High-level Expression, Purification And In Vitro Refolding Of Burkholderia Cepacia Lipase

As one of the renewable and clean fuels, biodiesel provides an impotant solution to the energy crisis, and it has been regarded as one of the main branches in new energy research in the future. Compared to the chemical technologies, lipase catalyzed biodiesel production presents many advantages, such as mild reaction conditions, high efficiency and environmental friendship. However, commercial lipase has too high price to put enzymatically catalyzed biodiesel production technique into industrialization..Lipase's price has been becoming the main bottleneck of the development of biodiesel industry. In this sense, developing cheap lipase is of enormous significance for biodiesel production.In this research, lipase and its cognate foldase genes have been cloned from Burkholderia cepacia and overexpressed in Escherichia coli BL21(DE3) separately. Then, the denatured lipase refolding in vitro under the assistance of foldase was studied, which would give a pilot study for the potential use of the refolded lipase.The main work and results are listed as below:1. Burkholderia cepacia lipase and its cognate foldase genes were cloned and overexpressed. The presence and position of signal peptide of the lipase was predicted according to bioinformatics method. The results showed that a 40-amino-acids sequence from the N terminal was probably the signal peptide.Then, N terminal truncated lipase was overexpressed in E.coli using expression vector pET-28a (+). The expression of the truncated lipase yielded insoluble inclusion bodies of the lipase, and it was highly purified by simplified washing with sodium deoxycholate treatment. then, dissolved in 8M urea. The measured protein concentration reached 4.085mg/mL. The transmembrane region of the foldase was predicted and the N terminal hydrophobic transmembrane truncated foldase was also expressed in E.coli using pET-28(a) vector. The truncated foldase was expressed in soluble form, and was highly purified as a His6-tagged fusion protein using Ni-NTA affinity column chromatography. The protein concentration of the foldase after dialysis was 0.67 mg/mL.2. The in vitro refolding of lipase in the assistance of foldase was studied. The effects of foldase/lipase molar ratio, temperature, time length, pH and initial protein concentration on the refolding of the lipase were examined. The optimal refolding conditions were: a low lipase concentration of 0.8μg/mL, in the presence of equal molar amount of foldase in 15℃and pH7.0 buffer, would get the highest enzymatic activity of 1514.0 U/mg after refolding for 5 hours; however, in a high concentration of 80μg/mL, lipase would need 40 hours to refold and got the highest enzymatic activity of 2284.0 U/mg.3. The refolded lipase was treated by vacuum freeze drying and immobilization so as to improve its application potential. Under vacuum freeze drying, the refolded lipase exhibited enzymatic activities of 694 U/mg and 213 U/mg with the substrate of pNPP and olive oil, respectively. The immobilized refolded lipase showed an enzymatic activity of 770.75 U/mg with the substrate of pNPP. The exploration on refolded lipase was an attempt to facilitate refolded lipase in industrial production, which will provide methodological choice.