Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

Abstract

Background: Pseudomonas lipases are widely used in industrial applications due to its unique biochemical properties, but one of the biggest limitations are the low yields obtained in native strains therefore, organisms as E. coli are used for the recombinant lipase overexpression. However, the recombinant lipase is accumulated as inclusion bodies and it affects biological activity, making that researchers evaluate different fermentation conditions to improve the activity of recombinant enzymes. 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, an optimization by a Response Surface Methodology was applied to maximize the lipase production. Results: The Box-Behnken designs revealed different optimal fermentation conditions for each osmolyte experiment. The glycerol showed the highest specific lipase activity compared to the other osmolytes and 0.1 M of osmolyte glycerol,5°C and 110 rpm showed the highest significant increase on the specific lipase activity and the data fitted the model very well. The validation showed that 452.01 U/mg of specific lipase activity was obtained which was significantly higher compared to the group where no glycerol was added (271.38 U/mg). The relative recombinant lipase expression was 2.7-fold lower at 5°C compared to 25 °C, but at 5°C the lipase activity was significantly higher. In addition, when the 3 L shaken Erlenmeyer Bioreactor was used to produce the recombinant lipase based on the power input parameter, the specific lipase activity was not significantly different from that found in Schott (408,4 U/mg and 452 U/mg, respectively), which means that this Bioreactor platform should be used for future scale-up processes.Conclusion: Low temperatures, low agitation rates and 0.1 M of glycerol in the autoinduction media enhanced the activity of the recombinant lipase produced in E. coli BL21(DE3). The optimized conditions and the 3 L shaken Erlenmeyer Bioreactor can be used to produce the recombinant enzyme in a higher volume based on the power input parameter. Further studies using this strategy may lead to the identification of optimal culture conditions for a given recombinant enzyme facilitating the large-scale bioprocess implementation.

The role of temperature and carbon dioxide climatic stress factors on the growth kinetics ofEscherichia coli

“…This difference might occur due to the different sizes of the plasmid. Another study by López et al (2021) used the E. coli BL21(DE3) strain and estimated the μmax at three different temperatures of 5, 15, and 25 o C in LB. They concluded that increasing the temperature, increased the μmax to a maximum level of 0.16 h -1 at 25 o C, which is lower by 0.12 h -1 than the μmax reported in this study at 27 o C, i.e.…”