HETEROLOGOUS GENE EXPRESSION OF THERMOSTABLE LIPASE LK4 IN PICHIA PASTORISYEAST

• Main Author: Munawaroh, Laelia
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/41388
• Institution: Institut Teknologi Bandung

Several isolated microorganism s from compost are well known for having a good lipase activity. Lipase is an enzyme that has broad applications in various industrial biotechnology processes, for example in the food industr y, specially in the making process of cheese or chocolate; in the pharmaceutical industry as enantioselective directors in the manufacture of drugs from a racemic; and in the biodiesel industry as a catalyst for transesterification reactions. The high potent ial of lipase as a biocatalyst in vari ous industrial processes encourages the development of various new studi es to increase lipase prod uction. Particular strategy to thi s challenge is by utilizing the recombinant DNA technology. Recombinant DNA technology is a series of biotechnological processes that make it possible to modi fy genes, isolate genes, and express genes in other organisms. The gene encoding lipase named lk-1 derived from organic compost isolates has been successfully cloned and expressed intracellularly into heterologous cells Escherichia coli BL21 (DE3) using pET30 (a) as an expression vector. Recombinant lipases produced from these studies have low acti vity due to the inclusion bodies. To overcome this probl em, the expressi on of the lk-1 encoding gene using Pichia pastoris host cell was carried out, with the hope that an active and functional recombinant lipase could be obtained. Pichia pastoris i s a methy l otropi c yeast that utilizes methanol as its carbon source. The expression of Pichia pastoris has several ad vantages for example be able to do post-translation modifications, can grow to achieve high cell d ensi ty without the accumul ation of ethanol, the technique used i s relatively easy and the effortless process of scaling-up using fermenters. The strategy conducted at the beginning of the stud y was the process of gene subcloning whi ch aimed to obtain a recombinant plasmid carrying the lk-1 insert gene. The process of recom binant plasmid construction went through a series of stages of i solation of the lk-1 gene previousl y contained in the pET30(a) vector, then amplified the gene using two pa irs of primers. The result showed lk-1 gene whi ch has a size of 936 bp. The gene /k-1 that had been obtai ned then ligated into the pGEM-T easy vector and u sed to transform Escherichia coli TOPlO F '. From these results. i solati on and amplifi cati on of the lk..:f. gene were carri ed out and then ligated Ill into pPJ CZaA. The pPICZaA-Ik-1 plasmid was used to transform Escherichia coli TOP IOF'. The recombinant pl asmid pPICZaA-Ik-1 was fused with the secretion signal of a-mating factor and und er the regulation of inducibl e AOX I promoter. The recombinant pl asmid pPICZaA-lk-1 was successfully constructed and it was confirmed that the recombinant plasmid contained lk-1 insertion genes. It was verifi ed by the present of a band 1 500 base pairs on the recombinant plasmid PCR el ectrophoregram using a pair of AOX primers. Furthermo re. the reconstructed pPICZaA-lk-1 plasmid that had been constructed was used to transform Pichia pastoris. Some transformants obtained were selected on YPD media containing zeocin with varying concentrations of 1 000 flg/ml and :2000tg/ml. Transformants that grow well on media with zeocin concentrations of 2000 flg/ml was suspected to carry multicopy cassette expression. The selected transformant then expressed to obtain recombinant lipase. The expression of lipase encod ing gene occurs when induction is carried out with methanol every 24 hours by utilizing the use of the AOX promoter. Analysis the results of recombinant protein expression was achieved through an analysi s of recombinant protein activity by measuring the ability of lipase hydrolysis to PNP-Laurate substrate. In this expression stage. several steps have been completed including optimization of colony selection. optimization of optimum expression time, and optimization of methanol concentration. The purpose of this optimization is to obtain the highest activit y recombinant lipase. From the results of this optimization, colony number 1 2 i s a colony that expresses recombinant lipase with the highest optimum expression, with the best expression time on the fifth day using 0.5% methanol. The profile of the recombinant protein was analyzed by SDS PAGE electrophoresis. Through the electrophorogram, SDS PAGE results were observed with the appearance of a band of about 35 kDa whi ch was thought to be a recombinant lipase protein band. Recombinant lipase obtained was purified usi ng Ni-NTA chromatography. and the resulting lipase activity was 0,9313 U/mg, higher than cr ude extract lipase which has activity 0,0699 U/mg.