Optimization of culture parameters to improve the recombinant lipase activity in e. Coli bl21 (de3) and preliminary scale-up process in two bioreactor platforms

Abstract

Pseudomonas lipases are widely used in industrial applications due to their unique biochemical properties. Due to low yields when the lipase is produced in the original strain, a recombinant lipase production strategy was used with E. coli. However, due to the incorrect folding, the recombinant enzyme only achieves the secondary structure which aggregates and form inclusion bodies that seriously reduce the biological activity, therefore the evaluation of different fermentation conditions are necessary to improve the activity of the enzyme and to decrease the inclusion bodies formation. In this study, a statistical experimental design was implemented to evaluate the effects of temperature, agitation rate and osmolyte concentration on the recombinant lipase activity produced in E. coli BL21 (DE3). Once the significant variables were identified, a Response Surface Methodology was performed to determine the optimal fermentation conditions for lipase production. As a result, the growth at 5°C, 110 rpm, and 0.1 M of glycerol significantly increased the specific lipase activity and showed that the data fitted the model very well. These culture conditions were validated against experimental results, and 452.01 U/mg of specific lipase activity was obtained, which was significantly higher than the control group where no glycerol was added (271.38 U/mg). Besides, it could be demonstrated that when E. coli BL21(DE3) was grown at the optimal culture condition at 5°C, the relative recombinant lipase expression was 2.7-fold lower compared to 25 °C. However, at 5°C the lipase activity was significantly higher compared to 25°C. Furthermore, when the 2 L Bioreactor (equipped with anchor impeller) was used to perform a preliminary scale-up process, the specific lipase activity was significantly different from that found at the 100 mL Schott scale (337,91 U/mg and 452,01 U/mg, respectively). Nevertheless, when the 3 L shaken Erlenmeyer Bioreactor was used, the specific lipase activity was not significantly different to that found at the Schott scale (408,4 U/mg and 452,0 U/mg, respectively), meaning that the optimal growth conditions used for the scale-up process can be a guide for future productions. This study represents a reliable and low-cost strategy for recombinant lipase production through the optimization of fermentation conditions. In our case, the recombinant lipase activity was enhanced at low temperatures, low agitation rates and the addition of glycerol to the auto-inducing media. It also demonstrates the utility of using the design of experiments to optimize the fermentation conditions at small scale before scaling-up the production of the recombinant enzyme in E. coli BL21 (DE3). Further studies using the strategy used here may lead to identifying optimal culture conditions for a given recombinant enzyme facilitating the large-scale bioprocess implementation and enhancing the biological activity of the target enzyme.