Applied Microbiology and Biotechnology

, Volume 79, Issue 4, pp 579–587 | Cite as

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

  • Ping Lu
  • Ming-Guang Feng
Biotechnologically Relevant Enzymes and Proteins


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.


Bifunctional 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).


  1. Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 14:529–532CrossRefGoogle Scholar
  2. Arai R, Wriggers W, Nishikawa Y, Nagamune T, Fujisawa T (2004) Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering. Proteins 57:829–838CrossRefGoogle Scholar
  3. Bai Y, Shen WC (2006) Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharm Res 23:2116–2121CrossRefGoogle Scholar
  4. Blazyk JL, Lippard SJ (2004) Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem 279:5630–5640CrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Chang HC, Kaiser CM, Hartl FU, Barral JM (2005) De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria. J Mol Biol 353:397–409CrossRefGoogle Scholar
  7. Crasto CJ, Feng J (2000) LINKER: a program to generate linker sequences for fusion proteins. Protein Eng 13:309–312CrossRefGoogle Scholar
  8. Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258–266CrossRefGoogle Scholar
  9. Doi N, Yanagawa H (1999) Insertional gene fusion technology. FEBS Letters 457:1–4CrossRefGoogle Scholar
  10. 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
  11. Gray NK, Hentze MW (1994) Regulation of protein synthesis by mRNA structure. Mol Biol Rep 19:195–200CrossRefGoogle Scholar
  12. Gustavsson M, Lehtio J, Denman S, Teeri TT, Hult K, Martinelle M (2001) Stable linker peptides for a cellulose-binding domain-lipase fusion protein expression in Pichia pastoris. Protein Eng 14:711–715CrossRefGoogle Scholar
  13. Gutman GA, Hatfield GW (1989) Nonrandom utilization of codon pairs in Escherichia coli. Proc Natl Acad Sci USA 86:3699–3703CrossRefGoogle Scholar
  14. Hoedemaeker FJ, Signorelli T, Johns K, Kuntz DA, Rose DR (1997) A single chain Fv fragment of p-glycoprotein-specific monoclonal antibody C219 design, expression, and crystal structure at 2.4 Å resolution. J Biol Chem 272:29784–29789CrossRefGoogle Scholar
  15. Hong SY, Lee JS, Cho KM, Math RK, Kim YH, Hong SJ, Cho YU, Kim H, Yun HD (2006) Assembling a novel bifunctional cellulose-xylanase from Thermotoga maritima by end-to-end fusion. Biotechnol Lett 28:1857–1862CrossRefGoogle Scholar
  16. Irwin B, Heck JD, Hatfield GW (1995) Codon pair utilization biases influence translational. J Biol Chem, 270:22801–22806CrossRefGoogle Scholar
  17. Karnielya S, Rayznera A, Sass E, Pines O (2006) a-Complementation as a probe for dual localization of mitochondrial proteins. Exp Cell Res 312:3835–3846CrossRefGoogle Scholar
  18. Levasseur A, Navarro D, Punt PJ, Belaich JP, Asther M, Record E (2005) Construction of engineered bifunctional enzymes and their overproduction in Aspergillus niger for improved enzymatic tools to degrade agricultural by-products. Appl Environ Microbiol 71:8132–8140CrossRefGoogle Scholar
  19. Lois AC, Barbara AS, John RM, Janet EP, Sharon LA, Andrew B, Glenn FP, Houston LL (1998) Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein. Int J Cancer 78:106–111CrossRefGoogle Scholar
  20. Longland AC, Theodorou MK, Sanderson R, Lister SJ, Powell CJ, Morris P (1995) Non-starch polysaccharide composition and in vitro fermentability of tropical forage legumes varying in phenolic content. Anim Feed Sci Technol 55:161–177CrossRefGoogle Scholar
  21. Lu P, Feng MG, Li WF, Hu CX (2006) Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced β-glucanase and xylanase expressed in Escherichia coli. FEMS Microbiol Lett 261:224–230CrossRefGoogle Scholar
  22. Maeda Y, Ueda H, Kazami J, Kawano G, Suzuki E, Nagamune T (1997) Engineering of functional chimeric protein G-Vargula luciferase. Anal Biochem 249:147–152CrossRefGoogle Scholar
  23. Mathlouthi N, Saulnier L, Quemener B, Larbier M (2002) Xylanase, b-glucanase, and other side enzymatic activities have greater effects on the viscosity of several feedstuffs than xylanase and b-glucanase used alone or in combination. J Agric Food Chem 50:5121–5127CrossRefGoogle Scholar
  24. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  25. Moura G, Pinheiro M, Silva R, Miranda I, Afreixo V, Dias G, Freitas A, Oliveira JL, Santos MAS (2005) Comparative context analysis of codon pairs on an ORFeome scale. Genome Biol 6:R28CrossRefGoogle Scholar
  26. Petriz J, Gottesman MM, Aran JM (2004) An MDR-EGFP gene fusion allows for direct cellular localization, function and stability assessment of p-glycoprotein. Curr Drug Deliv 1:43–56CrossRefGoogle Scholar
  27. 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
  28. Remy I, Wilson IA, Stephen WM (1999) Erythropoietin receptor activation by a ligand-induced conformation change. Science 283:990–993CrossRefGoogle Scholar
  29. 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
  30. 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
  31. Seo HS, Koo YJ, Lim JY, Song JT, Kim CH, Kim JK, Lee JS, Choi YD (2000) Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Appl Environ Microbiol 66:2484–2490CrossRefGoogle Scholar
  32. 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
  33. Trinh R, Gurbaxani B, Morrison SL, Seyfzadeh M (2004) Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression. Mol Immunol 40:717–722CrossRefGoogle Scholar
  34. Trujillo M, Duncan R, Santi DV (1997) Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme. Protein Eng 10:567–573CrossRefGoogle Scholar
  35. Volkel T, Korn T, Bach M, Muller R, Kontermann RE (2001) Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. Protein Eng 14:815–823CrossRefGoogle Scholar
  36. Wang WWS, Das D, McQuarrie SA, Suresh MR (2007) Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein. Eur J Pharm Biopharm 65:398–405CrossRefGoogle Scholar
  37. Werner S, Marillonnet S, Hause G, Klimyuk V, Gleba Y (2006) Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc Natl Acad Sci USA 103:17678–17683CrossRefGoogle Scholar
  38. 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
  39. Xue F, Gu Z, Feng J (2004) LINKER: a web server to generate peptide sequences with extended conformation. Nucleic Acids Res 32:562–565CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Institute of Microbiology, College of Life SciencesZhejiang UniversityHangzhouPeople’s Republic of China

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