Purification and improvement of the functional properties of Rhizopus oryzae lipase using immobilization techniques

Abstract

The adsorption/immobilization of Rhizopus oryzae lipase (ROL) has permitted the development of several strategies to improve the properties of this industrial enzyme. The enzyme can be purified, immobilized, hyperactivated and stabilized, though its enantioselectivity could still be improved. A moderately hydrophobic support (e.g., phenyl-Toyopearl) allows the almost-quantitative immobilization of the enzyme via selective hydrophobic adsorption, while any contaminant proteins are not adsorbed. A further desorption of the adsorbed enzyme with surfactants (e.g., 0.5% sucrose laurate) allows the complete purification of the enzyme in only one step, with a 90% purification yield and an 8-fold purification factor. By using a more hydrophobic support, the ROL can be immobilized and hyperactivated. The enzyme is completely adsorbed on octyl-Sepharose and C18-Sepabeads. The pure enzyme (when adsorbed on these supports) becomes hyperactivated to approximately 250% of the activity of the soluble enzyme. In this study, two protocols for the covalent immobilization of ROL were developed: a one-point immobilization on CNBr-activated Sepharose and a multipoint covalent immobilization on highly activated glyoxyl-agarose supports. The multipoint attached enzyme decreased in activity to 50% of the activity of the soluble enzyme, but the derivatives were 300-fold more stable than the soluble enzyme. The hydrolysis of racemic 2-O-butyryl-2-phenylacetic acid was modulated by the different immobilized derivatives. In fact, the most active and selective preparation was demonstrated to be the C18-SB-ROL derivative (0.05UI/mg and E=22) when compared with the CNBr-ROL enzyme preparation (0.0073UI/mg and E=3.5).