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Cloning, Expression and Purification of Halophilic Lipase from Micrococcus luteus
- Author(s)
- JOHNSON, JERVIAN JERDIAN
- Advisor
- Kwon-Young Choi
- Department
- 일반대학원 환경공학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2017-08
- Language
- eng
- Keyword
- Halophilic; lipase; lipase activity; enzyme; DNA cloning; protein purification
- Alternative Abstract
- Lipase is an enzyme that catalyzes the hydrolysis of triglycerides and fatty acids and has diverse applications in the biochemical industry. Lipase is currently used for catalyzing reactions in the food, oil and fat, detergent, leather, pulp and paper, and cosmetic industries; as well as in biodiesel production and organic chemistry. As such, cloning and purifying lipase displaying attractive properties is important. Lipase from the halophilic bacterium Micrococcus luteus was selected for cloning and purification because of its excellent salt resistance properties (reported by J. Johnson et. al). That is, it displayed a salt resistance of 2M NaCl, which is over 3 times higher than that of sea water. During the cloning and purification process, there were challenges that were encountered. Firstly, the size of the gene is small compared to the genomic DNA (800 bp compared to 3.86 Mbp) making primers that were designed less effective. However, using a combination of forward and reverse primers, the lipase gene was successfully cloned and determined to be 800 base pairs (bp). In addition, due to the incompatibility of M. luteus and E. coli BL21 (DE3)'s triplet coding, expression of the protein was also difficult. However, with optimization of the coding sequence for M. luteus lipase, it was successfully expressed and the protein was determined to be 35 kilo Daltons (kD) in size. The level of protein expression was low and as such various methods were employed in an effort to improve the amount of lipase expressed. These include: variation of IPTG concentration at induction; co-expression of lipase gene with vector chaperones; culturing the cells containing the recombinant gene using various growth media; and applying a wide range of chemical chaperones to the cell cultures. Through the optimization experiments it was determined that when the lipase gene was co-expressed with pG-Tf16 vector chaperone and grown with Reseinberg medium and Magnesium Chloride and induced at 0.05mM IPTG, optimum lipase expression was achieved. The gene was later purified using heat shock and immobilized metal-affinity chromatography (IMAC) methods. The activity of the lipase gene was tested using lipase and esterase assays. The lipase activity was determined to be 553.35 U/mg when tested with substrate, p-nitrophenylpalmitate (pNPP). This was compared to the activity of commercial lipase (lipase from Candida rugosa), which produced a specific activity of 25.66 U/mg, indicating that the lipase from M. luteus has comparatively excellent activity. Esterase activity was however not detected in lipase from M. luteus when p-nitrophenylbutyrate (pNPB) was used as the substrate. Based on the high lipase activity obtained through experiments, it is fair to say that the cloned and purified lipase from this research has commercial potential. Further research is however necessary to characterize the lipase and produce it in a form that allows it to be applied industrially.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Environmental Engineering > 3. Theses(Master)
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