89] Formaldehyde Dehydrogenase from Candida boidinii

Abstract

Publisher Summary This chapter describes the assay method, purification, and properties of formaldehyde dehydrogenase from Candida boidinii . Formaldehyde dehydrogenase is measured spectrophotometrically by following the rate of nicotinamide adenine dinucleotide dehydrogenase (NADH) formation at 340 nm in the presence of saturating amounts of formaldehyde, glutathione, and nicotinamide adenine dinucleotide (NAD). For enzyme preparations, Candida boidinii is grown in fermentors on a minimal medium with methanol as the sole carbon and energy source. Cells are harvested by centrifugation at the end of the exponential growth phase and stored frozen until used. The steps involved in the purification of the enzyme are (1) the preparation of crude extract, (2) streptomycin sulfate precipitation, (3) diethylaminoethyl (DEAE)-cellulose chromatography, and (4) hydroxyapatite colamn chromatography. All operations are carried out at 4°C and with 0.2% (v/v) 2-mercaptoethanol in all buffers to maintain the enzyme activity. The molecular weight of the formaldehyde dehydrogenase is determined by sedimentation diffusion equilibrium. In Candida boidinii, formaldehyde dehydrogenase is a key enzyme of the dissimilatory pathway of the methanol metabolism.