Engineering the substrate specificity of Alcaligenesd-aminoacylase useful for the production of d-amino acids by optical resolution

d-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of d-amino acid from the racemic mixture of N-acyl-dl-amino acids. However, its substrate specificity is biased toward certain N-acyl-d-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other d-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (kcat/Km) of mutant F191W toward N-acetyl-d-Trp and N-acetyl-d-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (kcat/Km) of mutant L298A toward N-acetyl-d-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of d-Trp which is an important building block of some therapeutic drugs.