Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

Authors Affiliation(s)

  • 1Department of Biotechnology, National Institute of Technology Durgapur, Mahatma Gandhi Avenue, Durgapur 713209, INDIA
  • 2Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City 57701, USA

Can J Biotech, Volume 1, Issue 1, Pages 19-25, DOI: https://doi.org/10.24870/cjb.2017-000105

Received: Feb 13, 2017; Revised: Mar 15, 2017; Accepted: Mar 20, 2017

Abstract

Cellulolytic enzymes are promising candidates for the use of cellulose in any bioprocess operations and for the disposal of the cellulosic wastes in an environmentally benign manner. Cellulases from thermophiles have the advantage of hydrolyzing cellulose at wider range of operating conditions unlike the normal enzymes. Herein we report the modeled structures of cellulolytic enzymes (endoglucanase, cellobiohydrolase and ß-glucosidase) from a thermophilic bacterium, Clostridium thermocellum and their validation using Root Mean Square Deviation (RMSD) and Ramachandran plot analyses. Further, the molecular interactions of the modeled enzyme with cellulose were analyzed using molecular docking technique. The results of molecular docking showed that the endoglucanase, cellobiohydrolase and ß-glucosidase had the binding affinities of -10.7, -9.0 and -10.8 kcal/mol, respectively. A correlation between the binding affinity of the endoglucanase with cellulose and the enzyme activity was also demonstrated. The results showed that the binding affinities of cellulases with cellulose could be used as a tool to assess the hydrolytic activity of cellulases. The results obtained could be used in virtual screening of cellulolytic enzymes based on the molecular interactions with the substrate, and aid in developing systems biology models of thermophiles for industrial biotechnology applications.

References

  1. Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W. and Foust, T.D. (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315: 804-807. Crossref
  2. Navanietha Krishnaraj, R., Berchmans, S. and Pal, P. (2014) Symbiosis of photosynthetic microorganisms with non photosynthetic ones for the conversion of cellulosic mass into electrical energy and pigments. Cellulose 21: 2349-2355. Crossref
  3. Navanietha Krishnaraj, R., Berchmans, S. and Pal, P. (2015) The three-compartment microbial fuel cell: a new sustainable approach to bioelectricity generation from lignocellulosic biomass. Cellulose 22: 655- 662. Crossref
  4. U.S. Department of Energy. 2011. U.S., Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. R.D. Perlack and B.J. Stokes (Leads), ORNL/TM-2011/224. Oak Ridge National Laboratory, Oak Ridge, TN. 227p.
  5. Van Dyk, J.S. and Pletschke, B.I. (2012) A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnol Adv 30: 1458-1480. Crossref
  6. Rastogi, G., Bhalla, A., Adhikari, A., Bischoff, K.M., Hughes, S.R., Christopher, L.P. and Sani, R.K. (2010) Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresour Technol 101: 8798-8806. Crossref
  7. Bhuvaneswari, A., Navanietha Krishnaraj, R. and Berchmans, S. (2013) Metamorphosis of pathogen to electrigen at the electrode/electrolye interface: Direct electron transfer of Staphylococcus aureus leading to superior electrocatalytic activity. Electrochem Commun 34: 25-28. Crossref
  8. Béguin, P., Millet, J., Chauvaux, S., Salamitou, S., Tokatlidis, K., Navas, J., Fujino, T., Lemaire, M., Raynaud, O., Daniel, M.K. and Aubert, J.P. (1992) Bacterial cellulases. Biochem Soc Trans 20: 42-46. Crossref
  9. Chang, C.J., Lee, C.C., Chan, Y.T., Trudeau, D.L., Wu, M.H., Tsai, C.H., Yu, S.M., David Ho, T.H., Wang, A.H.J., Hsiao, C.D., Arnold, F.H. and Chao, Y.C. (2016) Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination. PLoS One 11: e0147485. Crossref
  10. Hakamada, Y., Hatada, Y., Ozawa, T., Ozaki, K., Kobayashi, T. and Ito, S. (2001) Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis. FEMS Microbiol Lett 195: 67-72. Crossref
  11. Hakamada, Y., Hatada, Y., Koike, K., Yoshimatsu, T., Kawai, S., Kobayashi, T. and Ito, S. (2000) Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an Alkaliphilic bacillus strain. Biosci Biotechnol Biochem 64: 2281-2289. Crossref
  12. Dodda, S.R., Sarkar, N., Aikat, K., Navanietha Krishnaraj, R., Bhattacharjee, S., Bagchi, A. and Mukhopadhyay, S.S. (2016) Insights from the Molecular Dynamics Simulation of Cellobiohydrolase Cel6A Molecular Structural Model from Aspergillus fumigates NITDGPKA3. Comb Chem High Throughput Screen 19: 325-333. Crossref
  13. Akinosho, H., Yee, K., Close, D. and Ragauskas, A. (2014) The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications. Front Chem 2: 66. Crossref
  14. Zertuche, L. and Zall, R.R. (1982) A study of producing ethanol from cellulose using Clostridium thermocellum. Biotechnol Bioeng 24: 57-68. Crossref
  15. Liu, Y., Yu, P., Song, X. and Qu, Y. (2008) Hydrogen production from cellulose by co-culture of Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17. Int J Hydrogen Energy 33: 2927-2933. Crossref
  16. Sparling, R., Islam, R., Cicek, N., Carere, C., Chow, H. and Levin, D.B. (2006) Formate synthesis by Clostridium thermocellum during anaerobic fermentation. Can J Microbiol 52: 681-688. Crossref
  17. Hirano, K., Kurosaki, M., Nihei, S., Hasegawa, H., Shinoda, S., Haruki, M. and Hirano, N. (2016) Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass. Sci Rep 6: 35709. Crossref
  18. Bairoch, A. and Apweiler, R. (2000) The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res 28: 45-48.
  19. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402.
  20. Berman, H., Henrick, K. and Nakamura, H. (2003) Announcing the worldwide Protein Data Bank. Nat Struct Mol Biol 10: 980. Crossref
  21. Thompson, J.D., Gibson, T.J. and Higgins, D.G. (2002) Multiple sequence alignment using ClustalW and ClustalX., Curr Protoc Bioinformatics Chapter 2: Unit 2.3. Crossref
  22. Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26: 283-291. Crossref
  23. Morris, A.L., MacArthur, M.W., Hutchinson, E.G. and Thornton, J.M. (1992) Stereochemical quality of protein structure coordinates. Proteins: Struct, Funct, Bioinf 12: 345-364. Crossref
  24. Trott, O. and Olson, A.J. (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 3: 455-461. Crossref
  25. Tong, C.C., Cole, A.L. and Shepherd, M.G. (1980) Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus. Biochem J 191: 83-94. Crossref
  26. Pirota, R.D.P.B., Miotto, L.S., Delabona, P.S. and Farinas, C.S. (2013) Improving the extraction conditions of endoglucanase produced by Aspergillus niger under solid-state fermentation. Braz J Chem Eng 30. Crossref
  27. Haq, I., Akram, F., Khan, M.A., Hussain, Z., Nawaz, A., Iqbal, K. and Shah, A.J. (2015) CenC, a multidomain thermostable GH9 processive endoglucanase from Clostridium thermocellum: cloning, characterization and saccharification studies. World J Microbiol Biotechnol 31: 1699-1710. Crossref
  28. Navanietha Krishnaraj, R., Chandran, S., Pal, P. and Berchmans, S. (2013) Screening of Photosynthetic Pigments for Herbicidal Activity with a New Computational Molecular Approach. Comb Chem High Throughput Screen 16: 777-781.
  29. Mahato, D., Samanta, D., Mukhopadhyay, S.S. and Navanietha Krishnaraj, R. (2016) A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia. J Recept Signal Transduct 37: 276-282. Crossref
  30. Navanietha Krishnaraj, R., Sreeja Kumari, S.S., and Mukhopadhyay, S.S. 2016. Antagonistic Molecular Interactions of Photosynthetic Pigments with Molecular Disease Targets-A New Approach to Treat AD and ALS. J Recept Signal Transduct 36: 67-71. Crossref
  31. Navanietha Krishnaraj, R., Chandran, S., Pal, P. and Berchmans, S. (2014) Investigations on the Antiretroviral Activity of Carbon Nanotubes using Computational Molecular Approach. Comb Chem High Throughput Screen 17: 531-535. Crossref
  32. Navanietha Krishnaraj, R., Chandran, S., Pal, P., Varalakshmi, P. and Malliga, P. (2014) Molecular Interactions of Graphene with HIV-Vpr, Nef and Gag Proteins. Korean J Chem Eng 31: 744-747. Crossref
  33. Navanietha Krishnaraj, R., Chandran, S., Pal, P. and Berchmans, S. (2014) Molecular Modeling and Assessing the Catalytic Activity of Glucose dehydrogenase of Gluconobacter suboxydans with a New Approach for Power Generation in a Microbial Fuel Cell. Curr Bioinform 9: 327-330. Crossref
  34. Navanietha Krishnaraj, R., and Pal, P. (2017) Enzyme-Substrate Interaction based Approach for Screening Electroactive Microorganisms for Microbial Fuel Cell Applications. Indian J Chem Technol 24: 93-96.