Cloning of Lipase Gene Fragment from Thermophilic Bacteria of Pasir Impun Isolated

• Main Author: AKBAR NUGRAHA (NIM 10502038), YUDA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/12796
• Institution: Institut Teknologi Bandung

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are enzymes that catalyze the hydrolysis of triacylglyceride and long-chain fatty acids esters. Today, lipases are utilized in various industry such as detergent, food, drugs, and synthesis of novel chemicals. Many of lipases application reported the new source of the enzyme. In previous research, several thermophilic bacteria with high lipases activity have been isolated from thermogenic compost at Pasir Impun. Two isolates namely PI6A and PI13C have been identified as lipase producing strains. The growth curve of these isolates showed that PI6A growth rate was 0.131 OD/hour meanwhile the PI13C growth rate was 0.090 OD/hour. Maximum growth for PI6A isolate was at ODmax = 1.380 and PI13C at ODmax = 1.419. Morphology of bacteria and Gram Staining test showed that the two isolates of Pasir Impun are a bacil Gram-positive bacteria. FP9 primer and RP7 primer has been designed to amplify lipase gene fragment based on two conserve region at amino acid level representing oxyanion region and active site. Agarose gel electrophoregram of PCR product at annealing temperature 48oC showed that the amplified lipase gene fragment have same migration distance with 200 bp of pUC19/HinfI DNA marker. BLASTn results indicated that the sequence of nucleotide from PI6A lipase gene fragment shows 99% homology to the sequence of nucleotide encoded Bacillus licheniformis thermostable lipase. Hence lipase gene fragment of PI6A isolate has been successfully amplified. The results of this research is expected to be a reference to design and continue the next research in order to obtain full lipase gene from both Pasir Impun isolates.