Applied Microbiology and Biotechnology

, Volume 99, Issue 21, pp 8903–8915 | Cite as

Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose

  • Fang Yang
  • Xiaofeng Yang
  • Zhe Li
  • Chenyu Du
  • Jufang Wang
  • Shuang Li
Biotechnologically relevant enzymes and proteins
First Online:
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Revised:
Accepted:

Abstract

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na+ or K+. The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Keywords

β-Glucosidase Thermoanaerobacterium aotearoense P8G3#4 Glucose tolerance Cellobiose degradation Chaperones 
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Notes

Acknowledgments

The authors gratefully appreciate the financial support by the National Natural Science Foundation of China (NSFC 21276096), and the Open Project Program of Guangdong Key Laboratory of Fermentation and Enzyme Engineering, SCUT (FJ2013006). And Dr. Shuang Li was funded by the Pearl River New-Star of Science & Technology supported by Guangzhou City (2012 J2200012).

Conflict of interest

No conflict of interest exits in the submission of this manuscript.

Supplementary material

253_2015_6619_MOESM1_ESM.pdf (506 kb)
ESM 1 (PDF 506 kb)

References

  1. Badieyan S, Bevan DR, Zhang C (2012) Probing the active site chemistry of β-glucosidases along the hydrolysis reaction pathway. Biochemistry 51:8907–8918CrossRefPubMedGoogle Scholar
  2. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408CrossRefPubMedGoogle Scholar
  3. Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407CrossRefPubMedGoogle Scholar
  4. Borges DG, Baraldo A, Farinas CS, Giordano RDC, Tardioli PW (2014) Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized β-glucosidase. Bioresour Technol 167:206–213CrossRefPubMedGoogle Scholar
  5. Cai Y, Lai C, Li S, Liang Z, Zhu M, Liang S, Wang J (2011) Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense. Enzym Microb Technol 48:155–161CrossRefGoogle Scholar
  6. Chen L, Li N, Zong M-H (2012) A glucose-tolerant β-glucosidase from Prunus domestica seeds: purification and characterization. Process Biochem 47:127–132CrossRefGoogle Scholar
  7. Decker CH, Visser J, Schreier P (2001) β-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance. Appl Microbiol Biotechnol 55:157–163CrossRefPubMedGoogle Scholar
  8. Gilbert HJ, Stålbrand H, Brumer H (2008) How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation. Curr Opin Plant Biol 11:338–348CrossRefPubMedGoogle Scholar
  9. Gloster TM, Davies GJ (2010) Glycosidase inhibition: assessing mimicry of the transition state. Org Biomol Chem 8:305–320PubMedCentralCrossRefPubMedGoogle Scholar
  10. Gunata Z, Vallier MJ (1999) Production of a highly glucose-tolerant extracellular β-glucosidase by three Aspergillus strains. Biotechnol Lett 21:219–223CrossRefGoogle Scholar
  11. Harnpicharnchai P, Champreda V, Sornlake W, Eurwilaichitr L (2009) A thermotolerant β-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars. Protein Expr Purif 67:61–69CrossRefPubMedGoogle Scholar
  12. Hartl FU (2011) Chaperone-assisted protein folding: the path to discovery from a personal perspective. Nat Med 17:1206–1210CrossRefPubMedGoogle Scholar
  13. Hartl FU, Hayer-Hartl M (2002) Protein folding - molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852–1858CrossRefPubMedGoogle Scholar
  14. Hassan N, Nguyen T-H, Intanon M, Kori L, Patel BC, Haltrich D, Divne C, Tan T (2015) Biochemical and structural characterization of a thermostable β-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis. Appl Microbiol Biotechnol 99:1731–1744PubMedCentralCrossRefPubMedGoogle Scholar
  15. Herpoël-Gimbert I, Margeot A, Dolla A, Jan G, Mollé D, Lignon S, Mathis H, Sigoillot J-C, Monot F, Asther M (2008) Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol Biofuels 1:18PubMedCentralCrossRefPubMedGoogle Scholar
  16. Hodge DB, Karim MN, Schell DJ, McMillan JD (2008) Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresour Technol 99:8940–8948CrossRefPubMedGoogle Scholar
  17. Hoff JH v’t, Lehfeldt RA (1898) Lectures on theoretical and physical chemistry. E. Arnold, LondonGoogle Scholar
  18. Jabbour D, Klippel B, Antranikian G (2012) A novel thermostable and glucose-tolerant β-glucosidase from Fervidobacterium islandicum. Appl Microbiol Biotechnol 93:1947–1956CrossRefPubMedGoogle Scholar
  19. Jeng W-Y, Wang N-C, Lin M-H, Lin C-T, Liaw Y-C, Chang W-J, Liu C-I, Liang P-H, Wang AHJ (2011) Structural and functional analysis of three β-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis. J Struct Biol 173:46–56CrossRefPubMedGoogle Scholar
  20. Kengen SW, Luesink EJ, Stams AJ, Zehnder AJ (1993) Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem 213:305–312CrossRefPubMedGoogle Scholar
  21. Krogh KBRM, Harris PV, Olsen CL, Johansen KS, Hojer-Pedersen J, Borjesson J, Olsson L (2010) Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillium brasilianum. Appl Microbiol Biotechnol 86:143–154CrossRefPubMedGoogle Scholar
  22. Lai Z, Zhu M, Yang X, Wang J, Li S (2014) Optimization of key factors affecting hydrogen production from sugarcane bagasse by a thermophilic anaerobic pure culture. Biotechnol Biofuels 7:119PubMedCentralPubMedGoogle Scholar
  23. Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  24. Li S, Lai C, Cai Y, Yang X, Yang S, Zhu M, Wang J, Wang X (2010) High efficiency hydrogen production from glucose/xylose by the ldh-deleted Thermoanaerobacterium strain. Bioresour Technol 101:8718–8724CrossRefPubMedGoogle Scholar
  25. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666CrossRefGoogle Scholar
  26. Lu J, Du L, Wei Y, Hu Y, Huang R (2013) Expression and characterization of a novel highly glucose-tolerant β-glucosidase from a soil metagenome. Acta Biochim Biophys Sin 45:664–673CrossRefPubMedGoogle Scholar
  27. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577PubMedCentralCrossRefPubMedGoogle Scholar
  28. Mai Z, Yang J, Tian X, Li J, Zhang S (2013) Gene cloning and characterization of a novel salt-tolerant and glucose-enhanced β-glucosidase from a marine Streptomycete. Appl Biochem Biotechnol 169:1512–1522CrossRefPubMedGoogle Scholar
  29. Martínez-Alonso M, García-Fruitós E, Ferrer-Miralles N, Rinas U, Villaverde A (2010) Side effects of chaperone gene co-expression in recombinant protein production. Microb Cell Fact 9:64PubMedCentralCrossRefPubMedGoogle Scholar
  30. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of ruducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  31. Nam KH, Sung MW, Hwang KY (2010) Structural insights into the substrate recognition properties of β-glucosidase. Biochem Biophys Res Commun 391:1131–1135CrossRefPubMedGoogle Scholar
  32. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699PubMedCentralPubMedGoogle Scholar
  33. Nishihara K, Kanemori M, Yanagi H, Yura T (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 66:884–889PubMedCentralCrossRefPubMedGoogle Scholar
  34. Park TH, Choi KW, Park CS, Lee SB, Kang HY, Shon KJ, Park JS, Cha J (2005) Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana. Appl Microbiol Biotechnol 69:411–422CrossRefPubMedGoogle Scholar
  35. 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 5:31PubMedCentralCrossRefPubMedGoogle Scholar
  36. Rajasree KP, Mathew GM, Pandey A, Sukumaran RK (2013) Highly glucose tolerant β-glucosidase from Aspergillus unguis: NII 08123 for enhanced hydrolysis of biomass. J Ind Microbiol Biotechnol 40:967–975CrossRefPubMedGoogle Scholar
  37. Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM (2009) Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A 106:1760–1765PubMedCentralCrossRefPubMedGoogle Scholar
  38. Riou C, Salmon JM, Vallier MJ, Gunata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 64:3607–3614PubMedCentralPubMedGoogle Scholar
  39. Saha BC, Bothast RJ (1996) Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Appl Environ Microbiol 62:3165–3170PubMedCentralPubMedGoogle Scholar
  40. Sali A, Potterton L, Yuan F, Vanvlijmen H, Karplus M (1995) Evaluation of comparative protein modeling by MODELLER. Proteins 23:318–326CrossRefPubMedGoogle Scholar
  41. Sansenya S, Mutoh R, Charoenwattanasatien R, Kurisu G, Ketudat Cairns JR (2015) Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase from Thermoanaerobacterium xylanolyticum. Acta Crystallogr F 71:41–44CrossRefGoogle Scholar
  42. 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 127:500–507CrossRefPubMedGoogle Scholar
  43. Sørensen A, Lübeck M, Lübeck PS, Ahring BK (2013) Fungal β-glucosidases: a bottleneck in industrial use of lignocellulosic materials. Biomolecules 3:612–631PubMedCentralCrossRefPubMedGoogle Scholar
  44. Uchima CA, Tokuda G, Watanabe H, Kitamoto K, Arioka M (2012) 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 78:4288–4293PubMedCentralCrossRefPubMedGoogle Scholar
  45. Vuong TV, Wilson DB (2010) Glycoside hydrolases: catalytic base/nucleophile diversity. Biotechnol Bioeng 107:195–205CrossRefPubMedGoogle Scholar
  46. Yan TR, Lin CL (1997) Purification and characterization of a glucose-tolerant β-glucosidase from Aspergillus niger CCRC 31494. Biosci Biotechnol Biochem 61:965–970CrossRefPubMedGoogle Scholar
  47. Yang X, Lai Z, Lai C, Zhu M, Li S, Wang J, Wang X (2013) Efficient production of L-lactic acid by an engineered Thermoanaerobacterium aotearoense with broad substrate specificity. Biotechnol Biofuels 6:124PubMedCentralCrossRefPubMedGoogle Scholar
  48. Zanoelo FF, Polizeli M, Terenzi HF, Jorge JA (2004) β-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett 240:137–143CrossRefPubMedGoogle Scholar
  49. Zhao L, Pang Q, Xie J, Pei J, Wang F, Fan S (2013a) Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose. BMC Biotechnol 13:101PubMedCentralCrossRefPubMedGoogle Scholar
  50. Zhao L, Xie J, Zhang X, Cao F, Pei J (2013b) Overexpression and characterization of a glucose-tolerant β-glucosidase from Thermotoga thermarum DSM 5069T with high catalytic efficiency of ginsenoside Rb1 to Rd. J Mol Catal B-Enzym 95:62–69CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Fang Yang
    • 1
  • Xiaofeng Yang
    • 1
    • 3
  • Zhe Li
    • 1
  • Chenyu Du
    • 2
  • Jufang Wang
    • 1
  • Shuang Li
    • 1
    • 4
  1. 1.Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and BioengineeringSouth China University of TechnologyGuangzhouChina
  2. 2.School of Applied SciencesThe University of HuddersfieldHuddersfieldUK
  3. 3.School of Energy and EnvironmentCity University of HongHong KongChina
  4. 4.Guangzhou Higher Education Mega CenterGuangzhouPeople’s Republic of China

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