Synthesis and characterization of Ogataea thermomethanolica alcohol oxidase immobilized on barium ferrite magnetic microparticles.

Alcohol oxidase catalyzes the oxidation of primary alcohols into the corresponding aldehydes, making it a potential biocatalyst in the chemical industry. However, the high production cost and poor operational stability of this enzyme are limitations for industrial application. Immobilization of enzyme onto solid supports is a useful strategy for improving enzyme stability. In this work, alcohol oxidase from the thermotolerant methylotrophic yeast Ogataea thermomethanolica (OthAOX) was covalently immobilized onto barium ferrite (BaFe12O19) magnetic microparticles. Among different conditions tested, the highest immobilization efficiency of 71.0 % and catalytic activity of 34.6 U/g was obtained. Immobilization of OthAOX onto magnetic support was shown by Fourier-Transformed infrared microscopy, scanning electron microscopy and X-ray diffraction. The immobilized OthAOX worked optimally at 55 °C and pH 8.0. Immobilization also improved thermostability, in which >65% of the initial immobilized enzyme activity was retained after 24 h pre-incubation at 45 °C. The immobilized enzyme showed a greater catalytic efficiency for oxidation of methanol and ethanol than free enzyme. The immobilized enzyme could be recovered by magnetization and recycled for at least three consecutive batches, after which 70% activity remained. The properties of the immobilized enzyme suggest its potential industrial application for synthesis of aldehyde.

Purification, characterization, and stabilization of alcohol oxidase from Ogataea thermomethanolica.

Alcohol oxidase (AOX) functions in oxidation of primary alcohols into the corresponding aldehydes with potential on catalyzing synthesis reactions in chemical industry. In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50 °C in an alkaline range (pH 9.0). According to kinetic analysis, OthAOX showed a higher affinity toward short-chain aliphatic primary alcohol with the Vmax, Km, and kcat of 0.24 nmol/min, 0.27 mM, and 3628.8 min-1, respectively against methanol. Addition of alginic acid (0.35%) showed a protective effect on enhancing thermal stability of the enzyme, resulting in 72% increase in its half-life at 40 °C under the operational conditions. This enzyme represents a promising candidate for conversion of bioethanol to acetaldehyde as secondary chemical in biorefinery.