CLONING OF LIPASE GENE FROM LOCAL HALOPHILIC BACTERIA ISOLATE AG18

• Main Author: Melani Ms, Ela
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34212
• Institution: Institut Teknologi Bandung

Halostabil enzyme produced from halophilic microorganisms has become the center of attention in the field of research because of the increasing need for halostabil enzymes in industrial processes, particularly industries that require high levels of salt. Lipase is one of the halostabil enzyme. From previous studies, it has been found seven local halophilic bacterium isolated from a salt water mud from Bleduk Kuwu Crater located in Kuwu Village, Central Java. Seven isolates were identified to be potentially lipase producer. One of the isolate is AG18. From previous studies, 16S sRNA gene of AG18 have been the isolate using PCR approach, and based on its 16S sRNA gene sequence isolated AG18 has known to have highest homology with bacteria Halomonas elongate str.NY-12. In this study, we have check lipase activity against of AG18 by growing the culture on medium containing olive oil, rhodamine B and NaCl. Colonies that has lipase activity will gave a red-orange fluorescence under UV light with a wavelength 350 nm. From media with variation of NaCl concentration 0-30%, bacterial growth and lipase activity only observed on media with 5% and 10% NaCl. Optimal activity of lipase was observed in medium with 5% NaCl concentration. In addition, bacterial growth was observed on media with NaCl content of 5% and 10% indicate that halophilic bacterial isolates AG18 included into the moderate halophilic bacteria. Cloning of AG18 lipase gene was conducted in order to obtain lipase with a relatively high of amount and purity. Strategy of cloning were based on amplification of AG18 lipase gene using PCR, by using two pairs of primers (primer internal and external primary). Internal primer used to amplify the gene fragments in the area of conserved lipase, while the external primer used to amplify the whole of lipase gene. Lipase gene fragment with length of ± 450 bp was amplified by use the internal primer, as well as full-sized lipase gene ± 850 has been successfully amplified using primers external. Chromosomal DNA used was AG18 chromosomal DNA. Whole gene of lipase subsequently cloned into pGEM-T vector and transformed into host cells E. coli TOP10. Transformants were selected based on their resistance of ampicillin. The Confirmation of positive transformants obtained using size screening and restriction analysis. The positive transformed colonies will carry recombinant plasmids. Cutting the recombinant plasmid with EcoRI restriction enzyme produced two bands on agarose gel, with band of 3015 bp which is the vector pGEM-T and band of ± 850 bp which is the whole lipase gene. Two universal primers T7 and SP6 promoter used in the determination of the nucleotide sequence of the AG18 lipase. The alignment results of AG18lipase amino acid sequence showed that AG18 lipase has the highest homology with lipolytic proteins of Halomonas elongate DSM 2581 by 98% of homology. Alignment between the lipases of isolates AG18 with Halomonas elongata DSM2581 lipase give some differences in the nucleotide sequences of the two lipases. This residual difference is located on the important part of the enzyme that could cause differences in the characteristics of these two enzymes despite having high homology. Until now characteristics of both lipases are not yet available, because determination of the 3- dimensional structure of the two lipases has not yet performed.