PURIFICATION AND PROPERTIES OF CATECHOL 1,2-DIOXYGENASE-II FROM PSEUDOMONAS-PUTIDA STRAIN-87

Modified ortho-pathway enzymes (catechol 1,2-dioxygenase II, muconate cycloisomerase II, dienelactone hydrolase, and maleylacetate reductase ) were induced in Pseudomonas putida strain 87 grown on 3-chlorobenzoi c acid as the only carbon and energy source. Catechol 1,2-dioxygenase II, the key enzyme of clorocatechol cleavage, was purified The molecul ar weight of enzyme determined by gel filtration was 65 kD. The molecu lar weight determined by electrophoresis in the presence of sodium dod ecyl sulfate was 33 kD. The pH optimum for enzymatic activity was 7.2- 7.8 and the temperature optimum 35-degrees-C. The catechol 1,2-dioxyge nase II was most stable on storage in 50 mM Tris-HCl buffer (pH 7.8) a t 4-degrees-C. The relative V(max) values for the catechol 1,2-dioxyge nase II with 3-chloro-, 4-chloro-, and 3,5-dichlorocatechol were 28, 5 0, and 41%, respectively, of the value with catechol. The affinity of the enzyme for chlorocatechols was 3-9-fold higher than for methylcate chols and 10-20-fold higher than for unsubstituted catechol.