PURIFICATION AND CHARACTERIZATION OF RECOMBINANT LIPASE FROM LOCAL ISOLATE

• Main Author: AKMALIA HIDAYATI (NIM : 10508034); Pembimbing 1 : Fida Madayanti Warganegara, Ph.D; Pembimbing , NUR
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/16737
• Institution: Institut Teknologi Bandung

Lipase (triacylglycerol acylhydrolases, EC 3.1.1.3) is an enzyme catalyze the hydrolisis of ester bonds of triacylglycerols at the interface between an insoluble substrate and water. The importance of lipase in industry and biotechnology research, has been encouraged the screening of microorganism strains producing lipase. Therefore cloning the gene of such thermophilic enzymes into more suitable mesophilic hosts has been achieved in order to produce high level of thermostable proteins. From the previous research, the gen encoded thermostable lipase from local isolate PPD2 had been cloned and successfully expressed intracellularly in E.coli BL21(DE3). In this research, expression and purification optimization of the recombinant lipase PPD2 isolate in heterologue cell had been conducted. Overexpression of recombinant lipase was conducted after the cell culture was induced by IPTG 1 mM for 4 hours in optical density (OD600) of 0.60. Optimum lysis condition of E.coli BL21(DE3)-pET30(a)-lipITB1.2 cell was achieved by adding lysis buffer with composition of 4 mL of sodium-phospate buffer 0.05 M pH 7.5 and 5 mg of lisozyme for a gram of wet cell, continued with sonication in 8 minutes during interval of 30 seconds on and 30 seconds off. Specific activity of expressed crude extract enzyme was 0.02076 U/mg respectively. Purification with heat method dan batch DEAE-cellulose ion exchange chromatography has been successfully increased each specific activity about 3 fold dan 16 fold compared to crude extract enzyme. Optimization of Ni-NTA affinity chromatography purification was successfully purified the recombinant lipase which was showed by dominant band sized 46 kDa appear in the SDS-PAGE electroforegram. Determination of optimum pH and temperature of purified enzyme showed that highest specific activity obtained in pH 9.0 and 70 oC.