CATECHOL 2,3-OXYGENASE PRODUCTION BY GENETICALLY ENGINEERED ESCHERICHIA-COLI AND ITS APPLICATION TO CATECHOL DETERMINATION

Catechol 2,3-oxygenase was produced by Escherichia coli, harbouring the recombinant plasmid pBH100 which contained the pheB gene cloned from phenol-degrading Pseudomonas putida BH, and was applied for the determination of catechol in the liquor. E. coli JM103 (pBH100) and C600 (pBH100) showed, respectively, about 5 and 8.5 times higher activities than that of P. putida BH. Using the crude extract prepared from the culture broth of the recombinant, catechol between 0.1 and 3.0-mu-g/ml could be determined quantitatively in phosphate buffer, synthetic sewage and in mixtures of phenol, benzoate and salicylate, and also in sodium pyruvate solution. In addition to catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol could be determined. Oxygenase activity of the crude extract was maintained completely during the 100-day storage at -20-degrees-C after being freeze-dried with 10% acetone.