ISOLATION AND MUTAGENESIS OF A BACTERIAL STRAIN FOR ENHANCED PRODUCTION AND CHARACTERIZATION OF ORGANOPHOSPHATE HYDROLASE

Abstract

Organophosphate pesticides are of common interest due to increased envionmenal contamination. Such agents cause several undesired and un-solved problems in non-target species. The enzyme organophosphate hydrolase (OPH) producing bacterial strain Brevibacillus parabrevis SR2729 (B. parabrevis SR2729) was isolated from soil contaminated with chlorpyrifos. Brevibacillus parabrevis SR2729 was subjected to strain improvement by physical and chemical mutagenesis; where two strains B. parabrevis SR2729-A and B. parabrevis SR2729-B were selected after classical screening through Triton-x-100 and 3,5,6-trichloro-2-pyridinol. The mutant derived strains Brevibacillus parabrevis SR2729-A and Brevibacillus parabrevis SR2729-B showed 229 and 240 % greater OPH production as compared to parental strain. The optimum levels of six variables (pH, temperature, incubation time, carbon source, nitrogen source and pesticide concentration) were obtained after statistical analysis by RSM. After process optimization, the activities of OPH from B. parabrevis SR2729-A and B. parabrevis SR2729-B were 195 and 178 % greater than OPH from parental strain B. parabrevis SR2729. The specific activity for purified enzyme was observed to be 935, 1521 1911 U mg-1 from parental and mutant derived strains B. parabrevis SR2729-A and B. parabrevis SR2729-B, respectively. After kinetic and thermodynamic analysis, the enzyme from mutant derived strains was observed to be more suitable for utilizing chlorpyrifos as substrate, with higher thermostability as compared to the enzyme from parental strain. The molecular weight of OPH enzymes from parental and mutant derived strains were 37 kDa. In soil, the OPH from mutant derived strains B. parabrevis SR2729-A and B. parabrevis SR2729-B showed 206 and 208 % greater degradation of chlorpyrifos as compared to parental strain. The enzyme from mutant derived strains B. parabrevis SR2729-A and B. parabrevis SR2729-B degraded 28 and 65 % greater insecticide in water compared to enzyme from parental strain. In conclusion, the strain improvement by physical and chemical mutagenesis resulted in the selection of potential mutant derived strains which were more significant agents for the degradation of chlorpyrifos in soil and water samples.