Optimization for refolding condition of pseudomonas fluorescens lipase expressed as inclusion body in E.coli by response surface methodology

Abstract

To enhance in vitro refolding yield of Pseudomonas fluorescens SIK W1 lipase expressed as inclusion bodies in E.coli, response surface methodology(RSM) was applied to optimize three factors such as GdnHCl concentration, protein concentration and pH simultaneously. The refolding yield could be increased to about 90 % (9000 U/mg) by this method and this was about 150 % increase compared with the traditional optimization. The optimization of refolding was executed at the various conditions by RSM. Refolding yield was increased by lowering refolding temperature from 25℃ , to 4℃ and to -15℃ but the rate of refolding was decreased by lowering temperature. The refolding yield at 4℃ was 325% higher than the yield at 25℃. The refolding yield at -15℃ was increased slightly as compared with the yield at 4℃ but much longer time was needed to complete refolding than 4℃. Interestingly, the optimum was changed by adding the refolding additives and it could be traced by RSM. Addition of 40% glycerol shifted the optimum concentration of GdnHCl from 0.7 M to 1.4 M and addition of 0.5 M Arginine shifted the optimum concentration of GdnHCl to 0.5 M. From this result the mechanism of refolding additives (glycerol and arginine) could be investigaed. In addition, fluorescence were also used to monitor the folding transition of lipase and this was compared with the restoration of the enzyme activity. There was consistency at the high range of GdnHCl concentration but disagreement at the low range.