SITE DIRECTED MUTAGENESIS (H110F) AND ITS ROLE TOWARD ACTIVITY OF LK_ITB5a LIPASE

• Main Author: FAUZIAH MA’RUF (NIM : 21116026), ILMA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/22531
• Institution: Institut Teknologi Bandung

Microbial lipases are enzymes that are widely used for commercial purpose such as in chemical industries, food, pharmaceuticals, detergents and other industrial applications. The aim of this research was to study the effect of mutation at one of oxyanion hole residu (H110) on the activity of LK_ITB5a lipase. Computational analysis showed that mutatiot that can produce interest characteristic is substitution from H110 to F110. The wild type and mutant gene was constructed through Polymerase Chain Reaction method and the amplicons were inserted into pET-30a expression vector. Recombinant protein expression was perfomed at 37 ⁰C, IPTG 1mM, agitation 150 rpm and incubated for 4 hours. Overexpression of lipases were analyzed by SDS-PAGE and produced two recombinant protein bands of approximately 36 and 32 kD. Densitometric analysis using ImageJ software showed that 36 kD lipase expressed approximately 26,4 % for wild type lipase and 23,01 % for mutant lipase. While the 32 kD lipase was expressed about 14 % for wild type lipase and 14,67 % for mutant lipase. Both lipases have been partially purified using Ni-NTA affinity chromatography. LK_ITB5a wild type dan mutant H110F have same optimum substrate , temperature and pH ( pNP-Decanoate, 40 ⁰C and pH 9). Mutant lipase has higher specific activity than wild type at all kind of substrates(pNP-Decanoate, pNP-Laurate, pNP-Miristate dan pNP-Palmitate). Mutant lipase also has four and a half fold specific activity than wild type lipase at optimum temperature and pH . Therefore can be concluded that mutation H110F on LK_ITB5a lipase cause enzyme-substrate interaction change and cause increase of the specific activity of the enzyme.