Purification and properties of chlorocatechol 1,2-dioxygenase from Alcaligenes denitrificans BRI 6011

1 January 1993

journal article
Published by Canadian Science Publishing in Canadian Journal of Microbiology

Vol. 39 (1) , 1-5
https://doi.org/10.1139/m93-001

Abstract

The specific activity of chlorocatechol 1,2-dioxygenase from Alcaligenes denitrificans BRI 6011 was found to be maximal in the early logarithmic growth phase. The enzyme was purified from cultures at mid-log phase of growth using ammonium sulfate fractionation, and phenyl-Sepharose and DEAE-Sepharose chromatography. The protein gave a single band by SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 33 000, and the temperature and pH optima were 30 °C and 7.5, respectively. Catechol, 3-chlorocatechol (3-CC), 4-CC, 3,4-dichlorocatechol (3,4-DCC), 3,5-DCC, 3,6-DCC, 3-methylcatechol (3-MC), and 4-MC served as substrates for the enzyme. The V_max values for the dichlorocatechols were similar, while those for the monochlorinated and methylated catechols were higher. The K_m values for all the chlorinated catechols were typically below 1 μM, while those for catechol and the methylated catechols were above 10 μM.Key words: chlorocatechol 1,2-dioxygenase, Alcaligenes denitrificans, purification, characterization, chlorobenzoic acid degradation.

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