Sll BCLONING AND HETEROLOGOUS GENE EXPRESSION OF THERMOSTABLE LIPASE LK2 IN PICHIA PASTORIS

• Main Author: Ananda, Annisa
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/41389
• Institution: Institut Teknologi Bandung

Lipase is one of commercial enzymes that widely used in many industrial fields as biocatalyst. A high production of lipase is necessary to fulfil industrial demand. In the previous study, thermostable lipase gene LK2 isolated from compost has been cloned into pET-30a(+) expression vector and expressed in Escherichia coli BL21 (DE3) intracellularly. However, the lipase was formed inclusion bodies so that the activity is low. This study aims to obtain soluble lipase by expressing lipase gene IX2 in Pichw puston\ GS115 extracellularly. In the first step, pPICZaA-LK2 recombinant plasmid is obtained by subcloning of thermostable lipase gene !.K2 from pET-30a(+) vector to pPfCZa A. The gene is amplified using PCR with pET30a( +)-LK2 recombinant plasmid from the previous study as the template. Then, the amplicons of gene !X2 are inserted into pGEM-T Easy vectors to obtain pGEMT-LK2 recombinant plasmids. pGEMT­ LK2 recombinant plasmids are double digested with EcoRI and ..-¥hal to obtain lipase genes LK2 that will be inserted into pPICZa A shuttle vectors. Afterwards, pPICZaA-LK2 recombinant plasmid is replicated in Eschenchia coli TOPIOF' and the copies are isolated by alkaline lysis method. pPICZaA-LK2 recombinant plasmids isolated from Escherichw co/r TOP I OF' are single digested with S'acl. Then , P1chia puston.<; GS115 is transformed with the linear recombinant plasmids by electroporation method. The recombinant plasmid is integrated into the yeast genome by homologous recombination, so that yeast transformants integrated with lipase gene !.K2 are obtained. Thereafter, thermostable lipase gene LK2 is expressed in Pu::hiu pastoris and induced by adding methanol every 24 hours until 5 days. Lipase LK2 supernatant is analysed by hydrolysis activity assay using p-nitrophenol laurate as the substrate and SDS-PAGE. Before scaling up the expression of the gene, optimization of the expression is carried out by transformant optimization and methanol optimization. The activity assay result of supernatant from some transformants shows that transformant 3 (Mut) has the highest specific activity, that is 0,06529 U/mg. The activity assay result of methanol optimization shows that the highest specific activity is obtained on the fifth induction day by adding 1% methanol every 24 hours. SDS-PAGE electrophoref,rram of lipase LK2 supernatant shows a thick band around 35 kDa. The band size is correspond to the theoretical molecular weight of lipase LK2 (35,7 kDa), so the protein band on the electrophoregram can be concluded as lipase LK2 expressed in P. paston.•\.