special functional group, washing and drying it, and impregnating it with an aliphatic acid. Even by this method, however, the amount of the adsorbed enzyme and the enzyme activity remain still insufficient.
Further, an immobilized enzyme prepared by immobilizing a lipolytic enzyme called lipase onto a carrier is used as an enzyme mainly for use in reactions for the object of ester-exchanging (ester-interchanging or transesterifying) fats and oils and esterifying aliphatic acids and alcohols. These reactions are advantageously conducted at concentrations of water as low as possible (1000 ppm or less) to inhibit hydrolysis, and thus the immobilized enzyme is forcibly dried to give only several % water content in the carrier, since the immobilized enzyme is prepared.
However, the adsorbed enzyme tends to inactivate in the step of drying the immobilized enzyme, and there are many cases where the enzyme does not exhibit the maximum activity upon adsorption and when the enzyme exhibits activity thereof actually.
JP-A 62-134090 describes that after immobilization, the immobilized enzyme is dried under contact with aliphatic acid derivatives thereby raising its activity expression, but this method is neither practical nor efficient because expensive facilities are necessary for drying the immobilized enzyme and furthermore it is complicated to set up conditions etc. for slow drying.
Under these circumstances, it is desired that a lipolytic enzyme is prepared so as to exhibit its activity expression sufficiently and to prevent the enzyme from being left (or removed) or inactivated, whereby the amount of the enzyme used for lipolysis is reduced and the esterification reaction is promoted.
DISCLOSURE OF THE INVENTION
To solve this problem, it is desirable that a larger amount of a lipolytic enzyme is adsorbed at adsorption step so as to express high activity and that an atmosphere for promoting the reaction is created around the immobilized enzyme. The present invention relates to a process for preparing an immobilized enzyme for a lipolysis (or decomposition of fats and oils), which comprises absorbing and immobilizing the enzyme onto a porous, anion-exchanging resin as an immobilizing carrier, and which is treated with fats and oils, and the problem described above was thereby solved.
As a result of eager study by the present inventors to solve this problem, they found that it is necessary to confer a stable state on the enzyme adsorbed onto the carrier, for which it is effective to bring a reaction substrate. (or reactant) into contact with the enzyme rapidly after immobilization.
That is, the present invention relates to a process for esterification reaction, which comprises immobilizing a lipolytic enzyme on a carrier for immobilization by adsorption and, without drying, directly bringing the immobilized enzyme into contact with its substrate.
That is, in the present invention, the lipolytic enzyme in a un-dried state after immobilization is brought into contact with its substrate there by providing the immobilized enzyme with a higher degree of adsorption and a higher activity.
The invention provides a process for preparing an immobilized enzyme, which comprises the steps of:
immobilizing a lipolytic enzyme on a porous, anion-exchanging resin for a carrier by adsorption and,
without drying, treating the immobilized enzyme with fats and oils or a derivative of fats and oils.
The process may preferably further comprise the step of treating the carrier's resin with a lypophylic (or fat and/or oil-solving) aliphatic acid or a derivative of a lypophylic aliphatic acid in advance to the immobilization step.
The immobilized enzyme may be treated with fats and oils and the obtained immobilized enzyme is usable for hydrolysis.
Alternatively the immobilized enzyme may be treated with the derivative of fats and oils and the obtained immobilized enzyme is usable for esterification.
The used enzyme is preferably lipase.
The invention provides also use of the immobilized enzyme as defined above for hydrolysis or esterification of fats and oils or a derivative of fats and oils, for example hydrolysis of fats and oils, esterification of derivatives of fats and oils such as partial glycerides, glycerol and an aliphatic acid.
In addition, the invention provides a process for esterifying reaction substrates, which comprises the steps of:
immobilizing a lipolytic enzyme on a porous, anion-exchanging resin for a carrier by adsorption and,
without drying, bringing the immobilized enzyme into contact with the reaction substrates.
The invention moreover provides a process for hydrolysis of reaction substrates, which comprises the steps of:
immobilizing a lipolytic enzyme on a porous, anion-exchanging resin for a carrier by adsorption and,
without drying, treating the immobilized enzyme with reaction substrates and
hydrolyzing the reaction substrates.
In esterification and hydrolysis, the reaction substrates may be fats and oils, such as triglycerides, or a derivative of fats and oils. The derivative of fats and oils may be an aliphatic acid, glycerol or partial glycerides such as monoglycerides and diglycerides.
According to the invention, the immobilized enzyme can be treated with the reaction substrates for hydrolysis or esterification, immediately being subject to the reaction. Alternatively, the immobilized enzyme can be stored after treatment with fats and oils or a derivative of fats and oils.
MODES FOR CARRYING OUT THE INVENTION
The carrier used in the present invention is preferably a porous, anion-exchanging resin. The particle diameter of the resin is desirably 400 to 1000 &mgr;m, and the diameter of its pore is desirably 100 to 1500 Å.
The resin materials include phenol formaldehyde based, polystyrene based, acrylamide based, divinyl benzene based. In particular, phenol formaldehyde-based resin (e.g. tradename: Duolite A-568) is desirable. Its pores give a large surface area for adsorption of the enzyme to obtain a larger amount for adsorption.
In the present invention, the carrier is treated preferably with a lypophylic aliphatic acid or a lypophylic aliphatic acid derivative for pre-treatment before immobilization, thus creating a state of adsorption to exhibit a high activity. The lypophylic aliphatic acid or lypophylic aliphatic acid derivative used has preferably 8 to 18 carbon atoms. For example, said aliphatic acid includes linear and saturated aliphatic acids such as capric acid, lauric acid and myristic acid, unsaturated aliphatic acids such as oleic acid and linoleic acid, hydroxy aliphatic acids such as ricinoleic acid, or branched aliphatic acids such as isostearic acid. The aliphatic acid derivative includes esters between C
8
to C
18
aliphatic acids and compounds having a hydroxyl group, and examples thereof include mono-alcohol monohydric alcohol or monovalent alcohol) esters, polyhydric alcohol polyol or polyvalent alcohol) esters, phospholipids, or derivatives of these esters to which ethylene oxide has been added. The mono-alcohol esters include methyl ester, ethyl ester, and the polyvalent alcohol esters include monoglyceride, digl
Kase Minoru
Komatsu Toshiteru
Shimizu Masami
Shimizu Masao
Kao Corporation
Naff David M.
LandOfFree
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