HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF LIPASE FROM Pseudoxanthomonas sp (AL17)

• Main Author: SARI (NIM:20516004), JERVITA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28265
• Institution: Institut Teknologi Bandung

Lipase (triacylglyserol acylhidrolase E.C.3.1.1.3) is an enzyme that catalyzing <br /> <br /> the hydrolysis reaction of triglycerides and other esters that are not soluble in <br /> <br /> water. Thermostable lipases isolated from thermophilic microorganisms are <br /> <br /> currently receiving attention since the potential use in biotechnological <br /> <br /> applications. Lipases are widely used in various industrial fields, such as <br /> <br /> detergent, food, textile, pharmaceuticals, paper and cosmetics industries. Some <br /> <br /> thermophilic bacteria from the compost were succesfully cultivated in the <br /> <br /> laboratory, one of which is AL17. The isolate identified closed to <br /> <br /> Pseudoxanthomas sp. The lipase gene was succesfully cloned from the isolate into <br /> <br /> pJET 1.2 / blunt vector. In this study, the gene was expressed in Eschericia coli <br /> <br /> BL21 (DE3) as host using pET-30a (+) vector. Recombinant protein expression <br /> <br /> was performed at 37° C, with induction of Isopropyl &#946;-D-1-thiogalactopyranoside <br /> <br /> (IPTG) 1 mM, 150 rpm agitation and incubated for 4 h. The results showed that <br /> <br /> 34.5 kDa of protein overekspresed on Sodium Dodecyl Sulfate-Polyacrylamide <br /> <br /> Gel Electrophoresis (SDS-PAGE). Based on densitometric analysis using imageJ <br /> <br /> software, Lipase AL17 was expressed at around 42.07% of total protein. The <br /> <br /> protein was successfully purified using Nickel-Nitroleacetic Acid (Ni-NTA) <br /> <br /> affinity chromatography. The enzyme still showed specific activity of 0.043 units / <br /> <br /> mg, after purification hence increase of 1.58x compared to that the crude extract. <br /> <br /> Based on assaying of substrate specificity, AL17 lipase activity was best on the <br /> <br /> use of p-nitrophenyl dekanoate substrate (C10). The optimum temperature and pH <br /> <br /> of AL17 lipase are 70oC and pH 8. Based on the characterization results, Lipase <br /> <br /> AL17 has the potential to be used as an industrial high temperature biocatalyst.