Purification and Properties of Two Different Azoreductases from a Yeast Candida curvata AN723.

Abstract

Two azoreductases from Candida curvata AN723 have been purified and characterized. The yeast cells cultured in a GYP medium were disruped with French press. From the acetone fraction of the cell free extract, two different azoreductases (NADPH-dependent azoreductase 1 and NADH-dependent azoreductasese 2) were separated by DEAE-Toyopearl 650M chromatography followed by Toyopearl HW50F chromatography. Azoreductase 1 was further purified by ion exchange chromatography using TSK gel DEAE 5PW. Azoreductase 2 was further purified by affinity chromatography using AF-Blue Toyopearl 650 and chromatofocusing using Toyopearl HW50F and Rotofor system. Each final preparation was homogenous on polyacrylamide gel disc electrophoresis. Azoreductase 1 and 2 were purified 295- and 240-fold, respectively. The molecular mass of azoreductase 1 and 2 were calculated to be about 47,000 daltons and 56,000 daltons, respectively. The isoelectric points of azoreductase 1 and 2 were 4.5 and 4.9, respectively. Azoreductase 1 and 2 had the same optimum pH 5.5-6.0, while they were stable between pH 4.0 and 6.5, and between pH 5.0 and 6.0, respectively. The optimum temperature of azoreductase 1 was 50°C, and that of azoreductase 2 was 40°C, while the former was stable below 75°C, and the latter below 50°C. Azoreductase 1 and 2 had low substrate specificity. Spectrum of these two purified enzymes showed no characteristic absorption band of flavine in the visible range of wavelengths.