Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island

  • Marcel Suleiman
  • Carola Schröder
  • Barbara Klippel
  • Christian Schäfers
  • Anna Krüger
  • Garabed AntranikianEmail author
Biotechnologically relevant enzymes and proteins


Already-characterized microbial cellulases have proven to be highly useful for industrial processes, since they can withstand harsh industrial conditions with characteristics such as high thermo- and acid stability. These properties provide promising features for the process of plant biomass degradation and biofuel generation. Nevertheless, the number of known extremely thermoactive archaeal cellulases is low. Hence, the discovery of archaeal cellulases with different characteristics is crucial for the development of efficient and sustainable biorefinery. In this work, the metagenome of a high-temperature enrichment culture from marine environment of Vulcano Island was screened for the presence of novel endoglucanase-encoding genes of archaeal origin. The ORF vul_cel5A was detected, and the deduced protein was characterized as the most thermoactive endoglucanase described to date. Vul_Cel5A was identified as a thermoactive glycoside hydrolase family 5 endoglucanase, with the highest sequence identity (72–75%) to putative endoglucanases from archaeal genera. Vul_Cel5A showed the highest activity at notable 115 °C towards barley β-glucan (210.7 U/mg), and lichenan (209.9 U/mg), and further towards carboxymethyl cellulose (38.6 U/mg) and locust bean gum (83.0 U/mg). The endoglucanase exhibited a half-life time of 46 min at 100 °C and did not show any loss of activity after incubation for 48 h at 75 °C. Furthermore, Vul_Cel5A showed high affinity to barley β-glucan with a Km of 0.52 mg/mL and showed tolerance against various chemical reagents. Due to the outstanding high thermoactivity and thermostability and tolerance to acidic conditions, Vul_Cel5A represents a promising novel archaeal endo-β-glucanase for application in biorefineries for an efficient biomass pre-treatment.


Archaea Endoglucanase Heat activity Thermostability Barley β-glucan Carboxymethyl cellulose 


Author contributions

MS and GA designed the experiments. MS carried out all experiments in the lab. MS, CS, BK, CS, and AK screened for new endoglucanases in the metagenome and supported the experiments in the lab. MS, CS, AK, and GA drafted the manuscript. All authors contributed to the final manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_9542_MOESM1_ESM.pdf (151 kb)
ESM 1 (PDF 150 kb)


  1. Ando S, Ishida H, Kosugi Y, Ishikawa K (2002) Endoglucanase from Pyrococcus horikoshii. Appl Environ Microbiol 68:430–433.
  2. Antranikian G, Suleiman M, Schäfers C, Adams MWW, Bartolucci S, Blamey JM, Birkeland NK, Bonch-Osmolovskaya E, da Costa MS, Cowan D, Danson M, Forterre P, Kelly R, Ishino Y, Littlechild J, Moracci M, Noll K, Oshima T, Robb F, Rossi M, Santos H, Schönheit P, Sterner R, Thauer R, Thomm M, Wiegel J, Stetter KO (2017) Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island. Extremophiles 21:733–742. CrossRefPubMedGoogle Scholar
  3. Aspeborg H, Coutinho PM, Wang Y, Brumer III H, Henrissat B (2012) Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol 12.
  4. Bai Y, Wang J, Zhang Z (2010) Biotechnologically relevant enzymes and proteins. A novel family 9 β-1,3 (4) -glucanase from thermoacidophilic Alicyclobacillus sp. A4 with potential applications in the brewing industry. Appl Microbiol 3:251–259. CrossRefGoogle Scholar
  5. Bauer MW, Driskill LE, Kelly RM (1999) An endoglucanase, EglA, 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 181:284Google Scholar
  6. Bruins ME, Janssen AEM, Boom RM (2001) Thermozymes and their applications. Appl Biochem Biotechnol 90:155–186. CrossRefPubMedGoogle Scholar
  7. Chhabra SR, Shockley KR, Ward DE, Kelly RM (2002) Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides. Appl Environ Microbiol 68:545–554. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Choi JM, Han SS, Kim HS (2015) Industrial applications of enzyme biocatalysis: current status and future aspects. Biotechnol Adv 33:1443–1454. CrossRefPubMedGoogle Scholar
  9. Eichler J (2001) Biotechnological uses of archaeal extremozymes. Biotechnol Adv 19:261–278. CrossRefPubMedGoogle Scholar
  10. Elleuche S, Schröder C, Sahm K, Antranikian G (2014) Extremozymes- biocatalysts with unique properties from extremophilic microorganisms. Curr Opin Biotechnol 29:116–123CrossRefGoogle Scholar
  11. Gilad R, Rabinovich L, Yaron S, Bayer EA, Lamed R, Gilbert HJ, Shoham Y (2003) Cell, a noncellulosomal family 9 enzyme from Clostridium thermocellum, is a processive enduglucanase that degrades crystalline cellulose. J Bacteriol 185:391–398. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Graham JE, Clark ME, Nadler DC, Huffer S, Chokhawala HA, Rowland SE, Blanch HW, Clark DS, Robb FT (2011) Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment. Nat Commun 2:375–379. CrossRefPubMedGoogle Scholar
  13. Han Y, Dodd D, Hespen CW, Ohene-Adjei S, Schroeder CM, Mackie RI, Cann IKO (2010) Comparative analyses of two thermophilic enzymes exhibiting both β-1,4 mannosidic and β-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus. J Bacteriol 192:4111–4121.
  14. Henrissat B, Vegetales M, Grenoble F (1991) A classification of glycosyl hydrolases based sequence similarities amino acid. Biochem J 280:309–316. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Huang Y, Krauss G, Cottaz S, Driguez H, Lipps G (2005) A highly acid-stable and thermostable endo-beta-glucanase from the thermoacidophilic archaeon Sulfolobus solfataricus. Biochem J 385:581–588. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hurst LD, Merchant AR (2001) High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proc R Soc B Biol Sci 268:493–497. CrossRefGoogle Scholar
  17. Jaenicke R, Böhm G (1998) The stability of proteins in extreme environments. Curr Opin Struct Biol 8:738–748. CrossRefPubMedGoogle Scholar
  18. Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kim HW, Ishikawa K (2009) Structure of hyperthermophilic endocellulase from Pyrococcus horikoshii. Proteins Struct Funct Bioinforma 78:496–500. CrossRefGoogle Scholar
  20. Kim HW, Ishikawa K (2013) The role of disulfide bond in hyperthermophilic endocellulase. Extremophiles 17:593–599. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. CrossRefGoogle Scholar
  22. Li S, Sauer WC, Huang SX, Gabert VM (1996) Effect of β-glucanase supplementation to hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, β-glucans, and amino acids in young pigs. J Anim Sci 74:1649–1656CrossRefGoogle Scholar
  23. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:490–495. CrossRefGoogle Scholar
  24. Loow YL, Wu TY, Jahim JM, Mohammad AW, Teoh WH (2016) Typical conversion of lignocellulosic biomass into reducing sugars using dilute acid hydrolysis and alkaline pretreatment. Cellulose 23:1491–1520. CrossRefGoogle Scholar
  25. Meng DD, Ying Y, Chen X-H, et al (2015) Distinct Roles for Carbohydrate-Binding Modules of Glycoside. AEM 81:2006–2014.
  26. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. CrossRefGoogle Scholar
  27. Moreira LRS, Filho EXF (2008) An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biotechnol 79:165–178. CrossRefPubMedGoogle Scholar
  28. Nielsen H (2017) Predicting Secretory Proteins with SignalP. In: Kihara D (ed) Protein function prediction, Methods in Molecular Biology, vol 1611. Humana Press, New YorkCrossRefGoogle Scholar
  29. Pei J, Pang Q, Zhao L, Fan S, Shi H (2012) Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose. Biotechnol Biofuels 5:1–10.
  30. Schröder C, Elleuche S, Blank S, Antranikian G (2014) Characterization of a heat-active archaeal β-glucosidase from a hydrothermal spring metagenome. Enzym Microb Technol 57:48–54. CrossRefGoogle Scholar
  31. Shi R, Li Z, Ye Q, Xu J, Liu Y (2013) Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresour Technol 142:338–344. CrossRefPubMedGoogle Scholar
  32. Sigrist CJA, de Castro E, Cerutti L, Cuche BA, Hulo N, Bridge A, Bougueleret L, Xenarios I (2012) New and continuing developments at prosite. Nucleic Acids Res 41:344–347CrossRefGoogle Scholar
  33. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Yi Z, Su X, Revindran V, Mack RI, Cann I (2013) Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose. PLoS One 8:1–15. CrossRefGoogle Scholar
  35. Zhang J, Shi H, Xu L, Zhu X, Li X (2015) Site-directed mutagenesis of a hyperthermophilic endoglucanase Cel12B from Thermotoga maritima based on rational design. PLoS One 10:1–14. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Marcel Suleiman
    • 1
  • Carola Schröder
    • 1
  • Barbara Klippel
    • 1
  • Christian Schäfers
    • 1
  • Anna Krüger
    • 1
  • Garabed Antranikian
    • 1
    Email author
  1. 1.Institute of Technical MicrobiologyUniversity of TechnologyHamburgGermany

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