Immobilized cholinesterase enzyme preparations and a process for

Details Immobilized cholinesterase enzyme preparations and a process for Immobilized cholinesterase enzyme preparations and a process for

1983-03-08

1985-12-03

Rosen, Sam

Chemistry: molecular biology and microbiology

Carrier-bound or immobilized enzyme or microbial cell;...

Enzyme or microbial cell is immobilized on or in an organic...

435180, C12N 1106, C12N 1108

Patent

active

045566372

DESCRIPTION:

BRIEF SUMMARY
The invention relates to new immobilized cholinesterase enzyme preparations as well as to a process for the preparation thereof.
It is known that cholinesterase enzyme, isolated most frequently from horse serum, can be applied to detect neurotoxins of phosphate ester, carbamate and sulfate ester type, and to determine the quantity of neurotoxins occurring in air or water. The basis of detection and measurement is the inhibiting effect exerted on the enzyme by neurotoxins. Thus, immobilized cholinesterase enzyme preparations may play the central role of continuously operating measuring systems. The principles of such measuring systems are described, among others, in Anal. Chem. 37, 1378 (1965) and Anal. Biochem. 51, 362 (1973).
Several methods have been described in the literature for the immobilization of cholinesterase isolated from horse serum. According to Anal. Chem. 37, 1378 (1965), Anal. Biochem. 19, 587 (1967), Anal. Biochem. 33, 341 (1970) and U.S. Pat. No. 3,223,593 the enzyme is entrapped in various gels, primarily starch gels. U.S. Pat. No. 3,223,593 also discloses the entrapment of the enzyme in agar and carrageen gels. The use of polyacrylamide as entrapping medium is suggested in Biochim. Biophys. Acta 212, 362 (1970) and Anal. Biochem. 33, 341 (1970). The enzyme can also be coupled through covalent bonds to polymeric macromolecules with appropriate functional groups. Thus e.g. according to Biochim. Biophys. Acta 191, 478 (1969) the enzyme is immobilized on Sepharose 2B activated previously with cyanogen bromide, whereas according to Biochim. Biophys. Acta 377, 297 (1975) polymaleic anhydride is applied as support. According to Clin. Chim. Acta 121, 125 (1980) the enzyme is immobilized on non-porous glass by carbodiimide or glutaric aldehyde coupling, whereas according to the USSR patent specification No. 707,923 the enzyme is coupled to porous glass or silica gel activated with cyanuric acid chloride. As described in Can. J. Biochem. 48, 1314 (1970), cholinesterase isolated from horse serum can also be attached to a Procion brilliant orange DEAE-cellulose complex through covalent bonds.
A disadvantage of the known cholinesterase enzyme preparations in which the enzyme is entrapped in a gel is that the enzyme dissolves continuously from it. Therefore, such preparations are not suitable for continuous, prolonged application. The known cholinesterase enzyme preparations immobilized on the substrate by covalent bonds have adverse throughflow properties from the aspects of application in automatic systems, moreover some of the supports, such as Sepharose 2B, are sensitive to microbial effects.
The invention aims at the elimination of the above disadvantages. According to the invention immobilized cholinesterase enzyme preparations are provided which meet the requirements of prolonged use, and contain the enzyme bound to a chemically and microbially inert support with appropriate mechanical properties ensuring a high throughflow rate. As a further advantage, the enzyme can be coupled to the support under mild reaction conditions.
It has been observed that polymeric resins built up from acrylic acid and/or methacrylic acid and acryl amide and/or methacryl amide monomers with acryl or allyl type cross-linking agents [such as N,N'-methylene-bis(acrylamide), ethylene diacrylate or N,N'-diallyl-tartaric amide], containing at least 0.1 meq/g, preferably 2 to 8 meq/g, of --COOH functional groups, completely meet the requirements set forth in connection with the supports. These supports can be prepared by methods known per se. One can proceed e.g. so that acryl amide and/or methacryl amide is copolymerized with acrylic acid and/or methacrylic acid in the presence of a cross-linking monomer, whereas according to another possible method a cross-linked polymer is prepared first from acryl amide and/or methacryl amide using the above cross-linking agents, and the acid amide groups of the resulting polymer are subjected then to partial hydrolysis to provide the required amount of --COOH functional groups. Akri

REFERENCES:
patent: 4332694 (1982-06-01), Kalal
Li et al., Chem. Abst., vol. 97 (1982), p. 18737h.
Aizenberg et al., Chem. Abst., vol. 97 (1982), p. 213278r.

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