Production and Characterization of Whole-Cell Rhizopus oryzae CCT3759 to be Applied as Biocatalyst in Vegetable Oils Hydrolysis

Abstract

The present study aims to produce a mycelium-bound lipase of the fungus Rhizopus oryzae CCT3759 by submerged fermentation in order to be applied as biocatalyst in the hydrolysis of different vegetable oils. Optimal cultivation conditions have been achieved in a medium containing olive oil as inducer for 72 h of fermentation, thus obtaining 30.5 g/L of dry biomass concentration and hydrolytic activity of 389.1 U/g, which corresponds to a total lipase activity around of 12,000 U/L. Maximum hydrolytic activity was observed at pH 6.0 and 40 °C. Kinetic parameters concerning apparent Michaelis–Menten constant (Km = 50.5 mM) and maximum reaction rate (Vmax = 815.4 µmol/g min) have been determined in olive oil emulsion hydrolysis. Thermal stability tests revealed that the enzyme retained 75% of its initial activity after 4 h at 50 °C, whose thermal inactivation constant (Kd) and half-life (t1/2) was 0.073 h−1 and 9.4 h, respectively. The effect of biocatalyst concentration, expressed as activity units—U (200 and 400 U), on the hydrolysis of vegetable oils was investigated under fixed conditions: oil/buffer mass ratio of 25% (m/m), 100 mM buffer sodium phosphate pH 6.0, 40 °C and the mechanical stirring frequency of 600 rpm. As expected, increasing the initial activity from 200 to 400 U leads to higher values of initial reaction rates and hydrolysis percentage. However, initial reaction rate values were similar for six different vegetable oils due to the high accessibility of the lipase to the substrate under such experimental conditions. A complete hydrolysis of olive, cottonseed, sunflower and canola oils has been achieved after 26–30 h of reaction using 400 U of activity. These results suggest a promising application of the produced biocatalyst in the production of free fatty acids, an important class of compounds for oleochemical industries.