Cloning Of Termophilic Lipase Gene From A Bacterium Isolated From Hot Spring At Seram Island, Molluccas
Kloning Gen Penyandi Lipase Termofilik dari Isolat Bakteri Asal Mata Air Panas di Pulau Seram, Maluku.
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Date
2012Author
Apituley, Edwin Thomas
Suwanto, Antonius
Mubarik, Nisa Rachmania
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Thermophilic bacteria often possess enzymes that are stable and active at high temperature which are valuable for some biotechnological and industrial applications such as lipase enzyme for lipid hydrolysis and esterification reactions. The objectives of this research were to isolate and characterize lipase produced by the thermophilic bacterium, and to isolate and cloned its lipase encoding gene. Isolat T I.2 was thermophilic bacterium isolated from hot spring at Seram island, which had temperature 89°C and pH 6,3. Comparison of 16SrDNA sequence to GenBank database using Basic Local Alignment Search Tools for nucleotides (BLASTn) showed that isolat T I.2 was cosely related to Geobacillus stearothermophilus with 99% homology. The phylogenetic tree for 16S rRNA which was constructed using Neighbour Joining Method and distance was estimated using Kimura’s two parameter method with one hundred bootstrap replicates showed that the thermophile isolat T I.2 is clustered to other members of Geobacillus and closely related to Geobacillus stearothermophilus. Titrimetric method was used to measure activity of crude lipase preparation of isolat T I.2 at various temperature and pH with the result showed that optimum temperature and pH of lipase activity were 80°C and 6,5. Isolation of lipase gene was conducted using degenerate primers pair, primers pairs F1-R1 and primers pair F2-R1. Primer pair F1-R1 could amplified only 600 bp of DNA fragment however 1180 bp of isolat T I.2 lipase gene amplified by primer pair F2-R1 was successfully cloned to pGEM-T Easy and transformed to E.coli TOP 10. Crude lipase preparation of E. coli TOP 10 transforman which carry recombinan plasmid, and measured under optimal temperature 80°C and optimal pH 8,5, was 3,11 Unit/ml. Sequence of 390 amino acids deduced from lipase gene of isolat T I.2 was compared to GeneBank database using Basic Local Alignment Search Tools for protein (BLASTp) and the result showed that it was closely related to lipase of Geobacillus stearothermophilus L1 with 90% homology.