Publication Date

12-1975

Advisor(s) - Committee Chair

Martin Houston, Lewis Lockwood, James Skean

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

NADP+-dependent alcohol dehydrogenase (alcohol:NADP+:oxidoreductase, EC 1.1.1.2.) was partially purified from a cell-free extract of Phycomyces blakesleeanus (+ mating type, Vanderbilt strain). The fungus was grown in shake cultures containing glucose-asparagine broth, and crude cull-free enzyme extracts were prepared. The crude enzyme extracts were partially purified by a combination of ammonium sulfate precipitations and heat precipitation. Purification resulted in a 40;5 recovery of the enzyme with an increase in specific activity from 0.64 units per milligram of protein to 0.25 units per milligram. The partially-purified enzyme extracts were used to determine the kinetic properties and substrate specificity of the enzyme.

Michaelis-Menten constants (Km) and maximum velocities (Vmax) were calculated by Lineweaver-Burk plots for the primary alcohol series ethyl through octyl alcohol. A decrease in Km value and an increase in Vmax resulted as the carbon chain length of the alcohols were increased to seven carbons. These results indicate that alcohol dehydrogensse of Phicomyces blakesleeanus has a greater affinity for short-chained alcohols than do those alcohol dehydrogenases previously isolated from other sources.

The substrate specificity of this alcohol dehydrogenase was quite high. Employing a variety of alcohols and aldehydes, alcohol dehydrogenase activity was obtained only for primary alcohols and one secondary alcohol, 2-butanol. Formaldehyde also functioned as a substrate for this enzyme in a manner similar to primary alcohols. Stoichiometry studies using disc-gel electrophoresis revealed that alcohol dehydrogenase was the enzyme catalyzing all the reactions reported in this thesis.

Disciplines

Biology | Life Sciences

Download

Included in

Biology Commons

Share

COinS