GENE EXPRESSION OF PSEUDOMONAS STUTZERI LIPASE BK-AB12 AND CHARACTERIZATION GENE PRODUCT
Lipase is one the of the enzyme that widely applied in industry as a biocatalyst. Lipases are typically applied as bioactive component in commercial detergent, in pharmaceuticals, biodiesel, and food industries. For industrial application, lipase must be supplied in large quantity and also ac...
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| Format: | Theses |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/33954 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Lipase is one the of the enzyme that widely applied in industry as a biocatalyst. Lipases are typically applied as bioactive component in commercial detergent, in pharmaceuticals, biodiesel, and food industries. For industrial application, lipase must be supplied in large quantity and also active and stabile in various industrial processes. One way to comply with the industrial demands, many lipases were isolated from extremophiles which are type of microorganisms that able to live in extreme conditions, such as high temperature, high salinity, pH acid / alkaline, high pressure and other extreme condition. In the previous study, lipase has been isolated and characterized from halophilic bacterial Pseudomonas stutszeri BK-AB12 that displayed high stability and activity in various organic solvent. In order to obtained lipase with high quantity the gene of lipase from this bacteria has been isolated and cloned. In this current work, the isolated lipase gene was expressed in Escherichia coli BL21 (DE3) to obtain large quantity of enzyme. Prior the expression was carried out, the recombinant plasmid was verified by agarose gel electrophoresis and sequencing. Electrophoregram displayed positive result that the recombinant plasmid carrying a lipase gene. This analysis was also supported by the sequencing results confirming that the lipase gene containing in the recombinant plasmid. After the verification stage, the gene expression was carried out by inducing E. coli BL21 (DE3) carrying the lipase gene with IPTG (isopropyl-?-D-thiogalactoside) at a final concentration of 1 mM when the bacterial growth reach the optical density at 600 nm (OD600) about 0,6. The expression product was analyzed by SDS-PAGE and zymography. Based on this analysis, the molecular weitght of the over expression product was about 37 kDa. The activity assay showed that the crude of lipase has a specific activity about 1.484 unit / mg and 17.26 mg of total protein per 3 mililitre crude enzymes. The optimum conditions of enzyme activity was observed at
temperature of 50 oC, pH 9 and 1 M NaCl. The unit of activity is defined as the
amount of enzymes used to produce 1 mmol product (p-nitrophenol) per minute per mililitre enzyme.
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