Shaking Rate during Production Affects the Activity of Escherichia coli Surface-Displayed Candida antarctica Lipase A

Abstract

In this study, a surface-display system was applied for the expression of lipase A in an E. coli expression system. Since the target protein was exposed on the cell membrane, the shaking rate during culturing might have increased the oxygen mass transfer rate and the shear stress, both of which would be detrimental to the surface-displayed protein. The shaking rate did indeed have an effect on the properties of the surface-displayed lipase A from Candida antarctica (sdCALA). When cultivated at a shaking rate of less than 50 rpm, the specific activity of sdCALA was low, which was due to the limited amount of dissolved oxygen. When the shaking rate was greater than 100 rpm, the specific activity decreased as a result of shear stress. When cultivating CALA and sdCALA at various temperatures and values of pH, both proteins displayed the same activity profile, with the optimum conditions being 60 °C and pH 6. A kinetic study revealed that the sdCALA cultivated at 100 rpm gave a higher value of νm (0.074 μmol/mL/min) and a lower value of Km (0.360 μmol/mL) relative to those obtained at 200 rpm and relative to those of the free CALA. sdCALA retained over 80% of its activity after treatment at 70 °C for 30 min, but its activity decreased rapidly when the temperature was above 80 °C. The specific activity of sdCALA decreased in the presence of acetonitrile and acetone relative to that of the control (50% ethanol), regardless of the solvent concentration. The highest activity (0.67 U/mL) was obtained when the ethanol concentration was 30%.