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Site‐Directed Mutagenesis of Asp313, Glu315, and Asp391 Residues in Chitinase of Aeromonas Caviae
Author(s) -
Lin FuPang,
Chen HsingChen,
Lin ChungSaint
Publication year - 1999
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/713803487
Subject(s) - chitinase , aeromonas caviae , hydrolysis , mutagenesis , aeromonas , enzyme kinetics , mutant , biochemistry , biology , site directed mutagenesis , substrate (aquarium) , chemistry , enzyme , microbiology and biotechnology , active site , bacteria , genetics , gene , ecology
Site‐directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae .Kinetic parameters for 4‐methylumbelliferyl‐N, N‐diacetylchitobiose or 4‐methylumbelliferyl‐N,N, N‐triacetylchitotriose hydrolysis were determined with wild‐type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the K m values were different. The function ofthe carboxylgroup of Asp313 was partially replaced by the amide ofAsn when the 4‐methylumbelliferyl‐N, N, N‐triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.