Skip to main content
Log in

A Novel Member of GH16 Family Derived from Sugarcane Soil Metagenome

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Glycoside hydrolases (GHs) are enzymes found in all living kingdoms that are involved in multiple physiological functions. Due to their multiple enzymatic activities, GHs are broadly applied in bioethanol, food, and paper industry. In order to increase the productivity of these industrial processes, a constant search for novel and efficient enzymes has been proved to be necessary. In this context, metagenomics is a powerful approach to achieve this demand. In the current study, we describe the discovery and characterization of a novel member of GH16 family derived from the sugarcane soil metagenome. The enzyme, named SCLam, has 286 amino acid residues and displays sequence homology and activity properties that resemble known laminarases. SCLam is active against barley beta-glucan, laminarin, and lichenan (72, 33, and 10 U mg−1, respectively). The optimal reaction conditions were identified as 40 °C and pH 6.5. The low-resolution structure was determined using the small-angle X-ray scattering technique, revealing that SCLam is a monomer in solution with a radius of gyration equal to 19.6 Å. To the best of our knowledge, SCLam is the first nonspecific (1,3/1,3:1,4)-β-d-glucan endohydrolase (EC 3.2.1.6) recovered by metagenomic approach to be characterized.

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

Access this article

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

Similar content being viewed by others

References

  1. Amann, R. I., Ludwig, W., & Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59, 143–169.

    CAS  Google Scholar 

  2. Handelsman, J. (2004). Metagenomics: application of genomics to uncultured microorganisms. Microbiology and Molecular Biology Reviews, 68, 669–685.

    Article  CAS  Google Scholar 

  3. Schloss, P. D., & Handelsman, J. (2003). Biotechnological prospects from metagenomics. Current Opinion in Biotechnology, 14, 303–310.

    Article  CAS  Google Scholar 

  4. Kallifidas, D., Kang, H.-S., & Brady, S. F. (2012). Tetarimycin A, an MRSA-active antibiotic identified through induced expression of environmental DNA gene clusters. Journal of the American Chemical Society, 134, 19552–19555.

    Article  CAS  Google Scholar 

  5. MacNeil, I. A., Tiong, C. L., Minor, C., August, P. R., Grossman, T. H., Loiacono, K. A., Lynch, B. A., Phillips, T., Narula, S., Sundaramoorthi, R., Tyler, A., Aldredge, T., Long, H., Gilman, M., Holt, D., & Osburne, M. S. (2001). Expression and isolation of antimicrobial small molecules from soil DNA libraries. Journal of Molecular Microbiology and Biotechnology, 3, 301–308.

    CAS  Google Scholar 

  6. Wang, G. Y., Graziani, E., Waters, B., Pan, W., Li, X., McDermott, J., Meurer, G., Saxena, G., Andersen, R. J., & Davies, J. (2000). Novel natural products from soil DNA libraries in a streptomycete host. Organic Letters, 2, 2401–2404.

    Article  CAS  Google Scholar 

  7. Yun, J., Kang, S., Park, S., Yoon, H., Kim, M.-J., Heu, S., & Ryu, S. (2004). Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library. Applied and Environmental Microbiology, 70, 7229–7235.

    Article  CAS  Google Scholar 

  8. Jiang, C., Li, S.-X., Luo, F.-F., Jin, K., Wang, Q., Hao, Z.-Y., Wu, L.-L., Zhao, G.-C., Ma, G.-F., Shen, P.-H., Tang, X.-L., & Wu, B. (2011). Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning. Bioresource Technology, 102, 3272–3278.

    Article  CAS  Google Scholar 

  9. Alvarez, T. M., Goldbeck, R., dos Santos, C. R., Paixão, D. A. A., Gonçalves, T. A., Franco Cairo, J. P. L., Almeida, R. F., de Oliveira Pereira, I., Jackson, G., Cota, J., Büchli, F., Citadini, A. P., Ruller, R., Polo, C. C., de Oliveira Neto, M., Murakami, M. T., & Squina, F. M. (2013). Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome. PLoS One, 8, e70014.

    Article  CAS  Google Scholar 

  10. Liu, J., Liu, W.-D., Zhao, X.-L., Shen, W.-J., Cao, H., & Cui, Z.-L. (2011). Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library. Applied Microbiology and Biotechnology, 89, 1083–1092.

    Article  CAS  Google Scholar 

  11. Brennan, Y., Callen, W. N., Christoffersen, L., Dupree, P., Goubet, F., Healey, S., Hernández, M., Keller, M., Li, K., Palackal, N., Sittenfeld, A., Tamayo, G., Wells, S., Hazlewood, G. P., Mathur, E. J., Short, J. M., Robertson, D. E., & Steer, B. A. (2004). Unusual microbial xylanases from insect guts. Applied and Environmental Microbiology, 70, 3609–3617.

    Article  CAS  Google Scholar 

  12. Alvarez, T. M., Paiva, J. H., Ruiz, D. M., Cairo, J. P. L. F., Pereira, I. O., Paixão, D. A., de Almeida, R. F., Tonoli, C. C., Ruller, R., Santos, C. R., Squina, F. M., & Murakami, M. T. (2013). Structure and function of a novel cellulase 5 from sugarcane soil metagenome. PLoS One, 8, e83635.

    Article  Google Scholar 

  13. Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemistry Journal, 280, 309–316.

    Article  CAS  Google Scholar 

  14. Mamo, G., Faryar, R., & Karlsson, E. N. (2013). Microbial glycoside hydrolases for biomass utilization in biofuels applications. In V. K. Gupta & M. G. Tuhoy (Eds.), Biofuel technologies: Recent developments (pp. 171–188). Heidelberg: Springer.

    Chapter  Google Scholar 

  15. Juturu, V., & Wu, J. C. (2012). Microbial xylanases: engineering, production and industrial applications. Biotechnology Advances, 30, 1219–1227.

    Article  CAS  Google Scholar 

  16. Zheng, H., Guo, B., Chen, X.-L., Fan, S.-J., & Zhang, Y.-Z. (2011). Improvement of the quality of wheat bread by addition of glycoside hydrolase family 10 xylanases. Applied Microbiology and Biotechnology, 90, 509–515.

    Article  CAS  Google Scholar 

  17. Peberdy, J. F. (1990). Fungal cell walls. A review. In P. J. Kuhn, A. P. J. Trinci, M. J. Jung, & M. W. Goosey (Eds.), Biochemistry of cell walls and membranes in fungi (pp. 5–30). New York: Springer.

    Chapter  Google Scholar 

  18. Burton, R. A., & Fincher, G. B. (2009). (1,3;1,4)-beta-D-glucans in cell walls of the poaceae, lower plants, and fungi: a tale of two linkages. Molecular Plant, 2, 873–882.

    Article  CAS  Google Scholar 

  19. Olafsdottir, E. S., & Ingólfsdottir, K. (2001). Polysaccharides from lichens: structural characteristics and biological activity. Planta Medica, 67, 199–208.

    Article  CAS  Google Scholar 

  20. Davis, T. A., Volesky, B., & Mucci, A. (2003). A review of the biochemistry of heavy metal biosorption by brown algae. Water Research, 37, 4311–4330.

    Article  CAS  Google Scholar 

  21. Gueguen, Y., Voorhorst, W. G., van der Oost, J., & de Vos, W. M. (1997). Molecular and biochemical characterization of an endo-beta-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. Journal of Biological Chemistry, 272, 31258–31264.

    Article  CAS  Google Scholar 

  22. Kawai, R., Igarashi, K., Yoshida, M., Kitaoka, M., & Samejima, M. (2006). Hydrolysis of beta-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-beta-glucanase from the basidiomycete Phanerochaete chrysosporium. Applied Microbiology and Biotechnology, 71, 898–906.

    Article  CAS  Google Scholar 

  23. Iakiviak, M., Mackie, R. I., & Cann, I. K. (2011). Functional analyses of multiple lichenin-degrading enzymes from the rumen bacterium Ruminococcus albus 8. Applied and Environmental Microbiology, 77, 7541–7550.

    Article  CAS  Google Scholar 

  24. Masuda, S., Endo, K., Koizumi, N., Hayami, T., Fukazawa, T., Yatsunami, R., Fukui, T., & Nakamura, S. (2006). Molecular identification of a novel beta-1,3-glucanase from alkaliphilic Nocardiopsis sp. strain F96. Extremophiles, 10, 251–255.

    Article  CAS  Google Scholar 

  25. Strohmeier, M., Hrmova, M., Fischer, M., Harvey, A. J., Fincher, G. B., & Pleiss, J. (2004). Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae. Protein Science, 13, 3200–3213.

    Article  CAS  Google Scholar 

  26. Parrish, F. W., Perlin, A. S., & Resse, E. T. (1960). Selective enzymolysis of poly-J3-D-glucans, and the structure of the polymers. Canadian Journal of Chemistry, 38, 2094–2104.

    Article  CAS  Google Scholar 

  27. Pang, Z., Otaka, K., Maoka, T., Hidaka, K., Ishijima, S., Oda, M., & Ohnishi, M. (2005). Structure of beta-glucan oligomer from laminarin and its effect on human monocytes to inhibit the proliferation of U937 cells. Bioscience Biotechnology and Biochemistry, 69, 553–558.

    Article  CAS  Google Scholar 

  28. Zhan, X.-B., Lin, C.-C., & Zhang, H.-T. (2012). Recent advances in curdlan biosynthesis, biotechnological production, and applications. Applied Microbiology and Biotechnology, 93, 525–531.

    Article  CAS  Google Scholar 

  29. Bamforth, C. W. (2009). Current perspectives on the role of enzymes in brewing. Journal of Cereal Science, 50, 353–357.

    Article  CAS  Google Scholar 

  30. Humbert-Goffard, A., Saucier, C., Moine-Ledoux, V., Canal-Llaubères, R.-M., Dubourdieu, D., & Glories, Y. (2004). An assay for glucanase activity in wine. Enzyme and Microbial Technology, 34, 537–543.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein identification and analysis tool on the ExPASy server. In J. M. Walker (Ed.), The proteomics protocols handbook (pp. 571–607). Totowa: Humana.

    Chapter  Google Scholar 

  33. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.

    Article  CAS  Google Scholar 

  34. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.

    CAS  Google Scholar 

  35. 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, D490–D495.

    Article  CAS  Google Scholar 

  36. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  37. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  38. Evangelista, R. A., Liu, M.-S., & Chen, F.-T. A. (1995). Characterization of 9-aminopyrene-1,4,6-trisulfonate derivatized sugars by capillary electrophoresis with laser-induced fluorescence detection. Analytical Chemistry, 67, 2239–2245.

    Article  CAS  Google Scholar 

  39. Hammersley, A. P., Svensson, S. O., Hanfland, M., Fitch, A. N., & Hausermann, D. (1996). Two-dimensional detector software: from real detector to idealised image or two-theta scan. High Pressure Research, 14, 235–248.

    Article  Google Scholar 

  40. Guinier, A., Fournet, G., Walker, C., & Yudowitch, K. (1955). Small angle scattering of X-rays. New York: Wiley.

    Google Scholar 

  41. 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 

  42. Fischer, H., de Oliveira Neto, M., Napolitano, H. B., Polikarpov, I., & Craievich, A. F. (2009). 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, 101–109.

    Article  Google Scholar 

  43. Svergun, D. I., Petoukhov, M. V., & Koch, M. H. (2001). Determination of domain structure of proteins from X-ray solution scattering. Biophysical Journal, 80, 2946–2953.

    Article  CAS  Google Scholar 

  44. Volkov, V. V., & Svergun, D. I. (2003). Uniqueness of ab initio shape determination in small-angle scattering. Journal of Applied Crystallography, 36, 860–864.

    Article  CAS  Google Scholar 

  45. 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, 768–773.

    Article  CAS  Google Scholar 

  46. Bleicher, L., Prates, E. T., Gomes, T. C. F., Silveira, R. L., Nascimento, A. S., Rojas, A. L., Golubev, A., Martínez, L., Skaf, M. S., & Polikarpov, I. (2011). Molecular basis of the thermostability and thermophilicity of laminarinases: X-ray structure of the hyperthermostable laminarinase from Rhodothermus marinus and molecular dynamics simulations. Journal of Physical Chemistry B, 115, 7940–7949.

    Article  CAS  Google Scholar 

  47. Pang, Z., Otaka, K., Suzuki, Y., Goto, K., & Ohnishi, M. (2004). Purification and characterization of an endo-1,3-β-glucanase from Arthrobacter sp. Journal of Biological Macromolecules, 4, 57–66.

    CAS  Google Scholar 

  48. Cota, J., Alvarez, T. M., Citadini, A. P., Santos, C. R., de Oliveira Neto, M., Oliveira, R. R., Pastore, G. M., Ruller, R., Prade, R. A., Murakami, M. T., & Squina, F. M. (2011). Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-β-1,3-glucanase from Thermotoga petrophila. Biochemical and Biophysical Research Communications, 406, 590–594.

    Article  CAS  Google Scholar 

  49. Cheng, Y.-M., Hong, T.-Y., Liu, C.-C., & Meng, M. (2009). Cloning and functional characterization of a complex endo-beta-1,3-glucanase from Paenibacillus sp. Applied Microbiology and Biotechnology, 81, 1051–1061.

    Article  CAS  Google Scholar 

  50. Hong, T. Y., Cheng, C. W., Huang, J. W., & Meng, M. (2002). Isolation and biochemical characterization of an endo-1,3-beta-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-beta-glucan. Microbiology, 148, 1151–1159.

    CAS  Google Scholar 

  51. Bacic, A., Fincher, G. B., & Stone, B. A. (2009). Chemistry, biochemistry, and biology of 1-3 beta glucans and related polysaccharides. St. Louis: Elsevier.

    Google Scholar 

  52. Wood, P. J., Weisz, J., & Blackwell, B. A. (1994). Structural studies of (1-3)(1-4)-beta-D-glucans by 13C-nuclear magnetic resonance spectroscopy and by rapid analysis of cellulose-like regions using high-performance anion-exchange chromatography of oligosaccharides released by lichenase. Cereal Chemistry, 7, 301–307.

    Google Scholar 

  53. Cheng, R., Chen, J., Yu, X., Wang, Y., Wang, S., & Zhang, J. (2013). Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09. BMC Biotechnology, 13, 105.

    Article  CAS  Google Scholar 

  54. Krah, M., Misselwitz, R., Politz, O., Thomsen, K. K., Welfle, H., & Borriss, R. (1998). The laminarinase from thermophilic eubacterium Rhodothermus marinus-conformation, stability, and identification of active site carboxylic residues by site-direct mutagenesis. European Journal of Biochemistry, 257, 101–111.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge FAPESP for financial support to FMS (08/58037-9) and TMA (2010/11469-1); CNPq for financial support to FMS (442333/2014-5 and 310186/2014-5), MVL (158882/2014-8), and MON (478900/2012-0); and SAXS2 (LNLS/CNPEM) and CTBE/CNPEM for technical support.

Conflict of Interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Fabio Marcio Squina.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S1

SDS–PAGE analysis of SCLam purification. A) Affinity chromatography. Lane MW – molecular weight marker (labelled in kDa). Lane 1 – soluble fraction. Lane 2 – Flow-through. Lane 3 -Wash. Lanes 4 to 8 – Elutions. B) Size-exclusion chromatography. Lane MW - molecular weight marker (labelled in kDa). Lanes 1 to 3 - elution fractions from size-exclusion chromatography, in 20 mM sodium phosphate pH 7.4 containing 50 mM NaCl buffer. (JPEG 268 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alvarez, T.M., Liberato, M.V., Cairo, J.P.L.F. et al. A Novel Member of GH16 Family Derived from Sugarcane Soil Metagenome. Appl Biochem Biotechnol 177, 304–317 (2015). https://doi.org/10.1007/s12010-015-1743-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1743-7

Keywords

Navigation