MODIFICATION OF LOCAL ISOLATE LIPASE FOR BIODIESEL SYNTHESIS

• Main Author: Kartika Sari, Diah
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35415
• Institution: Institut Teknologi Bandung

Lipase (Triacylglicerol acylhydrolase, E.C. 3.1.1.3) derived from thermophilic bacteria can be used for various industrial purposes. Thermostable lipase derived from thermophilic bacteria such as KHA-P12, isolated from Kawah Hujan Crater, West Java, is predicted to be able to overcome existing problems in industry such as high temperature and longer time process. Lipase is excessively used in fat and oil processing, detergent ingredients, food processing, chemical substance synthesis, pharmacy, paper synthesis, cosmetic products, and biodiesel industry. Biodiesel is one of renewable, environmental-friendly, no-health-effect alternative fuels and can be used as motor vehicle fuel. Lipase can catalyze transesterification reaction in biodiesel synthesis by suitable reagent and with limited water presence. Modification of lipase enzyme to organic soluble lipase is applied to reduce water content that will impede biodiesel synthesis process. This research is aimed to modify thermostable KHA-P12 lipase to organic soluble lipase and to characterize modified lipase in biodiesel synthesis. Lipase from KHA-P12 (EU784082) was analysed qualitatively using CaCl2 media test. Extracellular lipase expressed by 70oC cultured KHA-P12 was isolated and precipitated using either acetone or ammonium sulphate fractionation. Both salt-free 40-60% ammonium sulphate fraction and aceton extract were lyophilized and the corresponding activity was quantified spectrophotometrically using para-nitrophenol palmitate (pNPP) as substrate. Lipase powder with highest activity was then modified to organic soluble lipase using decanoil chloride. The activity of the modified lipase was also determined. Biodiesel synthesis were catalysed by both modified and non-modified lipase enzyme. Transesterification reaction in this research was applied toward coconut oil and 2 types of alcohol that were methanol and ethanol. Transesterification product was analyzed using mass spectrometry analysis. Lipase qualitative test toward KHA-P12 showed positive result indicated by the formation of monolaurate calcium precipitate. Specific activity of acetone precipitate was 1.52 unit/mg proteins (2.7 grams), while those of ammonium sulphate 40-60% fraction was 0.82 unit/mg protein with total precipitate of 30 mg. Since acetone precipitate has higher specific activity and has more precipitate compared to that of ammonium sulphate fractionation, then acetone precipitate was futher modified with decanoil chloride. The specific activity of modified enzyme was 2.61 unit/mg protein, which was increased by 1.72 times compared to that of non modified enzyme (1,52 unit/mg protein) with the yield of 53%. Mass spectrometry result showed that transesterification reaction took place by both catalysts. The intensity of the product showed in spectrogram of modified enzyme has higher value compared to those of non modified enzyme for both types of alcohols. This data also showed that transesterification activity of organic soluble lipase also increase compared to those of natural lipase. This enzyme also showed higher specificity to ethanol compared to that of methanol.