Cloning and Sequencing of Haloacid Dehalogenase Gene from Klebsiella Pneumoniae ITB1

Monochloroacetic acid (MCA) production continues to grow every year and it causes environmental problems. Klebsiella pneumoniae ITB1 is identified to be able to grow on minimum medium with MCA as a sole of carbon source. This strain breaks down MCA to produce 2-hydroxyacetic acid and releases chloride ion into the medium, which can be detected by colorimetric method. Enzyme responsible for this activity is haloacid dehalogenase. This experiment amplified the haloacid dehalogenase gene of Klebsiella pneumoniae ITB1 by PCR. The result was 0.69 kb DNA fragment. This fragment was cloned to pGEM-T easy and then transformed to competent E. coli TOP10 cells. Recombinant colonies were selected based on ampicillin resistant and β-galactosidase activity. Recombinant plasmid confirmation was conducted by size screening, re-PCR, and restriction analysis. The restriction analysis showed positive result as the EcoRI digests of pGEM-HAD recombinant produced two DNA fragments of 3.15 kb and 0.71 kb. Nucleotide sequence of hakp1 DNA fragment confirmed that it is a haloacid dehalogenase gene with 99% similarity to Klebsiella pneumonia. Amino acids sequence deduced by in silico analysis of hakp1 showed 100% sequence similarity with haloacid dehalogenase of multispecies Klebsiella (Accession No. WP_004179015.1). Conserve residues analysis using ClustalW2 of Hadkp1 protein compared to another three haloacid dehalogenases i.e. from Burkholderia cepacia MBA4, Xanthobacter autotrophicus GJ10, and Pseudomonas sp. YL showed 20 conserve residues. Five of the 20 conserve residues were catalytic residues, which are aspartate (D), threonine (T), serine (S), lysine (K) and tyrosine (Y). The 3D structure of Hadkp1 protein predicted by in silico analysis has been conducted by I TESSER. Blastp analysis also showed that Hadkp1 protein of Klebsiella pneumoniae ITB1 has high similarity to enolase-phosphatase of Klebsiella pneumonia. Enolase-phosphatase is an enzyme that catalyzes dephosphorylation and enolization of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-methylthiopentane to 1,2-dihydroxy3-oxo-5-(methylthio)-pent-1-ene. This enzyme has important role in methionine salvage pathway of Klebsiella pneumoniae metabolism.