OVEREXPRESSION OF PSEUDOMONAS-PUTIDA CATECHOL 2,3-DIOXYGENASE WITH HIGH SPECIFIC ACTIVITY BY GENETICALLY-ENGINEERED ESCHERICHIA-COLI

The cloned xylE gene encoding catechol 2,3-dioxygenase (metapyrocatech ase) from TOL plasmid in Pseudomonas putida mt-2 has been expressed in Escherichia coli W3110 to a level of similar to 15% of the total solu ble protein. Of the total iron in the crude extract, 45% was on the en zyme. The crystallized enzyme from E. coli had higher iron content (3. 7 mol/mol enzyme) and specific activity (536 U/mg) than the enzyme fro m P. putida mt-2. However, no differences were observed in physicochem ical, protein-chemical, and kinetic properties between the two enzymes , The enzyme was a homotetramer, and no changes were observed in the v alues of M(r) (136,000+/-5,000) and Stokes radius (4.26 nm) in the con centration range from 0.36 nM to 2.8 mu M, indicating that the native enzyme neither dissociated into subunits nor polymerized in this range , The catalytic center activity and the K-m values for catechol and di oxygen were 278 s(-1), 1.87 and 7.45 mu M, respectively, at pH 7.5 and 25 degrees C, The enzyme showed a broad substrate specificity. Among substrates, 4-methylcatechol and 4-chlorocatechol showed specificity c onstants (similar to 200 mu M(-1).s(-1)) higher than that for catechol , Acetone and phenol derivatives competitively inhibited the activity against catechol. The relationship between specific activity and iron content was not linear, suggesting some conformational changes in the partially iron-depleted enzyme.