| Lipase (triacylglycerol acyhydrolases, EC 3.1.1.3) are an important class of enzymes which are widely used in detergents, food, medicine and other fields, because of few by-products, mild reaction condition, less consumption and low demands on instrument, etc. In recent years, microbial lipases have become an important source of lipase. Microorganism extracted lipases is the main sources of industrial enzymes. With broaden of the applied range of microbial lipase, the require of enzyme tolerance is becoming much higher. Therefore, it is of great value to exploit lipases with special enzymatic properties.Thermophilic lipases are the hotspot, because of special physical and chemical properties, and the unique catalytic activity and stability, promoting the development of enzyme engineering and laying the foundation of industrial applications. In this study we cloned a lipase from a thermophilic bacterium, which considered to be a thermophilic lipase. The pET22b (+) was used as an expression vector to produce the target protein without using the leading peptide and was transformed into E. coli BL21 (DE3) cells for overexpression. After purified through Ni-chelating affinity chromatography, a band about 25kDa corresponding to the enzyme was isolated from induced crude supernatant which the protein content is 2.89 ng·mL-1 using coomassie brilliant blue. After adding his-tag into pET22b-EST by gene mutation, the gene is successfully expressed in E.coli.The enzyme exhibited catalytic activity in the temperature range of 40-90℃. With the increase of temperature, the enzyme activity was increased, and showed an optimum temperature at of 70℃. When the temperature rose to 80℃, although the enzyme activity was decreased, the relative enzyme activity remained still nearly 70%. The lipaseTt could still showed good catalytic activity at the elevated temperature, which was the advantage of our lipase than others. As expected, the lipaseTt was observed resistant at high temperature and maintained 70% activity at 80℃. This character of thermal tolerance might be highly useful for industrial application. Surprisingly, our enzyme showed about half-life at 80℃ to be 3h.We explored activity of the lipaseTt in two different solvents i.e. DMSO and methanol. Surprisingly, the enzyme activity of the lipaseTt was increased obviously by adding a certain concentration of organic solvents. When the concentration of DMSO and methanol was increased, the lipaseTt relative enzyme activity would be gradually increased. The highest relative enzyme activity was found in 56% DMSO and 45% methanol. In addition, the lipaseTt was activated up to 7-fold by 56% DMSO and 3-fold by 45% methanol at 60min than in the absent of organic solvent.In order to analyze the influence of organic solvents on enzyme conformation, we measured the kinetic parameters of the lipaseTt and protein secondary structure, the kinetic parameters under various conditions with or without polar organic solvent were subsequently determined, and the Km was1.4 mM-1,1.23 mM-1, and 3.38 mM-1 under phosphate buffer, phosphate buffer with 56% DMSO and phosphate buffer with 45% methanol, respectively. The Kcat was 0.19s-1,4.42 s-1, and 0.62 s-1 under phosphate buffer, phosphate buffer with 56% DMSO and phosphate buffer with 45% methanol, respectively. However, the organic solvents were not considered to change the enzyme active center within the allowed error range. Organic solvent molecules changed the substance around the active center, resulting in high affinity with substrate molecule. Measuring and calculating the secondary structure of the EST protein utilizing CD, the results showed that the content of α-helix was 35.7%. But after processing the enzyme with 10%,20%, 44% and 45% methanol, the content of α-helix decreased to 29.2%、 30.8%、25.4% and 21.7%. |
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