Najavand, Saeed, Sahebghadam Lotfi, Abbass, Mohsenifar, Afshin, Arjmand, Sareh, Mota, Ali (2012). Expression and Characterization of Bacterial Organophosphorus Hydrolase in Pichia pastoris with the Intent to Degrade Organophosphate Neurotoxins. , 15(1), 61-72.
Saeed Najavand; Abbass Sahebghadam Lotfi; Afshin Mohsenifar; Sareh Arjmand; Ali Mota. "Expression and Characterization of Bacterial Organophosphorus Hydrolase in Pichia pastoris with the Intent to Degrade Organophosphate Neurotoxins". , 15, 1, 2012, 61-72.
Najavand, Saeed, Sahebghadam Lotfi, Abbass, Mohsenifar, Afshin, Arjmand, Sareh, Mota, Ali (2012). 'Expression and Characterization of Bacterial Organophosphorus Hydrolase in Pichia pastoris with the Intent to Degrade Organophosphate Neurotoxins', , 15(1), pp. 61-72.
Najavand, Saeed, Sahebghadam Lotfi, Abbass, Mohsenifar, Afshin, Arjmand, Sareh, Mota, Ali Expression and Characterization of Bacterial Organophosphorus Hydrolase in Pichia pastoris with the Intent to Degrade Organophosphate Neurotoxins. , 2012; 15(1): 61-72.

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

Pathobiology Reserach
Article 6, Volume 15, Issue 1, 2012, Pages 61-72    PDF (1.77 M)
Authors
Saeed Najavand1; Abbass Sahebghadam Lotfi* 2; Afshin Mohsenifar3; Sareh Arjmand4; Ali Mota1
1Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
21- Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 2- Department of Animal and Marine Biotechnology, National Institute of Genetics Engineering and Biotechnology, Tehran, Iran
3Assistant Professor, Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4Department of Animal and Marine Biotechnology, National Institute of Genetics Engineering and Biotechnology, Tehran, Iran
Abstract
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 7.49-fold to a specific activity of 0.421×103 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 µM/min (421 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 40 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
Keywords
Organophosphorus compounds; Organophosphorus hydrolase; Pichia pastoris
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