Abstract: In order to determine the relationship between amino acid residues at the special site and specific activity of Microbacterium sp.OU01 chitosanase,p CT7- CHISP6H- m Mschito plasmid was used as a template for the sitedirected mutagenesis from Asp69 to Asn69 and Glu51 to Gln51.The specific activity of chitosanase with and without mutation was measured. Mutation of Glu51 into Gln51 resulted in a drastic loss of specific activity. Through the determination of kinetic constants of enzyme,it was found that the mutant enzyme hadalmostthe same Kmvalue as the native enzyme,but the Vmaxvalue from the mutant enzyme( Glu51→Gln51) varied obviously compared with that of the native enzyme. According to the difference between per- mutant and mutant c DNA sequences,the secondary structure and three- dimensional structure of chitosanase were also predicted using DNA Star and Deep view analysis software. The results showed that the amino acid Glu51 was critical significant for maintaining the activity of chitosanase from Microbacterium sp. OU01. The site- directed mutagenesis of Microbacterium sp. OU01 chitosanase at position 51 changed the properties of secondary structure as well as hydrogen- bonding networks,thus probably resulting in the loss of specific activity.