Expression And Characterization Of A Lipase From Bacillus licheniformis IBRL-CHS2
| dc.contributor.author | Subba Reddy, Nidyaletchmy | |
| dc.date.accessioned | 2018-01-15T07:09:58Z | |
| dc.date.available | 2018-01-15T07:09:58Z | |
| dc.date.issued | 2017-08 | |
| dc.description.abstract | Lipases are employed in a number of industrial applications as very useful biocatalysts. Therefore, it is always worthwhile to study new lipases in detail to find out their novel properties and a way to exploit their potential as a biocatalyst. Bacillus licheniformis IBRL-CHS2 in this study was obtained from the culture collection of Industrial Biotechnology Research Laboratory (IBRL), Universiti Sains Malaysia. The lipase gene of the bacterium was cloned into the cloning vector, pGEM-T Easy and the nucleotide and amino acid sequences of the lipase were deposited into the GeneBank with accession numbers KU984433 and AOT80658 respectively. Alignment of the amino acid sequence with other Bacillus lipases sequences revealed that it belongs to subfamily 1.4 of bacterial lipases. Expression of the lipase was attempted using several combinations of vector and host. The usage of pCold I as the expression vector and E. coli BL21 (DE3) as the host, coupled with the removal of the enzyme’s signal peptide proved to be the best expression system for this lipase. Optimum expression conditions were determined to be at 15°C for 24 hours incubation with 0.2 mM IPTG. The His-tagged enzyme was purified 13 folds with 68% recovery and specific activity of 331.3 U/mg by a one-step affinity purification method. The optimum temperature for the lipase was determined to be 35°C and the optimum pH is 7. Interestingly, the enzyme was stable even after 24 hours of incubation in various 25% (v/v) organic solvents tested namely isooctane (log P= 4.5), n-hexane (log P= 3.6), DMSO (log P= -1.22) and methanol (log P= -0.76). The mentioned organic solvents had a stimulatory effect on the lipase activity compared to the control. Chloroform (log P= 2) and diethylether (log P= 0.85) meanwhile inhibited lipase activity. Substrate specificity study revealed that the lipase possess highest affinity towards p-nitrophenol laurate (C12:0) with Km of 0.36 mM and Vmax was 357 μmol min-1 mg-1. Among the natural oils tested, the lipase showed optimum activity towards coconut oil and lowest activity towards olive oil. The lipase was stable in the presence of 1 mM and 5 mM of Ca2+, K+, Na+, Mg2+ and Ba2+ but the activity decreased in the presence of Zn2+ and Al3+. The lipase was relatively stable in the presence of various 1 mM effector molecules although marginal decrease in activity was detected at the higher concentration of 5 mM. EDTA and ascorbic acid slightly inhibited the lipase activity at both concentrations. Lipase activity was stimulated in the presence of 1 mM non-ionic surfactants like Triton X-100, Nonidet P40, Tween 20 and Tween 40. The anionic surfactant, SDS inhibited lipase activity. Site-directed mutagenesis of the catalytic triad residues- Ser-77, Asp-129 and His-152 resulted in the lipase losing all of its activity and further re-affirmed their importance for catalysis to take place. Due to the high homology (69%) that this lipase shares with the previously solved structure of lipase A from Bacillus subtilis, its tertiary structure was predicted with high confidence using the homology modelling software, SWISS-MODEL. The result revealed that the lipase shares similar fold structure with subfamily 1.4 lipases. This study has laid a strong foundation and reason for future studies of the lipase to enhance some of its properties especially its thermostability. Characterization of the lipase has highlighted some unique properties of the lipase that differs from other members of subfamily 1.4. It is very stable in 25% (v/v) organic solvent and thus can be employed in the area of organic synthesis and other suitable industries. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/5385 | |
| dc.language.iso | en | en_US |
| dc.publisher | Universiti Sains Malaysia | en_US |
| dc.subject | Expression and characterization of | en_US |
| dc.subject | a lipase from Bacillus licheniformis | en_US |
| dc.title | Expression And Characterization Of A Lipase From Bacillus licheniformis IBRL-CHS2 | en_US |
| dc.type | Thesis | en_US |
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