Purification and Characterization of Formate Dehydrogenase in a Methanol-utilizing Yeast, Kloeckera sp. No. 2201

Abstract

An NAD linked formate dehydrogenating enzyme which catalyzed the last step of methanol oxidation system was extracted from the methanol-grown Kloeckera sp. No. 2201. The specific activity of the enzyme in the extract of methanol-grown cells was found to be considerably higher than that of the glucose-grown cells. The enzyme was purified about 35-fold from the extract of methanol-grown cells by heat treatment, column chromatographies on DEAE-cellulose and on hydroxylapatite, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous by analyses with electrophoresis and ultracentrifugation. The purified enzyme was a kind of NAD: formate oxidoreductase (EC, 1. 2. 1. 2) which catalyzed specifically the oxidation of formate to carbon dioxide. The Km values were 22mM for formate and 0.1mM for NAD. The enzyme was inactivated by potassium cyanide, sodium azide, and p-chloromercuribenzoate but not by any metal-chelating reagents tested. Other general properties of the enzyme were also investigated.