PURIFICATION AND PROPERTIES OF ALDOSE REDUCTASE FROM PACHYSOLEN-TANNOPHILUS

Aldose reductase (EC 1.1.1.21) from Pachysolen tannophilus IFO 1007 was purified 15 fold from the crude enzyme in a yield of 0.9% by pH 5 treatment, protamine sulfate precipitate, ammonium sulfate fractionation, and G-100 gel chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. The optimum pH and temperature were 5-6 and 50.degree. C, and it was stable at pH 6-8 and up to 35.degree. C. Its activity was enhanced slightly by Na2SO4, glycylglycine, glutathione, and cysteine, and inhibited remarkably by SH inhibitors such as AgNO3, HgCl2, lead acetate and iodoacetate. Its Km values were determined as follows: 0.97 mM for D-glyceraldehyde, 1.7 mM for DL-glyceraldehyde, 3.5 mM for D-erythrose, 12 mM for D-xylose, 18 mM for L-arabinose, 25 mM for galactose, 33 mM for valeraldehyde, 33 mM for 2-deoxy-D-glucose, 50 mM for propionaldehyde, 67 mM for D-ribose, 200 mM for D-mannose, and 280 mM for acetaldehyde. The enzyme also reduced glucose, L-sorbose, butylaldehyde, and benzaldehyde. Its molecular weight was estimated to be 40,650 by sedimentation equilibrium, 40,000 by SDS polyacrylamide gel electrophoresis and 43,000 by Sephadex G-200 column chromatography.