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

• Published in: Biotechnology and bioengineering 2000-09, Vol.69 (6), p.591-596
• Main Authors: Richins, Richard D., Mulchandani, Ashok, Chen, Wilfred
• Format: Article
• Language: English
• Subjects: Aryldialkylphosphatase; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Biodegradation; Biodegradation of pollutants; Biological and medical sciences; Biotechnology; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; CBD; Cellulose; Cellulose - metabolism; cellulose-binding protein; Chemical bonds; Crystalline materials; decontamination; Environment and pollution; Enzyme kinetics; Enzyme Stability; Enzymes, Immobilized - genetics; Enzymes, Immobilized - metabolism; Esterases - genetics; Esterases - isolation & purification; Esterases - metabolism; Flavobacterium - enzymology; Fundamental and applied biological sciences. Psychology; hydrolase; Hydrolysis; Industrial applications and implications. Economical aspects; Kinetics; Molecular Sequence Data; nerve agents; OPH; organophosphates; Paraoxon - metabolism; Phosphates; Purification; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Solutions
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/1097-0290(20000920)69:6<591::AID-BIT2>3.0.CO;2-X

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 Km. Immobilization of the enzymes onto microcrystalline cellulose resulted in a further increase in the Km 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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 591–596, 2000.