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Applied Microbiology and Biotechnology

, Volume 103, Issue 23–24, pp 9505–9514 | Cite as

Characterization of an extremely thermo-active archaeal β-glucosidase and its activity towards glucan and mannan in concert with an endoglucanase

  • Carola Schröder
  • Daniela Eixenberger
  • Marcel Suleiman
  • Christian Schäfers
  • Garabed AntranikianEmail author
Biotechnologically relevant enzymes and proteins

Abstract

A metagenome from an enrichment culture of a hydrothermal vent sample taken at Vulcano Island (Italy) was sequenced and an endoglucanase-encoding gene (vul_cel5A) was identified in a previous work. Vul_Cel5A with maximal activity at 115 °C was characterized as the most heat-active endoglucanase to date. Based on metagenome sequences, genomes were binned and bin4 included vul_cel5A as well as a putative GH1 β-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant β-glucosidase Vul_Bgl1A produced in E. coli BL21 pQE-80L exhibited highest activity at 105 °C and pH 7.0 (76.12 ± 5.4 U/mg, 100%) using 4NP β-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active β-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP β-D-mannopyranoside in comparison with 4NP β-D-glucopyranoside. Moreover, nearly complete hydrolysis of cellobiose was demonstrated. The enzyme exhibited a high glucose tolerance with 26% residual activity in presence of 2 M glucose and was furthermore activated at glucose concentrations of up to 0.5 M. When the endoglucanase Vul_Cel5A and the β-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley β-glucan and 215% towards mannan were achieved compared with the activity of Vul_Cel5A alone (100%). Consequently, a significant increase in glucose formation was observed when both enzymes were incubated with β-glucan and mannan suggesting a synergistic effect. Hence, the two archaeal extremozymes are ideal candidates for complete glucan and mannan saccharification at temperatures above the boiling point of water.

Keywords

β-Glucosidase Extremozymes Heat-active Glucose-tolerant Synergism 

Notes

Author contribution

Carola Schröder planned the study and drafted the manuscript with Garabed Antranikian. Characterization of β-glucosidase was carried out by Daniela Eixenberger. Concerted endoglucanase and β-glucosidase experiments were performed by Marcel Suleiman and Christian Schäfers performed the genome binning and drafted the respective paragraphs. All authors commented on previous versions of the manuscript and 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.

References

  1. Adlakha N, Sawant S, Anil A, Lali A, Yazdani S (2012) Specific fusion of a β-1,4-endoglucanase and β-1,4-glucosidase enhances celluloytic activity and helps in channeling of intermediates. Appl Environ Microbiol 78:7447–7454.  https://doi.org/10.1128/AEM.01386-12 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410.  https://doi.org/10.1016/S0022-2836(05)80360-2 CrossRefGoogle Scholar
  3. 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.  https://doi.org/10.1007/s00792-017-0938-y CrossRefPubMedGoogle Scholar
  4. Bauer MW, Bylina EJ, Swanson RV, Kelly RM (1996) Comparison of a β-glucosidase and a β-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 271:23749–23755.  https://doi.org/10.1074/jbc.271.39.23749 CrossRefPubMedGoogle Scholar
  5. Bhatia Y, Mishra S, Bisaria VS (2002) Microbial beta-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol. 22:375–407.  https://doi.org/10.1080/07388550290789568 CrossRefGoogle Scholar
  6. 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.  https://doi.org/10.1006/abio.1976.9999 CrossRefGoogle Scholar
  7. Britton HTS, Robinson RA (1931) Universal buffer solutions and the dissociation constant of veronal. J Chem Soc:1456–1473.  https://doi.org/10.1039/JR9310001456 CrossRefGoogle Scholar
  8. Gupta P, Verma S, Vakhlu J (2014) Comparative analysis of beta-glucosidase thermostability: differences in amino acids composition and distribution amon mesostable and thermostable beta-glucosidases. J Adv Bioinf Appl Res 5:215–227Google Scholar
  9. Henrissat B (1991) A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem J 280:309–316.  https://doi.org/10.1042/bj2800309 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Hwa K-Y, Subramani B, Shen S-T, Lee Y-M (2014) An intermolecular disulfide bond is required for thermostability and thermoactivity of β-glycosidase from Thermococcus kodakarensis KOD1. Appl Microbiol Biotechnol 98:7825–7836CrossRefGoogle Scholar
  11. Kengen SWM, Luesink EJ, Stams AJM, Zehnder AJB (1993) Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem 213:305–312.  https://doi.org/10.1111/j.1432-1033.1993.tb17763.x CrossRefPubMedGoogle Scholar
  12. Ketudat Cairns JR, Esen A (2010) β-Glucosidases. Cell. Mol. Life Sci 67:3389–3405CrossRefGoogle Scholar
  13. Kim HJ, Park AR, Lee JK, Oh DK (2009) Characterization of an acid-labile, thermostable beta-glycosidase from Thermoplasma acidophilum. Biotechnol Lett 31:1457–1462.  https://doi.org/10.1007/s10529-009-0018-1 CrossRefPubMedGoogle Scholar
  14. Kim HW, Ishikawa K (2010) Complete saccharification of cellulose at high temperature using endocellulase and β-glucosidase from Pyrococcus sp. J. Microbiol Biotechnol 20:889–892.  https://doi.org/10.4014/jmb.0912.12020 CrossRefGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.  https://doi.org/10.1038/227680a0 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Li D, Li X, Dang W, Tran PL, Park SH, Oh BC, Hong WS, Lee JS, Park KH (2013) Characterization and application of an acidophilic and thermostable β-glucosidase from Thermofilum pendens. J Biosci Bioeng 115:490–496.  https://doi.org/10.1016/j.jbiosc.2012.11.009 CrossRefPubMedGoogle Scholar
  17. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495.  https://doi.org/10.1093/nar/gkt1178 CrossRefPubMedGoogle Scholar
  18. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH (2015) CDD: NCBI’s conserved domain database. Nucleic Acids Res 43:D222–D226.  https://doi.org/10.1093/nar/gku1221 CrossRefPubMedGoogle Scholar
  19. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428.  https://doi.org/10.1021/ac60147a030 CrossRefGoogle Scholar
  20. Murphy L, Bohlin C, Baumann MJ, Olsen SN, Sørensen TH, Anderson L, Borch K, Westh P (2013) Product inhibition of five Hypocrea jecorina cellulases. Enzym Microb Technol 52:163–169.  https://doi.org/10.1016/j.enzmictec.2013.01.002 CrossRefGoogle Scholar
  21. Narayanasamy S, Jarosz Y, Muller EEL, Heintz-Buschart A, Herold M, Kaysen A, Laczny CC, Pinel N, May P, Wilmes P (2016) IMP: a pipeline for reproducible reference-independent integrated metagenomic and metatranscriptomic analyses. Genome Biol 17(206).  https://doi.org/10.1186/s13059-016-1116-8
  22. Park AR, Kim HJ, Lee JK, Oh DK (2010) Hydrolysis and transglycosylation activity of a thermostable recombinant beta-glycosidase from Sulfolobus acidocaldarius. Appl Biochem Biotechnol 160:2236–2247.  https://doi.org/10.1007/s12010-009-8705-x CrossRefPubMedGoogle Scholar
  23. Park SH, Park KH, Oh BC, Alli I, Lee BH (2011) Expression and characterization of an extremely thermostable β-glycosidase (mannosidase) from the hyperthermophilic archaeon Pyrococcus furiosus DSM3638. New Biotechnol 28:639–648.  https://doi.org/10.1016/j.nbt.2011.05.002 CrossRefGoogle Scholar
  24. Santos Salgado JC, Parras Meleiro L, Carli S, Ward RJ (2018) Glucose tolerant and glucose stimulated β-glucosidases - a review. Bioresour Technol 267:704–713.  https://doi.org/10.1016/j.biortech.2018.07.137 CrossRefGoogle Scholar
  25. 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.  https://doi.org/10.1016/j.enzmictec.2014.01.010 CrossRefGoogle Scholar
  26. Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30:2068–2069.  https://doi.org/10.1093/bioinformatics/btu153 CrossRefGoogle Scholar
  27. Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A (2013) Role and significance of β-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresour Technol 127:500–507.  https://doi.org/10.1016/j.biortech.2012.09.012 CrossRefPubMedGoogle Scholar
  28. Suleiman M, Schröder C, Klippel B, Schäfers C, Krüger A, Antranikian G (2019) Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island. Appl Microbiol Biotechnol 103(3):1267–1274.  https://doi.org/10.1007/s00253-018-9542-z CrossRefPubMedGoogle Scholar
  29. Teugjas H, Väljamäe P (2013) Selecting β-glucosidases to support cellulases in cellulose saccharification. Biotechnol Biofuels 6:105.  https://doi.org/10.1186/1754-6834-6-105 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Uchima CA, Tokuda G, Watanabe H, Kitamoto K, Arioka M (2018) Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant β-glucosidase from the termite Nasutitermes takasagoensis. Appl Environ Microbiol 78:4288–4293.  https://doi.org/10.1128/AEM.07718-11 CrossRefGoogle Scholar
  31. Vlasenko E, Schülein M, Cherry J, Xu F (2010) Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases. Bioresour Technol 101:2405–2411.  https://doi.org/10.1016/j.biortech.2009.11.057 CrossRefPubMedGoogle Scholar
  32. Vorhoorst WGB, Eggen RIL, Luesink EJ, de Vos WM (1995) Characterization of the celB gene coding for β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli. J Bacteriol 177(24):7105–7111.  https://doi.org/10.1128/jb.177.24.7105-7111.1995 CrossRefGoogle Scholar
  33. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 2,46(W1):W296-W303.  https://doi.org/10.1093/nar/gky427 CrossRefGoogle Scholar
  34. Wu Y-W, Tang Y-H, Tringe SG, Simmons BA, Singer SW (2014) MaxBin: an automated binning method recover individual genomes from metagenomes using an expectation-maximization algorithm. Microbiome 2(26).  https://doi.org/10.1186/2049-2618-2-26
  35. Yin YR, Sang P, Xian WD, Li X, Jiao JY, Liu L, Hozzein WN, Xiao M, Li WJ (2018) Expression and characteristics of two glucose-tolerant GH1 β-glucosidases from Actinomadura amylolytica YIM 77502T for promoting cellulose degradation. Front Microbiol 9:3149.  https://doi.org/10.3389/fmicb.2018.03149 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Carola Schröder
    • 1
  • Daniela Eixenberger
    • 1
  • Marcel Suleiman
    • 1
  • Christian Schäfers
    • 1
  • Garabed Antranikian
    • 1
    Email author
  1. 1.Institute of Technical MicrobiologyHamburg University of Technology (TUHH)HamburgGermany

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