Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes.
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.
Richins
R D
RD
Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA.
Mulchandani
A
A
Chen
W
W
D043303:Aryldialkylphosphatase; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004950:Esterases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005417:Flavobacterium / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010261:Paraoxon / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D012996:Solutions; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D004950:Esterases; D043303:Aryldialkylphosphatase; D010261:Paraoxon