Purification and characterization of LipA from Bacillus amyloliquefaciens

• Main Authors: Po-Shu Shih, 施博書
• Other Authors: 吳蕙芬
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/58ww9c

碩士 === 國立臺灣大學 === 農業化學研究所 === 106 === Microbial lipases are mainly applied in food industry, detergents and the other chemical industry. Because the isolation and purification of lipases from microorganisms is easier than those of animals and plants, the mass production and study of lipases from microorganisms are really important. Lipases of Bacillus spp. are heat and alkaline tolerant, so the lipase activity under heat and alkaline conditions can remain stable. Besides, lipases from the Bacillus spp. belong to those of the smallest molecular weight, and this make it a benefit for application in biotechnology. However, the original bacterial strain usually can not produce large amount of lipases, so it is better to select a transformant carrying a lipase gene in a plasmid which can produce large amount of lipases with special and specific characteristics for effective application in industry. Although many lipase genes form Bacillus spp. have been sequenced, studies on lipases of Bacillus amyloliquefaciens are still not comprehensive. A strain of Bacillus amyloliquefaciens was isolated from the environment and its lipase gene was selected, and cloned into pET21a, for expressing in E.coli by an induction with IPTG. Through the purification of the lipase protein (LipA) by the nickle column, a protein whose molecular weight is about 25kDa was obtained. The optimal temperature and pH value for LipA lipase activity are 35℃ and pH-10, respectively. Also, the LipA lipase is stable under 35℃ and pH value 9-10. The LipA lipase prefers p-nitrophenyl laurate as its substrate. The buffer containing a final concentration of 10 mM Zn2+, Fe2+, Cu2+, Ca2+, SDS or Triton-X-100 would strongly suppress the enzymatic activity. 10% DMSO and isopropanol would slightly enhance the enzyme activity, but 25% of most organic solvent could inhibit LipA activity. The results of the thin-layer-chromatography (TLC) showed that the longer the incubation time, the more degradation product of olive oil, including fatty acid and diglyceride. The degradation rate of olive oil after 96 hrs by B. amyloliquefaciens, the transformant expressing and LipA lipase was 38.8%, 45.3% and 53.5%, respectively, whereas for soybean oil, it was about 35.5%, 43% and 62%, respectively. Taken together, the above results revealed that LipA lipase has the potential to be applied in biodiesel production, recycled oil treatment and oil modification.