Optimation of Monochloroacetic Acid (MCA) Degradation by Haloacid Dehalogenase from Bacillus cereus Local Strain in pET-bcfd1 Recombinant Clone in E. coli BL21(DE3)

• Main Author: Ade Pratama , Rachmad
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/23755
• Institution: Institut Teknologi Bandung

Monochloroacetic acids (MCAs) are organic compounds industrially important. These compounds are obtained as the chlorination products of acetic acid and have been widely used as building blocks in the production of carboxymethyl cellulose (CMC), 2,4dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) herbicide, paint and graffiti removal, plastic, and other forms as its ester or amide. However, these compounds cause water environmental pollution and harm to human health as a result of their toxicity, corrosive, persistence, and carsinogenic effects. These compounds are commonly found as pollutants in drinking water and wastewater. Biodegradation is one way to reduce the pollution caused by these compounds, utilizing an enzyme called haloacid dehalogenase produced by the microorganisms. In the previous research, the haloacid dehalogenase gene from Bacillus cereus local strain has been successfully isolated in E. coli BL21(DE) pET-bcfd1 recombinant clone. This research aims to optimize the MCA degradation by this living E. coli BL21(DE3) carrying pET-bcfd1 recombinant clone in LB medium containing MCA. Liberation of chloride ion in medium as a result of enzimatic reaction catalyzed by haloacid dehalogenase to MCA as the substrate was followed spectrophotometrically as described by Bergmann and Sanik. The MCA degradation perform by the recombinant clone living cells is performed in the ranges of 0-1 mM of MCA concentrations, 30 oC and 37 oC of incubation temperatures, 0-12 hours of incubation periods before adding IPTG, 0-1 mM of IPTG concentrations, 1-8 hours of induction periods, and 30 oC and 37 oC of induction temperatures in medium pHs 6-10. The results indicated that the optimum MCA degradation is achieved in 0,2 mM MCA concentration, incubation at 37 oC for 6 hours, 0,01 mM IPTG, induction at 30 oC for 2 hours, and in medium of pH 7. On the other hand, the enzimatic reaction using haloacid dehalogenase crude enzyme is conducted for 10 minutes in the ranges of MCA concentrations of 0-1 mM, reaction temperatures of 25 oC-57 oC, and bufer pHs of 6-11 suggests that in MCA concentration of 0,1 mM, reaction temperature of 37 oC, and buffer of pH 10 provides optimum condition of MCA degradation.