Construction and characterization of a bifunctional fusion enzyme ofBacillus-sourced β-glucanase and xylanase expressed inEscherichia coli

Abstract

A chimeric gene, Glu-Xyl, encoding Bacillus amyloliquefaciens glucanase (Glu, 24.4 kDa) and Bacillus subtilis xylanase (Xyl, 21.2 kDa), was constructed via end-to-end fusion and expressed successfully in Escherichia coli. The purified fusion protein (46.1 kDa) exhibited both glucanase and xylanase activities. Compared with parental enzymes, the Glu moiety was characterized by kinetic parameters of decreased K(m) (0.66-fold) and increased K(cat) (2.75-fold), whereas the Xyl moiety had an increased K(m) (1.37-fold) and decreased K(cat) (0.79-fold). These indicate a 3.15-fold net increase and a 31% decrease in catalytic efficiency (K(cat)/K(m)) of the Glu and Xyl moieties. Activities and stabilities of both moieties at 40-90 degrees C or pH 3.0-10.0 were compared with those of the parental enzymes. Despite some variations, common optima were 40 degrees C and pH 9.0 for the Glu moiety and parent, and 50-60 degrees C and pH 9.0 for the Xyl counterparts. Thus, the fusion enzyme Glu-Xyl was bifunctional, with greatly enhanced glucanase activity associated with a decrease in xylanase activity.

In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

“…In the study of Lu et al (2006), Glu moiety temperature stability decreased in comparison with the parental protein. Such results were consistent with ours.…”

“…Activity varia- tions of fusion enzymes are likely attributed to unpredicted, complicated folding of each protein domain (Seo et al 2000). Anyway, Lu et al (2006) had developed a significant exploration of bifunctional enzyme of β-1,3-1,4-glucanase and β-xylanase.…”

“…At the same time, in view of the results by Lu et al (2006), different linkers can be projected for inserting into β-xylanase and β-1,3-1,4-glucanase. Thus, linker 1 in this study was designed considering that alanine has a smaller molecular structure than most other amino acids.…”

“…Two natural bifunctional enzymes with xylanase and β-1,3-1,4-glucanase activities were reported by Flint et al (1993) and Chen et al (2006), respectively. A bifunctional enzyme of β-1,3-1,4-glucanase and β-xylanase, in which a chimeric gene, Glu-Xyl, encoding Bacillus amyloliquefaciens glucanase and Bacillus subtilis xylanase, was constructed via endto-end fusion and expressed successfully in E. coli, and the purified fusion protein exhibited greatly enhanced β-1,3-1,4-glucanase activity associated with a decrease in β-xylanase activity (Lu et al 2006). Activity varia- tions of fusion enzymes are likely attributed to unpredicted, complicated folding of each protein domain (Seo et al 2000).…”

“…In previous study, Lu et al (2006) constructed a bifunctional enzyme via end to end fusion, which expressed successfully in Escherichia coli, and the purified fusion protein exhibited with greatly enhanced β-1,3-1,4-glucanase activity associated with a decrease in β-xylanase activity. Studies showed that proper linkages using linker peptides may help the moieties to function independently for optimization of their features (Carlsson et al 1996;Gustavsson et al 2001).…”