Expression and Characterization of Bacterial Organophosphorus Hydrolase in Pichia pastoris with the Intent to Degrade Organophosphate Neurotoxins

Objective: Organophosphorus hydrolase (OPH) is a homodimeric enzyme that can hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. This makes OPH a suitable element for the biodegradation of these compounds. Methods: We successfully cloned the OPH gene from Pseudomonas diminuta, after optimization for Pichia pastoris, into a yeast expression vector (pPICZαB). After transformation and induction of recombinant yeasts, the expressed enzyme was investigated for its biochemical and kinetical parameters. Results: The enzyme was purified ۷.۴۹-fold to a specific activity of ۰.۴۲۱×۱۰۳ U/mg protein from the supernatant with a yield of ۳۳%. The purified enzyme was able to degrade organophosphates. It had an optimal activity and stability up to ۵۰°C, and a pH range of ۷.۰-۱۰.۰. The enzyme had a Km of ۴۵.۹۶ µM and a Vmax of ۱۱.۲۳ µM/min (۴۲۱ µM/min/mg) for paraoxon as a substrate. This enzyme was sensitive to divalent cations and inactivated by denaturing compounds such as SDS. The molecular mass of the purified enzyme as estimated by SDS–PAGE analysis was approximately ۴۰ kDa. Conclusion: In this study, the purified enzyme effectively hydrolyzed paraoxon, an organophosphorus compound. The activity and stability of this enzyme at high temperatures and pH, and low Km in comparision with bacterial isolates could make it an attractive biocatalyst for applied bioremediation and biosensing

کلیدواژه ها:

Organophosphorus compounds ، Organophosphorus hydrolase ، Pichia pastoris ، پیکیا پاستوریس ، ترکیبات ارگانوفسفره ، ارگانوفسفر هیدرولاز