| Lipases(triacylglycerol hydrolases EC 3.1.1.3) are a class of enzymes that can catalyze both the hydrolysis and the synthesis of esters across an oil-water interface. Lipase can catalyze various reactions, which includes hydrolysis of esters, ester exchange and ester synthesis. Thus, lipases play an important role in a number of industrial applications, including detergents, cosmetics, foods, pharmaceuticals and flavor industry, and it has important industrial value. Here, we successfully cloned lipase gene mas1 from marine microorganism and expressed in Pichia pastoris host X-33. The enzymology properties, high density fermentation conditions and the effects of molecular chaperones on expression were studied. The results were showed as follows:1. The expression and purification of lipase MAS1MAS1 gene was cloned into the expression vector of p PICZa A, and then integrated into the genome of Pichia pastoris host X-33 by electroporation. The recombinant genetic engineering strain of MAS1- p PICZαA /X33 was obtained successfully. The supernatant was purified by Nickel column and got a single band at 29 KDa by SDS-PAGE,indicating that mas1 gene has successfully expressed in Pichia pastoris X33. The supernatant was purified by Nickel column and got a single bland at 29 KDa.2. Optimization of high density fermentation conditionsIn this study, the fermentation conditions for the 30- L fermentation tank were optimized, mainly including the induction temperature, p H and feed mode. 1)Under cultivation temperatures of 30 ℃, 28 ℃, 26 ℃, 24 ℃ and 22 ℃ with 144 h, enzyme activity significantly decreased when the temperature increased. The maximum enzyme activity, total protein concentration and wet cell weight were 107 U/m L, 0.678 g/L and 450 g/L at 22 ℃, which were 2.2, 1.45 and 1.18 fold higher than that of 30℃. 2)The fermentation was also carried out at different p H(p H4.0-8.0), the highest enzyme activity( 240 U/m L) was obtained at p H 6.0, and total protein content was 0.867 g/L,which were 2.47 and 1.34-folds of p H5.0. It had little impact on the growth of yeast under the p H 4.0-7.0, the wet cell weight were all about 450 g/L, but when the condition was set at p H 8.0, the cells could’t growth normaly, and lipase enzyme activity could hardly be detected.3) Results showed that the method of methanol- sorbitol mixed induction could’t improve the expression compared to methanol as the sole carbon source.Under this condition, the lipase activity from 240 U/m L to 280 U/m L, the biomass was increased from 458 g/L to 480 g/L.3. The effects of different chaperones on the protein expression of MAS1 lipaseChaperones(endoplasmic reticulum stress response proteins HAC1, disulfide bond isomerase PDI, vitreoscilla hemoglobin VHB and heavy chain binding protein Bip) were cloned into the recombinant strains MAS1- p PICZαA /X33, respectively. Results indicated that the co-expression of MAS1-HAC1, MAS1-PDI and MAS1-VHB could facilitate the expression of proteins. After 144 h of cultivation, the lipase activityies of recombinant strains were 330, 440 and 340 U/m L, respectively. It was 1.37, 1.81 and 1.41-folds higher than that of original strains. The activity of recombinant strain harboring MAS1 and Bip genes was only 160 U/m L, indicating that Bip gene restrained the expression of MAS14. Study on the enzymatic properties of MAS1MAS1 showed the optimum temperature of 40 ℃, and it had a wide range of temperature stability; The optimum p H of MAS1 was 7.0, and it showed a good stability in the range of p H5.0- p H9.0. At the same time, the tolerance of different metal ions were well studied. Most of esidual enzyme activities were more than 83% after treated with different metal ions, including Cu2 +, Ca2 +, Ni2 +and Mg2 +, MAS1 enzyme activities were still kept 90% of the initial enzyme activity under different metal ions. It showed a good tolerance in different organic solvents. Different surfactants showed the inhibition to the enzyme activity, including the Tween-20, Tween-60, Tween-80 and Triton 100, the residual enzyme activities were only 6.21%, 5.4%, 7.33% and 23.9%, respectively. The optimal artificial substrate was p-nitrophenyl octanoate, it could hydrolyze triglyceride and diglyceride,and it showed no position selectivity to diglyceride. |
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