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GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
Published on: October 29, 2013
Purification, characterization, and stabilization of alcohol oxidase from Ogataea thermomethanolica
Natthaya Mangkorn1, Pattanop Kanokratana2, Niran Roongsawang3
1Joint Graduate School for Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi, Bangmod, Bangkok 10140, Thailand.
Abstract:
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.
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