CONSTRUCTION AND EXPRESSION OF RECOMBINANT Staphylococcus WL1 LIPASE

• Main Author: Khairunnisa, Fatiha
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34382
• Institution: Institut Teknologi Bandung

Lipase (EC 3.1.1.3) is an enzyme which has ability to catalyze hydrolysis reaction and synthesis of triacylglycerol. Lipase from Staphylococcus WL1 isolate (LipFWS) catalyzes the transesterification of CPO to produce biodiesel. The characteristics of LipFWS with KM value of 9.8 mM and optimum activity at 55 ?C and pH 7 are suitable for the application in biodiesel industry. In previous study, gene encoding Staphylococcus WL1 lipase (lipFWS) has been identified. Based on the gene information, research on the over expression of LipFWS is essential. The aim of the present research was to construct and express the recombinant LipFWS either extracellularly or intracellularly. The methods included the designing of specific primers and amplification of lipFWS by PCR, cloning DNA fragments obtained from PCR into expression vectors pMM1525 and pET-30a, expressing pMM-lipFWS in Bacillus megaterium MS941 and pET- lipFWS in Escherichia coli BL21 (DE3), in silico analysis of the constructions of PMM-lipFWS and pET-lipFWS, and characterizing of recombinant LipFWS. The results showed that fragments of lipFWS, with ~2,200 bp fragment for construction in pMM1525 and ~1,200 bp fragment for construction in pET-30a, were obtained. ProlipFWS fragment containing SacI and BglII recognition sites were inserted into pMM1525, and mature lipFWS fragment with recognition sites of EcoRI and HindIII was introduced to pET-30a. The construction of pMM- ipFWS had ~9,600 bp in size. The restiction analysis of pMM-lipFWS with SacI and BglII showed a fragment of pMM ~7,400 bp and a lipFWS fragment of ~2,200 bp indicating the lipFWS has been successfully constructed into pMM1525. The recombinant LipFWS deduced composed of 723 amino acid residues with Mr of 81.143 kDa. Bacillus megaterium MS941 harbouring pMM- LipFWS expressed an extracellular LipFWS with a molecular mass of ~45 kDa. The expressed protein was shorter due to the proteolytic activity during the LipFWS was secreted. On the other hand, the lipFWS fragment inserted in the pET-30a expression vector produced pET-lipFWS possessing ~6,600 bp in size. The restriction analysis of pET-lipFWS with EcoRI and HindIII showed a pET linear fragment of ~5,400 bp and a mature lipFWS fragment of ~1,200 bp indicating lipFWS has successfully been constructed into pET-30a. The deduced amino acid of LipFWS was correct, consisting of 442 residues with a relative molecular mass of ~49.7 kDa. SDS-PAGE showing a band of ~49.7 kDa confirmed the expression of recombinat LipFWS. The lipase activity was 0.327 U/mg.