title: Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced beta-glucanase and xylanase expressed in Escherichia 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.
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Lu
authors.forename: Ming-Guang
authors.initials: MG
authors.lastname: Feng
authors.forename: Wei-Fen
authors.initials: WF
authors.lastname: Li
authors.forename: Chun-Xia
authors.initials: CX
authors.lastname: Hu
mesh_terms: D019677:Artificial Gene Fusion; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014990:Xylans / Q000378:metabolism; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014990:Xylans; D047071:beta-Glucans; D006026:Glycoside Hydrolases