The hydrolytic activity of a thermophilic cyclomaltodextrinase (CMD) from Anoxybacillus flavithermus ZNU-NGA and a representative single mutant were investigated against soluble substrates including α-, β- and γ-cyclomaltodestrines (CDs). Based on the occurrence of arginine (Arg) at position 403 in some homologue proteins, His403 in Wild-type (WT) CMD was replaced with Arg (H403R variant) with site-directed mutagenesis procedures. According to bioinformatics data, Arg403 in mutant protein is located near Glu357 as one of the catalytic residues in a manner that they are able to create a medium-range attractive electrostatistic interaction. Structural studies by Far UV-CD showed that this mutation is accompanied by conversion of a small fraction of α-helix to β-form structure. Fluorescence data reveals that, the hydrophobic regions at the surface of protein, as the binding sites for ANS (8-Anilinonaphthalene-1-sulfonic acid) increase in mutant protein, demonstrating relative inflation of H403R variant compared with WT protein. However, the polarity of microenvironment around chromophores did not change upon mutation. Activity measurement in different ranges of pH and temperatures showed that the optimum values of pH and temperature in mutant enzyme is the same as WT enzyme, however; the activity at optimum points increased in H403R variant. It was also revealed that the H403R variant had slightly improved catalytic efficiency for γ-CD. The same value of activation parameters for both protein variants indicates that mutation does not alter the mechanism of catalysis during enzyme-substrate formation.
Keywords: Catalytic efficiency; Cyclomaltodestrine; Cyclomaltodextrinase; Mutant; Spectroscopy; Substrate specificity.
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