HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF THIOESTERASE

• Main Author: MAHARDIKA (NIM:20516030), GITA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/27551
• Institution: Institut Teknologi Bandung

Thioesterase (thioester hydrolase, EC 3.1.2.-) is an enzyme that catalyzing hydrolysis <br /> <br /> <br /> <br /> reaction of thioester bonds between carbonyl and sulfuric groups. In previous research, <br /> <br /> <br /> <br /> thioesterase gene was cloned through metagenome approach from Domas Crater with the <br /> <br /> <br /> <br /> size at around 500 base pairs. Based on bioinformatic analysis using Basic Local Alignment <br /> <br /> <br /> <br /> Search Tool (BLAST) in National Center for Biotechnology Information (NCBI) programs, <br /> <br /> <br /> <br /> the amplicon carry thioesterase gene with the highest homology of 66% with putative <br /> <br /> <br /> <br /> thioesterase gene from uncultured Acidilobus sp. JCHS. In this study, the gene was <br /> <br /> <br /> <br /> successfully overexpressed in E. coli BL21(DE3) through pET-30a expression vector. The <br /> <br /> <br /> <br /> expression was confirmed by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis <br /> <br /> <br /> <br /> (SDS-PAGE) analysis. The protein showed highly expressed at around 17 kDa with the level <br /> <br /> <br /> <br /> of expression at around 32.25%. The data was supported by zimographic analysis showing <br /> <br /> <br /> <br /> the hydrolysis activity. Protein thioesterase was also successfully purified using Nickel- <br /> <br /> <br /> <br /> Nitrilotriacetic Acid (Ni-NTA) affinity chromatography. The specific activity of purified <br /> <br /> <br /> <br /> thioesterase showed 0.055 U/mg protein with purity times of 6.8 compared to that the crude <br /> <br /> <br /> <br /> extract. Thioesterase showed optimum activity against p-NP decanoate as substrate with <br /> <br /> <br /> <br /> temperature at 80 °C and pH 8. In addition, thioesterase was known to be active in various <br /> <br /> <br /> <br /> polar organic solvents. All of the data obtain suggested that the enzyme might be classified as <br /> <br /> <br /> <br /> thermostable and alkaline tolerant enzyme.