Characterization and in vitro refolding of pseudomonas fluorescens lipase expressed as inclusion bodies in escherichia coli

Abstract

(Part 1) Purification and Characterization of Pseudomonas fluorescens SIK W1 Lipase Expressed in Escherichia coli Pseudomonas fluorescens SIK W1 lipase was expressed as a form of inclusion bodies in Escherichia coli, which was equivalent to 46 % of total cell protein. The inclusion bodies isolated from other cell components were solubilized in the buffer containing 8 M urea and then refolded by diluting urea. The lipase with active conformation was purified by hydrophbic interaction chromatography, gel filtration, anion-exchange chromatography and hydroxyapatite chromatography from the refolded sample. By these purification steps, a single band for active lipase was detected on non-reducing SDS-PAGE and 10-fold purification was attained on the basis of specific activity. Specific activity of the purified lipase toward olive oil emulsion was found to be 7,395 units per mg protein. The optimum pH and temperature of the lipase were pH 8.5 and 45-55℃, respectively. The lipase showed higher lipolytic activity toward tricaproin ($C_6$) and tricaprylin ($C_8$) among the triglycerides examined and preferentially hydrolyzed ester bond of 1- and 3- position of triolein. Lipase activity was greatly increased approximately 6-fold and stability for pH was shifted to alkaline pH by $Ca^{2+}$ ion. The lipase was inhibited by $Hg^{2+}$, $Ag^{2+}$, p-chloromercuribenzoate, diethylpyrocarbonate and sodium dodecyl sulfate. (Part 2) Enhancing in vitro Refolding Yied of Pseudomonas fluorescens Lipase Expressed as Inclusion Bodies in E. coli To enhance in vitro refolding yield of Pseudomonas fluorescence SIK W1 lipase expressed as inclusion bodies in E. coli, various refolding conditions were investigated, protein concentration, oxidoshuffling condition, guanidine hydrochloride (GdnHC1) concentration pH, activator (calcium ion), temperature and dilution mode. Refolding efficiency was optimal at protein concentration of 40-100 and 20-50 ㎍/ml in the presence of 0.8 and 0...