DOI QR코드

DOI QR Code

Characterization of Heterologously Expressed Acetyl Xylan Esterase1 Isolated from the Anaerobic Rumen Fungus Neocallimastix frontalis PMA02

  • Kwon, Mi (InfoBoss Incorporation) ;
  • Song, Jaeyong (Department of Agricultural Science, Korea National Open University) ;
  • Park, Hong-Seog (GnCBio Incorporation) ;
  • Park, Hyunjin (Department of Agricultural Science, Korea National Open University) ;
  • Chang, Jongsoo (Department of Agricultural Science, Korea National Open University)
  • Received : 2016.04.27
  • Accepted : 2016.06.09
  • Published : 2016.11.01

Abstract

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

Keywords

Acetyl Xylan Esterase;Neocallimastix frontalis;Hemicellulose;Anaerobic Rumen Fungus

Acknowledgement

Supported by : Korea National Open University

References

  1. Altaner, C., B. Saake, M. Tenkanen, J. Eyzaguirre, C. B. Faulds, P. Biely, L. Viikari, M. Siika-aho, and J. Puls. 2003. Regioselective deacetylation of cellulose acetates by acetyl xylan esterases of different CE-families. J. Biotechnol. 105:95-104. https://doi.org/10.1016/S0168-1656(03)00187-1
  2. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3:286-290. https://doi.org/10.1016/0167-7799(85)90004-6
  3. Biely, P., M. Cziszarova, I. Uhliarikova, J. W. Agger, X. L. Li, V. G. H. Eijsink, and B. Westereng. 2013. Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase. Biochim. Biophys. Acta 1830:5075-5086. https://doi.org/10.1016/j.bbagen.2013.07.018
  4. Blum, D. L., X. L. Li, H. Chen, and L. G. Ljungdahl. 1999. Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Appl. Environ. Microbiol. 65:3990-3995.
  5. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  6. Coutinho, P. M. and B. Henrissat. 1999. Carbohydrate-active enzymes: an integrated database approach. In: Recent Advances in Carbohydrate Bioengineering (Eds. H. J. Gilbert, G. J. Davies, B. Henrissat, and B. Svensson). The Royal Society of Chemistry, Cambridge, UK. pp. 3-12.
  7. Cybinski, D. H., I. Layton, J. B. Lowry, and B. P. Dalrymple.1999. An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans. Appl. Microbiol. Biotechnol. 52:221-225. https://doi.org/10.1007/s002530051512
  8. Dalrymple, B. P., D. H. Cybinski, I. Layton, C. S. McSweeney, G. P. Xue, Y. J. Swadling, and J. B. Lowry.1997. Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology 143:2605-2614. https://doi.org/10.1099/00221287-143-8-2605
  9. Drzewiecki, K., A. Angelov, M. Ballschmiter, K. J. Tiefenbach, R. Sterner, and W. Liebl. 2010. Hyperthermostable acetyl xylan esterase. Microb. Biotechnol. 3:84-92. https://doi.org/10.1111/j.1751-7915.2009.00150.x
  10. Fujino, Y., K. Ogata, T. Nagamine, and K. Ushida. 1998. Cloning, sequencing, and expression of an endoglucanase gene from the rumen anaerobic fungus Neocallimastix frontalis MCH3. Biosci. Biotechnol. Biochem. 62:1795-1798. https://doi.org/10.1271/bbb.62.1795
  11. Garcia-Vallve, S., A. Romeu, and J. Palau. 2000. Horizontal gene transfer of glycosyl hydrolases of the rumen fungi. Mol. Biol. Evol. 17:352-361. https://doi.org/10.1093/oxfordjournals.molbev.a026315
  12. Garcia-Campayo, V. and T. M. Wood. 1993. Purification and characterisation of a beta-D-xylosidase from the anaerobic rumen fungus Neocallimastix frontalis. Carbohydr. Res. 242:229-245. https://doi.org/10.1016/0008-6215(93)80037-F
  13. Gasteiger, E., C. Hoogland, A. Gattiker, S. E. Duvaud, M. R. Wilkins, R. D. Appel, and A. Bairoch. 2005. Protein identification and analysis tools on the ExPASy server. In: The Proteomics Protocols Handbook (Ed. J. M. Walker). Humana Press, Totowa, NJ, USA. pp. 571-607.
  14. Guruprasad, K, B. V. B. Reddy, and M. W. Pandit. 1990. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng. 4:155-161. https://doi.org/10.1093/protein/4.2.155
  15. Gutierrez, R., E. Cederlund, L. Hjelmqvist, A. Peirano, F. Herrera, D. Ghosh, W. Duax, H. Jornvall, and J. Eyzaguirre. 1998. Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain. FEBS Lett. 423:35-38. https://doi.org/10.1016/S0014-5793(98)00055-6
  16. Ho, Y. W., N. Abdullah, and S. Jalaludin. 1988. Penetrating structures of anaerobic rumen fungi in cattle and swamp buffalo. J. Gen. Microbiol. 134:177-181.
  17. Ikai, A. 1980. Thermostability and aliphatic index of globular proteins. J. Biochem. 88:1895-1898.
  18. Johnson, K. G., J. D. Fontana, and C. R. MacKenzie. 1988. Measurement of acetylxylan esterase in Streptomyces. Meth. Enzymol. 160:551-560. https://doi.org/10.1016/0076-6879(88)60168-6
  19. Jue, C. K. and P. N. Lipke. 1985. Determination of reducing sugars in the nanomole range with tetrazolium blue. J. Biochem. Biophys. Methods 11:109-115. https://doi.org/10.1016/0165-022X(85)90046-6
  20. Kwon, M., J. Song, J. K. Ha, H. Park, and J. Chang. 2009. Analysis of functional genes in carbohydrate metabolic pathway of anaerobic rumen fungus Neocallimastix frontalis PMA02. Asian Australas. J. Anim. Sci. 22:1555-1565. https://doi.org/10.5713/ajas.2009.80371
  21. Li, X. L. and R. E. Calza. 1991. Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188. Appl. Environ. Microbiol. 57:3331-3336.
  22. McDermid, K. P., C. R. Mackenzie, and C. W. Forsberg. 1990. Esterase activities of Fibrobacter succinogenes subsp. succinogenes S85. Appl. Environ. Microbiol. 56:127-132.
  23. Mountfort, D. O. and R. A. Asher. 1985. Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 49:1314-1322.
  24. Orpin, C. G. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J. Gen. Microbiol. 91:249-262. https://doi.org/10.1099/00221287-91-2-249
  25. Orpin, C. G. 1981. Isolation of cellulolytic Phycomycete fungi from the caecum of the horse. J. Gen. Microbiol. 123:287-296.
  26. Selig, M. J., E. P. Knoshaug, W. S. Adney, M. E. Himmel, and S. R. Decker. 2008. Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities. Bioresour. Technol. 99:4997-5005. https://doi.org/10.1016/j.biortech.2007.09.064
  27. Tenkanen, M. 1998. Action of Trichoderma reesei and Aspergillus oryzae esterases in the deacetylation of hemicelluloses. Biotechnol. Appl. Biochem. 27:19-24. https://doi.org/10.1111/j.1470-8744.1998.tb01370.x
  28. Thomson, J. A. 1993. Molecular biology of xylan degradation. FEMS Microbiol. Rev. 104:65-82. https://doi.org/10.1111/j.1574-6968.1993.tb05864.x
  29. Wood, T. M., C. A. Wilson, and S. I. McCrae. 1995. The cellulase system of the anaerobic rumen fungus Neocallimastix frontalis: studies on the properties of fractions rich in endo-(1$\rightarrow$4)-beta-D-glucanase activity. Appl. Microbiol. Biotechnol. 44:177-184. https://doi.org/10.1007/BF00164499

Cited by

  1. strain C1A vol.6, pp.2167-8359, 2018, https://doi.org/10.7717/peerj.4276