Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-02-12
2001-08-28
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, C435S177000, C435S178000, C435S182000, C435S280000
Reexamination Certificate
active
06280983
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a gel containing an immobilised enzyme, a method for preparing such a gel and its application in enzyme-catalysed reactions in hydrophobic solvents.
2. Description of the Related Art
Enzyme-catalysed reactions are in widespread use particularly in foodstuffs and pharmacological chemistry. Some enzymes being very expensive, it is essential that they are carefully recovered for re-use in subsequent reactions. This can be done by immobilising the enzymes on solid supports e.g. in the form of small solid beads which are introduced in the reaction mixture and separated after the reaction has been accomplished. Continuous reactions may be performed by placing these beads in a column and by allowing the reaction mixture to flow through the bed of beads. In such beads, the enzyme is covalently bound into a compound, e.g. a polymer containing reactive groups reacting with the amino groups in the protein part of the enzyme.
The work Koskinen A. M. P. & Klibanov, A. M. Blackie Academic & Professional, Glasgow 1996 describes methods for immobilising an enzyme on a solid support in the section “Modes of using enzymes in organic media” by P. Adlercreutz. The methods may be summarised as follows:
an aqueous solution containing the enzyme is brought into contact with a solid support, water is removed under reduced pressure and the enzyme is precipitated onto the solid support;
the enzyme is precipitated from an aqueous solution in the presence of a solid support, the precipitation taking place with the addition of a cooled, water-miscible solvent, such as acetone;
the enzyme in the aqueous solution is allowed to spontaneously adsorb on a solid support;
the enzyme is covalently bound to a solid support, and
the enzyme, which is adsorbed or precipitated on a solid support, is cross-linked with glutaraldehyde.
The publication S. Backlund et al.,
Kamia
-
Kemi
Vol. 20 (1993) 197-201 describes an enzyme-catalysed synthesis of a number of esters by means of the enzyme lipase incorporated in a water-in-oil microemulsion. The publication S. Backlund et al., Colloid Polym Sci 274:540-547 (1996) describes a lipase-catalysed synthesis of optically active esters from racemic 2-octanol and various carboxylic acids. The lipase is incorporated in a microemulsion-based gel, in which the microemulsion is either a water-in-oil microemulsion or a microemulsion having a bicontinuous structure.
However, the above methods for immobilising enzymes involve a large number of drawbacks. Above all, the immobilising methods described above are very sophisticated, laborious and expensive.
Precious enzymes are used e.g. in the preparation of optically active pharmaceutical compounds, which obviously have to be perfectly pure and above all, free from toxic substances. In this conjunction, polymers and similar enzyme-binding substances may involve an undesired risk. Hydrocarbons are for instance used as a hydrophobic component in the preparation of microemulsions. Surfactants are used to stabilise microemulsions, AOT (sodium 1,4-bis (2-ethylhexyl)sulfosuccinate) being probably the most common of these. The AOT surfactant is not suitable for use in pharmaceutical contexts.
EP patent application 68594 describes a gel consisting of water, enzyme, gelatiniser, buffer, and albumine or protein. The albumine or protein component is allegedly added to stabilise the enzymic system. The gel thus produced will have the form of small beads. Judging by the disclosure, the mixture used is unlikely to have resulted in a gel which is mechanically divisible into fragments at a temperature equalling at least approximately the gelatination temperature of the gelatiniser or at the ambient temperature. The disclosure indicates that the mixture was cooled down to 5° C. Cooling to such a low temperature implies that bead formation would have been impossible at a higher temperature.
The publication Biotech. Bioeng. 21 (1979) 1697-1709 (Tosa et al) describes a gel with carrageenan as a gelatiniser. The gel was shaped as a cube at a low temperature (10° C.). There is no suggestion of the possibility to divide this gel mechanically at the gelatination temperature of carrageenan or in the proximity of this.
SUMMARY OF THE INVENTION
The purpose of the present invention is to achieve a straightforward gel providing ease of production and handling and containing an enzyme immobilised within it. A further purpose is that such a gel be free from toxic substances and thus suitable for pharmaceutical synthesis. Moreover, none of the components in the gel should act as a substrate for the enzyme. Still a further purpose is to provide a gel having a surface with open pores such that the enzyme immobilised within the gel is well contacted with the surrounding reaction mixture. The purpose is also to enable the reaction to be performed at the ambient temperature without stirring, which is an economical procedure in terms of energy supply.
Thus, the invention relates to a gel containing an immobilised enzyme. The gel is characterised in that it consists of a water-soluble enzyme, a water-soluble gelatiniser and water, the ratio of the amount of gelatiniser to the amount of water having been selected such that the gel formed can be mechanically divided into dimensionally substantially stable fragments at a temperature which can reach the lower limit of the gelatination temperature range of the gelatiniser.
REFERENCES:
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patent: 4912032 (1990-03-01), Hoffman et al.
patent: 4978619 (1990-12-01), Kajiwara et al.
patent: 5292649 (1994-03-01), Kosugi et al.
patent: 5378627 (1995-01-01), Shibatani et al.
patent: 5776741 (1998-07-01), Pedersen et al.
patent: 0 056 111 A1 (1982-07-01), None
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patent: 1 500 456 (1978-02-01), None
Koch et. al., “Enzymatic Catalysis in Organic Media: Reactors with a Solidified Water-Phase”, Biochem. Eng. vol. 3, pp. 58-60 (3rd Int. Symp. 1995).*
“Immobilized Biocatalysts”, Ullmann's Encyclopedia of Industrial Chemistry. (5th ed), vol. A14, pp. 40-41 (1989).*
Tosa et al., “Immobilization of Enzymes and Microbial Cells . . . ”Biotech. and Bioeng., vol. XXI 1697-1709 (1979).
Koch et al., “Enzymatic Catalysis in Organic Media: . . . ” Aachen University of Technology, Dept. of Biotechnology, D-52076 Aachen (Germany).
Ullmann's Encyclopedia of Industrial Chemistry, (5th ed) vol. A14, pp. 40-41.
Backlund Sune
Eriksson Folke
Hedström Gun
Karlsson Stefan
Lydon James C.
Naff David M.
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