High secretory production of an alkaliphilic actinomycete xylanase and functional roles of some important residues.

Using native signal peptide, an alkaliphilic actinomycete xylanase XynK was overexpressed in Escherichia coli and secreted into the culture medium completely. At its optimum catalytic temperature of 55 °C, the cellulose-free xylanase exhibits high activity and stability at pH 7.0-11.0. In comparison with the well-studied actinomycete xylanase from Streptomyces lividans, as an alkaliphilic xylanase, XynK exhibited different biochemical and catalytic characteristics. With the aid of site-directed mutagenesis, some residues were demonstrated to be important to the activity, stability, or substrate binding of the enzyme. The pH stability of mutants H131S and W135A both decreased obviously under high pH values. Combined with their K(m) parameters and homology model analysis, His131 was proposed to be important to both substrate binding and enzyme catalyzing, whereas Trp135 significantly influenced enzyme stability. Good stability under alkaline condition, as well as high secretory expression implies good potentials of the alkaline xylanase in various industrial applications. In addition, results from site-directed mutagenesis provide useful information for further pH stability mechanisms investigation.