Applied Microbiology and Biotechnology

, Volume 103, Issue 21–22, pp 8899–8909 | Cite as

Cloning and heterologous expression of a novel halo/alkali-stable multi-domain xylanase (XylM18) from a marine bacterium Marinimicrobium sp. strain LS-A18

  • Hao Yu
  • Shuxue Zhao
  • Yaqin Fan
  • Chunhui Hu
  • Weidong LuEmail author
  • Lizhong GuoEmail author
Biotechnologically relevant enzymes and proteins


Halophilic bacteria are good bioresources for halotolerant alkaline enzymes. A multi-domain high-molecular-weight endo-β-1,4-xylanase gene, xylM18, was cloned from a halophilic marine bacterium Marinimicrobium sp. LS-A18. XylM18 is different from any of the functionally reported xylanases. It has a glycosyl hydrolase (GH) 43 domain, a GH10 domain, and two serine-rich linkers, representing a novel family. The gene, encoding 1022 residues, was cloned and heterologously expressed in Escherichia coli BL21(DE3) cells. Purified XylM18 was proved to be a xylanase. It showed diminished activity without salt and showed activity with a broad NaCl range from 0.2 to 25% (w/v). NaCl can increase the optimal temperature from 30 °C (0% NaCl) to 50 °C (10% NaCl). The purified XylM18 was active between pH 6.0 and 10.0 and was optimally active at pH 7.0. The xylanase activities were basically unchanged at a NaCl concentration range from 10 to 20% or pH from 7 to 10 after 24 h incubation. The apparent Km and Vmax values of XylM18 for xylan were 2.76 mg/mL and 60.0 U/mg, respectively. The GH10 domain of this enzyme, XylM18-GH10, was expressed and characterized. XylM18-GH10 also showed xylanase activity and maintained halo-stable property. The apparent Km and Vmax values of XylM18-GH10 for xylan were 1.60 mg/mL and 130.1 U/mg, respectively. Other domains of XylM18 showed no xylanase activity. In summary, XylM18 is a halo-tolerant and alkali-stable endoxylanase which is a suitable candidate for xylan biodegradation in high-salt and alkali conditions. To our knowledge, this is the first report of a multidomain high-molecular-weight xylanase.


Endo-β-1,4-xylanase Glycosyl hydrolase family 10 Halophilic bacterium Marinimicrobium 



This study was funded by the Shandong Provincial Natural Science Foundation (ZR2016CQ06), Key Project of Edible Fungus Genetic Breeding System of Modern Agriculture of Shandong Province, and the Qingdao Agricultural University Scientific Research Foundation (6631115052). We are very grateful to Dr Joel Rankin (Michigan State University) for valuable comments on the manuscript.

Author contributions

H.Y., S.Z., W.L., and L.G. conceived and designed the project. H. Y., S.Z., and Y.F. performed the experiments. H.Y., W.L., and L.G. contributed reagents and materials. H.Y., C.H., Y.F., and S.Z. analyzed data. H.Y., C.H., and L.G. wrote the manuscript. All of the authors have read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Anthony T, Chandra Raj K, Rajendran A, Gunasekaran P (2003) High molecular weight cellulase-free xylanase from alkali-tolerant Aspergillus fumigatus AR1. Enzym Microb Technol 32:647–654Google Scholar
  2. Bu Y, Cui Y, Peng Y, Hu M, Ye T, Tao Y, Wu B (2018) Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design. Appl Microbiol Biotechnol 102:3675–3685PubMedGoogle Scholar
  3. Carli S, Meleiro LP, Rosa JC, Moraes LAB, Jorge JA, Masui DC, Furriel RPM (2016) A novel thermostable and halotolerant xylanase from Colletotrichum graminicola. J Mol Catal B Enzym 133:S508–S517Google Scholar
  4. Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23PubMedGoogle Scholar
  5. Dheeran P, Nandhagopal N, Kumar S, Jaiswal YK, Adhikari DK (2012) A novel thermostable xylanase of Paenibacillus macerans IIPSP3 isolated from the termite gut. J Ind Microbiol Biotechnol 39:851–860PubMedGoogle Scholar
  6. Diogo JA, Hoffmam ZB, Zanphorlin LM, Cota J, Machado CB, Wolf LD, Squina F, Damásio ARL, Murakami MT, Ruller R (2015) Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance. Enzym Microb Technol 69:31–37Google Scholar
  7. Dwivedi P, Vivekanand V, Ganguly R, Singh RP (2009) Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation. Biomass Bioenergy 33:581–588Google Scholar
  8. Fernandes P (2014) Marine enzymes and food industry: insight on existing and potential interactions. Front Mar Sci 1:1–18Google Scholar
  9. Ferrer M, Golyshina O, Beloqui A, Golyshin PN (2007) Mining enzymes from extreme environments. Curr Opin Microbiol 10:207–214PubMedGoogle Scholar
  10. Fu G, Wehr G, Edwards J, Hauge B (2008) Rapid one-step recombinational cloning. Nucleic Acids Res 36:e54PubMedPubMedCentralGoogle Scholar
  11. Ghadikolaei KK, Sangachini ED, Vahdatirad V, Noghabi KA, Zahiri HS (2019) An extreme halophilic xylanase from camel rumen metagenome with elevated catalytic activity in high salt concentrations. AMB Express 9:86PubMedPubMedCentralGoogle Scholar
  12. Graziano G, Merlino A (2014) Molecular bases of protein halotolerance. Biochim Biophys Acta 1844:850–858PubMedGoogle Scholar
  13. Graciano L, Correa JM, Vieira FG, Bosetto A, Loth EA, Kadowaki MK, Gandra RF, Simao Rde C (2015) Cloning and expression of the xynA1 gene encoding a xylanase of the GH10 group in Caulobacter crescentus. Appl Biochem Biotechnol 175:3915–3929PubMedGoogle Scholar
  14. Guo B, Chen XL, Sun CY, Zhou BC, Zhang YZ (2009) Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-beta-1,4-xylanase from marine Glaciecola mesophila KMM 241. Appl Microbiol Biotechnol 84:1107–1115PubMedGoogle Scholar
  15. Hu RF, Liu L, Liu TJ, Ouyang PK, He BH, Liu SJ (2008) Reducing sugar content in hemicellulose hydrolysate by DNS method: a revisit. J Biobased Mater Bioenerg 2:156–161Google Scholar
  16. Hung K-S, Liu S-M, Tzou W-S, Lin F-P, Pan C-L, Fang T-Y, Sun K-H, Tang S-J (2011) Characterization of a novel GH10 thermostable, halophilic xylanase from the marine bacterium Thermoanaerobacterium saccharolyticum NTOU1. Process Biochem 46:1257–1263Google Scholar
  17. Irfan M, Guler HI, Belduz AO, Shah AA, Canakci S (2016) Cloning, purification and characterization of a cellulase-free xylanase from Geobacillus thermodenitrificans AK53. Prikl Biokhim Mikrobiol 52:296–305PubMedGoogle Scholar
  18. Khalili Ghadikolaei K, Akbari Noghabi K, Shahbani Zahiri H (2017) Development of a bifunctional xylanase-cellulase chimera with enhanced activity on rice and barley straws using a modular xylanase and an endoglucanase procured from camel rumen metagenome. Appl Microbiol Biotechnol 101:6929–6939PubMedGoogle Scholar
  19. Khandeparker R, Verma P, Deobagkar D (2011) A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing. New Biotechnol 28:814–821Google Scholar
  20. Konkit M, Kim JH, Kim W (2016) Marimicrobium arenosum gen. nov., sp. nov., a moderately halophilic bacterium isolated from sea sand. Int J Syst Evol Microbiol 66:856–861PubMedGoogle Scholar
  21. Li AX, Guo LZ, Lu WD (2012) Alkaline inulinase production by a newly isolated bacterium Marinimicrobium sp. LS-A18 and inulin hydrolysis by the enzyme. World J Microbiol Biotechnol 28:81–89PubMedGoogle Scholar
  22. Liao H, Sun S, Wang P, Bi W, Tan S, Wei Z, Mei X, Liu D, Raza W, Shen Q, Xu Y (2014) A new acidophilic endo-beta-1,4-xylanase from Penicillium oxalicum: cloning, purification, and insights into the influence of metal ions on xylanase activity. J Ind Microbiol Biotechnol 41:1071–1083PubMedGoogle Scholar
  23. Liao H, Zheng H, Li S, Wei Z, Mei X, Ma H, Shen Q, Xu Y (2015) Functional diversity and properties of multiple xylanases from Penicillium oxalicum GZ-2. Sci Rep 5:12631PubMedPubMedCentralGoogle Scholar
  24. Lim JM, Jeon CO, Lee JC, Song SM, Kim KY, Kim CJ (2006) Marinimicrobium koreense gen. nov., sp. nov. and Marinimicrobium agarilyticum sp. nov., novel moderately halotolerant bacteria isolated from tidal flat sediment in Korea. Int J Syst Evol Microbiol 56:653–657PubMedGoogle Scholar
  25. Li Q, Sun B, Li X, Xiong K, Xu Y, Yang R, Hou J, Teng C (2018) Improvement of the catalytic characteristics of a salt-tolerant GH10 xylanase from Streptomyce rochei L10904. Int J Biol Macromol 107:1447–1455PubMedGoogle Scholar
  26. Liu CJ, Suzuki T, Hirata S, Kawai K (2003a) The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60. J Biosci Bioeng 95:95–101PubMedGoogle Scholar
  27. Liu CJ, Suzuki T, Hirata S, Kawai K (2003b) Processing of XynE (110-kDa) of Aeromonas caviae ME-1 to 72-kDa xylanase in Escherichia coli transformant. J Biosci Bioeng 96:406–408PubMedGoogle Scholar
  28. Liu T, Zhang J (2018) High-level expression and characterization of Aspergillus niger ATCC 1015 xylanase B in Komagataella phaffii. Appl Biol Chem 61:373–381Google Scholar
  29. Liu X, Huang Z, Zhang X, Shao Z, Liu Z (2014) Cloning, expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda. Extremophiles 18:441–450PubMedGoogle Scholar
  30. Long C, Liu J, Gan L, Zeng B, Long M (2017) Optimization of xylanase production by Trichoderma orientalis using corn cobs and wheat bran via statistical strategy. Waste Biomass Valoriz 10:1277–1284Google Scholar
  31. Long L, Xu M, Shi Y, Lin Q, Wang J, Ding S (2018) Characterization of two new endo-β-1,4-xylanases from Eupenicillium parvum 4–14 and their applications for production of feruloylated oligosaccharides. Appl Biochem Biotechnol 186:816–833PubMedGoogle Scholar
  32. Lu WD, Pang HQ, Guo LZ (2013) Genome sequence of the fructan-degrading organism Marinimicrobium sp. strain LS-A18, isolated from a marine solar saltern. Genome Announc 1:e00776–e00713PubMedPubMedCentralGoogle Scholar
  33. Menon G, Mody K, Keshri J, Jha B (2010) Isolation, purification, and characterization of haloalkaline xylanase from a marine Bacillus pumilus strain, GESF-1. Biotechnol Bioprocess Eng 15:998–1005Google Scholar
  34. Moller MF, Kjeldsen KU, Ingvorsen K (2010) Marinimicrobium haloxylanilyticum sp. nov., a new moderately halophilic, polysaccharide-degrading bacterium isolated from Great Salt Lake, Utah. Anton Leeuw 98:553–565Google Scholar
  35. Nigam PS (2013) Microbial enzymes with special characteristics for biotechnological applications. Biomolecules 3:597–611PubMedPubMedCentralGoogle Scholar
  36. Paes G, Berrin JG, Beaugrand J (2012) GH11 xylanases: Structure/function/properties relationships and applications. Biotechnol Adv 30:564–592PubMedGoogle Scholar
  37. Prade RA (1996) Xylanases: from biology to biotechnology. Biotechnol Genet Eng Rev 13:101–132PubMedGoogle Scholar
  38. Shah V, Charlton T, Kim JR (2018) Laboratory evolution of Bacillus circulans xylanase inserted into pyrococcus furiosus maltodextrin-binding protein for increased xylanase activity and thermal stability toward alkaline pH. Appl Biochem Biotechnol 184:1232–1246PubMedGoogle Scholar
  39. Shao W, Deblois S, Wiegel J (1995) A high-molecular-weight, cell-associated xylanase isolated from exponentially growing Thermoanaerobacterium sp. strain JW/SL-YS485. Appl Environ Microbiol 61:937–940PubMedPubMedCentralGoogle Scholar
  40. Teo SC, Liew KJ, Shamsir MS, Chong CS, Bruce NC, Chan KG, Goh KM (2019) Characterizing a halo-tolerant GH10 xylanase from Roseithermus sacchariphilus strain RA and its CBM-truncated variant. Int J Mol Sci 20:2284PubMedCentralGoogle Scholar
  41. Uday USP, Choudhury P, Bandyopadhyay TK, Bhunia B (2016) Classification, mode of action and production strategy of xylanase and its application for biofuel production from water hyacinth. Int J Biol Macromol 82:1041–1054PubMedGoogle Scholar
  42. Wang C-Y, Chan H, Lin H-T, Shyu Y-T (2010) Production, purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06. Ann Appl Biol 156:187–197Google Scholar
  43. Wejse PL, Ingvorsen K, Mortensen KK (2003) Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium. Extremophiles 7:423–431PubMedGoogle Scholar
  44. Xiao Z, Grosse S, Bergeron H, Lau PC (2014) Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae. Appl Microbiol Biotechnol 98:8211–8222PubMedGoogle Scholar
  45. Yoon JH, Kang SJ, Jung YT, Oh TK (2009) Marinimicrobium locisalis sp. nov., isolated from a marine solar saltern, and emended description of the genus Marinimicrobium. Int J Syst Evol Microbiol 59:2260–2263PubMedGoogle Scholar
  46. Zhao K, Guo LZ, Lu WD (2012) Extracellular production of novel halotolerant, thermostable, and alkali-stable carboxymethylcellulase by marine bacterium Marinimicrobium sp. LS-A18. Appl Biochem Biotechnol 168:550–567PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Shandong Provincial Key Laboratory of Applied Mycology, College of Life SciencesQingdao Agricultural UniversityQingdaoPeople’s Republic of China

Personalised recommendations