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J Bacteriol. 1974 December; 120(3): 1133-1143
Copyright © 1974 American Society for Microbiology. All Rights Reserved.

Purification and Characterization of the Nocardial Acetylesterase Involved in 2-Butanone Degradation

^a Department of Microbiology and Agricultural Experiment Station, Louisiana State University, Baton Rouge, Louisiana 70803

ABSTRACT

An inducible acetylesterase (EC 3.1.1.6) that hydrolyzes ethyl acetate, an intermediate in the degradation of 2-butanone by Nocardia strain LSU-169, was purified. The polypeptide molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 39,500, and the enzyme molecular weight determined by sucrose density gradient centrifugation was 84,000. The purified enzyme demonstrated aggregation in polyacrylamide gels. The esterase hydrolyzed p-nitrophenyl acetate, ethyl acetate, and methyl acetate; however, enzymatic hydrolysis of phosphates, sulfates, dipeptides, lactones, or the ethyl esters of N-benzoyl-L-tyrosine could not be detected. The apparent K_m for esterase activity with p-nitrophenyl acetate as the substrate was 6.7 x 10^–5 M, and the maximal velocity (V) was 1,223 µmol/min per mg of protein at 30 C. With ethyl acetate as the substrate, the apparent K_m was 3.6 x 10^–4 M and V was 1,026 µmol/min per mg of protein. No significant inhibition of esterase activity was obtained with organophosphates, mercuric compounds, eserine sulfate, sodium arsanilate, NaF, CaCl₂, CoCl₂, or MnCl₂. At concentrations from 7 x 10^–4 to 4 x 10^–3 M, 2-butanol and primary alcohols with chain lengths of four or more carbons inhibited esterase activity from 59 to 86%. Linear noncompetitive inhibition of esterase activity by 3-methyl-1-butanol with a K_i of 1.0 x 10^–3 M was demonstrated.

¹ Present address: Department of Microbiology, University of Texas, Austin, Tex. 78753.

² Present address: Department of Bacteriology, University of Southwestern Louisiana, Lafayette, La. 70501.

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