CLONING AND HETEROLOGOUS EXPRESSION OF PATATIN LIKE PHOSPHOLIPASE GENE FROM LOCAL ISOLATE AL17

• Main Author: Qisti, Aulia
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/87415
• Institution: Institut Teknologi Bandung

Patatin-like phospholipase is an enzyme that plays a role in hydrolyzing phospholipid ester bonds. Local isolate AL17 is known to contain a gene encoding patatin-like phospholipase. This study focuses on obtaining patatin-like phospholipase from the AL17 and characterize its properties. The research was conducted in four main steps, namely gene cloning, bioinformatics and biocomputation analysis of the gene, expression, and characterization of the enzyme. For the gene cloning, two specific primer pairs were designed to amplify the target genes from the bacterial genome. The amplification results obtained two gene fragments, namely patatin-like phospholipase 1 (ITBPLP1) and patatin-like phospholipase 2 (ITBPLP2), with the size of 2,3 and 1.1 kb, respectively. These gene fragments were then ligated to cloning vectors, resulting in recombinant plasmids, namely pJET-ITBPLP1 and pJET-ITBPLP2, used for transformation of Escherichia coli TOP10. The plasmids were further analyzed through sequencing and bioinformatics. The sequence was translated to amino acids, which showed the presence of four important conserved regions. In addition, the sequence showed similarity levels at 46% and 39% to similar genes from Pseudomonas aeruginosa. For the expression, the genes were reamplified using expression primers containing NdeI and SalI. Following the amplification, the amplicons were inserted to pET30a(+) at the NdeI and SalI restriction site, resulting pET-ITBPLP1.1, pET ITBPLP1.2, and pET-ITBPLP2. The combined expression vectors were used to transform Escherichia coli BL21 (DE3). The genes were expressed on Luria Bertani at 37 °C by induction of IPTG. The proteins were isolated using the alkaline lysis method with 0.1% SDS. Following SDS-PAGE, ITBPlp1.1 and ITBPlp1.2 were overexpressed, while ITBPlp2 was expressed at a low level. All the three enzymes exhibited hydrolysis activity on para-nitrophenyl decanoate. In silico characterization using molecular docking showed that the enzyme ITBPlp1 prefer para-nitrophenyl butyrate, while ITBPlp2 prefer para-nitrophenyl decanoate.