Molecular Biology Reports

, Volume 45, Issue 6, pp 2201–2211 | Cite as

Expression and characterisation of a thermophilic endo-1,4-β-glucanase from Sulfolobus shibatae of potential industrial application

  • Angela BoyceEmail author
  • Gary Walsh
Original Article


An endo-1,4-β-d-glucanase gene was cloned from the thermophilic archaea Sulfolobus shibatae and expressed in E. coli. The recombinant enzyme was purified by heat denaturation and affinity chromatography prior to characterisation. The purified recombinant enzyme exhibited maximum activity at 95–100 °C and displayed a broad pH profile with over 91% of its maximum activity observed at pH 3–5. Upon assessment of enzyme thermal stability, full activity was observed after 1 h incubation at 75, 80 and 85 °C while 98%, 90% and 84% of original activity was detected after 2 h at 75, 80 and 85 °C, respectively. Maximum activity was observed on barley β-glucan and lichenan and the purified enzyme also hydrolysed CMC and xylan. Endoglucanase activity was confirmed by viscometric assay with a rapid decrease in substrate viscosity observed immediately upon incubation with barley β-glucan or CMC. The crude enzyme released reducing sugars from acid-pretreated straw at 75–85 °C. The thermophilic nature and biochemical properties of the enzyme indicate its potential suitability in industrial applications undertaken at high temperature, such as the production of second-generation bioethanol from lignocellulosic feedstocks and in the brewing industry. This is the first known report of an endoglucanase from S. shibatae.


Endoglucanase Sulfolobus shibatae Thermophilic Lignocellulose Brewing 



This work was supported by Science Foundation Ireland/Enterprise Ireland Technology Innovation Development Award.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Raddadi N, Cherif A, Daffonchio D, Neifar M, Fava F (2015) Biotechnological applications of extremophiles, extremozymes and extremolytes. Appl Microbiol Biotechnol 99(19):7907–7913. CrossRefPubMedGoogle Scholar
  2. 2.
    Akram F, Haq IU, Imran W, Mukhtar H (2018) Insight perspectives of thermostable endoglucanases for bioethanol production: a review. Renewable Energy 122:225–238. CrossRefGoogle Scholar
  3. 3.
    Haq I, Akram F (2017) Enhanced production of a recombinant multidomain thermostable GH9 processive endo-1,4-β-glucanase (CenC) from Ruminiclostridium thermocellum in a mesophilic host through various cultivation and induction strategies. Appl Biochem Biotechnol 183(1):171–188. CrossRefPubMedGoogle Scholar
  4. 4.
    Sharma A, Tewari R, Rana SS, Soni R, Soni SK (2016) Cellulases: classification, methods of determination and industrial applications. Appl Biochem Biotechnol 179(8):1346–1380. CrossRefPubMedGoogle Scholar
  5. 5.
    Bhalla A, Bansal N, Kumar S, Bischoff KM, Sani RK (2013) Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour Technol 128:751–759. CrossRefPubMedGoogle Scholar
  6. 6.
    Dos Santos LV, De Barros Grassi MC, Gallardo JCM, Pirolla RAS, Calderón LL, De Carvalho-Netto OV, Parreiras LS, Camargo ELO, Drezza AL, Missawa SK, Teixeira GS, Lunardi I, Bressiani J, Pereira GAG (2016) Second-generation ethanol: the need is becoming a reality. Ind Biotechnol 12(1):40–57. CrossRefGoogle Scholar
  7. 7.
    Kuhad RC, Deswal D, Sharma S, Bhattacharya A, Jain KK, Kaur A, Pletschke BI, Singh A, Karp M (2016) Revisiting cellulase production and redefining current strategies based on major challenges. Renewable Sustain Energy Rev 55:249–272. CrossRefGoogle Scholar
  8. 8.
    Irfan M, Tayyab A, Hasan F, Khan S, Badshah M, Shah AA (2017) Production and characterization of organic solvent-tolerant cellulase from Bacillus amyloliquefaciens AK9 isolated from hot spring. Appl Biochem Biotechnol 182(4):1390–1402. CrossRefPubMedGoogle Scholar
  9. 9.
    Jain KK, Kumar S, Deswal D, Kuhad RC (2017) Improved production of thermostable cellulase from Thermoascus aurantiacus RCKK by fermentation bioprocessing and its application in the hydrolysis of office waste paper, algal pulp, and biologically treated wheat straw. Appl Biochem Biotechnol 181(2):784–800. CrossRefPubMedGoogle Scholar
  10. 10.
    Kazeem MO, Shah UKM, Baharuddin AS, AbdulRahman NA (2017) Prospecting agro-waste cocktail: supplementation for cellulase production by a newly isolated thermophilic B-licheniformis 2D55. Appl Biochem Biotechnol 182(4):1318–1340. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Elleuche S, Schroder C, Sahm K, Antranikian G (2014) Extremozymes-biocatalysts with unique properties from extremophilic microorganisms. Curr Opin Biotechnol 29:116–123. CrossRefPubMedGoogle Scholar
  12. 12.
    Guerriero G, Hausman J-F, Strauss J, Ertan H, Siddiqui KS (2015) Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases. Plant Sci 234:180–193. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kallioinen A, Puranen T, Siika-Aho M (2014) Mixtures of thermostable enzymes show high performance in biomass saccharification. Appl Biochem Biotechnol 173(5):1038–1056. CrossRefPubMedGoogle Scholar
  14. 14.
    Szijarto N, Horan E, Zhang J, Puranen T, Siika-aho M, Viikari L (2011) Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw. Biotechnol Biofuels 4:2CrossRefGoogle Scholar
  15. 15.
    Viikari L, Vehmaanpera J, Koivula A (2012) Lignocellulosic ethanol: from science to industry. Biomass Bioenerg 46:13–24. CrossRefGoogle Scholar
  16. 16.
    Miller PS, Blum PH (2010) Extremophile-inspired strategies for enzymatic biomass saccharification. Environ Technol 31(8–9):1005–1015. CrossRefPubMedGoogle Scholar
  17. 17.
    Bai Y, Wang J, Zhang Z, Shi P, Luo H, Huang H, Luo C, Yao B (2010) A novel family 9 beta-1,3(4)-glucanase from thermoacidophilic Alicyclobacillus sp A4 with potential applications in the brewing industry. Appl Microbiol Biotechnol 87(1):251–259. CrossRefPubMedGoogle Scholar
  18. 18.
    Wang J, Niu C, Liu X, Chen X, Li Q (2014) Characterization of a new 1,3–1,4-β-glucanase gene from Bacillus tequilensis CGX5-1. Appl Biochem Biotechnol 173(3):826–837. CrossRefPubMedGoogle Scholar
  19. 19.
    Grogan D, Palm P, Zillig W (1990) Isolate-B12, which harbors a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp-nov. Arch Microbiol 154(6):594–599. CrossRefPubMedGoogle Scholar
  20. 20.
    Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 3 edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  21. 21.
    Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72(1–2):248–254. CrossRefGoogle Scholar
  22. 22.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Boyce A, Walsh G (2015) Characterisation of a novel thermostable endoglucanase from Alicyclobacillus vulcanalis of potential application in bioethanol production. Appl Microbiol Biotechnol 99(18):7515–7525. CrossRefPubMedGoogle Scholar
  24. 24.
    Miller GL (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. CrossRefGoogle Scholar
  25. 25.
    Huang YW, Krauss G, Cottaz S, Driguez H, Lipps G (2005) A highly acid-stable and thermostable endo-beta-glucanase from the thermoacidophilic archaeon Sulfoloblus solfataricus. Biochem J 385:581–588. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Girfoglio M, Rossi M, Cannio R (2012) Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-beta-1-4-glucanase. J Bacteriol 194(18):5091–5100. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Klose H, Roeder J, Girfoglio M, Fischer R, Commandeur U (2012) Hyperthermophilic endoglucanase for in planta lignocellulose conversion. Biotechnol Biofuels. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Maurelli L, Giovane A, Esposito A, Moracci M, Fiume I, Rossi M, Morana A (2008) Evidence that the xylanase activity from Sulfolobus solfataricus O alpha is encoded by the endoglucanase precursor gene (sso1354) and characterization of the associated cellulase activity. Extremophiles 12(5):689–700. CrossRefPubMedGoogle Scholar
  29. 29.
    Zeldes BM, Keller MW, Loder AJ, Straub CT, Adams MWW, Kelly RM (2015) Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front Microbiol. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Skovgaard PA, Jorgensen H (2013) Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes. J Ind Microbiol Biotechnol 40(5):447–456. CrossRefPubMedGoogle Scholar
  31. 31.
    Eckert K, Zielinski F, Lo Leggio L, Schneider E (2002) Gene cloning, sequencing, and characterization of a family 9 endoglucanase (CeIA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009. Appl Microbiol Biotechnol 60(4):428–436. CrossRefPubMedGoogle Scholar
  32. 32.
    Eckert K, Schneider E (2003) A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases. Eur J Biochem 270(17):3593–3602. CrossRefPubMedGoogle Scholar
  33. 33.
    Huang XP, Monk C (2004) Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp nov. World J Microbiol Biotechnol 20(1):85–92. CrossRefGoogle Scholar
  34. 34.
    Wang J, Gao G, Li Y, Yang L, Liang Y, Jin H, Han W, Feng Y, Zhang Z (2015) Cloning, Expression, and characterization of a thermophilic endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B. Int J Mol Sci 16(10):25080–25095. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wang K, Luo H, Bai Y, Shi P, Huang H, Xue X, Yao B (2014) A thermophilic endo-1,4-beta-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials. Appl Microbiol Biotechnol 98(16):7051–7060. CrossRefPubMedGoogle Scholar
  36. 36.
    Hua C, Li W, Han W, Wang Q, Bi P, Han C, Zhu L (2018) Characterization of a novel thermostable GH7 endoglucanase from Chaetomium thermophilum capable of xylan hydrolysis. Int J Biol Macromol 117:342–349. CrossRefPubMedGoogle Scholar
  37. 37.
    Ando S, Ishida H, Kosugi Y, Ishikawa K (2002) Hyperthermostable endoglucanase from Pyrococcus horikoshii. Appl Environ Microbiol 68(1):430–433. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Bauer MW, Driskill LE, Callen W, Snead MA, Mathur EJ, Kelly RM (1999) An endoglucanase, eg1A, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes β-1,4 bonds in mixed-linkage (1→3),(1→4)-β-D-glucans and cellulose. J Bacteriol 181(1):284–290PubMedPubMedCentralGoogle Scholar
  39. 39.
    Plascencia-Espinosa M, Santiago-Hernández A, Pavón-Orozco P, Vallejo-Becerra V, Trejo-Estrada S, Sosa-Peinado A, Benitez-Cardoza CG, Hidalgo-Lara ME (2014) Effect of deglycosylation on the properties of thermophilic invertase purified from the yeast Candida guilliermondii MpIIIa. Process Biochem 49(9):1480–1487. CrossRefGoogle Scholar
  40. 40.
    Zou S, Huang S, Kaleem I, Li C (2013) N-glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris. J Biotechnol 164(1):75–81. CrossRefGoogle Scholar
  41. 41.
    Han YM, Lei XG (1999) Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Arch Biochem Biophys 364(1):83–90. CrossRefPubMedGoogle Scholar
  42. 42.
    Hoiberg-Nielsen R, Fuglsang CC, Arleth L, Westh P (2006) Interrelation ships of glycosylation and aggregation kinetics for Peniophora lycii phytase. Biochemistry 45(15):5057–5066. CrossRefPubMedGoogle Scholar
  43. 43.
    Méndez Arias J, de Oliveira Moraes A, Modesto LFA, de Castro AM, Pereira N (2017) Addition of surfactants and non-hydrolytic proteins and their influence on enzymatic hydrolysis of pretreated sugarcane bagasse. Appl Biochem Biotechnol 181(2):593–603. CrossRefPubMedGoogle Scholar
  44. 44.
    Parnthong J, Kungsanant S, Chavadej S (2018) The influence of nonionic surfactant adsorption on enzymatic hydrolysis of oil palm fruit bunch. Appl Biochem Biotechnol 1–14. CrossRefGoogle Scholar
  45. 45.
    Rocha-Martín J, Martinez-Bernal C, Pérez-Cobas Y, Reyes-Sosa FM, García BD (2017) Additives enhancing enzymatic hydrolysis of lignocellulosic biomass. Bioresour Technol 244:48–56. CrossRefPubMedGoogle Scholar
  46. 46.
    Deep K, Poddar A, Das SK (2016) Cloning, overexpression, and characterization of halostable, solvent-tolerant novel β-endoglucanase from a marine bacterium Photobacterium panuliri LBS5T (DSM 27646T). Appl Biochem Biotechnol 178(4):695–709. CrossRefPubMedGoogle Scholar
  47. 47.
    Huang XM, Li QQ, Chen XL, Fan JX, Xu XH, Sun XD, Li DY, Zhao HX (2017) Expression and characteristics of an endoglucanase from Trichoderma atroviride (TaEGII) in Saccharomyces cerevisiae. Appl Biochem Biotechnol 182(3):1158–1170. CrossRefPubMedGoogle Scholar
  48. 48.
    Xu X, Li J, Zhang W, Huang H, Shi P, Luo H, Liu B, Zhang Y, Zhang Z, Fan Y, Yao B (2015) A neutral thermostable beta-1,4-glucanase from Humicola insolens Y1 with potential for applications in various industries. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Miotto LS, de Rezende CA, Bernardes A, Serpa VI, Tsang A, Polikarpov I (2014) The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on beta-glucan. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    McCarthy T, Hanniffy O, Savage AV, Tuohy MG (2003) Catalytic properties and mode of action of three endo-beta-glucanases from Talaromyces emersonii on soluble beta-1,4- and beta-1,3–1,4-linked glucans. Int J Biol Macromol 33(1–3):141–148. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Chemical Sciences and Bernal InstituteUniversity of LimerickLimerickIreland

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