To improve the efficiency and to investigate the molecular determinants that direct substrate specificity of chlorocatechol 2,3-dioxygenase CbzEGJ31, several mutant enzymes were constructed. Loci for substitutions of amino acids were selected by sequence comparisons as well as by homology modelling of known chlorocatechol 2,3-dioxygenases (CbzEBASF, CbzESK1 and CbzE16-6A). Activity measurements with various catechols showed that most of the modifications influenced activity only to a minor degree. The amino acid at position 154 seems to be located at a non-important position in the enzyme with minor extension into the substrate tunnel. Similarly, the change of related amino acids such as D95E and Y223F did not influence the catalysis since both residues are far away from the catalytic centre and the substrate tunnel. Even the modification of isoleucine to threonine in position 310, located at the outer substrate tunnel, showed a significant alteration of activities. Position 196 seems to be of higher relevance since the modification of valine to alanine, i.e. the reduction of the side-chain, produced much alteration. The amino acid is located at the interface of inner to outer substrate tunnel. CbzEV196A showed high relative kcat for 3-chlorocatechol. A pronounced increase in activity for 3-chlorocatechol resulted by the change from alanine to valine and from aspartic acid to glycine laying in the outer substrate tunnel at position 211 and 212 respectively.