D7A MUTAGENESIS ON HALOACID DEHALOGENASE GENE FROM PSEUDOMONAS AERUGINOSA ITB1 IN ET PAED-D RECOMBINANT CLONE

• Main Author: Uma Khanifah, Sahda
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/82777
• Institution: Institut Teknologi Bandung

Organohalogens are organic compounds that contain at least one halogen atom. Organohalogens, one of which is haloalkanoic acid, are widely synthesized for various industrial purposes, such as chemical industries, textiles, pharmaceuticals, and for agricultural purposes. Available data indicates that the demand for organohalogens are steadily rising each year, which also creates large quantities of organohalogen wastes dumped to the environment. Unfortunately, these organohalogen wastes are commanly persistent and toxic to many organisms, including humans. Therefore, organohalogen wastes managements, including haloalkanoic acid waste, are continuesly developed. Bioremediation is considered as one of the safe and environmentally friendly techniques to reduce this xenobiotic compound. Bioremediation and biodegradation of haloalkanoic acids could be performed by microorganisms that produce haloacid dehalogenase, a hydrolytic enzyme that catalyzes the cleavage of carbon-halogen covalent bonds in haloalkanoic acids. One microorganism known to produce haloacid dehalogenase is Pseudomonas aeruginosa ITB1. Research on haloacid dehalogenase from P. aeruginosa ITB1 has been continuesly develop. The gene encoding haloacid dehalogenase from P. aeruginosa ITB1, referred to as paed-d, has been successfully cloned and expressed in E. coli host cells. The haloacid dehalogenase produced by this recombinant clone, referred to as Paed-d, has also been successfully purified and its activity against monochloroacetic acid (MCA) has been determined. In-silico mutagenesis studies on paed-d have also been conducted which indicated that aspartate residue at position 7 (D7) plays an important role as nucleophile in catalysis. To validate this finding, this research was performed, employed PCR site-directed mutagenesis to investigate the D7 role in the catalysis of MCA degradation. Initialy, a single colony of P. aeruginosa ITB1 was isolated, followed by chromosomal DNA isolation, which then used as a template to isolate paed-d gene by PCR approach using Fr1 (5’-GAATTCATGCGCGCGATCCTGTTCGA-3’) as the forward primer and Rv1 (5’ AAGCTTTCAGGCCGAGGCCGCCAGTT-3’) as the reverse primer. The obtained amplicon was cloned into pGEM-T in E. coli TOP10, then subcloned into the pET-30a(+) for expression analysis in E. coli BL21 (DE3). Site directed mutagenesis on paed-d to create paed-D7A was performed by PCR approached on pET-paed-d recombinant clone using MutF (5’ GCGATCCTGTTCGCTGTGTTCGGTACC-3’) as the forward primer and MutR (5’ GGTACCGAACACAGCGAACAGGATCGC-3’) as the reverse primer. The nucleotide sequences of paed-d and paed-D7A were then determined and analyzed. The expression of pET paed-d and pET-paed-D7A was studied in E. coli BL21(DE3) and its haloacid dehalogenase activities were studied againts MCA. The Paed-d and Paed-D7A activities were measured using Bergmann and Sanik method by determining the amount of chloride ions released to the medium in the enzymatic reaction. The results showed that each paed-d and paed-D7A have a size of ~700 bp. The success of obtaining paed-D7A mutant was proved through the analysis of the nucleotide sequence, which showed that base A at the 20th position of the gene had been successfully changed to C, indicated that the GAT codon had been successfully changed to GCT, hence changing the aspartic acid at 7th position to alanine, means that D7 in Paed-d has been changed to A7 in Paed-D7A. Expacy Protparam analysis on the protein mass prediction translated from paed-d and paed D7A provide about 26.352 kDa of proteins. This result was in line with the SDS-PAGE analysis of Paed-d and Paed-D7A proteins produced in E. coli BL21(DE3) recombinant clones, which both gave ~28 kDa proteins. Paed-d was able to degrade 89% of MCA with a specific activity of 27.69 (µmol Cl-/mg of protein) whereas Paed-D7A was able to degrade 34% of MCA with a specific activity of 10.97 (µmol Cl-/mg of protein), meant that the D7A mutant had 2.5 times lower specific activity compared to the wild-type. The presence of haloacid dehalogenase activity in Paed-D7A is different from the in-silico analysis, which showed that the D7A mutation would eliminate its activity. This fenomena indicates the possibility of another aspartic acid presence in the haloacid dehalogenase molecule from P. aeruginosa ITB1 that might replace the D7 nucleophile function in catalysis. Nevertheless, the decrease of Paed D7A activity compared to Paed-d, indicates that D7 in the haloacid dehalogenase of P. aeruginosa ITB1 plays an important role in the catalysis. The results of this study are in line with the function of aspartate as a nucleophile in the haloacid dehalogenase of Pseudomonas sp. YL, where the D10A mutation has been observed to eliminate its activity against L-2 chloropropionate.