PROTEIN CHARACTERIZATION OF HETEROLOGOUS EXPRESSION RESULT OF GENE ENCODING THERMOSTABLE LIPASE FROM LOCAL ISOLATE (LK1)

• Main Author: WIDI NURFADILAH (NIM:20516051), AJENG
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/25248
• Institution: Institut Teknologi Bandung

Thermostable lipases are widely used in various industries, such as foods, pharmaceuticals, <br /> <br /> <br /> <br /> detergents and biodiesel industries as well as continue to be developed new variants of lipase <br /> <br /> <br /> <br /> from various sources. The previous study were obtained several clones of gene encoding <br /> <br /> <br /> <br /> thermostable lipases based on metagenom approach. This study is focused to obtain a <br /> <br /> <br /> <br /> functional thermostable lipase through heterologous expression of gene encoding <br /> <br /> <br /> <br /> thermostable lipase from local isolate (LK1). Therefore, the gene was successfully subcloned <br /> <br /> <br /> <br /> from pJET1.2 vector into pET30a vector and expressed using Eschericia coli BL21 (DE3) as <br /> <br /> <br /> <br /> host. The purificatiom of the protein was carried out by Ni-NTA affinity chromatography. <br /> <br /> <br /> <br /> The gene was inserted into pET30a vector at SalI and NdeI restriction sites. The expression <br /> <br /> <br /> <br /> was induced by addition of isopropyl B-D-thiogalactopyranoside (IPTG) 1 mM for 4 hours <br /> <br /> <br /> <br /> incubation time at 37?C. The expression result was analyzed using Sodium Dodecyl Sulphat- <br /> <br /> <br /> <br /> Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed two bands that are 38 kDa and 33 <br /> <br /> <br /> <br /> kDa, respectively. LK1 lipase has been successfully purified using Ni-NTA affinity <br /> <br /> <br /> <br /> chromatography and still showed specific activity at 0.0035 U/mg which is 2.3 times purified. <br /> <br /> <br /> <br /> LK1 lipase has high substrate specificity to paranitrophenyl-decanoate (pNP-decanoate) with <br /> <br /> <br /> <br /> optimum activity at 70?C, pH 8 and still retains its activity for 2 hours. From all of the data <br /> <br /> <br /> <br /> obtained LK1 lipase might be categorized as thermostable and alkaline tolerance enzyme.