Optimization of haloacid dehalogenase production by recombinant E. coli BL21 (DE3)/pET-hakp1 containing haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 using Response Surface Methodology (RSM)

Abstract

Organohalogens, including monochloroacetic acid (MCA), are abundantly synthesized compounds for various industrial purposes. MCA is widely used as a raw material or as an intermediate compound for the production of pesticides, herbicides, fungicides, plastics, surfactants, shampoos, liquid soaps, and emulsion agents. Nonetheless, widespread and large-scale utilization of organohalogens might negatively impact life quality as these compounds are toxic to organisms and persistently present in the environment. An effort to decrease the effect of MCA pollutant is by performing bioremediation, taking advantage of microorganisms that produce haloacid dehalogenases, a class of enzymes that catalyze the breakage of carbon halogen bonds. In this sense, we have isolated Klebsiella pneumoniae ITB1 that could degrade MCA. The haloacid dehalogenase gene from this bacterium has been successfully cloned into pGEM-T vector and subcloned into pET-30a(+) expression vector to yield pET-hakp1 recombinant clone in Escherichia coli BL21 (DE) host cell. This research aimed to find an optimum condition for producing haloacid dehalogenase from this recombinant clone using Response Surface Methodology (RSM). Among the independent variables studied were the concentration of inducer, incubation temperature after the induction, and incubation period after the induction. We obtained the crude extract of the enzyme as cells' lysate after sonicating the bacterial cells. Haloacid dehalogenase activity against MCA substrate was determined by measuring the amount of chloride ions released into the medium of the enzymatic reaction using the colorimetry method, according to Bergmann and Sanik. The result indicated that the optimum condition for haloacid dehalogenase production by E. coli BL21 (DE3)/pET-hakp1 was observed when using 1.8 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) as the inducer, followed by 4 h incubation with shaking at 37 °C, which was predicted to result in a maximum of 0.48 mM chloride ions from 0.50 mM of MCA substrate. This report provides an insight into applying RSM for optimization of enzyme production from E. coli recombinant clones.

Keywords

• RSM
• Haloacid dehalogenase
• Organohalogen
• Monochloroacetic acid