CLONING AND SEQUENCING HALOACID DEHALOGENASE GENE FROM Klebsiella pneumoniae LOCAL STRAIN

• Main Author: Yulius Tahya, Candra
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/33968
• Institution: Institut Teknologi Bandung

Monochloroacetic acid is a xenobiotic compound used world wide for many utilities, such as herbicide, surfactant, plastic stabilizer, textile, anaesthetic, and pharmaceutical. Monochloroacetic acid production continues to grow every year and it causes environmental problems. Klebsiella pneumoniae local strain is identified to be able to grow in minimum medium with monochloroacetic acid as a sole of carbon source. The Klebsiella pneumoniae local strain has been confirmed by 16S rDNA typing utilizing two universal primers i.e. BactF1 (5’- monochloroacetic acid to produce 2-hydroxyacetic acid and releases chloride ion into the medium which can be detected by colorimetric method. Klebsiella pneumoniae is also known as a nitrogen fixation bacterium, in which the nitrogen from the atmosphere is reduced to ammonia and certain amino acid, so it could improve crops production. In this research, the gene that codes haloacid dehalogenase was amplified by PCR using primer pairs designed based on haloacid dehalogenase genes from another strains of Klebsiella pneumoniae that have been published in GeneBank. Primer ForKA.p (5’- ATGATCCGCGCCATCGTG-3’) with Tm = 58.4 oC and primer RevHAK.p (5’- TCATGCTGGGATCTGCTCC-3’) with Tm = 57.1 oC were used for gene amplification. The resulted 0.69 kb DNA fragment was cloned to pGEM-T easy using DNA ligation kit and then transformed to competent E. coli TOP10 cells by heat shocked method. Recombinant colonies were selected based on ampicillin resistant and ?-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 pGEM-HAD recombinant produced two DNA fragments of 3.15 kb and 0.71 kb. Nucleotide sequencing of hakp1 DNA fragment confirmed is haloacid dehalogenase gene with 99% similarity to Klebsiella pneumoniae PMK1 (Accession No. CP008929.1), Klebsiella pneumoniae subsp. pneumoniae PittNDM01 (Accession No. CP006798.1), Klebsiella pneumoniae subsp. pneumoniae strain KPNIH31 (Accession No. CP009876.1), Klebsiella pneumoniae subsp. pneumoniae 1158 (Accession No. CP006722.1), Klebsiella pneumoniae HK787 (Accession No. CP006738.1), Klebsiella pneumoniae strain XH209 (Accession No. CP009461.1), Klebsiella pneumoniae subsp. pneumoniae Kp13 (Accession No. CP003999.1), Klebsiella pneumoniae CG43 (Accession No. CP006648.1), and Klebsiella pneumoniae KCTC 2242 (Accession No. CP002910.1). Amino acids sequence deduced by in silico analysis of hakp1 showed sequence similarity 100% with haloacid dehalogenase of multispecies Klebsiella (Accession No. WP_004179015.1). Conserve residues analysis using ClustalW2 of Hadkp1 protein compared to three haloacid dehalogenases i.e. from Burkholderia cepacia MBA4 (PDB: 2NO4_A), Xanthobacter autotrophicus GJ10 (PDB: 1QQ5_A), and Pseudomonas sp. YL (PDB: 1ZRN_A) 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 prediction in silico analysis of Hadkp1 protein, showed 42% similarity to enolase-phosphatase E-1 Homo sapiens, 28% similarity to deflourinating L-haloacid dehalogenase Rha0230 Rhodococcus jostii RHA1, and 24% similarity to dehalogenase Burkholderia cepacia. Blastp analysis also showed that Hadkp1 protein of Klebsiella pneumoniae local strain also has high similarity to enolase-phosphatase of Klebsiella pneumoniae. 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.