Purification, crystallization and some properties of .BETA.-hydroxyisobutyrate dehydrogenase from Candida rugosa IFO 0750.

Abstract

β-Hydroxyisobutyrate dehydrogenase was purified and crystallized from Candida rugosa IFO 0750. The overall purification was about 3700-fold with a yield of 34.5%. The molecular weight was estimated to be 80, 000 by the gel filtration method and 75, 000 by the sedimentation velocity method. The enzyme consisted of two identical subunits with molecular weights of 40, 000. The enzyme dehydrogenated both L- and D-β-Hiydroxyisobutyric acid (β-HIBA) with NAD+ as a coenzyme. The specific activities of the enzyme for L- and D-β-HIBA were 36.1 and 8.94 units per mg protein, respectively. The Km values for L-, D-β-HIBA and NAD+ were 3.70x 10-4M, 1.25x 10-3M and 5.00x 10-5M, respectively. It was suggested from kinetic analysis that the dehydrogenations of L- and D-β-HIBA were catalyzed at a single active center of the enzyme.