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

Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241


Although a lot of xylanases are studied, only a few xylanases from marine microorganisms have been reported. A new xylanase gene, xynA, was cloned from marine bacterium Glaciecola mesophila KMM 241. Gene xynA contains 1,272 bp and encodes a 423-amino acid xylanase precursor. The recombinant xylanase, XynA, expressed in Escherichia coli BL21 is a monomer with a molecular mass of 43 kDa. Among the characterized xylanases, XynA shares the highest identity (46%) to the xylanase from Flavobacterium sp. strain MSY2. The optimum pH and temperature for XynA is 7.0 and 30 °C. XynA retains 23% activity and 27% catalytic efficiency at 4 °C. XynA has low thermostability, remaining 20% activity after 60-min incubation at 30 °C. Its apparent melting temperature (T m) is 44.5 °C. These results indicate that XynA is a cold-active xylanase. XynA shows a high level of salt-tolerance, with the highest activity at 0.5 M NaCl and retaining 90% activity in 2.5 M NaCl. It may be the first salt-tolerant xylanase reported. XynA is a strict endo-β-1,4-xylanase with a demand of at least four sugar moieties for effective cleavage. It efficiently hydrolyzes xylo-oligosaccharides and xylan into xylobiose and xylotriose without producing xylose, suggesting its potential in xylo-oligosaccharides production.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Akila G, Chandra TS (2003) A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase. FEMS Microbiol Lett 219:63–67

  2. Ali MK, Rudolph FB, Bennett GN (2004) Thermostable xylanase 10B from Clostridium acetobutylicum ATCC824. J Ind Microbiol Biotechnol 31:229–234

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

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

  5. Biely P, Vrsanská M, Tenkanen M, Kluepfel D (1997) Endo-β-1, 4-xylanase families: differences in catalytic properties. J Biotechnol 57:151–166

  6. Blanco J, Coque JJR, Velasco J, Martín JF (1997) Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1, 4-xylanase of Thermomonospora alba ULJB1 with cellulose-binding ability. Appl Microbiol Biotechnol 48:208–217

  7. 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 72:248–254

  8. Bradner JR, Sidhu RK, Gillings M, Nevalainen KM (1999) Hemicellulase activity of antarctic microfungi. J Appl Microbiol 87:366–370

  9. Collins T, Meuwis MA, Stals I, Claeyssens M, Feller G, Gerday C (2002) A novel family 8 xylanase: functional and physico-chemical characterization. J Biol Chem 277:35133–35139

  10. Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23

  11. Collins T, Hoyoux A, Dutron A, Georis J, Genot B, Dauvrin T, Arnaut F, Gerday C, Feller G (2006) Use of glycoside hydrolase family 8 xylanases in baking. J Cereal Sci 43:79–84

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

  13. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, Warren RA (1991) Domains in microbial 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev 55:303–315

  14. Gruppen H, Hamer RJ, Voragen AGJ (1992) Water-unextractable cell wall material from wheat flour. 2. Fractionation of alkali-extracted polymers and comparison with waterextractable arabinoxylans. J Cereal Sci 16:53–67

  15. 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

  16. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316

  17. Hou YH, Wang TH, Long H, Zhu HY (2006) Novel cold-adaptive Penicillium strain FS010 secreting thermo-labile xylanase isolated from Yellow Sea. Acta Biochim Biophys Sin 38(2):142–149

  18. Hu Y, Zhang G, Li A, Chen J, Ma L (2008) Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach. Appl Microbiol Biotechnol 80:823–830

  19. Humphry DR, George A, Black GW, Cummings SP (2001) Flavobacterium frigidarium sp. nov., an aerobic, psychrophilic, xylanolytic and laminarinolytic bacterium from Antarctica. Int J Syst Evol Microbiol 51:1235–1243

  20. Inglis GD, Popp AP, Selinger LB, Kawchuk LM, Gaudet DA, McAllister TA (2000) Production of cellulases and xylanases by low-temperature basidiomycetes. Can J Microbiol 46:860–865

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

  22. Kormelink FJM, Voragen AG (1993) Degradation of different [(glucurono) arabino] xylans by a combination of purified xylan-degrading enzymes. Appl Microbiol Biotechnol 38:688–695

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

  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  25. Lee CC, Smith M, Kibblewhite-Accinelli RE, Williams TG, Wagschal K, Robertson GH, Wong DW (2006a) Isolation and characterization of a cold-active xylanase enzyme from Flavobacterium sp. Curr Microbiol 52:112–116

  26. Lee CC, Kibblewhite-Accinelli RE, Wagschal K, Robertson GH, Wong DW (2006b) Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library. Extremophiles 10:295–300

  27. Li N, Meng K, Wang Y, Shi P, Luo H, Bai Y, Yang P, Yao B (2008) Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9. Appl Microbiol Biotechnol 80:231–240

  28. 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

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

  30. Morosoli R, Bertrand JL, Mondou F, Shareck F, Kluepfel D (1986) Purification and properties of a xylanase from Streptomyces lividans. Biochem J 239:587–592

  31. Petrescu I, Lamotte-Brasseur J, Chessa JP, Ntarima P, Claeyssens M, Devreese B, Marino G, Gerday C (2000) Xylanase from the psychrophilic yeast Cryptococcus adeliae. Extremophiles 4:137–144

  32. Romanenko LA, Zhukova NV, Rohde M, Lysenko AM, Mikhailov VV, Stackebrandt E (2003) Glaciecola mesophila sp. nov., a novel marine agar-digesting bacterium. Int J Syst Evol Microbiol 53:647–651

  33. Ruiz-Arribas A, Fernández-Abalos JM, Sánchez P, Garda AL, Santamariá RI (1995) Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8. Appl Environ Microbiol 61:2414–2419

  34. Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629

  35. Turkiewiz M, Kalinowska H, Zielinska M, Bielecki S (2000) Purification and characterisation of two endo-1, 4-xylanases from Antarctic krill, Euphasia superba Dana. Comp Biol Physiol Part B 127:325–335

  36. Wu S, Liu B, Zhang X (2006) Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific. Appl Microbiol Biotechnol 72:1210–1216

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

  38. Yuan KP, Vrijmoed LL, Feng MG (2005) Survey of coastal mangrove fungi for xylanase production and optimized culture and assay conditions. Wei Sheng Wu Xue Bao 45(1):91–96

  39. Zhang G, Huang J, Huang G, Ma L, Zhang X (2007) Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Appl Microbiol Biotechnol 74:339–346

Download references


The work was supported by Hi-Tech Research and Development program of China (2007AA091504, 2007AA091903), COMRA Program (DYXM-115-02-2-6), and Specialized Research Fund for the Doctoral Program of Higher Education (20060422053).

Author information

Correspondence to Xiu-Lan Chen.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.


(DOC 347 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guo, B., Chen, X., Sun, C. et al. Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241. Appl Microbiol Biotechnol 84, 1107–1115 (2009). https://doi.org/10.1007/s00253-009-2056-y

Download citation


  • Xylanase
  • XynA
  • Cold-active
  • Salt-tolerant
  • Glaciecola mesophila
  • Marine
  • Xylan