Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1999-06-23
2001-10-16
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C435S209000
Reexamination Certificate
active
06303356
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a modified enzyme and its modification process and more particularly, to the modified enzyme and its modification process, wherein amine group (—NH2) from amino acid residues of cellulase or hemicellulase, carbohydrase, is covalently coupled with the Polyethylene glycol fatty alcohol ether expressed by the following formula 1 and then the modified enzyme exhibits a high stability of activities against pH and temperature as well as better enzyme activity.
RO—(CH
2
CH
2
O)
n
—H (1)
Where, R is an alkyl group of 14 to 18 carbon atoms, and n is an integer of 3 to 12.
2. Description of the Related Art
Enzymes, biochemical molecules produced from fungi or bacteria, catalyze various reactions due to a high degree of specificity for their substrates. Now that excessive use of chemicals is mainly involved in more severe environmental contamination, many researches have focused on the utilization of environment-friendly enzymes.
For example, the use of enzyme for degradation of carbohydrate is advantageous in that reactions can be performed under very mild conditions, compared with the conventional acid hydrolysis, without any corrosion of devices and environmental problems. Nevertheless, the degradation method of enzyme is not easy for its actual application in an industrial field, since (1) the degradation rate by enzyme is slow, (2) enzymes lose their apparent activity during hydrolysis due to tight adsorption of enzymes to carbohydrate, (3) the compliance and stability of enzymes are poor against the outer environmental conditions (e.g., pH, temperature, foreign materials in an enzymatic system, etc.).
To overcome these shortcomings, the following conventional methods designed to immobilize enzymes have been reported: (1) coupling methods such as physical adsorption, ionic bonding, biochemical bonding or covalent bonding, (2) microencapsulated methods, (3) cross-linking methods by a cross-linking agent, and (4) combination methods thereof. Some literatures have disclosed a method of modifying some enzymes (e.g., catalase, lipase, peroxidase, and chymotrypsin) into a copolymer of polyethylene glycol and cyaruric chloride [Trends Biotechnol., 4, 190 (1986); Biotechnol. Lett. 9, 187 (1987)].
Up to now, studies on the chemical modification of cellulase and hemicellulase, carboyhdrase, have not been reported. The methods for treating cellulase have been mainly confined either to the enzymological studies designed to control the activity of each enzyme component via purification and separation of cellulase, or to researches designed to extract new enzymes from animals or plants.
In addition, some literatures have disclosed that the concurrent use of surfactant contributes much to enhancement of enzyme activity [Biotechnol. Bioeng., 23, 1365 (1981); Biotechnol. Bioeng., 28, 1727 (1986)]. A majority of recently commercialized enzymes contain the surfactant. However, a simple mixture containing enzyme and surfactant is highly sensitive to the outer environmental conditions (e.g., temperature, pH, etc.), thus restricting its wide use.
To enhance the stability of enzyme activity, some researchers have recently modified enzymes with a synthetic copolymer of polyethyleneglycol alkylallylether and maleic acid anhydride [J. Chem. Eng. Japan, 25(2), 202 (1992)]. The stability of enzyme activity has been modified to some extent but the nominal activity of modified enzyme is lower than that of unmodified native enzyme.
As described above, the conventional modification methods of enzyme, which are chiefly directed towards a simple mixing of enzyme with surfactants, are entirely different from the present invention. Further, the chemical modification of the present invention is significantly different from that of the above mentioned literatures [J. Chem. Eng. Japan, 25(2), 202(1992)] in terms of chemical structure of materials in use and performance of modified enzyme in improving the activity and stability.
SUMMARY OF THE INVENTION
Through intensive efforts to achieve an improvement in enzyme activity, the inventors have finally come to know that if a single substance comprising enzyme and surfactant is prepared via chemical coupling of enzyme and surfactant instead of a simple physical mixture of both enzyme and surfactant, the stability of enzyme activity can be significantly enhanced in a specific range of modification. Further, the inventors have noted that the significant activity of modified enzyme may be ensured by selecting polyoxyethylene fattyalcoholether (that is, a polyethylene glycol fatty alcohol ether) as a surfactant, which allows enzyme and substrate to be readily associated and dissociated. Thus, the inventors have completed the present invention.
Therefore, an object of the present invention is to provide a novel modified enzyme and the process for preparing such enzyme, wherein amine group (—NH2) from amino and residues of cellulase or hemicellulase, carbohydrase, is covalently coupled with the polyethylene glycol fatty alcohol either expressed by the following formula 1 and then the modified enzyme exhibits a high stability of activities against pH and temperature as well as better enzyme activity.
REFERENCES:
Trends Biotechnol. 4, 190 (1986).
Biotechnol. Lett., 9, 187 (1987).
Biotechnol, Bioeng., 23, 1365 (1981).
Biotechnol. Bioeng., 28 1727 (1986).
J. Chem. Eng. Japan, 25 (2), 202 (1992).
Kim Jeong Eun
Ow Steven Say-kyoun
Shin Jong-Ho
Guttman Harry J
Korea Research Institute of Chemical Technology
Weber Jon P.
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