Covalent immobilization of recombinant organophosphorus hydrolase on spores of Bacillus subtilis.

Organophosphorus pesticides are widely used in agriculture. Accordingly, decontamination of these pesticides and their residual in environment is an important aim of researchers. One of the best approaches is enzymatic detoxification of these compounds with organophosphorus hydrolase (OPH). The immobilization of OPH on environmentally friendly supports is of great importance for developing stabilized enzymes for degradation of organophosphorus compounds. In this study, Bacillus subtilis spores were applied as a new matrix for immobilizing OPH for the first time; this enzyme was covalently bound to the spores by using EDC-NHS as coupling reagents and the immobilization was confirmed by enzymatic activity, Western blot, flow cytometry and fluorescence microscopic analysis. The immobilization yield was about 55% and the immobilized OPH hydrolysed paraoxon, an organophosphate substrate, without significant loss of activity was six times. The spores with immobilized OPH on their surface were successfully characterized using FT-IR analysis and SEM imaging. Thermal and pH stability was improved by immobilization of OPH on the spore surface. Owing to safety, environmentally friendly and low cost of spores, these spores can be employed in biosensors for monitoring and biodegradation of organophosphate contaminants in the environment and detoxification processes in bioreactors with high reusability without decrease in the activity. We believe that the spore, an environmentally friendly matrix, can be used for covalent immobilization of OPH efficiently and can be applied for detoxification of organophosphorus compounds under adverse environmental conditions.