Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina

  • 569 Accesses

  • 60 Citations


A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25°C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml−1. The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40°C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The K m and V max values for birchwood xylan are 2.06 mg ml−1 and 0.49 mmol min−1mg−1, respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

  2. Apel PC, Panaccione DG, Holden FR, Walton JD (1993) Cloning and targeted gene disruption of XYL1, a beta 1,4-xylanase gene from the maize pathogen Cochliobolus carbonum. Mol Plant Microbe Interact 6:467–473

  3. Araki T, Tani S, Maeda K, Hashikawa S, Nakagawa H, Morishita T (1999) Purification and characterization of beta-1, 3-xylanase from a marine bacterium, Vibrio sp. XY-214. Biosci Biotechnol Biochem 63:2017–2019

  4. Atkins SD, Clark IM, Sosnowska D, Hirsch PR, Kerry BR (2003) Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selectRive media, and baiting. Appl Environ Microbiol 69:4788–4793

  5. Badal CS (2002) Production, purification and properties of xylanase from a newly isolated Fusarium proliferatum. Process Biochem 37:1279–1284

  6. Biswas MJ, Jana SC, Mishra AK, Nanda G (1990) Production, purification and characterization of xylanase from hyperxylanolytic mutant of Aspergillus ochraceus. Biotechnol Bioeng 35:244–251

  7. Chandrakant P, Bisaria VS (1998) Simultaneous bioconversion of cellulose and hemicellulose to ethanol. Crit Rev Biotechnol 18:295–331

  8. Degefu Y, Paulin L, Lubeck PS (2001) Cloning, sequencing and expression of a xylanase gene from the maize pathogen Helminthosporium turcicum Pass. Eur J Plant Pathol 107:457–465

  9. Fialho MB, Carmona EC (2004) Purification and characterization of xylanase from Aspergillus giganteus. Folia Microbiol 49:13–18

  10. Gorbacheva IV, Rodionova NA (1997) Studies on xylan degrading enzymes. I. Purification and characterization of endo-1,4-β-xylanase from Aspergillus niger str. 14. Biochim Biophys Acta 484:79–93

  11. Gupta S, Bhushan B, Hoondal GS (2000) Isolation, purification and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J Appl Microbiol 88:325–334

  12. Heinz P, Walter S (1995) Induction of endo-xylanase in the fungus Thermomyces lanuginosus. Enzyme Microb Technol 17:114–118

  13. Henrissat B, Teeri TT, Warren RAJ (1998) A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. FEBS Lett 425:352–354

  14. Huang YH, Long ZE, Li SZ, Pei LL, Wang SX, Yu M (2002) Isolation and identification of prolific microorganism in xylanase. Journal of Jiangxi Normal University (Natural science ed.) (Chinese) 26:245–248

  15. Iefuji H, Chino M, Kato M, Iimura Y, (1996) Acid xylanase from yeast Cryptococcus sp. S-2: purification, characterization, cloning, and sequencing. Biosci Biotechnol Biochem 60:1331–1338

  16. Irena R, Jacek P, Stanislaw B (2006) Isolation and properties of Aspergillus niger IBT-90 xylanase for bakery. Appl Microbiol Biotechnol 69:665–671

  17. Jeffrey AR, Garret BN, Katrina LH (1991) DNA isolation from small tissue samples using salt and spermine. Nucleic Acids Res 19:6053

  18. John M, Schmidt B, Schmidt J (1979) Purification and some properties of five endo-1,4-β-xylanases and β-xylosidases produced by strain of A. niger. Can J Biochem 157:125–134

  19. Krisana A, Rutchadaporn S, Jarupan G, Lily E, Sutipa T, Kanyawim K (2005) Endo-1,4-beta-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation. J Biochem Mol Biol 38:17–23

  20. Liu R, Qu Y, Jiang Y, Gao P (1999) Purification and characterization of alkaline xylanases from Pseudomonas G6-2. Wei Sheng Wu Xue Bao 39:132–136

  21. Marais JP, De Wit JL, Quicke GV (1966) A critical examination of the Nelson–Somogyi method for the determination of reducing sugars. Anal Biochem 15:373–381

  22. Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of carboxymethyl cellulase activity. Anal Biochem 2:127–132

  23. Morris DD, Gibbs MD, Chin CW, Koh MH, Wong KK, Allison RW, Nelson PJ, Bergquist PL (1998) Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp. Appl Environ Microbiol 64:1759–1765

  24. Morris DD, Gibbs MD, Ford M, Thomas J, Bergquist PL (1999) Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. Rt69B.1. Extremophiles 3:103–111

  25. Neeta K, Abhay S, Mala R (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411–456

  26. Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591

  27. Rogalski J, Oleszek M, Tokarzewska-Zadora J (2001) Purification and characterization of two endo-1,4-β-xylanases and β-xylosidases from Phelbia radiata. Acta Microbiol Pol 50:117–128

  28. Sapag A, Wouters J, Lambert C, de Ioannes P, Eyzaguirre J, Depiereux E (2002) The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships. J Biotechnol 95:109–131

  29. Schlacher A, Holzmann K, Hayn M, Steiner W, Schwab H (1996) Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. J Biotechnol 49:211–218

  30. Yamaura I, Koga T, Matsumoto T, Kato T (1997) Purification and some properties of endo-1,4-beta-D-xylanase from a fresh-water mollusc, Pomacea insularus (de Ordigny). Biosci Biotechnol Biochem 61:615–620

Download references


This study was supported by the National Nature Science Foundation (30600014), Hubei Province Nature Science Foundation (2005ABA201) and the Open Project of State Key Laboratory of Agriculture Microorganism (AML-0303).

Author information

Correspondence to Li Xin Ma or Xian En Zhang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhang, G.M., Huang, J., Huang, G.R. et al. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina . Appl Microbiol Biotechnol 74, 339–346 (2007). https://doi.org/10.1007/s00253-006-0648-3

Download citation


  • Xylanase
  • Plectosphaerella cucumerina
  • Genome-walking PCR
  • Heterologous expression
  • Pichia pastoris