Expression and Characterisation of Lipase from Halomonas elongata BK-AB8 Halophilic Bacateria from Bledug Kuwu Mud Crater Central Java

• Main Author: Dewa Made Kresna, I
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38226
• Institution: Institut Teknologi Bandung

In recent years, the demand of lipase has increased dramatically. Lipase is an enzyme that catalyzes hydrolysis reaction of trigliceride to form glicerol and fatty acid and it has an important role in digestion, transportaion and lipid processing inside human body. In industries, lipase has been used in organic sinthetic process, detergen formulation, pharmacy fields, foods, oil industries include biodisel. Lipase has to be provided in a huge number and also has to be stable in various industries process. An effort that has been done to fullfil the industrial demand, some lipases have been isolated from extremophilic bacteria. Enzyme from this kind of bacteria has the ability to maintain its activity for extreme conditions. One of extremophilic bacteria that generally used as a source of this enzyme is halophilic bacteria. There is an issue about enzyme that isolated directly from nature bacteria, its quantity is always in small amount. To solve the present condition, this research aims to produce over-expression lipase gene from Halomonas elongata BK-AB8 using E. coli BL21 (DE3) as host cell. Before introducing the gene into expression system, the recombinant plasmid that has available from the previous research is verified utilizing restriction enzymes and PCR, then the gene was confirmed using agarose gel electrophoresis. Electroforegram showed positive result about the recombinant palsmid has been inserted lipase gene. After verification procedure, this gene expression was done by induction the host cell using IPTG (isopropyl-?-D-thiogalactoside) when optical density (OD600) has reached 0.6-0.8. Result of lipase expression was visualized using SDS-PAGE and Zymogram. Analysis on molecular weight of the enzyme showed a band on 33.3 kDa with pI is 6.13. The electroforegram of the expression of the lipase revealed a positive result and its activity was confirmed occur in solution of debris cell. This result is consistent to the in-silico prediction that predicted this enzyme will have low solubility that lead its precipitation in lipid and non-polar-debris enrichment. Determination of its optimum conditions, optimation in production of this enzyme was done. The parameters used in this research are temperature, IPTG concentration, and time of induction. Parameters analysis was done by observing the thickness of the band on the SDS-PAGE gel. The results from this optimation procedure had results that the optimum temperature is 25oC on IPTG concentration is 1.2 mM and induction time is 5 hours after addition of inducer. Optimalization was also done using two different reagents which were by addition of lysozyme and SDS. The results on this step showed that by additioning SDS 0.5% (b/v) when lysis process gave better activity of the lipase than addition of lisozyme 0.1% (b/v). Crude extract of the lipase from expression procedure then through further characterization process and gave optimum activity to pNPD (4-Nitrophenyl decanoat) substrate. Optimum pH and temperature on this step were 8.5 and 50oC after addition of ion Fe3+. The present of NaCl up to 12% (b/v) concentration seems does not give a significant activity. Organic solvents such as methanol, ethanol, n-buthanol, and acetonitrile had also shown increasing of the activity significantly. Addition of inhibitors showed that EDTA, PMSF, and SDS can decrease the activity of lipase AB8. Tm value of lipase AB8 was 70oC. From the slope of ln activity/time curve, obtained value of kD(45oC) and kD(50oC) were 4,1 × 10–3 and 7,6 × 10–3 minutes–1 respectively. Meanwhile t1/2 of it at 50oC and 45oC were 91.675 and 169.57 minutes repectively. Secondary and tertairy structure prediction gave result !/" folding pattern and about its triad catalytic of this lipase lies on Serin-136, Aspartat-214 and Histidin-244 residues. Lipase AB8 is one of the HSL (Hormone Sensitive Lipase) group of enzyme with the family is IV.