Volume 15, Issue 1 (2012)
mjms 2012, 15(1): 61-72 |
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Najavand S, Sahebghadam Lotfi A, Mohsenifar A, Arjmand S, Mota A. Expression and Characterization of Bacterial Organophosphorus Hydrolase in Pichia pastoris with the Intent to Degrade Organophosphate Neurotoxins. mjms 2012; 15 (1) :61-72
URL: http://mjms.modares.ac.ir/article-30-2824-en.html
URL: http://mjms.modares.ac.ir/article-30-2824-en.html
1- Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2- 1- 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
3- Assistant Professor, Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4- Department of Animal and Marine Biotechnology, National Institute of Genetics Engineering and Biotechnology, Tehran, Iran
2- 1- 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
3- Assistant Professor, Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4- Department of Animal and Marine Biotechnology, National Institute of Genetics Engineering and Biotechnology, Tehran, Iran
Abstract: (7212 Views)
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
Article Type: Original Manuscipt |
Subject:
Molecular|Biochemistry
Received: 2011/10/14 | Accepted: 2012/05/16
Received: 2011/10/14 | Accepted: 2012/05/16
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