ISOLATION AND CHARACTERIZATION OF THERMOSTABLE LIPASES FROM THERMOPHILIC BACTERIA (ISOLATE AL96)

• Main Author: ( NIM : 90514007 ), SEPTIANI
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/24266
• Institution: Institut Teknologi Bandung

Lipase belongs to hydrolase enzyme that catalyzes hydrolysis reaction of esters in fats or triglycerides to produce fatty acids and glycerol. This enzyme plays an important role in biotechnology such as in pharmaceutical industry, chemical industry, food industry, detergents, and biodiesel industry. In industry, it often used in condition with high temperature and pH. Therefore, it is necessary lipase enzyme which has thermostable properties and stable at high pH. Lipase can be isolated from animals, plants, and microorganisms. Thermostable lipases can be obtained from thermophilic microorganisms or bacteria, some of which can be isolated from compost or hot springs. One of the isolates namely AL96 has a thermostable properties and alcohol tolerant. Lipase from isolate AL96 is not well characterized yet, therefore this study carries out the characterization of isolates lipase AL96. To produce lipase enzyme in great quantities and have high activity, it needs to examine the important factors such as growth conditions, isolation method and purification which performed in order to obtain lipase enzyme with high purity level. In addition, the characterization of enzymes includes as temperature and pH optimization in order to know the enzyme optimum activity. In this study it has done the isolation and characterization of lipase from isolate AL96. This study was started with construction of bacterial growth curve and lipase activity curve to obtain the best incubation time for isolates AL96 in producing lipases. Observations of lipolytic enzyme activity performed in conjunction with the cell growth. The results showed that the optimum lipase enzyme produced after 17 hours incubation. Isolation of lipase was done by growing isolate AL96 in 50 mL starter media then culture 1 mL put in 100 mL of new media and incubated for ± 17 hours until the OD value reached approximately 1.069. Crude extract of enzymes obtained through harvesting cells cultured bacteria by centrifugation for 45 min at 4000 g. Crude extract of enzymes was in the supernatant because enzymes has extracellular properties. Crude extract of thermostable lipase enzyme purified by using ammonium sulfate fractionation method. The result of determination for specific activity showed that the activity of lipase increased after ammonium sulfate fractionation and dialysis. Increasing of specific activity showed that increasing of lipase purity. The result of partially purification by using ammonium sulfate against to crude extract of enzymes from isolate AL96 showed that the fraction 50-70% has the highest activity as much 0,018 U/mg protein, followed by the fraction of 30-50% with the specific activity as much 0,015 U/mg protein. The result of SDS-PAGE and zymogram indicated that lipase band with size 85 kDa for three fractions namely fraction 50-70%, 30-50%, 0-30%. Lipases for fraction 30-50% have the optimum temperature at 65 0C and for fraction 50-70% have an optimum temperature at 75 0C and this lipases can be categorized as thermostable lipases. Characterization of the pH optimum in fraction of 50-70% and 30-50% indicated that both fractions have optimum in pH 10. <br />