Abstract
Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Kn higher kcat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- BMGY:
-
buffered glycerol-complex medium
- BMMY:
-
buffered methanol-complex medium
References
Bai Y. & Shen W.C. 2006. Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharm. Res. 23: 2116–2121.
Barrera M., Cervantes M., Sauer W.C., Araiza A.B., Torrentera N. & Cervantes M. 2004. Ileal amino acid digestibility and performance of growing pigs fed diets supplemented with xylanase. J. Anim. Sci. 82: 1997–2003.
Bülow L. & Mosbach K. 1991. Multienzyme systems obtained by gene fusion. Trends Biotechnol. 9: 226–231.
Cantarel B.L., Coutinho P.M., Rancurel C., Bernard T., Lombard V. & Henrissat B. 2009. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37(Database issue): D233–D238.
Cao Y.H., Qiao J.Y., Li Y.H. & Lu W.Q. 2007. De novo synthesis, constitutive expression of Aspergillus sulphureus β-xylanase gene in Pichia pastoris and partial enzymic characterization. Appl. Microbiol. Biotechnol. 76: 579–585.
Chen H., Yan X., Liu X., Wang M., Huang H., Jia X. & Wang J. 2006. Purification and characterization of novel bifunctional xylanase, XynIII, isolated from Aspergillus niger A-25. J. Microbiol. Biotechnol. 16: 1132–1138.
Flint H.J., Martin J., McPherson C.A., Daniel A.S. & Zhang J.X. 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: 2943–2951.
Grossmann M., Wong R., Szkudlinski M.W. & Weintraub B.D. 1997. Human thyroid-stimulating hormone (hTSH) subunit gene fusion produces hTSH with increased stability and serum half-life and compensates for mutagenesis-induced defects in subunit association. J. Biol. Chem. 272: 21312–21316.
Gustavsson M., Lehtiö J., Denman S., Teeri T.T., Hult K. & Martinelle M. 2001. SStable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris. Protein Eng. 114: 711–715.
Horton R.M., Hunt H.D., Ho S.N., Pullen J.K. & Pease L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68.
Li S., Sauer W.C., Huang S.X. & Gabert V.M. 1996. Effect of β-glucanase supplementation to hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, β-glucans, and amino acids in young pigs. J. Anim. Sci. 74: 1649–1656.
Lu P., Feng M.G., Li W.F. & Hu C.X. 2006. Construction and characterization of a bifunctional fusion enzyme of Bacillussourced β-glucanase and xylanase expressed in Escherichia coli. FEMS Microbiol. Lett. 261: 224–230.
Mathlouthi N., Mallet S., Saulnier L., Quemener B. & Larbier M. 2002. Effects of xylanase and β-glucanase addition on performance, nutrient digestibility, and physico-chemical conditions in the small intestine contents and caecal microflora of broiler chickens fed a wheat and barley-based diet. Anim. Res. 51: 395–406.
Qiao J.Y., Dong B., Li Y.H., Zhang B. & Cao Y.H. 2009. Cloning of a β-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli. Appl. Biochem. Biotechnol. 152: 334–342.
Qiao J.Y., Zhang B., Chen Y.Q. & Cao Y.H. 2000. Codon optimization, expression and characterization of Bacillus subtilis MA139 β-1,3-1,4-glucanase in Pichia pastoris. Biologia 65: 191–196.
Seo H.S., Koo Y.J., Lim J.Y., Song J.T., Kim C.H., Kim J.K., Lee J.S. & Choi Y.D. 2000. Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Appl. Environ. Microbiol. 66: 2484–2490.
St John F.J., González J.M. & Pozharski E. 2010. Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. FEBS Letters 584: 4435–4441.
Wu Y.B., Ravidran V., Thomas D.G., Birtles M.J. & Hendricks W.H. 2004. Influence of method of whole wheat inclusion and xylanase supplementation on the performance, apparent metabolisable energy, digestive tract measurements and gut morphology of broilers. Brit. Poultry Sci. 45: 385–394.
Zhao X., Huo K.K. & Li Y.Y. 2000. Synonymous codon usage in Pichia pastoris. Chin. J. Biotechnol. 16: 308–311.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Qiao, J., Cao, Y. In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris . Biologia 67, 649–653 (2012). https://doi.org/10.2478/s11756-012-0056-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.2478/s11756-012-0056-3