SUB CLONING AND EXPRESSION OF HALOACID DEHALOGENASE GENE FROM Bacillus cereus LOCAL STRAIN

• Main Author: Idris
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34039
• Institution: Institut Teknologi Bandung

Haloacid compounds are organic compounds widely used as pesticides active substances, plasticizer, precursors in chemicals syntheses, and as organic solvents in many chemical industries. These compounds cause environmental pollution and harmful to human health due to its toxicity, persistence, and its possible transformation to other toxic metabolites. In addition, these compounds can spread over the biosphere, seep into the grounds, accumulated in the ground water, and may cause long-term pollution. In long-term exposure, these compounds could caused kidneys difunction, liver and nervous systems damage, as well as decreasing the immunity and developing cancer. Microorganisms that produced dehalogenase could metabolized organohalogen compounds and transformed to harmless and non-toxic metabolite. Therefore, these microorganisms are potentially used to overcome organohalogen pollutans. However, dehalogenase generally expressed in low level, hence it is not efficient to reduce the pollutants. The development of molecular recombinant DNA technology provides a solution of this problem. Production of several potential enzymes in large quantities have been succesfully performed by this technique, using an approach of identifying the gene encoding the desire enzyme and then expressing that gene in the new expression system with higher expression ability. Previous studies had succesfully isolated and characterized the haloacid dehalogenase gene from Bacillus cereus local strain named bcfd1, obtained from Agriculture Research and Development Association of Indonesia. This gene had been cloned to pGEM-T vector and transformed into E. coli TOP10. This research aims to sub clone this bcfd1 gene to pET-30a expression vector and analyze its expression in E. coli BL21 (DE3). The research is started with designing specific primer to amplify the bcfd1 gene from Bacillus cereus chromosome. In order to obtaine the right cloned direction, the EcoRI and HindIII restriction sites were added on each site of primer. The amplified DNA fragment obtained from PCR was first cloned to cloning vector pGEMT-Easy, than sub cloned to expression vector pET-30a and transformed into E. coli BL21 (DE3). The recombinant pGEMT-Easy and pET-30a were confirmed by restriction analysis and its nucleotide sequences. Expression of bcfd1 gene in pET-30a was analysed by qualitative and quantitative activity assay. It was confirmed that the 870 bp of bcfd1 gene encodes haloacid dehalogenase has been successfully sub cloned into the pET-30a by restriction analyses, re-PCR, and determining the nucleotide sequences. The SDS PAGE analysis indicated that the recombinant protein of haloacid dehalogenase was successfully expressed, showed by a thick band approximately 37 kD in size. These expressed proteins have a larger sizes compared to original size due to the addition of several amino acids as His-tag, S- tag and enterokinase at the N terminal region. High expression of recombinant protein was obtained by inducing the culture at OD550 0,8-1,0 using 0.01 mM IPTG, then incubated at 30 oC for 2 hours at 200 rpm. Zymogram analysis proved that the recombinant protein possesed dehalogenase activity, where white precipitate AgCl was observed on gel after the gel was immersed in a solution of 0.1 M AgNO3. The quantitative assay of Bcfd1activity toward monochloro acetate gave specific activity 37 U/mg at 30 oC, pH9. The Bcfd1 expressed at 30 oC showed higher activity than that expressed at 37 oC. Suggest that lower temperature may increase protein solubility and suppress protease activity. Analysis of three-dimension structure of Bcfd1 showed that this protein has ?/ß- hydrolase folding motif with two domains, namely cop and core domain. This folding conserved among haloacid dehalogenase superfamilly. Bcfd1 active site is estimated to be located at the interface area between these two domains.