Expression and characterization of bacterial organophosphorus hydrolase in pichia pastoris with the intent to degrade organophosphate neurotoxins

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. We successfully cloned the OPH gene from Pseudomonas diminuta, after optimization for Pichia pastoris, into a yeast expression vector [pPICZlphaB]. After transformation and induction of recombinant yeasts, the expressed enzyme was investigated for its biochemical and kinetical parameters. The enzyme was purified 7.49-fold to a specific activity of 0.421×10[3] U/mg protein from the supernatant with a yield of 33%. The purified enzyme was able to degrade organophosphates. It had an optimal activity and stability up to 50°C, and a pH range of 7.0-10.0. The enzyme had a Km of 45.96 µM and a Vmax of 11.23 microM/min [421 microM/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 40 kDa. 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

Expression and purification of functionally active recombinant human alpha 1-antitrypsin in methylotrophic yeast pichia pastoris

Human alpha 1-antitrypsin [AAT] cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZ?-AAT, human AAT was expressed in the yeast Pichia pastoris [P.pastoris] in a secretary manner and under the control of inducible alcohol oxidase 1 [AOX1] promoter. The amount of AAT protein in medium was measured as 60 mg/l 72 hr after induction with methanol. Results indicated the presence of protease inhibitory function of the protein against elastase. Purification was done using His-tag affinity chromatography. Due to the different patterns of glycosylation in yeast and human, the recombinant AAT showed different SDS-PAGE patterns compared to that of serum-derived AAT while pI shifted from 4.9 in native AAT compared to 5.2 in recombinant AAT constructed in this study