#TITLE_ALTERNATIVE#

• Main Author: RIZKI NURUL AENI (NIM : 20512044) ; Pembimbing RukmanHertadi, D.Sc, SUCI
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/19502
• Institution: Institut Teknologi Bandung

Phospholipase B (PLB) is one of hydrolase class of enzymes that catalyzed bond breaking of two acyl chains of phospholipid simultanoeusly through sn-1 and sn-2 mechanisms. PLB produced by a halophilic microorganism, i.e. the microorganism that is able to adapt to environmental conditions with high levels of salt and lipids, has great potential to be developed as a catalyst in the industry, in particular the food industry such as palm oil and soy sauce that requires the stable enzyme on such conditions. Wide applications in food industries implicate to the high demand of this enzyme, unfortunately direct isolation of the enzyme from wild type is not too effective and economical. Recombinant DNA techniques is the answer to meet the requirement. Recombinant DNA technology can provide abundant enzyme production and cheaper. In the present study, we isolated and amplified plb gene from halophilic bacteria Pseudomonas stutzeri BK-AB12 by polymerase chain reaction (PCR) technique. A pair of the primers have been designed for such purpose with the following sequences: of 5’-ATGTCCACGCTCATCCAGCCCGAT-3’ for the forward primer and 5’-GACTTTCTGCGCGAGCGCCTGAAATAG-3’ for the reverse primer. Electrophoresis analysis of PCR product showed the band with the approximate length of 960 bp. The PCR product was then ligated into cloning vector pGEM-T easyto generate a recombinant plasmid and transformed into E. coli for cloning. Retriction analysis using the EcoR I and colony PCR showed positive colonies carrying recombinant plb. Sequencing result on recombinant plasmid using primer T7 and SP6 showed that the isolated genes has ± 941 bp. Sequence analysis by BLASTn program showed that the gene has high similarity with Lysophospholipase plb Pseudomonas stutzeri A1501 with identity level about 99%. PLB protein has putative signal sequences at positions of 1 to 49. Results of 3D structure prediction with comparative methods by Swis-model program showed that PLB from P. stutzeri has similar structure with Monoglyceride lipase (PDB id: 3hju) that has 23.70% identity with PLB. Based on analysis above, the plb genes from Pseudomonas Stutzeri BK-AB12 has successfully obtained.