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Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73

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Abstract

An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73 showed high enzymatic activity within a broad pH range (pH 4–10), with a peak activity at pH 6.5. The optimal temperature for enzymatic activity was observed at 55°C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins of B. thuringiensis.

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References

  1. Muzarelly R. A. A., Jeuniaux C., & Gooday, G. W. (1986). Chitin in nature and technology (pp. 39–50). New York: Pergamon Press.

    Google Scholar 

  2. Granados, R. R., Fu, Y., Corsaro, B., & Hughes, P. R. (2001). Enhancement of Bacillus thuringiensis toxicity to lepidopterous species with the enhancin from Trichoplusia ni granulovirus. Biological Control, 20, 153–159.

    Article  CAS  Google Scholar 

  3. Shabrukova, N. V., Shestakova, L. M., Zainetdinova, D. R., & Gamayurova, V. S. (2002). Research of acid hydrolysis of chitin-glucan and chitosan-glucan complexes. Chemistry and Computational Simulation. Butlerov Communication, 2, 57–59.

    Google Scholar 

  4. Regev, A., Keller, M., Strizhov, N., Sneh, B., Prudovski, E., Chet, I., Ginzberg, I., Koncz-Kalman, Z., Konzcz, Z., Schell, J., & Zilberstein, A. (1996). Synergistic activity of Bacillus thuringiensis δ-endotoxin and a bacterial endochitinase against Spodoptera littoralis. Applied and Environmental Microbiology, 62, 3581–3586.

    PubMed  CAS  Google Scholar 

  5. Reyes-Ramírez, A., Escudero-Abarca, B. I., Aguilar-Uscanga, G., Hayward-Jones, P. M., & Barboza-Corona, J. E. (2004). Antifungal activity of Bacillus thuringiensis chitinase and its potential for the biocontrol of phytopathogenic fungi in soybean seeds. Journal of Food Science, 69, M131–134.

    Article  Google Scholar 

  6. Morales de la Vega, L., Barboza-Corona, J. E., Aguilar-Uscanga, M. G., & Ramírez-Lepe, M. (2006). Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi. Canadian Journal of Microbiology, 52, 651–657.

    Article  CAS  Google Scholar 

  7. Casique-Arroyo, G., Bideshi, D., Salcedo-Hernández, R., & Barboza-Corona, J. E. (2007). Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74. Antonie van Leeuwenhoek, 92, 1–9.

    Article  PubMed  CAS  Google Scholar 

  8. Höfte, H., & Whiteley, H. R. (1989). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiological Reviews, 53, 242–255.

    PubMed  Google Scholar 

  9. Chernin, L., Ismailov, Z., Haran, S., & Chet, I. (1995). Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Applied and Environmental Microbiology, 61, 1720–1726.

    PubMed  CAS  Google Scholar 

  10. Bhattacharya, D., Nagpure, A., & Gupta, R. K. (2007). Bacterial chitinases: Properties and potential. Critical Reviews in Biotechnology, 27, 21–28.

    Article  PubMed  CAS  Google Scholar 

  11. Watanabe, T., Kimura, K., Sumiya, T., Nikaidou, N., Suzuki, K., Suzuki, M., Taiyoji, M., Ferrer, S., & Regue M. (1997). Genetic analysis of the chitinase system of Serratia marcescens 2170. Journal of Bacteriology, 179, 7111–7117.

    PubMed  CAS  Google Scholar 

  12. Wu, M. L., Chuang, Y. C., Chen, J. P., Chen, C. S., & Chang M. C. (2001). Identification and characterization of the three chitin-binding domains with the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Applied and Environmental Microbiology, 67, 5100–5106.

    Article  PubMed  CAS  Google Scholar 

  13. Susuki, K., Sugawara, N., Susuki, M., Uchiyama, T., Katouno, F., Nikaidou, N., & Watanabe T. (2002). Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: Enzymatic properties and synergism on chitin degradation. Bioscience, Biotechnology, and Biochemistry, 66, 1075–1083.

    Article  Google Scholar 

  14. Thamthiankul, S., Suan-Ngay, S., Tantimavanich, S., & Panbangred, W. (2001). Chitinase from Bacillus thuringiensis subsp. pakistani. Applied Microbiology and Biotechnology, 56, 395–401.

    Article  PubMed  CAS  Google Scholar 

  15. Barboza-Corona, J. E., Nieto-Mazzocco, E., Velázquez-Robledo, R., Salcedo-Hernández, R., Bautista, M., Jiménez, B., & Ibarra, J. E. (2003). Cloning, sequencing, and expresión of the chitinase gene chiA74 from Bacillus thuringiensis. Applied and Environmental Microbiology, 69, 1023–1029.

    Article  PubMed  CAS  Google Scholar 

  16. Zhong, W. F., Jiang, L. H., Yan, W. Z., Cai, P. Z, Zhang, Z. X., & Pei, Y. (2003). Cloning and sequence of chitinase gene from Bacillus thuringiensis subsp. israeliensis. Yi Chuan Xue Bao, 30, 364–369 (In Chinese).

    PubMed  CAS  Google Scholar 

  17. Arora, N., Ahmad T., Rajagopal R., & Bhatnagar R. K. (2003). A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochemical and Biophysical Research Communications, 307, 620–625.

    Article  PubMed  CAS  Google Scholar 

  18. Lin, Y., & Xiong, G. (2004). Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti. Biotechnology Letters, 26, 635–639.

    Article  PubMed  CAS  Google Scholar 

  19. Driss, F., Kallassy-Awad, M., Zouari, N., & Jaoua, S. (2005). Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki. Journal of Applied Microbiology, 99, 945–953.

    Article  PubMed  CAS  Google Scholar 

  20. Lereclus, D., Arantes, O., Chaufaux, J., & Lecadet, M. M. (1989). Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis. FEMS Microbiology Letters, 60, 211–218.

    CAS  Google Scholar 

  21. Pospiech, A., & Neumann, B. (1995). A versatile quick-prep of genomic DNA form gram-positive bacteria. Trends in Genetics, 11, 217–218.

    Article  PubMed  CAS  Google Scholar 

  22. Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 74, 5463–5467.

    Article  PubMed  CAS  Google Scholar 

  23. Barboza-Corona J. E., Contreras J.C., Velázquez-Robledo, R., Bautista-Justo, M., Cruz-Camarillo, R., & Ibarra J. E. (1999). Selection of chitinolytic strain of Bacillus thuringiensis. Biotechnology Letters, 21, 1125–1129.

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  25. Ruiz-Sanchez A, Cruz-Camarillo, R., Salcedo-Hernández, R., Ibarra, J., & Barboza-Corona, J. E. (2005). Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima). Molecular Biotechnology, 31, 103–112.

    Article  PubMed  CAS  Google Scholar 

  26. Hardt, M., & Laine, R. A. (2004). Mutation of active site residues in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: Use of a green fluorescent protein binding assay. Archives of Biochemistry and Biophysics, 426, 256–297.

    Article  CAS  Google Scholar 

  27. Chang, M. C., Lai, P. L., & Wu, M. L. (2004). Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromononas hydrophila JP101. FEMS Microbiology Letters, 232, 61–66.

    Article  PubMed  CAS  Google Scholar 

  28. Ferrandon, S., Sterzenbach, T., Mersha, F. B., & Xu, M. Q. (2003). A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag. Biochimica et Biophysica Acta, 1621, 31–40.

    PubMed  CAS  Google Scholar 

  29. Watanabe, T., Susuki, K., Oyanagi, W., Ohnishi, K., & Tanaka, H. (1990). Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. Journal of Biological Chemistry, 265, 15659–15665.

    PubMed  CAS  Google Scholar 

  30. Watanabe, T., Oyanagi, W., Suzuki, K., Ohnishi, K., Tanaka, H. (1992). Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases. Journal of Bacteriology, 174, 408–414.

    PubMed  CAS  Google Scholar 

  31. Chernin, L., de la Fuente, L., Sovoleb, V., Haran, S., Vorgias, C. E., Oppehneim, A. B., & Chet, I. (1997). Molecualr cloning, structural análisis, and expresión in Escherichia coli of a chitinase gene from Enterobacter agglomerans. Applied and Environmental Microbiology, 63, 834–839.

    PubMed  CAS  Google Scholar 

  32. Du, C., & Nickerson, K. W. (1996). Bacillus thuringiensis HD-73 spores have surface-localized Cry1Ac toxin: Physiological and pathogenic consequences. Applied and Environmental Microbiology, 62, 3722–3726.

    PubMed  CAS  Google Scholar 

  33. Brurberg, M. B., Eijsink, V. G. H., & Nes, I. F. (1994). Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification of the gene product. FEMS Microbiology Letters, 124, 399–404.

    Article  PubMed  CAS  Google Scholar 

  34. Lewin, B. (2000). Genes VII (pp. 147–149). New York: Oxford University Press Inc.

    Google Scholar 

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Acknowledgments

This research was supported by grant SEP-CONACYT (2003-C02-44990) from México. The authors are grateful for the technical assistance of Crysthian de Jesús Lázaro Que of the Instituto Tecnológico de la Zona Olmeca, Tabasco, Mexico, and to the Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), at Irapuato, Guanajuato, Mexico, for gene sequencing.

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Authors and Affiliations

  1. Instituto de Ciencias Agrícolas, Departamento de Ingeniería en Alimentos, Universidad de Guanajuato, Apartado postal 311, Irapuato, Guanajuato, 36500, Mexico

    J. Eleazar Barboza-Corona, Dora M. Reyes-Rios & Rubén Salcedo-Hernández

  2. Colegio de Estudios Científicos y Tecnológicos del Estado de Guanajuato, Plantel Irapuato Valle Verde, Irapuato, Guanajuato, 36568, Mexico

    Dora M. Reyes-Rios

  3. Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA, 92504, USA

    Dennis K. Bideshi

  4. Department of Entomology, University of California, Riverside, CA, 92521, USA

    Dennis K. Bideshi

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  1. J. Eleazar Barboza-Corona
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  2. Dora M. Reyes-Rios
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  3. Rubén Salcedo-Hernández
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Correspondence to J. Eleazar Barboza-Corona.

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Barboza-Corona, J.E., Reyes-Rios, D.M., Salcedo-Hernández, R. et al. Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73. Mol Biotechnol 39, 29–37 (2008). https://doi.org/10.1007/s12033-007-9025-4

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  • DOI: https://doi.org/10.1007/s12033-007-9025-4

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