Semi-rational site-directed mutagenesis of phyI1s from Aspergillus niger 113 at two residue to improve its phytase activity.

Through alignment of amino acid sequences among different phytases, we found that the amino acid at residues 53 and 91 vary broadly. To prove that the amino acid at residues 53 and 91 were related to phytase specific activity, two single mutant phyI1s Q53R and K91D were obtained by site-directed mutagenesis strategy. None of the single amino acid residues in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants (Q53R and K91D) indicated that the mutants were attributed to 2.2- and 1.5-fold increased specific activity, and a 1.47- and 1.16-fold increased affinity for sodium phytate. In addition, the overall catalytic efficiency (k(cat)/K(m)) of the two mutants was improved 4.08- and 2.84-fold compared to that of the wild type. Such mutants will be instrumental for the structure-function study of the enzyme and for industrial application.