ISOLATION AND CHARACTERIZATION OF THERMOSTABLE LIPASE FROM GEOBACILLUS LITUANICUS YTAE-13 ISOLATED FROM GEDONGSONGO

• Main Author: Herawati Permana, Anita
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34588
• Institution: Institut Teknologi Bandung

Lipase (EC 3.1.1.3 Triacylglycerol hydrolase) is an enzyme that hydrolizing lipid. Lipase is widely used in industries, such as in detergent, textiles, paper, food, beverages, biodiesel, stereospecific reaction in pharmaceutical and polymer synthetic industries. Beside of high catalytic activity, biocatalyst in industrial process required high resilience and stability during the catalysis process. Research on thermostable enzymes, including lipases are important matter to obtain a superior biocatalyst for industrial purposes. One of potential sources of thermostable lipase is thermophilic microorganism. The thermophilic microorganism used in this research was YTae-13 isolated from Gedongsongo hot spring. Previous research has identified that YTae-13 is Geobacillus lituanicus BGSCW9A89 based on sequence analysis of 16S rRNA gene. At the beginning of the research, media used for lipase production was varied. The variation was performed by adding surfactant, Tween, in the media. YTae-13 was cultivated in media with Tween 20, Tween 80, and without surfactant added. The highest specific activity was shown by the lipase from YTae-13 which was grown in media containing no Tween. This result suggested that the present of Tween was interfered the expression of YTae-13 lipase. Thus, the media without surfactant was further used as production media. The profile of bacterial growth and enzyme production in the selected media showed these were three times expression of lipases, which are in the middle of exponential phase, end of exponential phase, and end of stationary phase. Lipase isolated in this research was the ones that produced in the end of stationary phase. The isolation of lipase has been conducted by cultivating YTae-13 at conditions of 55oC, pH 8.3, and 120 rpm within 18 hours. Two active lipase fractions were obtained after amonium sulfate fractionation, which were at fraction of 60-80% and 80-100% fractions whose activities were detected of 17.1 and 16.5 unit/mg protein respectively. The specific activites were increased by 1.26 and 1.21 fold for 60-80% and 80-100% fractions respectively compared to those the crude extract (13.6 unit/mg protein). Protein yield after fractionation were 10.4% and 13.7% for 60-80% and 80-100% fraction respectively. The fractionated enzyme was then subjected for concentration treatment using polyethersulfone membrane. The specific activities and the yield after concentration treatment were 51.8 unit/mg protein; 9.08% for 60-80% fraction and 40.6 unit/mg protein; 11.3% for 80-100% fraction. These results showed that the specific activities were increased by 3.81 and 2.99 fold for 60-80% and 80-100% fractions respectively compared to those the crude extract. Activity assay in various temperatures and pH showed that each fraction has two optimum temperatures and pH, which were 60oC & 80oC and 8.0 & 10.0. Native-PAGE and zymogram profile showed that both fractions consist of several protein bands that appeared lipolytic activity. There were five lipase bands at 60-80% fraction and two lipase bands at 80-100% fraction. Electroelution result of two lipase bands in the 80-100% fraction, followed by SDS-PAGE analysis showed two sub unit proteins whose have molecular weight 71.1 kDa and 45.4 kDa, respectively. These results suggested that YTae-13 isolate was expressed several lipases.