ISOLATION AND CHARACTERIZATION OF DETERGENT STABLE LIPASE FROM HALOPHILIC BACTERIA Halomonas eurihalina BK-AB15

• Main Author: Rizki Pangesti, Ertanti
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/33996
• Institution: Institut Teknologi Bandung

The use of enzymes as biocatalysts in a variety of industries has increased rapidly. One of the biocatalysts is a lipase that acts as a biocatalyst in the hydrolysis reaction of long-chain triacylglycerol into free fatty acids and glycerol. Lipase applied to a variety of industries, including food industry, paper industry, cosmetics, detergents, biodiesel, biotechnology, and others. Variations in the application raise the need of lipase with certain characteristics, such as resistance to alcohol, organic solvents, high salinity, detergents, and high temperatures. This condition prompted to search other sources of lipase in various extremophile microorganisms in extreme habitat conditions. In this study, lipase isolated from Halomonas eurihalina BK-AB15, halophilic bacteria, which is able to live in high salinity environment. Halomonas eurihalina BK-AB15 comes from the source of brine mud crater "Bledug Kuwu" located in the village Kuwu Kradenan, Purwodadi, Central Java. Lipolytic potential of H. eurihalina BK-AB15 shown by fluorescent in orange colonies, when the bacteria are grown on solid medium containing rhodamine B. Lipase is produced in a liquid medium containing 0.5% peptone, 0.5% yeast extract, 0.05% CaCl2, and NaCl 8%. The crude extract of lipase was fractionated using ammonium sulfate 0- 30%, 30-60%, and 60-70%, wherein the fraction with the highest specific activity was obtained in 60-70% fraction. Native PAGE and zymography showed that the best fractions containing only one kind of lipase, with a molecular weight of 39 kDa resulted by SDS-PAGE characterization. Lipase has optimum activity at pH 9.5 and 45 oC. This enzyme activity was influenced by metal ions, where the addition of Ca2+, Mg2+ and Ba2+ gave effect to the increase in activity, while the addition of Zn2+, K+, and Ni2+ gave opposite effect. Because the effect of Ca2+ addition was more prominent in enhancing lipase activity, we further evaluated this effect toothers parameters, such as pH and temperature optimum, variations of NaCl concentration, inhibitor, organic solvents, variations of SDS concentration, and thermal stability. The addition of Ca2+ did not affect the pH and temperature optimum of lipase. Metal ion chelator, such as EDTA did not significantly decrease the activity, so that this enzyme may not belong to the group metalloenzim. On the contrary, in the presence of Ca2+, addition of EDTA decreased lipase activity significantly. In addition, this enzyme also may not belong to the group of serine hydrolases, because PMSF was not significantly inhibit lipase activity with the absence of Ca2+ but lipase activity increased with the addition of Ca2+. Effect of surfactant SDS (sodium dodecyl sulfate) was also not significantly decrease the lipase activity in the absence of Ca2+ but increased lipase activity with the presence of Ca2+. Whether there was Ca2+ added in lipase or not, showed that lipase has stability at various concentrations of SDS, so that this enzyme has the potential to be applied as bioactive components in detergents. The stability of lipase with or without the presence of Ca2+ also performed at various concentration of NaCl. The result showed highest activity of lipase without Ca2+ at 4% NaCl, while highest with addition of Ca2+ at 6% NaCl. So, lipase produced in this research can be categorized as a halophile lipase (stable at high salinity). In terms of stability in organic solvents, lipase obtained show good stability in polar solvents, such as propanol, butanol, isopropanol, acetonitrile and acetone, but not stable in nonpolar solvents, such as n-hexane and chloroform. Different result observed when the addition of Ca2+, this lipase showed good stability in a polar solvent, such as ethanol, butanol, acetone, and acetonitrile. In addition, the lipase also showed better stability in nonpolar solvents, such as n-hexane and chloroform. Therefore, this enzyme could potentially be developed as a biocatalyst in the production of biodiesel. Thermal stability showed that lipase with the addition of Ca2+ is more stable than the lipase without Ca2+. Melting temperature (Tm) of the lipase without Ca2+ was 64 oC, while the lipase by addition of Ca2+ has a Tm value of 66 oC. Overall, addition of Ca2+ ion is not only improved the stability and activity of lipase but also improved the adaptability of the enzyme in broader conditions.