Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers
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The flexible peptides (GGGGS)n (n ≤ 3), the α-helical peptides (EAAAK)n (n ≤ 3) and two other peptides were used as linkers to construct bifunctional fusions of β-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 β-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.
KeywordsBifunctional fusion enzyme β-glucanase Xylanase Peptide linker optimization
Chang HC (Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany) is thanked for providing the nucleotide sequences of α-helical peptide linkers. This study was supported jointly by the Ministry of Science and Technology of China (2007DFA3100), the Natural Science Foundation of China (30571250), the Ministry of Education of China (IRT0535), and Zhejiang R&D Program (2007C12035).
- Flint HJ, Martin J, McPherson CA, Daniel AS, Zhang JX (1993) A bifunctional enzyme, with separate xylanase and β(1, 3-1, 4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens. J Bacteriol 175:2943–2951Google Scholar
- Morag E, Bayer EA, Lamed R (1990) Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes. J Bacteriol 172:6098–6105Google Scholar
- Rosenblum MG, Cheung LH, Liu Y, Marks JW (2003) Design, expression, purification, and characterization, in vitro and in vivo, of an antimelanoma single-chain Fv antibody fused to the toxin gelonin. Cancer Res 63:3995–4002Google Scholar
- Salobir J (1998) Effect of xylanase alone and in combination with β-glucanase on energy utilization, nutrient utilization and intestinal viscosity of broilers fed diets based on two wheat samples. Arch Gefluegelkund 62:209–213Google Scholar
- Shan D, Press OW, Tsu TT, Hayden MS, Ledbetter JA (1999) Characterization of scFv-Ig constructs generated from theAnti-CD20 mAb 1F5 using linker peptides of varying lengths. J Immunol 162:6589–6595Google Scholar
- Xue GP, Goblus KS, Orpin CG (1992) A novel polysaccharide hydrolase cNAD (celD) from Neocallimastix pareiciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities. J Gen Microbiol 138:2397–2403Google Scholar