Skip to main content
SpringerLink
Log in

Purification and characterization of chitinases from Paecilomyces variotii DG-3 parasitizing on Meloidogyne incognita eggs

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.

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

Similar content being viewed by others

References

  1. Agrios GN (2005) Plant pathology. Elsevier Academic Press, New York

    Google Scholar 

  2. Baratto CM, Dutra V, Boldo JT, Leiria LB, Vainstein MH, Schrank A (2006) Isolation, characterization, and transcriptional analysis of the chitinase chi2 gene (DQ011663) from the biocontrol fungus Metarhizium anisopliae var. anisopliae. Curr Microbiol 53:217–221. doi:10.1007/s00284-006-0078-6

    Article  PubMed  CAS  Google Scholar 

  3. Bird AF, McClure MA (1975) The tylenchid (nematode) egg shell: structure, composition and permeability. Parasitology 72:19–28

    Article  Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive method for the quatitation of microgram quantities of protein utilizing the principle of protein-dye. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3

    Article  PubMed  CAS  Google Scholar 

  5. Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biotechnol 71:773–782. doi:10.1007/s00253-005-0183-7

    Article  PubMed  CAS  Google Scholar 

  6. Guthrie JL, Khalif S, Castle AJ (2005) An improved method for detection and quantification of chitinase activities. Can J Microbiol 51:491–495. doi:10.1139/w05-020

    Article  PubMed  CAS  Google Scholar 

  7. Henrissat B (1991) A classification of glycosyl hydrolates based on amino acid sequence similarities. Biochemistry 280:309–316

    CAS  Google Scholar 

  8. Hoell IA, Klemsdal SS, Vaaje-Kolstad G, Horn GJ, Eijsink VGH (2005) Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1. Biochem Biophysiol ACTA 1748:180–190

    CAS  Google Scholar 

  9. Hollis T, Monzingo AF, Bortone K, Ernst S, Cox R, Robertus JD (2000) The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis. Protein Sci 9:544–551

    PubMed  CAS  Google Scholar 

  10. Huang XAW, Zhao NH, Zhang KQ (2004) Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host. Res Microbiol 155:811–816. doi:10.1016/j.resmic.2004.07.003

    Article  PubMed  CAS  Google Scholar 

  11. Imoto T, Yamashita KY (1971) A simple activity measurement of lysozyme. Agric Biol Chem 35:1154–1156

    CAS  Google Scholar 

  12. Izume M, Ohtakara A (1987) Preparation of D-glucosamine oligosaccharides by the enzymatic hydrolysis of chitosan. Agric Biol Chem 51:1189–1191

    CAS  Google Scholar 

  13. Jung WJ, Kuk JH, Kim KY, Kim TH, Park RD (2005) Purification and characterization of chitinase from Paenibacillus illinoisensis KJA-424. J Microbiol Biotechnol 15:274–280

    CAS  Google Scholar 

  14. Kang SC, Park S, Lee DG (1999) Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae. J Invertebr Pathol 73:276–281. doi:10.1006/jipa.1999.4843

    Article  PubMed  CAS  Google Scholar 

  15. Khan A, Williams K, Mark P, Molloy MP, Nevalainen H (2003) Purification and characterization of a serine protease and chitinase from Paecilomyces lilacinus and detection of chitinase activity on 2D gels. Protein Expr Purif 32:210–220. doi:10.1016/j.pep.2003.07.007

    Article  PubMed  CAS  Google Scholar 

  16. Khan A, Williams KL, Nevalainen HKM (2004) Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles. Biol Control 31:346–352. doi:10.1016/j.biocontrol.2004.07.011

    Article  CAS  Google Scholar 

  17. Kuk JH, Jung WJ, Jo GH, Kim YC, Kim KY, Park RD (2005) Production of N- acetyl-β-D-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme. Appl Microbiol Biotechnol 68:384–389. doi:10.1007/s00253-004-1877-y

    Article  PubMed  CAS  Google Scholar 

  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–683. doi:10.1038/227680a0

    Article  PubMed  CAS  Google Scholar 

  19. Leger S, Joshi RJ, Bidochka L, Roberts DW (1996) Characterization and ultrastructural localization of Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during fungal invasion of host (Manduca sexta) cuticle. Appl Environ Microbiol 62:907–912

    Google Scholar 

  20. Li CD (1998) Review of fungal chitinases. Mycopathology 161:345–360

    Google Scholar 

  21. Luke KA, Higgins CL, Wittung-Stafshede P (2007) Thermodynamic stability and folding of proteins from hyperthermophilic organism. FEBS J 274:4023–4033. doi:10.1111/j.1742-4658.2007.05955.x

    Article  PubMed  CAS  Google Scholar 

  22. Marco JLD, Valadares-Inglis MC (2003) Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis perniciosa. Appl Microbiol Biotechnol 64:70–75. doi:10.1007/s00253-003-1490-5

    Article  CAS  Google Scholar 

  23. Marco JLD, Lima LHC, Sousa MV, Felix CR (2000) A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa, the causal agent of witches’ broom disease of cocoa. J Microbiol Biotechnol 16:383–386

    Article  Google Scholar 

  24. Mavromatis K, Woo MLSL, Bouriotis V (2003) Mode of action and antifungal properties of two cold-adapted chitinases. Extremophiles 7:385–390. doi:10.1007/s00792-003-0338-3

    Article  PubMed  CAS  Google Scholar 

  25. Monreal J, Reese ET (1968) The chitinase of Serratia marcescens. J Microbiol 15:689–696

    Google Scholar 

  26. Morton CO, Hirsch PR, Kerry BR (2004) Infection of plant-parasitic nematodes by nematophagous fungi—a review of the application of molecular biology to understand infection processes and to improve biological control. Nematology 6:161–170. doi:10.1163/1568541041218004

    Article  CAS  Google Scholar 

  27. Mukherjee G, Sen SK (2006) Purification, characterization, and antifungal activity of chitinase from Streptomyces venezuelae P10. Curr Microbiol 53:265–269. doi:10.1007/s00284-005-0412-4

    Article  PubMed  CAS  Google Scholar 

  28. Nguyen VN, Jung WJ, Hwangbo H, Kim KY, Nguyen AD, Park RD (2006) Screening of chitinolytic and chitosanolytic fungi and the application for controlling plant-parasitic nematode Meloidogyne sp. In: Kim SK, No HK, Park RD (eds) Advances in Chitin Science and Technology. Hanrimwon Printing, Seoul, pp 254–256

    Google Scholar 

  29. Nguyen VN, Kim YJ, Oh KT, Jung WJ, Park RD (2007) The role of chitinase from Lecanicillium antillanum B-3 in parasitism to root-knot nematode Meloidogyne incognita eggs. Biocontrol Sci Technol 17:1047–1058. doi:10.1080/09583150701668658

    Article  Google Scholar 

  30. Nguyen VN, Kim YJ, Oh KT, Jung WJ, Park RD (2008) The antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9. Curr Microbiol 56:28–32. doi:10.1007/s00284-007-9033-4

    Article  PubMed  CAS  Google Scholar 

  31. Patil RS, Ghormade V, Deshpande MV (2000) Chitinolytic enzymes: an exploration. Enzyme Microb Technol 26:473–483. doi:10.1016/S0141-0229(00)00134-4

    Article  PubMed  CAS  Google Scholar 

  32. Pohl T (1990) Concentration of protein and removal of solutes. In: Deutscher WP (ed) Guide to protein purification. Metho enzymol 182. Academic Press, London, pp 68–83

    Chapter  Google Scholar 

  33. Sahai AS, Manocha MS (1993) Chitinases of fungi and plants: their involvement in morphogenesis and host-parasite interaction. FEMS Microbiol Rev 11:317–338. doi:10.1111/j.1574-6976.1993.tb00004.x

    Article  CAS  Google Scholar 

  34. Seidl V, Huemer B, Seiboth B, Kubicek CP (2005) A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases. FEBS J 272:5923–5939. doi:10.1111/j.1742-4658.2005.04994.x

    Article  PubMed  CAS  Google Scholar 

  35. Tikhonov VE, Lopez-Llorca LV, Salinas J, Jansson HB (2002) Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium. Fungal Genet Biol 35:67–78. doi:10.1006/fgbi.2001.1312

    Article  PubMed  CAS  Google Scholar 

  36. Toharisman A, Suhartono T, Barth MS, Hwang JK, Pyun YR (2005) Purification and characterization of a thermostable chitinase from Bacillus licheniformis Mb-2. J Microbiol Biotechnol 21:733–738

    Article  CAS  Google Scholar 

  37. Tronsmo A, Harman GE (1993) Detection and quatification of N-acetyl-β-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal Biochem 208:74–79. doi:10.1006/abio.1993.1010

    Article  PubMed  CAS  Google Scholar 

  38. Trudel J, Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178:362–366. doi:10.1016/0003-2697(89)90653-2

    Article  PubMed  CAS  Google Scholar 

  39. Wang SY, Moyne AL, Thottappilly G, Wu SJ, Locy RD, Singh NK (2001) Purification and characterization of a Bacillus cereus exochitinase. Enzyme Microb Technol 28:492–498. doi:10.1016/S0141-0229(00)00362-8

    Article  PubMed  CAS  Google Scholar 

  40. Watanabe T (2002) Pictorial atlas of soil and seed fungi morphologies of cultured fungi and key to species, 2nd edn. CRC Press, New York

    Google Scholar 

  41. White TG, Brunst T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Guide to Methods and Applications. Academic press, London, pp 315–322

    Google Scholar 

  42. Zaldivar M, Velasquez JC, Contreras I, Perez LM (2001) Trichoderma aureoviride 7–121, a mutant with enhanced production of lytic enzymes: its potential use in waste cellulose degradation and/or biocontrol. Electron J Biotechnol 4:1–9

    Google Scholar 

  43. Zeilinger S, Galhaup C, Payer K, Woo SL, Mach RL, Fekete C, Lorito M, Kubicek CP (1999) Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host. Fungal Genet Biol 26:131–140. doi:10.1006/fgbi.1998.1111

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program funded by the Ministry of Science and Technology (No. 10300000322-06J0000-32210).

Author information

Authors and Affiliations

  1. Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University, Gwangju, 500-757, South Korea

    Van-Nam Nguyen, In-Jae Oh, Young-Ju Kim, Kil-Yong Kim & Ro-Dong Park

  2. Division of Applied Plant Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 500-757, South Korea

    Young-Cheol Kim

Authors
  1. Van-Nam Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. In-Jae Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Young-Ju Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kil-Yong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Young-Cheol Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ro-Dong Park
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ro-Dong Park.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nguyen, VN., Oh, IJ., Kim, YJ. et al. Purification and characterization of chitinases from Paecilomyces variotii DG-3 parasitizing on Meloidogyne incognita eggs. J Ind Microbiol Biotechnol 36, 195–203 (2009). https://doi.org/10.1007/s10295-008-0485-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-008-0485-8

Keywords

Search

Navigation