Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes.

Autor: Richins RD; Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA., Mulchandani A, Chen W
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2000 Sep 20; Vol. 69 (6), pp. 591-6.
DOI: 10.1002/1097-0290(20000920)69:6<591::aid-bit2>3.0.co;2-x
Abstrakt: Bifunctional fusion proteins consisting of organophosphate hydrolase (OPH) moieties linked to a Clostridium-derived cellulose-binding domain (CBD) were shown to be highly effective in degrading organophosphate nerve agents, enabling purification and immobilization onto different cellulose materials in essentially a single step. Enzyme kinetics studies were performed for the CBD-OPH fusions using paraoxon as the substrate. The kinetics values of the unbound fusion enzymes were similar to OPH with a modest increase in K(m). Immobilization of the enzymes onto microcrystalline cellulose resulted in a further increase in the K(m) values of approximately twofold. The pH profile of the cellulose-immobilized enzymes was also only minimally affected. The CBD-OPH fusion proteins could be immobilized onto a variety of cellulose matrixes, and retained up to 85% of their original activity for 30 days. The durability of the bound fusions increased with the amount of Avicel used, suggesting that protein/cellulose interactions may have a dramatic stabilizing effect. Repeated hydrolysis of paraoxon was achieved in an immobilized enzyme reactor with 100% degradation efficiency over 45 days. These fusion proteins should prove to be invaluable tools for the development of low cost, OPH-based cellulose materials for the simultaneous adsorption and degradation of stored or spilled organophosphate wastes.
(Copyright 2000 John Wiley & Sons, Inc.)
Databáze: MEDLINE
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