Optimization of Fermentation Conditions, Purification, Cloning and Expression of a Cold-active Lipase from Pseudomonas Sp.RT-1
|School||Fujian Normal University|
|Keywords||Cold-active Lipase Pseudomonas sp. Purification Optimization of fermentation conditions Cloning Expression|
Pseudomonas sp. RT-1, which isolated from a low temperature environment, secrete Cold-active Lipase (PL-1) when grow in a suitable medium. The previous study attracted us an interested in this enzyme for its low temperature activity and thermo-stability. In order to study the molecular basis of this feature, we optimized the fermentation medium and condition in the production of this lipase, purified, cloned and expressed the gene in the E.coli.The fermentation media and condition were optimized by single factorial experiments at the first stage, carbon, nitrogen, sample loading capacity, optimum fermentation time were screened for the obtain a condition that have the most positive influence on the lipase production. Based on the result, screening methodology Plackett-Burman design was used to evaluate the effects of eight factors related to lipase production and three statistically significant factors temperature, K2HPO4 and pH were selected. The path of steepest ascent was used to approach the optimal region of lipase production subsequently. Then, the optimal combined concentration for maximum enzyme activity were further optimized by response surface methodology and determined as follows:temperature 16.73℃, K2HPO40.37%and pH 9.46. The optimization of culture conditions of Pseudomonas sp. RT-1 led to a 5.77 fold increase in lipase production.The lipase PL-1 was purified from culture supernatant by ammonium sulfate precipitation, dialysis, gel filtration chromatography. The apparent molecular mass of PL-1 estimated by SDS-PAGE was 44.3kDa. PL-1 shows high activity and stability at low temperature (10-40℃) and high pH (9-11), the optimum is 18℃and pH 10.2.PL-1 catalyzes shorter carbon chain length fatty acid triglycerides (C<12) and shows good organic solvent-tolerant. The activity of PL-1 was enhance by 10 mmol/L Ca2+ K+, Na+and Fe3+but was inhibited by 10 mmol/L Cu2+、Co2+、Mn2+、Mg2+、Zn+、Ba2+ and Al3+. Ca2+is the most important factor which can crease 146.07% of activity of the PL-1.PL-1 encoding gene was cloned by PCR using the primers which designed by the method of CODHOP according to conservative domains of lipase. The sequencing result indicates that the cDNA of PL-1 consist of 1410 bp, encoding a polypeptide of 470 amino acids. Consensus motif G-H-S-L-G was found in the putative protein sequences indicating that the protein is a lipase in nature.PL-1 cDNA was inserted into vector pET-28a and introduced into E.coli BL 21(DE3). The expression of the recombinant PL-1 was confirmed by SDS-PAGE and the activity was detected by the olive oil agar plate.