Advertisement

Biochemical and kinetic characterization of GH43 β-d-xylosidase/α-l-arabinofuranosidase and GH30 α-l-arabinofuranosidase/β-d-xylosidase from rumen metagenome

  • Jungang Zhou
  • Lei Bao
  • Lei Chang
  • Yufei Zhou
  • Hong LuEmail author
Original Paper

Abstract

The present study focuses on characterization of two hemicellulases, RuXyn1 and RuXyn2, from rumen bacterial metagenome and their capabilities for degradation of xylans. Glycosyl hydrolase (GH) family 43 β-d-xylosidase/α-l-arabinofuranosidase RuXyn1 can hydrolyze p-nitrophenyl-β-d-xylopyranoside (pNPX), p-nitrophenyl-α-l-arabinofuranoside (pNPA), and xylo-oligosaccharide substrates, while GH30 1,5-α-l-arabinofuranosidase/β-d-xylosidase RuXyn2, the first α-l-arabinofuranosidase assigned to this GH family, shows activities towards 1,5-α-l-arabinobiose and pNPX substrates but no activity for pNPA. Kinetic analysis for aryl-glycosides revealed that RuXyn2 had higher catalytic efficiency than RuXyn1 toward pNPX substrate. RuXyn1 shows high synergism with endoxylanase, elevating by 73% the reducing sugars released from brichwood xylans, and converted most intermediate xylo-oligosaccharide hydrolysate into xylose. The high xylose conversion capability of RuXyn1 suggests it has potential applications in enzymatic production of xylose and improvement of hemicellulose saccharification for production of biofuels. RuXyn2 shows no obviously synergistic effect in the endoxylanase-coupled assay for enzymatic saccharification of xylan. Further cosmid DNA sequencing revealed a neighboring putative GH43 α-l-arabinofuranosidase RuAra1 and two putative GH3 β-xylosidase/arabinosidases, RuXyn3 and RuXyn5, downstream of RuXyn2, indicating that this hemicellulase gene cluster may be responsible for production of end-product, xylose and arabinose, from hemicellulose biomass.

Keywords

Metagenome Xylosidase β-d-Xylosidase/α-l-arabinofuranosidase Xylan Synergistic 

Notes

Acknowledgments

This work was supported by Chinese High-tech Research and Development program 2007AA021302 and Sunhy Biology Co., Ltd.

References

  1. 1.
    An D, Dong XZ, Dong ZY (2005) Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses. Anaerobe 11:207–215PubMedCrossRefGoogle Scholar
  2. 2.
    Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56:326–338PubMedCrossRefGoogle Scholar
  3. 3.
    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 72:248–254PubMedCrossRefGoogle Scholar
  4. 4.
    Buck GE, O’Hara LC, Summersgill JT (1992) Rapid simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol 30:1331–1334PubMedGoogle Scholar
  5. 5.
    Coughlan MP, Hazlewood GP (1993) Beta-1, 4-d-xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol Appl Biochem 17:259–289PubMedGoogle Scholar
  6. 6.
    Dehority BA (1991) Effects of microbial synergism on fibre digestion in the rumen. Proc Nutr Soc 50:149–159PubMedCrossRefGoogle Scholar
  7. 7.
    Engel PC (1981) Enzyme kinetics: the steady-state approach, 2nd edn. Chapman and Hall, London, pp 26–36Google Scholar
  8. 8.
    Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, Davies G (1995) Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci USA 92:7090–7094PubMedCrossRefGoogle Scholar
  9. 9.
    Honda Y, Fushinobu S, Hidaka M, Wakagi T, Shoun H, Taniguchi H, Kitaoka M (2008) Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase. Glycobiology 18:325–330PubMedCrossRefGoogle Scholar
  10. 10.
    Huang LN, Hseu TH, Lee YJ (1995) EMBL database submission accession no. U38661. http://www.ebi.ac.uk/cgi-bin/emblfetch?style=html&id=U38661&Submit=Go
  11. 11.
    Jordan DB, Li XL (2007) Variation in relative substrate specificity of bifunctional beta-d-xylosidase/alpha-l-arabinofuranosidase by single site mutations: roles of substrate distortion and recognition. Biochim Biophys Acta 1774:1192–1198PubMedGoogle Scholar
  12. 12.
    Krause DO, Denmana SE, Mackiec RI, Morrisond M, Raea AL, Attwoode GT, McSweeneya CS (2003) Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics. FEMS Microbiol Rev 27:663–693PubMedCrossRefGoogle Scholar
  13. 13.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  14. 14.
    Lee RC, Hrmova M, Burton RA, Lahnstein J, Fincher GB (2003) Bifunctional family 3 glycoside hydrolases from barley with alpha-l-arabinofuranosidase and beta-d-xylosidase activity. Characterization, primary structures, and COOH-terminal processing. J Biol Chem 278:5377–5387PubMedCrossRefGoogle Scholar
  15. 15.
    Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666CrossRefGoogle Scholar
  16. 16.
    Liu JR, Yu B, Lin SH, Cheng KJ, Chen YC (2005) Direct cloning of a xylanase gene from the mixed genomic DNA of rumen fungi and its expression in intestinal Lactobacillus reuteri. FEMS Microbiol Lett 251:233–241PubMedCrossRefGoogle Scholar
  17. 17.
    Mai V, Wiegel J, Lorenz WW (2000) Cloning, sequencing, and characterization of the bifunctional xylosidase/arabinosidase from the anaerobic thermophile Thermoanaerobacter ethanolicus. Gene 247:137–143PubMedCrossRefGoogle Scholar
  18. 18.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  19. 19.
    Nummi M, Perrin JM, Niku-Paavola ML, Enari TM (1985) Measurement of xylanase activity with insoluble substrate. Biochem J 226:611–620Google Scholar
  20. 20.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  21. 21.
    Shallom D, Shoham Y (2003) Microbial hemicellulases. Curr Opin Microbiol 6:219–228PubMedCrossRefGoogle Scholar
  22. 22.
    Shin HY, Lee JH, Lee JY, Han YO, Han MJ, Kim DH (2003) Purification and charaterization of ginsenoside Ra-hydrolyzing beta-d-Xylosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium. Biol Pharm Bull 26:1170–1173PubMedCrossRefGoogle Scholar
  23. 23.
    St John FJ, González JM, Pozharski E (2010) Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. FEBS Lett 584:4435–4441PubMedCrossRefGoogle Scholar
  24. 24.
    Streit WR, Schmitz RA (2004) Metagenomics—the key to the uncultured microbes. Curr Opin Microbiol 7:492–498PubMedCrossRefGoogle Scholar
  25. 25.
    Wagschal K, Heng C, Lee CC, Robertson GH, Orts WJ, Wong DW (2009) Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08. Appl Biochem Biotechnol 155:1–10CrossRefGoogle Scholar
  26. 26.
    Wagschal K, Heng C, Lee CC, Wong DWS (2009) Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture. Appl Microbiol Biotechnol 81:855–863PubMedCrossRefGoogle Scholar
  27. 27.
    Wilkie KCB (1979) The hemicelluloses of grasses and cereals. Adv Carbohydr Chem Biochem 36:215–262CrossRefGoogle Scholar
  28. 28.
    You C, Huang Q, Xue H, Xu Y, Lu H (2010) Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix patriciarum. Biotechnol Bioeng 105:861–870PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2011

Authors and Affiliations

  • Jungang Zhou
    • 1
  • Lei Bao
    • 1
  • Lei Chang
    • 1
  • Yufei Zhou
    • 1
  • Hong Lu
    • 1
    • 2
    Email author
  1. 1.State Key Laboratory of Genetic Engineering, School of Life SciencesFudan UniversityShanghaiChina
  2. 2.Institutes of Biomedical SciencesFudan UniversityShanghaiChina

Personalised recommendations