A gene encoding Catechol 2,3-dioxygenase (CD-2,3) was cloned from 3,4-dichloroaniline degrading bacterium Pseudomonas sp. KB35B. Comparison of the deduced amino acids of the CD-2,3 with other CD-2,3 proteins revealed that only three amino acids (Q116, N142 and V215) were particularly different in the CD-2,3 of KB35B. To elucidate how these differences are related with the biochemical properties of the protein, these amino acids were converted into the corresponding residue of other CD-2,3 proteins or Gly, respectively. The effects of amino acid conversion on the catalytic properties of the altered enzymes were then determined, and the results showed that Q116 is a critical amino acid involved in both substrate affinity and catalysis since the conversion of Q116 into other amino acids resulted in the change of both $K_m$ and $V_{max}$. However, the substitution of N142 showed only change of $V_{max}$, suggesting that this domain may involve in substrate catalysis. The mutation of V215 domain led to the deficiency of expression, implying that this amino acid is essential for the expression of the CD-2,3.