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 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 Vmax and kcat were approximately 1/10(7) of those of the wild-type enzyme. The Asp98 --> Asn mutation did not affect the Km value significantly, but decreased kcat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 --> Glu mutation affected neither Km nor kcat. 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.
Yoon
H G
HG
Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, Seoul.
Yang
S W
SW
Kim
H Y
HY
Kim
H K
HK
Shin
D H
DH
Hong
B S
BS
Cho
H Y
HY
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D048271:Chitosan; D017124:Conserved Sequence; D004355:Drug Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D007930:Leucine / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013696:Temperature
D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D007930:Leucine; D019791:Guanidine