MODIFIED PURIFICATION AND HETEROLOG EXPRESSION OF LIPASE FROM Geobacillus thermoleovorans
Lipase (triacylglycerol acylhydrolases, EC 3.1.1.3) is an enzyme catalyze the hydrolisis of ester bonds of triacylglycerols at the interface between an insoluble substrate and water. Microorganisms isolated from Papandayan crater known to have a good lipase activity. Lipase from local isolates have...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/14943 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Lipase (triacylglycerol acylhydrolases, EC 3.1.1.3) is an enzyme catalyze the hydrolisis of ester bonds of triacylglycerols at the interface between an insoluble substrate and water. Microorganisms isolated from Papandayan crater known to have a good lipase activity. Lipase from local isolates have been successfully cloned and expressed intracellularly in E.coli BL21 (DE3) using pET30 (a) as an expression vector. The recombinant lipase was purified using Ni-NTA affinity chromatography, but the results have not been optimal because there were two protein bands in the SDS PAGE electrophoregram. This research aims to study the possibility of using hydrophobic interaction chromatography in the purification of recombinant lipase. In terms of structure, the recombinant lipase from local isolates have many hydrophobic regions, so by using hydrophobic interaction chromatography is expected to obtain only one active band in the SDS PAGE electrophoregram. In this research, production, expression, and isolation of the recombinant lipase from local isolates have been done, then proceed to step purification. Protein purification is done by using three steps, heating, batch DEAE-cellulose ion exchange and hydrophobic interaction chromatography. The activity of enzyme for each step of purification was determined using the substrate, p-Nitrophenylpalmitat. Specific activity of crude extract enzyme was 0.0059 U/mg. Purification with heat method and batch DEAE-cellulose ion exchange chromatography has been successfully increased each specific activity about 2 fold dan 7 fold compared to crude extract enzyme. The enzyme which was purified using hydrophobic column has specific activity increased by 28 fold compared to the crude extract. |
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