Skip to main content
SpringerLink
Log in

Biochemical Characterization and Low-Resolution SAXS Molecular Envelope of GH1 β-Glycosidase from Saccharophagus degradans

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure–function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans β-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C. The enzyme has high specificity toward short d-glucose saccharides with β-linkages with the following preferences β-1,3 > β-1,4 ≫ β-1,6. The enzyme kinetic parameters, obtained using artificial substrates p-β-NPGlu and p-β-NPFuc and also the disaccharides cellobiose, gentiobiose and laminaribiose, revealed SdBgl1B preference for p-β-NPGlu and laminaribiose, which indicates its affinity for glucose and also preference for β-1,3 linkages. To better understand structural basis of the enzyme activity its 3D model was built and analysed. The 3D model fits well into the experimentally retrieved low-resolution SAXS-based envelope of the enzyme, confirming monomeric state of SdBgl1B in solution.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Dias, M. O. S., Junqueira, T. L., Cavalett, O., Cunha, M. P., Jesus, C. D. F., Rossell, C. E. V., et al. (2012). Integrated versus stand-alone second generation ethanol production from sugarcane bagasse and trash. Bioresource Technology, 103(1), 152–161. doi:10.1016/j.biortech.2011.09.120.

    Article  CAS  Google Scholar 

  2. Singhania, R. R., Patel, A. K., Sukumaran, R. K., Larroche, C., & Pandey, A. (2013). Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology, 127, 500–507. doi:10.1016/j.biortech.2012.09.012.

    Article  CAS  Google Scholar 

  3. Phillips, C. M., Beeson, W. T., Cate, J. H., & Marletta, M. A. (2011). Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chemical Biology, 6, 1399–1406. doi:10.1021/cb200351y.

    Article  CAS  Google Scholar 

  4. Mohanram, S., Amat, D., Choudhary, J., Arora, A., & Nain, L. (2013). Novel perspectives for evolving enzyme cocktails for lignocellulose hydrolysis in biorefineries. Sustainable Chemical Processes, 1(1), 15. doi:10.1186/2043-7129-1-15.

    Article  Google Scholar 

  5. Ensor, L. A., Stosz, S. K., & Weiner, R. M. (1999). Expression of multiple complex polysaccharide-degrading enzyme systems by marine bacterium strain 2-40. Journal of Industrial Microbiology and Biotechnology, 23(2), 123–126. doi:10.1038/sj.jim.2900696.

    Article  CAS  Google Scholar 

  6. Taylor, L. E., Henrissat, B., Coutinho, P. M., Ekborg, N. A., Hutcheson, S. W., & Weiner, R. M. (2006). Complete cellulase system in the marine bacterium Saccharophagus degradans Strain 2-40(T). Journal of Bacteriology, 188(11), 3849–3861. doi:10.1128/JB.01348-05.

    Article  CAS  Google Scholar 

  7. Ekborg, N. A., Gonzalez, J. M., Howard, M. B., Taylor, L. E., Hutcheson, S. W., & Weiner, R. M. (2005). Saccharophagus degradans gen. nov., sp. nov., a versatile marine degrader of complex polysaccharides. International Journal of Systematic and Evolutionary Microbiology, 55(4), 1545–1549. doi:10.1099/ijs.0.63627-0.

    Article  CAS  Google Scholar 

  8. Shin, M. H., Lee, D. Y., Skogerson, K., Wohlgemuth, G., Choi, I.-G., Fiehn, O., et al. (2010). Global metabolic profiling of plant cell wall polysaccharide degradation by Saccharophagus degradans. Biotechnology and Bioengineering, 105(3), 477–488. doi:10.1002/bit.22557.

    Article  CAS  Google Scholar 

  9. Robert, X., & Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(W1), W320–W324. doi:10.1093/nar/gku316.

    Article  CAS  Google Scholar 

  10. Isorna, P., Polaina, J., Latorre-García, L., Cañada, F. J., González, B., & Sanz-Aparicio, J. (2007). Crystal structures of Paenibacillus polymyxa beta-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases. Journal of Molecular Biology, 371(5), 1204–1218. doi:10.1016/j.jmb.2007.05.082.

    Article  CAS  Google Scholar 

  11. Yamamoto, K., Miyake, H., Kusunoki, M., & Osaki, S. (2011). Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae. Journal of Bioscience and Bioengineering, 112(6), 545–550. doi:10.1016/j.jbiosc.2011.08.016.

    Article  CAS  Google Scholar 

  12. Hassan, N., Nguyen, T.-H., Intanon, M., Kori, L., Patel, B. C., Haltrich, D., et al. (2015). Biochemical and structural characterization of a thermostable β-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis. Applied Microbiology and Biotechnology, 99(4), 1731–1744. doi:10.1007/s00253-014-6015-x.

    Article  CAS  Google Scholar 

  13. Matsuzawa, T., Jo, T., Uchiyama, T., Manninen, J. A., Arakawa, T., Miyazaki, K., et al. (2016). Crystal structure and identification of a key amino acid for glucose tolerance, substrate specificity and transglycosylation activity of metagenomic β-glucosidase Td2F2. The FEBS Journal,. doi:10.1111/febs.13743.

    Google Scholar 

  14. Camilo, C. M., & Polikarpov, I. (2014). High-throughput cloning, expression and purification of glycoside hydrolases using ligation-independent cloning (LIC). Protein Expression and Purification, 99, 35–42. doi:10.1016/j.pep.2014.03.008.

    Article  CAS  Google Scholar 

  15. Petersen, T., Brunak, S., von Heij, H. G., & Nielson, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 8, 785–786.

    Article  CAS  Google Scholar 

  16. Neuhoff, V., Stamm, R., & Eibl, H. (1985). Clear background and highly sensitive protein staining with Coomassie blue dyes in polyacrylamide gels: A systematic analysis. Electrophoresis, 6(9), 427–448. doi:10.1002/elps.1150060905.

    Article  CAS  Google Scholar 

  17. Zhang, Y. H. P., & Lynd, L. R. (2004). Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnology and Bioengineering, 88, 797–824.

    Article  CAS  Google Scholar 

  18. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428. doi:10.1021/ac60147a030.

    Article  CAS  Google Scholar 

  19. Leatherbarrow, R. J. (1987). Enzfitter A non-linear regression data analysis program for the IBM PC. Amsterdam: Elsevier.

    Google Scholar 

  20. Provencher, S. W. (1982). CONTIN: A general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Computer Physics Communications, 27(3), 229–242. doi:10.1016/0010-4655(82)90174-6.

    Article  Google Scholar 

  21. Hammersley, A. (1997). FIT2D: An introduction and overview. Grenoble: European Synchrotron Radiation Source.

    Google Scholar 

  22. Petoukhov, M. V., Franke, D., Shkumatov, A. V., Tria, G., Kikhney, A. G., Gajda, M., et al. (2012). New developments in the ATSAS program package for small-angle scattering data analysis. Journal of Applied Crystallography, 45(2), 342–350. doi:10.1107/S0021889812007662.

    Article  CAS  Google Scholar 

  23. Svergun, D. I. (1992). Determination of the regularization parameter in indirect-transform methods using perceptual criteria. Journal of Applied Crystallography, 25, 495–503.

    Article  Google Scholar 

  24. Franke, D., & Svergun, D. I. (2009). DAMMIF, a program for rapid ab initio shape determination in small-angle scattering. Journal of Applied Crystallography, 42(2), 342–346. doi:10.1107/S0021889809000338.

    Article  CAS  Google Scholar 

  25. Petoukhov, M. V., & Svergun, D. I. (2013). Applications of small-angle X-ray scattering to biomacromolecular solutions. The International Journal of Biochemistry & Cell Biology, 45(2), 429–437. doi:10.1016/j.biocel.2012.10.017.

    Article  CAS  Google Scholar 

  26. Kozin, M. B., & Svergun, D. I. (2001). Automated matching of high- and low-resolution structural models. Journal of Applied Crystallography, 34, 33–41.

    Article  CAS  Google Scholar 

  27. Svergun, D., Barberato, C., & Koch, M. H. J. (1995). CRYSOL—A program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. Journal of Applied Crystallography, 28(6), 768–773.

    Article  CAS  Google Scholar 

  28. Fischer, H., de Oliveira Neto, M., Napolitano, H. B., Polikarpov, I., & Craievich, A. F. (2010). Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale. Journal of Applied Crystallography, 43(1), 101–109. doi:10.1107/S0021889809043076.

    Article  CAS  Google Scholar 

  29. Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9(1), 40. doi:10.1186/1471-2105-9-40.

    Article  Google Scholar 

  30. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., & Henrissat, B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Research, 42(D1), D490–D495. doi:10.1093/nar/gkt1178.

    Article  CAS  Google Scholar 

  31. Turan, Y. (2008). A pseudo-β-glucosidase in Arabidopsis thaliana: Correction by site-directed mutagenesis, heterologous expression, purification, and characterization. Biochemistry (Moscow), 73(8), 912–919. doi:10.1134/S0006297908080099.

    Article  CAS  Google Scholar 

  32. Gao, J., & Wakarchuk, W. (2014). Characterization of five β-glycoside hydrolases from Cellulomonas fimi ATCC 484. Journal of Bacteriology, 196(23), 4103–4110. doi:10.1128/JB.02194-14.

    Article  Google Scholar 

  33. Feigin, L. A., & Svergun, D. I. (1987). General principles of small-angle diffraction. In G. W. Taylor (Ed.), Structure analysis by small-angle X-ray and neutron scattering (pp. 25–55). New York: Springer. doi:10.1007/978-1-4757-6624-0.

    Chapter  Google Scholar 

  34. Schmidt, M., Nerger, D., & Burchard, W. (1979). Quasi-elastic light scattering from branched polymers: 1. Polyvinylacetate and polyvinylacetate-microgels prepared by emulsion polymerization. Polymer, 20(5), 582–588.

    Article  CAS  Google Scholar 

  35. Chen, Z.-Y., Weakliem, P. C., & Meakin, P. (1988). Hydrodynamic radii of diffusion-limited aggregates and bond-percolation clusters. The Journal of Chemical Physics, 89(9), 5887. doi:10.1063/1.455732.

    Article  CAS  Google Scholar 

  36. Senff, H., & Richtering, W. (1999). Temperature sensitive microgel suspensions: Colloidal phase behavior and rheology of soft spheres. The Journal of Chemical Physics, 111(4), 1705–1711.

    Article  CAS  Google Scholar 

  37. Dünweg, B., Reith, D., Steinhauser, M., & Kremer, K. (2002). Corrections to scaling in the hydrodynamic properties of dilute polymer solutions. The Journal of Chemical Physics, 117(2), 914. doi:10.1063/1.1483296.

    Article  Google Scholar 

  38. Rambo, R. P., & Tainer, J. A. (2011). Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers, 95(8), 559–571. doi:10.1002/bip.21638.

    Article  CAS  Google Scholar 

  39. Kachala, M. (2015). Development of methods to analyze and represent small-angle scattering data from interacting and flexible biological macromolecules. Universität Hamburg.

  40. Hammel, M. (2012). Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS). EBJ European Biophysics Journal, 41(10), 789–799. doi:10.1007/s00249-012-0820-x.

    Article  CAS  Google Scholar 

  41. Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., & Zhang, Y. (2014). The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12(1), 7–8. doi:10.1038/nmeth.3213.

    Article  Google Scholar 

  42. Wang, X., He, X., Yang, S., An, X., Chang, W., & Liang, D. (2003). Structural basis for thermostability of β-glycosidase from the thermophilic eubacterium Thermus nonproteolyticus HG102. Journal of Bacteriology, 185(14), 4248–4255.

    Article  CAS  Google Scholar 

  43. Nam, K. H., Kim, S.-J., Kim, M.-Y., Kim, J. H., Yeo, Y.-S., Lee, C.-M., et al. (2008). Crystal structure of engineered beta-glucosidase from a soil metagenome. Proteins, 73(3), 788–793. doi:10.1002/prot.22199.

    Article  CAS  Google Scholar 

  44. de Giuseppe, P. O., Souza, T. D. A. C. B., Souza, F. H. M., Zanphorlin, L. M., Machado, C. B., Ward, R. J., et al. (2014). Structural basis for glucose tolerance in GH1 β-glucosidases. Acta Crystallographica. Section D: Biological Crystallography, 70(6), 1631–1639. doi:10.1107/S1399004714006920.

    Article  Google Scholar 

  45. Zanphorlin, L. M., de Giuseppe, P. O., Honorato, R. V., Tonoli, C. C. C., Fattori, J., Crespim, E., et al. (2016). Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. Scientific Reports, 6, 23776. doi:10.1038/srep23776.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The work was supported by the Grants from Brazilian funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)#2008/56255-9; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)#490022/2009-0,#158752/2015-5 and by a collaborative Grant agreement between the University of Hamburg, Germany and the University of Sao Paulo (USP), Brazil.

Author information

Authors and Affiliations

  1. Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, São Carlos, SP, 13566-590, Brazil

    Hevila Brognaro, Evandro Ares de Araujo, Vasily Piyadov, Maria Auxiliadora Morim Santos & Igor Polikarpov

  2. Instituto de Química, Universidade de São Paulo, Avenida Prof. Lineu Prestes, 748, Bloco 10, Sala 1054, São Paulo, SP, 05508-900, Brazil

    Vitor Medeiros Almeida & Sandro Roberto Marana

Authors
  1. Hevila Brognaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vitor Medeiros Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evandro Ares de Araujo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vasily Piyadov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maria Auxiliadora Morim Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sandro Roberto Marana
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Igor Polikarpov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Igor Polikarpov.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Brognaro, H., Almeida, V.M., de Araujo, E.A. et al. Biochemical Characterization and Low-Resolution SAXS Molecular Envelope of GH1 β-Glycosidase from Saccharophagus degradans . Mol Biotechnol 58, 777–788 (2016). https://doi.org/10.1007/s12033-016-9977-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-016-9977-3

Keywords

Search

Navigation