Abstract: The catalytic domain and key amino acid residues of dextranase from Arthrobacter oxydans KQ11 (AoDex) were predicted by homologous sequence alignment. The mutants Gln418→Gly, Asp420→Gly, Glu423→Gly, Asp439→Gly, and Asp440→Gly were obtained from the catalytic domain 418-QTDGIELYKGSTMKNTFFNANDD-440 by site-directed mutagenesis. The mutant dextranases Q418GDex, D420GDex, E423GDex, and D439GDex had almost no enzymatic activity, while D440GDex had equal enzymatic activity to AoDex. However, at temperatures between 25 and 40 ℃, D440GDex activity increased by 2–3 folds compared to AoDex activity. The optimum pH for D440GDex was 6.5 while that for AoDex was 5.5. Q418, D420, E423, D439 were the key amino acid residues in the catalytic domain of AoDex. Mutation of D440 had a great impact on the enzymatic properties, suggesting it to be not the general base of the AoDex catalytic domain. These findings suggest that AoDex and the GH family 49 share a similar catalytic mechanism, which will provide theoretical support for improving the enzymatic properties of AoDex.

Key words: dextranase, site-directed mutagenesis, catalytic domain, key amino acid residues, structure prediction