D7A MUTATION OF HALOACID DEHALOGENASE GENE FROM PSEUDOMONAS AERUGINOSA IN RECOMBINANT CLONE

• Main Author: RAUDHANIA TRISNAHADI NIM:10513017, SAFFIRA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/30763
• Institution: Institut Teknologi Bandung

Monochloroacetic acid (MCA) is widely used in agriculture, mining, pharmaceutical, and <br /> <br /> <br /> other fields. The increased use of monocloroacetic acid causes more waste which is toxic to <br /> <br /> <br /> human. Pseudomonas aeruginosa is one of Gram negative bacteria that produces haloacid <br /> <br /> <br /> dehalogenase, which can be used to remediate organohalogenic pollutants. In previous <br /> <br /> <br /> research, the haloacid dehalogenase gene from Pseudomonas aeruginosa has been cloned into <br /> <br /> <br /> pGEM-T in E. coli TOP10 host cells, and subcloned into pET-30a (+) expression system E. <br /> <br /> <br /> coli BL21 (DE3). Expression was being studied, and it was suspected that D7 residue plays an <br /> <br /> <br /> important role in its activity in degrading monocloacetic acid. In this study, a directional <br /> <br /> <br /> mutation of D7A using PCR approach with mutagenic primers was performed. The <br /> <br /> <br /> polymerase used was Pfu polymerase and the DpnI restriction enzyme was used to cleave the <br /> <br /> <br /> methylated GMe6ATC sequence on plasmid template. The PCR result were then used to <br /> <br /> <br /> transform E. coli TOP10. The mutated gene in recombinant paed-d D7A was then sequenced, <br /> <br /> <br /> and its sequence was aligned with the initial gene showed a D7A mutation. Bioinformatics <br /> <br /> <br /> analysis showed a change in beta-sheet length on the haloacid dehalogenase D7A structure. <br /> <br /> <br /> The haloacid dehalogenase activity of D7A was then compared with its initial enzyme by <br /> <br /> <br /> quantitative analysis, measuring the amount of Cl- released from the catalytic reaction with <br /> <br /> <br /> MCA substrate. The results indicated that there was no difference of activity between haloacid <br /> <br /> <br /> dehalogenase with haloacid dehalogenase D7A. It can be concluded that D7 is not the catalytic <br /> <br /> <br /> residue in the haloacid dehalogenase from Pseudomonas aeruginosa local strain. <br />