ISSN:
1573-4943
Keywords:
Thermostable chitosanase
;
Bacillus coagulans CK108
;
site-directed mutagenesis
;
leucine residue
;
catalytic activity
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 → Arg and Leu64 → Gln mutations were essentially inactive and kinetic parameters such as V max and k cat were approximately 1/107 of those of the wild-type enzyme. The Asp98 → Asn mutation did not affect the K m value significantly, but decreased k cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K m nor k cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1007147214796
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