Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

Maheshwari D. T.; Thygaraj Varsha; N. S. Kumar

doi:10.14429/dlsj.2.12272

Maheshwari D. T. Defence Bioengineering and Electromedical Laboratory, Bengaluru – 560 093, India
Thygaraj Varsha Defence Bioengineering and Electromedical Laboratory, Bengaluru – 560 093, India
N. S. Kumar Defence Bioengineering and Electromedical Laboratory, Bengaluru – 560 093, India

DOI: https://doi.org/10.14429/dlsj.2.12272

Keywords: Organophosphorus compounds, Pseudomonas diminuta, Organophosphorus hydrolase, Chromatography

Abstract

Synthetic organophosphorus compounds are highly toxic hence, widely used as pesticides, insecticides and chemical warfare agents. Organophosphorus hydrolase (OPH) is an organophosphotriester hydrolyzing enzyme; effectively hydrolyze a range of organophosphate esters. The objective of the present study was extraction and purification of OPH enzyme from Pseudomonas diminuta bacteria (soil microorganism) and to study kinetic properties of the purified enzyme. Enzyme was extracted and purified from bacteria by ammonium sulphate precipitation and ion exchange chromatography. Purity of an enzyme was determined by sodium dodecyl sulphate-polyacryamide gel electrophoresis (SDS-PAGE). Purified OPH enzyme specific activity was found to be 27.7 fold of 32.8U/mg protein, molecular weight of 72 Kda and it is a homodimer since it has shown a single band in SDS-PAGE separation. Maximum activity of the free OPH enzyme was found at Optimum pH 7.5 and temperature 35^oC with the incubation time of 10 min. Michaelis constant (Km) and maximum velocity (Vmax) values of free OPH enzyme for methyl parathion as substrate was found to be 286.2μM and 2.5 μM/min respectively.

Author Biographies

Maheshwari D. T., Defence Bioengineering and Electromedical Laboratory, Bengaluru – 560 093, India

Dr Maheshwari D.T. has received her PhD in Life Sciences fromBharathiar University, Coimabatore, India, in 2013. Presentlyworking as Scientist ‘C’. She has published some research papers inpeer reviewed journals in the area of nutrition and biochemistry. Herareas of interests : Biosensors development, biodetection systems,
immunology and molecular biology.

Thygaraj Varsha, Defence Bioengineering and Electromedical Laboratory, Bengaluru – 560 093, India

Mr C. Thyagaraj, obtained his BSc from Govt Degree College,Kuppam, in 2013 and MSc (Biotechnology) from Dravidian University, in 2015. He worked as contract engineer in Defence Bioengineering and Electromedical Laboratory (DEBEL) for a year. His area of interests : Biodetection systems and molecular biology.

N. S. Kumar, Defence Bioengineering and Electromedical Laboratory, Bengaluru – 560 093, India

Dr N. S. Kumar, currently working as Scientist ‘F’ and heading the Department of Environmental Protection at DEBEL. He was involved in the design and development of NBC protective equipment,air quality management for submarine (INS Arihant) and NABL Accreditation. He has more than 25 publications in peer reviewed journal and posses some patents.