PURIFICATION AND PROPERTIES OF CHLOROCATECHOL 1,2-DIOXYGENASE FROM ALCALIGENES-DENITRIFICANS BRI 6011

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-degrees-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 muM, while those for catechol and the methylated catechols were above 10 muM.