| dc.description.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. | |
