Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis

Bacterial chitosanases share weak amino acid sequence similarities at certa in regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the func tional importance of the conserved region in a novel thermostable chitosana se (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the cons erved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was chan ged to aspartate and glutamine or asparagine and,glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydro lysis were determined with wild-type and 10 mutant chitosanases. The Leu64 --> Arg and Leu64 --> Gln mutations were essentially inactive and kinetic p arameters such as V-max and k(ear) were approximately 1/10(7) of those of t he wild-type enzyme. The Asp98 --> Asn mutation did not affect the K-m valu e significantly, but decreased k(eat) to 15% of that of wild-type chitosana se. On the other hand, the Asp98 --> Glu mutation affected neither K-m nor k(ear). The observation that approximately 15% of activity remained after t he substitution of Asp98 by Asn indicated that the carboxyl side chain of A sp98 is not absolutely required for catalytic activity. These results indic ate that the Leu64 residue is directly involved in the catalytic activity o f TCH-2.