PURIFICATION AND CHARACTERIZATION OF LIPASE FROM Geobacillus sp LOCAL ISOLATE DMS-3
Currently, lipase is widely used in many industrial areas, such as detergents, surfactants, agriculture, pharmaceuticals and cosmetics. However, hydrolyzing process of triglyceride into free fatty acids in the industry is usually conducted at high temperatures. Therefore, thermostable lipase and enz...
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| Format: | Theses |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/34421 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Currently, lipase is widely used in many industrial areas, such as detergents, surfactants, agriculture, pharmaceuticals and cosmetics. However, hydrolyzing process of triglyceride into free fatty acids in the industry is usually conducted at high temperatures. Therefore, thermostable lipase and enzymes with optimum activities at wide range of pHs can be a solution for this technical problem. New sources of thermostable lipase have to be explored for the industrial needs. Since Indonesia is located in the ring of fire, thermophilic microorganisms can be easily found and isolated from the hot springs.
Previously, we screened and isolated Geobacillus sp. DMS-3 that produced several thermostable lipases. Lipases of LipDMS3 have been studied and indicated that the enzymes consits of multisubunit proteins as shown by four active bands on zymogram. The purpose of this study is to purify and characterize the lipase DMS-3. This study was conducted as follows: regenerating isolate local DMS-3 and producing crude extract of lipase at 65 °C for 14 hours, purifying by ammonium sulphate precipitation technique followed by anion exchange and gel filtration chromatography, stability assay measurement at temperatures of 70 ºC for 1 hours and –20 ºC for 10 min, as well as biochemical characterization of lipase on pHs and temperatures.
Specific activity of crude extract of LipDMS3 as measured against pNPP substrate is 15 U/mg proteins. Purification by 20-40% ammonium sulphate precipitation showed the largest value of specific activity lipase, equal to 75,57 U/mg proteins and the zymography result showed each of ammonium sulfate fractions consisting of 3 lipase active bands, as size ~200, 100, and 72 kDa. Interestingly, zymography profile of lipase on stability assay showed that LipDSM3 still has a lipolytic activity. The optimum pH and temperature for 20-40% fractions of LipDMS3 were 9.0 and 70 ºC, respectively. Purification step using anion exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephacryl S200) increased the specific activity of lipase by 34-fold and 105-fold, respectively. The active fraction from DEAE-Toyopearl showed the highest lipase activity was tested on native-PAGE and the electrophoregram showed only two lipase bands with molecular size higher than 100 kDa. The active fraction at gel filtration run on SDS-PAGE showed a prominent single band with a molecular weight of ~60 kDa and its optimum pH is 10.0. In conclusion, LipDMS3 can easily to purify by gel filtration and contain a homopolymeric protein, whose protomer size was ~60 kDa. This lipase has optimum pH at 10.0.
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