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Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus

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Abstract

Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-d-cellobioside, 4-nitrophenyl-β-d-cellobioside and 4-nitrophenyl-β-d-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.

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Abbreviations

CMC:

Carboxymethyl cellulose

MUC:

4-methylumbelliferyl β-d-cellobioside

pNPC:

p-nitrophenol-β-d-cellobioside

pNPG3:

p-nitrophenol-β-d-cellotrioside

pNPL:

p-nitrophenol-β-d-lactoside

References

  1. Kudo, T. (2009). Termite-microbe symbiotic system and its efficient degradation of lignocellulose. Bioscience, Biotechnology and Biochemistry, 73, 2561–2567.

    Article  CAS  Google Scholar 

  2. Ohkuma, M. (2003). Termite symbiotic systems: Efficient bio-recycling of lignocellulose. Applied Microbiology and Biotechnology, 61, 1–9.

    Article  CAS  Google Scholar 

  3. US DOE. (2007). Biofuels: Bringing biological solutions to energy challenges. US Department of Energy Office of Science. http://genomicscience.energy.gov/pubs/Biofuels_Flyer_2007-2.pdf.

  4. King, P. J. H., Mahadi, N. M., Bong, C. F. J., Ong, K. H., & Hassan, O. (2014). Bacterial microbiome of Coptotermes curvignathus (Isoptera: Rhinotermitidae) reflects, the coevolution of species and dietary pattern. Insect Science, 21, 584–596.

    Article  CAS  Google Scholar 

  5. Brune, A. (1998). Termite guts: The world’s smallest bioreactors. Trends in Biotechnology, 16, 16–21.

    Article  CAS  Google Scholar 

  6. Brune, A. (2014). Symbiotic digestion of lignocellulose in termite guts. Nature Reviews, 12, 168–180.

    CAS  Google Scholar 

  7. Ni, J., & Tokuda, G. (2013). Lignocellulose-degrading enzymes from termites and their symbiotic microbiota. Biotechnology Advances, 31, 838–850.

    Article  CAS  Google Scholar 

  8. Lo, N., & Eggleton, P. (2011). Termite phylogenetics and co-cladogenesis with symbionts. In D. E. Bignell, Y. Roisin, & N. Lo (Eds.), Biology of termites: A modern synthesis (pp. 27–50). Berlin: Springer.

    Google Scholar 

  9. Tho, Y. P. (1992). Termites of Peninsular Malaysia in Malayan Forest Records No. 36. (Kirton, L.G. ed.). Forest Research Institute Malaysia, Kuala Lumpur, 36, 1–224.

    Google Scholar 

  10. Woon, J. S. K., Mackeen, M. M., Sudin, A. H., Mahadi, N. M., Ilias, R. M., Abdul Murad, A. M., et al. (2016). Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus Thielavia terrestris. Biotechnology Letters, 38(5), 825–832.

    Article  CAS  Google Scholar 

  11. Mansfield, S. D., Mooney, C., & Saddler, J. N. (1999). Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnology Progress, 15, 804–816.

    Article  CAS  Google Scholar 

  12. Woon, J. S. K., Mackeen, M. M., Mahadi, N. M., Ilias, R. M., Abdul Murad, A. M., & Abu Bakar, F. D. (2016). Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris. Biotechnology and Applied Biochemistry, 63(5), 690–698.

    Article  CAS  Google Scholar 

  13. Teeri, T. T. (1997). Crystalline cellulose degradation: New insight into the function of cellobiohydrolases. Trends in Biotechnology, 15, 160–167.

    Article  Google Scholar 

  14. Nakashima, K., Watanabe, H., & Azuma, J. I. (2002). Cellulase genes from the parabasalian symbiont Pseudotrichonympha grassii in the hindgut of the wood-feeding termite Coptotermes formosanus. Cellular and Molecular Life Sciences, 59, 1554–1560.

    Article  CAS  Google Scholar 

  15. Tokuda, G., & Watanabe, H. (2007). Hidden cellulases in termites: Revision of an old hypothesis. Biology Letters, 3, 336–339.

    Article  CAS  Google Scholar 

  16. Sethi, A., Kovaleva, E. S., Slack, J. M., Brown, S., Buchman, G. W., & Scharf, M. E. (2013). A GHF7 cellulase from the protist symbiont community of Reticulitermes flavipes enables more efficient lignocellulose processing by host enzymes. Archives of Insect Biochemistry and Physiology, 84(4), 175–193.

    Article  CAS  Google Scholar 

  17. Den Haan, R., Mcbride, J. E., Grange, D. C. La, Lynd, L. R., & Van Zyl, W. H. (2007). Functional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme and Microbial Technology, 40, 1291–1299.

    Article  Google Scholar 

  18. Wang, G., Zhang, X., Wang, L., Wang, K., Peng, F., & Wang, L. (2012). The activity and kinetic properties of cellulases in substrates containing metal ions and acid radicals. Advances in Biological Chemistry, 2(11), 390–395.

    Article  CAS  Google Scholar 

  19. Cregg, J. M., Vedvick, T. S., & Raschke, W. C. (1993). Recent advances in the expression of foreign genes in Pichia pastoris. Bio-Technology, 11, 905–910.

    CAS  Google Scholar 

  20. Valencia, J. A., Wang, H., & Siegfried, B. D. (2014). Expression and characterization of a recombinant endoglucanase from western corn rootworm, in Pichia pastoris. Journal of Insect Science, 1(14), 242.

    Article  Google Scholar 

  21. Sambrook, J. W., & Russell, D. (2001). Molecular cloning: A laboratory manual. New York: Cold Spring Harb Lab Press Cold Spring Harb.

    Google Scholar 

  22. Schulz, M. H., Zerbino, D. R., Vingron, M., & Birney, E. (2012). Oases: Robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics, 28(8), 1086–1092.

    Article  CAS  Google Scholar 

  23. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389–3402.

    Article  CAS  Google Scholar 

  24. Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., & Warren, R. A. (1991). Domains in microbial beta-1, 4-glycanases: Sequence conservation, function, and enzyme families. Microbiological Reviews, 55(2), 303–315.

    CAS  Google Scholar 

  25. 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 

  26. Lõoke, M., Kristjuhan, K., & Kristjuhan, A. (2011). Extraction of genomic DNA from yeast for PCR-based applications. Biotechniques, 50, 325–328.

    Google Scholar 

  27. Wan Seman, W. M. K., Bakar, S. A., Bukhari, N. A., Gaspar, S. M., Othman, R., Nathan, S., et al. (2014). High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions. Journal of Biotechnology, 184, 219–228.

    Article  CAS  Google Scholar 

  28. Bradford, M. (1976). Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye-binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociation constants. Journal of the American Chemical Society, 56(3), 658–666.

    Article  CAS  Google Scholar 

  31. Hoshino, E., Shiroishi, M., Amano, Y., Nomura, M., & Kanda, T. (1997). Synergistic actions of exo-type cellulases in the hydrolysis of cellulose with different crystallinities. Journal of Fermentation and Bioengineering, 84, 300–306.

    Article  CAS  Google Scholar 

  32. Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27(3), 297–306.

    Article  CAS  Google Scholar 

  33. Tomme, P., & Claeyssens, M. (1989). Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei: A chemical modification study. FEBS Letter, 243(2), 239–243.

    Article  CAS  Google Scholar 

  34. Humphrey, A. (1998). Shake flask to fermentor: What have we learned? Biotechnology Progress, 14(1), 3–7.

    Article  CAS  Google Scholar 

  35. Kyomuhendo, P., Myrnes, B., & Nilsen, I. W. (2007). A cold-active salmon goose-type lysozyme with high heat tolerance. Cellular and Molecular Life Sciences, 64, 2841–2847.

    Article  CAS  Google Scholar 

  36. Tokuda, G., Watanabe, H., Matsumoto, T., & Noda, H. (1997). Cellulose digestion in the wood-eating higher termite, Nasutitermes takasagoensis (Shiraki): distribution of cellulases and properties of endo-beta-1,4-glucanase. Zoological Science, 14(1), 83–97.

    Article  CAS  Google Scholar 

  37. Inoue, T., Moriya, S., Ohkuma, M., & Kudo, T. (2005). Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus. Gene, 349, 67–75.

    Article  CAS  Google Scholar 

  38. Ni, J., Tokuda, G., Takehara, M., & Watanabe, H. (2007). Heterologous expression and enzymatic characterization of β-glucosidase from the drywood-eating termite, Neotermes koshunensis. Applied Entomology and Zoology, 42(3), 457–463.

    Article  CAS  Google Scholar 

  39. Evans, T. A., Forschler, B. T., & Grace, J. K. (2013). Biology of invasive termites: A worldwide review. Annual Review of Entomology, 58, 455–474.

    Article  CAS  Google Scholar 

  40. Rouland, C., Lenoir-Rousseaux, J. J., Mora, P., & Renoux, J. (1989). Origin of the exocellulase and the beta-glucosidase purified from the digestive tract of the fungus-growing termite Macrotermes muelleri. Sociobiology, 15(1989), 237–246.

    Google Scholar 

  41. McEwen, S. E., Slaytor, M., & O’Brien, R. W. (1980). Cellobiase activity in three species of Australian termites. Insect Biochemistry, 10(5), 563–567.

    Article  CAS  Google Scholar 

  42. Ma, R. J., Wang, C. Y., Liu, Y. W., Sivakumar, T. R., Ren, Z. X., Fang, Y., et al. (2014). Identification and characterization of a novel endoglucanase (CMCase) isolated from the larval gut of Bombyx mori. Journal of Asia-Pacific Entomology, 17(1), 67–71.

    Article  CAS  Google Scholar 

  43. Beukes, N., & Pletschke, B. I. (2006). Effect of sulfur-containing compounds on Bacillus cellulosome-associated “CMCase” and “Avicelase” activities. FEMS Microbiology Letters, 264(2), 226–231.

    Article  CAS  Google Scholar 

  44. Boer, H., Teeri, T. T., & Koivula, A. (2000). Characterization of Trichoderma reesei cellobiohydrolase Cel7a secreted from Pichia pastoris using two different promoters. Biotechnology and Bioengineering, 69, 486–494.

    Article  CAS  Google Scholar 

  45. Akcapinar, G. B., Venturini, A., Martelli, P. L., Casadio, R., & Sezerman, U. O. (2015). Modulating the thermostability of Endoglucanase I from Trichoderma reesei using computational approaches. Protein Engineering, Design & Selection, 28(5), 127–135.

    Article  CAS  Google Scholar 

  46. Godbole, S., Decker, S. R., Nieves, R. A., Adney, W. S., Vinzant, T. B., Baker, J. O., et al. (1999). Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris. Biotechnological Progress, 15(5), 828–833.

    Article  CAS  Google Scholar 

  47. Soares, J. F., Dal Prá, V., Kempka, A. P., Prestes, R. C., Tres, M. V., Kuhn, R. C., et al. (2016). Cellulases for food applications. In V. Gupta (Ed.), New and future developments in microbial biotechnology and bioengineering: Microbial cellulase system properties and applications (pp. 201–208). Amsterdam: Elsevier.

    Chapter  Google Scholar 

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Acknowledgements

The authors would like to thank the Ministry of Science, Technology and Innovation (MOSTI) of Malaysia for providing the research grant 02-05-20-SF11118 and Shaman M. Gaspar (Infors South East Asia/Bumi Sains Sdn. Bhd.) for his help with the bioreactor.

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Author notes

  1. James Sy-Keen Woon

    Present address: Faculty of Medical Sciences, Newcastle University Medicine Malaysia, 79200, Iskandar Puteri, Johor, Malaysia

Authors and Affiliations

  1. School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia

    James Sy-Keen Woon, Abdul Munir Abdul Murad & Farah Diba Abu Bakar

  2. Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia Bintulu Sarawak Campus, 97008, Bintulu, Sarawak, Malaysia

    Patricia Jie Hung King

  3. School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia

    Mukram Mohamed Mackeen

  4. Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia

    Mukram Mohamed Mackeen & Nor Muhammad Mahadi

  5. Malaysia Genome Institute, Jalan Bangi Lama, 43000, Kajang, Selangor, Malaysia

    Nor Muhammad Mahadi & Wan Mohd Khairulikhsan Wan Seman

  6. Department 4 (Materials and Environment), Federal Institute of Materials Research and Testing, Unter den Eichen 87, 12205, Berlin, Germany

    William J. Broughton

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Correspondence to Farah Diba Abu Bakar.

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Woon, J.SK., King, P.J.H., Mackeen, M.M. et al. Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus . Mol Biotechnol 59, 271–283 (2017). https://doi.org/10.1007/s12033-017-0015-x

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