Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers.

The flexible peptides (GGGGS)n (n < or = 3), the alpha-helical peptides (EAAAK)n (n < or = 3) and two other peptides were used as linkers to construct bifunctional fusions of beta-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both beta-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304-426% and 82-143% of the parental ones, respectively. The peptide linker (GGGGS)(2) resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)(3) also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide linkers to separate the two moieties at a reasonable distance for beneficial interaction.