EXPLORATION AND CHARACTERIZATION OF THERMOSTABLE ALKALINE LIPASE FROM LOCAL ISOLATE WEST JAVA THERMOPHILIC MICROORGANISM

• Main Author: Febriani
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34556
• Institution: Institut Teknologi Bandung

Lipase (triacylglycerol hydrolase, E.C.3.1.1.3) is one of the most important class of hydrolytic enzymes. The enzyme serves significant role in industry due to its wide application, such as in food, paper, detergent, pharmaceutical, chemical synthetic with specific stereoisomer, biodiesel, waste treatment, and textile industries. The highly potential application of lipase could be increased futher finding unique biochemical characteristic of the enzymes which fits to industrial needs, particularly lipases which could stable and active in extreme condition such as temperature and pH. One of the methods to discover the unique characteristic is by isolating the enzyme from various microorganisms, expecifically from those that newly discovered strain or species. The goal of this study is to isolate alkaline thermostable lipase produced by thermophilic West Java local isolate with relatively high purity level and unique biochemical characteristic from lipase. Eight local thermophilic microorganisms have been isolated and cultivated in laboratory. All microorganisms were isolated from hot springs in West Java, namely Domas, Papandayan, Kahuripan, and Kawah Hujan A craters. Some of them are identified as unique Indonesian local isolate and have potential to produce alkaline thermostable lipase. The unique enviromental condition of the habitat of the isolates may expect to get various characteristic enzymes. From the organism, screening of lipolytic activity showed that all isolates exhibiting lipase activity when cultivated in agar media supplemented by olive oil and rodhamin B. DMS-2 and KHA-P12 exhibit high lipolytic activity based on the formation of orange red fluorescence hallo around the colonies observed by UV ray. Fluorescent intensity of the isolates showed highest intensity compared to that of other isolates. The quantitative analysis of lipase producing isolates was carried out by analyzing the growth and lipase activity of those eight isolates. Lipase activity was measured using spectrophotometry method with p-nitrophenyl palmitat as a substrate. DMS-3 isolate showed highest growth rate incubated at temperature 65 and 70 oC, with growth rate of 1.037 and 1.200 OD/jam respectively. Two isolates (DMS-3 and KHA-P12) showed higher activity of lipase when incubated at temperature of 70 oC, pH 9, compared to that of the other isolates. The highest lipase activity were show by DMS-3 and KHA-P12 which were 21.272 unit dan 11.982 unit respectively. Based on qualitative and quantitative analysis, these two isolates, DMS-3 and KHA-P12, were selected as potential isolate for further study. The lipases expressed from these isolates were then isolated, purifed and characterized. Based on Gram staining test and Scanning Electron Microscopy, the two isolate showed rod shape and positive gram bacteria. The result is in agreement with the previous researcher based on ribotyping analysis, the DMS-3 and KHA-P12 were closely related to Geobacillus kaustophilus and Thermus aquaticus respectively. Ammonium sulfate precipitation of DMS-3 lipase showed that the highest specific activity at 20-60% saturated fraction. The lipase was futher purified using chromatography of DEAE Sepharose Fast Flow and Sephacryl S-200 and exhibiting specific activity at 1200.48 unit/mg protein or 54 times higher compared to that the crude extract, with yield of 9.12%. Polyacryamide gel electrophoresis combining with zymografy analysis showed that there were 4 bands of protein exhibiting active lipase bands. Further analysis was conducted by eluting each of the bands followed by SDS PAGE showed that the first, second, and third bands revealed few bands similar of each other on the gel. Meanwhile the fourth band only showed single band. These result suggested that DMS-3 isolated expressed more than one type of lipases. The lipases might be multimers (heteropolymeric and homopolymeric). Based on unique characteristic of analysis suggested that lipase from DMS-3 were different with other known Geobacillus sp Meanwhile lipase produced by isolate KHA-P12 was purified using ammonium sulfate fractionation and hydrophobic interaction chromatography with HiPrep Butyl FF 16/10 matrix. The lipase of KHA-P12 showed highest activity at ammonium sulfate fraction of 0-60%. Futher purification with HiPrep Butyl FF 16/10 chromatography revealed a peak of lipase fractions. The highest specific activity was found at 19.46 unit/mg protein from fractions eluted with 80% ethylene glycol. The activity of the fraction was 6.55 times higher times compared to that the crude extract. The electrophoregram obtained from Native PAGE, SDS-PAGE, and Zimogram showed that KHA-P12 produce three types of lipase, however these three lipases seem to form an aggregate. Electroelusion of one of the active lipase followed by SDS-PAGE generated 4 bands and 1 band of native protein showed lipase activity. Character of lipase from KHA-P12 reveals that the enzyme exibited optimum activity at pH of 8.5, incubated at temperature 65 oC. The lipase activity assay at optimum pH 8.5 with variation of temperature showed 3 optimum activity at 55, 65, and 75 oC. The maximum temperature showed at 65 oC. The finding of three optimum temperatures informs that KHA-P12 lipases consisted more than one lipase types. At temperature 55 oC, there are two optimum pH activities : at pH 9 and 10. While at temperature 75 oC, only single optimum pH, was observed at pH 8.5. The results suggested that active enzyme at 75 oC is most likely consists of single type of lipase. Metal ion of Ca2+ and Mn2 + (10 mM) could increase lipase activity, while Zn2+ stabilize lipase activity. How ever metal ion such as Co2+ , K+, Na+, Ba2+, Cu2+, Fe3+, Sn2+, Fe3+ (10 mM) inhibited lipase activity. Lipase from KHA-P12 could be activated with metal ion of Ca2+ and Mn2+. The same effect was also demonstrated by EDTA, DTT, SDS, and PSMF 10 mM. Futher more PSMF 10 mM reduced 77.78% of lipase activity. The results showed that the lipase with optimum activity at 75 oC, pH 8.5 is hydrolase serin type lipase. Substrate spesificity analysis of the enzyme showed that could hydrolyse long chain fatty acid from C10-C18. This lipase has highest substrate specificity to p- nitrophenyl myristate and palmitate with specific activity at 9.35 dan 9.77 unit/mg protein respectively. This type of lipase is potential to be applied in detergent industry due to its capability to degrade various fatty acid chains. The high activity of lipase in long chain substrate suggested that the lipase is also potential for application as catalyst for biodiesel production. Local isolate lipase in this research was characterized as alkaline thermostable lipase in various long chain fatty acid from substrat specificity. Characterization of lipase from DMS-3 and KHA-P12 was proven unique biochemical characteristic of thermostable alkaline lipase from local isolate West Java. The lipase which were produced from local isolate thermophilic microorganism can be developed to increase knowledge of thermostable enzyme and applied in biotechnological industry.