, Volume 23, Issue 6, pp 681–686 | Cite as

Characterization and homology modelling of a novel multi-modular and multi-functional Paenibacillus mucilaginosus glycoside hydrolase

  • Ntsoaki Leticia Mosina
  • Wolf-Dieter Schubert
  • Don A. CowanEmail author
Original Paper


Glycoside hydrolases, particularly cellulases, xylanases and mannanases, are essential for the depolymerisation of lignocellulosic substrates in various industrial bio-processes. In the present study, a novel glycoside hydrolase from Paenibacillus mucilaginosus (PmGH) was expressed in E. coli, purified and characterised. Functional analysis indicated that PmGH is a 130 kDa thermophilic multi-modular and multi-functional enzyme, comprising a GH5, a GH6 and two CBM3 domains and exhibiting cellulase, mannanase and xylanase activities. The enzyme displayed optimum hydrolytic activities at pH 6 and 60 °C and moderate thermostability. Homology modelling of the full-length protein highlighted the structural and functional novelty of native PmGH, with no close structural homologs identified. However, homology modelling of the individual GH5, GH6 and the two CBM3 domains yielded excellent models based on related structures from the Protein Data Bank. The catalytic GH5 and GH6 domains displayed a (β/α)8 and a distorted seven stranded (β/α) fold, respectively. The distinct homology at the domain level but low homology of the full-length protein suggests that this protein evolved by exogenous gene acquisition and recombination.


Multi-modular Multi-functional Thermophilic enzyme Paenibacillus mucilaginosus 



Carbohydrate Active enZYme database


Carbohydrate binding module


Carboxymethyl cellulose


Dinitrosalicylic acid


Glycoside hydrolase


Global Model Quality Estimation


Immobilized Metal Affinity Chromatography


Isopropyl β–d-1-thiogalactopyranoside


Sodium chloride


Paenibacillus mucilaginosus glycoside hydrolase


Sodium dodecyl sulphate–polyacrylamide gel electrophoresis



The authors gratefully acknowledge the National Research Foundation for project funding. NLM wishes to thank the NRF for the Innovation Doctoral Scholarship.

Compliance with ethical standards

Conflict of interest

The authors declare they have no conflict of interest.

Supplementary material

792_2019_1121_MOESM1_ESM.docx (97 kb)
Supplementary file1 (DOCX 97 kb)


  1. Armenta S, Moreno-Menieta S, Sánchez-Cuapio Z, Sánchez S, Rodriguez-Sanoja R (2017) Advances in molecular engineering of carbohydrate-binding modules. Proteins 85:1602–1617CrossRefGoogle Scholar
  2. Aspeborg H, Coutinho PM, Wang Y, Brumer H, Henrissat B (2012) Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol 12:186CrossRefGoogle Scholar
  3. Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270CrossRefGoogle Scholar
  4. Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27:343–350CrossRefGoogle Scholar
  5. Chandel AK, Chandrasekhar G, Silva MB, Silvério da Silva S (2012) The realm of cellulases in biorefinery development. Crit Rev Biotechnol 32:187–202CrossRefGoogle Scholar
  6. Costa M, Fernandes VO, Ribeiro T, Serrano L, Cardoso V, Santos H, Lordelo M, Ferreira LM, Fontes CM (2014) Construction of GH16 β-glucanase mini-cellulosomes to improve the nutritive value of barley-based diets for broilers. J Agric Food Chem 62:7496–7506CrossRefGoogle Scholar
  7. Davies GJ, Dauter M, Brzozowski AM, Bjørnvad ME, Andersen KV, Schülein M (1998) Structure of the Bacillus agaradherans Family 5 Endoglucanase at 1.6 Å and its cellobiose complex at 2.0 Å resolution. Biochemistry 37:1926–1932CrossRefGoogle Scholar
  8. De Lano WL (2002) Accessed 1 June 2018
  9. Den W, Sharma VK, Lee M, Nadadur G, Varma RS (2018) Lignocellulosic biomass transformations via greener oxidative pretreatment processes: access to energy and value-added chemicals. Front Chem 6:141CrossRefGoogle Scholar
  10. Ding C, Li M, Hu Y (2018) High-activity production of xylanase by Pichia stipitis: purification, characterization, kinetic evaluation and xylooligosaccharides production. Int J Biol Macromol 117:72–77CrossRefGoogle Scholar
  11. Fusco AF, Ronca R, Fiorentino G, Pedone E, Contursi P, Bartolucci S, Limauro D (2018) Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum. Extremophiles 22:131–140CrossRefGoogle Scholar
  12. Gallardo O, Fernández-Fernáadez M, Valls C, Valenzuela M, Roncero B, Vidal T, Pastor FIJ (2010) Characterization of a family GH5 xylanase with activity on neutral oligosaccharides and evauluation as a pulp bleaching aid. Appl Environ Microbiol 76:6290–6294CrossRefGoogle Scholar
  13. Graham JE, Clark ME, Nadler DC, Huffer S, Chokhawala HA, Rowland SE, Blanch HW, Clark DS, Robb FT (2011) Identification and characterizationof a multidomain hyperthermophilic cellulase from an archaeal enrichment. Nat Commun 2:375CrossRefGoogle Scholar
  14. Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modelling with SWISS-MODEL and Swiss-PDBViewer: a historical perspective. Electrophoresis S:162–173CrossRefGoogle Scholar
  15. Guillén D, Sánchez S, Rodríguez-Sanoja R (2010) Carbohydrate-binding domains: multiplicity of biological roles. Appl Microbiol Biotechnol 85:1241–1249CrossRefGoogle Scholar
  16. Hernandez-Gomez MC, Rydahl MG, Rogowski A, Morland C, Cartmell A, Crouch L, Labourel A, Fontes CMGA, Willats WGT, Gilbert HJ, Knox PJ (2015) Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate binding module. FEBS Lett 589:2297–2303CrossRefGoogle Scholar
  17. Lombard V, Golaconda RH, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucl Acids 42:D490–495CrossRefGoogle Scholar
  18. Mayilraj S, Stackebrandt E (2014) The family Paenibacillaceae. In: Rosenberg E, DeLong EF, Lory S, Stackebrandte E, Thompson F (eds) The prokaryotes. Springer, Berlin, pp 267–280Google Scholar
  19. Mertz B, Kuczenski RS, Larsen RT, Hill AD, Reilly PJ (2005) Phylogenetic analysis of family 6 glycoside hydrolases. Biopolymer 79:197–206CrossRefGoogle Scholar
  20. Mota TR, de Oliveira DM, Marchiosi R, Ferrarese-Filho O, dosSantos WD (2018) Plant cell wall composition and enzymatic deconstruction. Bioengineering 5:63–77CrossRefGoogle Scholar
  21. Nacke H, Engelhaupt M, Brady S, Fischer C, Tautz J, Daniel R (2012) Identification and characterization of novel cellulolytic genes and enzymes derived from German grassland soil metagenomes. Biotechnol Lett 34:663–675CrossRefGoogle Scholar
  22. Naumoff DG (2016) GH10 family of glycoside hydrolases: structure and evolutionary connections. Bioinformatics 50:132–140Google Scholar
  23. Ribeiro DA, Cota J, Alvarez TM, Brüchli F, Bragato J, Pereira BM, Pauletti BA, Jackson G, Pimenta MT, Murakami MT et al (2012) The Penicillium echinulatum secretome on sugar cane bagasse. PLoS ONE 7:e50571CrossRefGoogle Scholar
  24. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, New YorkGoogle Scholar
  25. Sandgren M, Wu M, Karkehabadi S, Mitchinson C, Kelemen BR, Larenas EA, Ståhlberg J, Hansson H (2013) The structure of a bacterial cellobiohydrolase: the catalytic core of the Thermobifida fusca family GH6 cellobiohydrolase Cel6B. J Mol Biol 425:622–635CrossRefGoogle Scholar
  26. Shaw A, Bott R, Vonrhein C, Bricogne G, Power S, Day AG (2002) A novel combination of two classic catalytic schemes. J Mol Biol 320:303–309CrossRefGoogle Scholar
  27. Shoseyov O, Shani Z, Levy I (2006) Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev 70:283–295CrossRefGoogle Scholar
  28. Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and bacteria. Rev Biotechnol 17:39–67CrossRefGoogle Scholar
  29. Thomas CM, Nielsen KM (2005) Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat Rev Microbiol 3:711–721CrossRefGoogle Scholar
  30. Tshukudu O (2012) Cloning and characterisation of a cellulase from a metagenomic library. Dissertation, University of Western CapeGoogle Scholar
  31. van den Brink J, de Vries RP (2011) Fungal enzyme sets for plant polysaccharide degradation. Appl Microbiol Biotechnol 91:1477–1492CrossRefGoogle Scholar
  32. Varnai A, Makela MR, Djajadi DT, Rahikainen J, Hatakka A, Viikari L (2014) Carbohydratebinding modules of fungal cellulases: occurrence in nature, function, and relevance in industrial biomass conversion. Adv Appl Microbiol 88:103–165CrossRefGoogle Scholar
  33. Walia A, Guleria S, Mehta P, Chauhan A, Parkash J (2017) Microbial xylanases and their industrial application in pulp and paper biobleaching: a review. 3 Biotech 7:11CrossRefGoogle Scholar
  34. Wang R, Gong L, Xue X, Qin X, Ma R, Luo H, Zhang J, Yao B, Su X (2016) Identification of the C-terminal GH5 domain from CbCel9b/Man5A as the first glycoside hydrolase with thermal activation property from a multimodular bifunctional enzyme. PLoS ONE 11:1–11Google Scholar
  35. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L et al (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucl Acids Res. CrossRefPubMedGoogle Scholar
  36. Yaniv O, Morag E, Borovok I, Bayer EA, Lamed R, Frolow F, Shimon LJ (2013) Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA of Clostridium thermocellum with flanking linkers: implications for cellulosome structure. Acta Cryst 69:733–737Google Scholar
  37. Yi Z, Su X, Revindran V, Mackie RI, Cann I (2013) Molecular and biochemical analyses of CbCel9A/Cel48A, highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose. PLoS ONE 8:1–15Google Scholar
  38. Zhou Q, Jia J, Ji P, Han W (2017) Novel application potential of GH6 cellobiohydrolase ctcel6 from thermophilic Chaetomium thermophilum for gene cloning, heterologous expression and biological characterization. Int J Agric Biol 19:2Google Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department Biochemistry, Genetics and Microbiology, Centre for Microbial Ecology and GenomicsUniversity of PretoriaPretoriaSouth Africa

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