CLEA-Based Immobilization of Methylotropic Yeast Alcohol Oxidase: Influence on Storage Stability and Reaction Efficiency

There is still a great demand for sustainable oxidative systems for the conversion of alcohol to carbonyl compound, a pivotal reaction in daily organic synthesis, in the fine chemical and pharmaceutical industries. In this study we have focused on the reclamation of a biocatalytic process viable for laboratory and large scale applications. In the past, alcohol oxidase (AO), which is known to catalyze oxidation of short aliphatic alcohols, was trialed to perform oxidations, but its poor stability has limited the comprehensive applications of this enzyme. An immobilization technique by forming the cross-linked enzyme aggregates (CLEA) of this enzyme was selected as a bioengineering tool to overcome the stasis of AO application. It was demonstrated that inhibition of AO by hydrogen peroxide can be neglected due to the presence of catalase in both forms of enzyme: free and immobilized. However, AO was inactivated by the second reaction product, aldehyde. Two stages of inactivation were distinguished in the case of 1-propanol oxidation: reversible competitive inhibition followed by irreversible inactivation. Immobilization has greatly increased the storage stability of AO—after 12 weeks more than 50% of enzyme activity remained at 8 and 22 °C temperature. It was demonstrated that the application of biocatalysis for alcohol oxidation processes is not limited by the stability of enzymes, but rather due to lack of engineering solutions, and further development strategies should be directed toward creating engineering systems with an immediate elimination of the formed reaction product—aldehyde.