Annals of Microbiology

, Volume 65, Issue 1, pp 565–574 | Cite as

Chitin-degrading enzymes from an actinomycete ectosymbiont of Acromyrmex subterraneus brunneus (Hymenoptera: Formicidae)

  • Guilherme Duarte Rossi
  • Tiago Domingues Zucchi
  • Aline Sartori Guidolin
  • Aline Peruchi
  • Fernando Luis Cônsoli
Original Article

Abstract

Microbes have many mechanisms to exert their inhibitory activity against target pests. One such mechanism involves the production and secretion of hydrolytic enzymes, such as chitinases, which are produced naturally by plants in response to attack by insect herbivores and phytopathogens and have been sought as an additional factor to enhance pest management. Thus, our main aim was to screen the diverse actinomycete community associated with the integument of Acromyrmex subterraneus brunneus for a chitinase-producing strain and to characterize its chitinases. We identified isolate ENT-21—a Streptomyces sp.—as a chitinase-producer and our data indicate that this isolate produces a chitinolytic complex that contains a chitinase and a high-molecular-weight β-N-acetylglucosaminidase (>100 kDa) when cultured in Chitin-Czapek broth. The presence of chitinases in the genome of this isolate was checked by diagnostic PCR, and two chitinase genes belonging to family 18 group A and family 19 were verified. The chitinolytic activity of the crude extract was observed at pH values ranging from 3.8 to 11.0, with the highest chitinase activities recorded at pH 9.0 and 9.5, whereas optimum β-N-acetylglucosaminidase activity was observed over a narrow pH range, between pH 4.7 and 5.1. We describe some biochemical and molecular properties of the chitinase and β-N-acetylglucosaminidase produced by ENT-21, and discuss the potential for exploitation of these enzymes for pest control.

Keywords

Streptomyces Chitinases β-N-acetylglucosaminidase Biotechnology 

Notes

Acknowledgments

The authors appreciate the financial support provided by FAPESP to G.D.R. (PhD fellowship—process 2012/50021-1), T.D.Z. (PostDoctoral fellowship—processes 2007/58712-5 and 2011/14333-6), A.S.G. (PhD fellowship—process 2012/04287-0) and A.P. (Masters fellowship—process 2010/13675-8). F.L.C. also acknowledges FAPESP for funding this research (grants—processes 2007/59019-1; 2011/50877-0).

References

  1. Arakane Y, Muthukrishnan S (2010) Insect chitinase and chitinase-like proteins. Cell Mol Life Sci 67:201–216CrossRefPubMedGoogle Scholar
  2. Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen CW, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O'Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders D, Sharp S, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417:141–147CrossRefPubMedGoogle Scholar
  3. Bhattacharya D, Nagpure A, Gupta RK (2007) Bacterial chitinases: Properties and potential. Crit Rev Biotechnol 27:21–28CrossRefPubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Chandrasekaran R, Revathi K, Nisha S, Kirubakaran SA, Sathish-Narayanan S, Senthil-Nathan S (2012) Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab. Pestic Biochem Physiol 104:65–71CrossRefGoogle Scholar
  6. Chater KF, Biró S, Lee KJ, Palmer T, Schrempf H (2010) The complex extracellular biology of Streptomyces. FEMS Microbiol Rev 34:171–198CrossRefPubMedGoogle Scholar
  7. Currie CR, Poulsen M, Mendenhall J, Boomsma JJ, Billen J (2006) Coevolved crypts and exocrine glands support mutualistic bacteria in fungus-growing ants. Science 6:81–83CrossRefGoogle Scholar
  8. Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biotechnol 71:773–782CrossRefPubMedGoogle Scholar
  9. Das SN, Neeraja C, Sarma PVSRN, Prakash JM, Purushotham P, Kaur M, Dutta S, Podile AR (2012) Microbial chitinases for chitin waste management. In: Satyanarayana T, Johri BN, Prakash A (eds) Microorganisms in environmental management. Springer, Dordrecht, pp 135–150CrossRefGoogle Scholar
  10. Dawson RMC, Elliott DC, Elliott WH, Jones KM (eds) (1989) Data for biochemical research. Oxford University Press, NewYorkGoogle Scholar
  11. Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376CrossRefPubMedGoogle Scholar
  12. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  13. Filho BPD, Lemos FJA, Secundino NFC, Pascoa V, Pereira ST, Pimenta PFP (2002) Presence of chitinase and β-N-acetylglucosaminidase in the Aedes aegypti: a chitinolytic system involving peritrophic matrix formation and degradation. Insect Biochem Mol Biol 32:1723–1729CrossRefPubMedGoogle Scholar
  14. Fitch WM (1971) Toward defining the course of evolution: minimum change for specific tree topology. Syst Biol 20:406–416CrossRefGoogle Scholar
  15. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704CrossRefPubMedGoogle Scholar
  16. Guo YP, Zheng W, Rong XY, Huang Y (2008) A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics. Int J Syst Evol Microbiol 58:149–159CrossRefPubMedGoogle Scholar
  17. Hahn M, Hennig M, Schlesier B, Höhne W (2000) Structure of jack bean chitinase. Acta Crystallogr D 56:1096–1099CrossRefPubMedGoogle Scholar
  18. Herrera-Estrella A, Chet I (1999) Chitinases in biological control. Chitin Chitinases 87:171–184CrossRefGoogle Scholar
  19. Horsch M, Mayer C, Sennhauser U, Rast DM (1997) β-N-acetylhexosaminidase: a target for the design of antifungal agents. Pharmacol Ther 76:187–218CrossRefPubMedGoogle Scholar
  20. Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism, vol 3. Academic, New York, pp 21–123CrossRefGoogle Scholar
  21. Kawase T, Saito A, Sato T, Kanai R, Fujii T, Nikaidou N, Miyashita N, Watanabe T (2004) Distribution and phylogenetic analysis of famly 19 chitinases in Actinobacteria. Appl Environ Microbiol 70(2):1135–1144CrossRefPubMedCentralPubMedGoogle Scholar
  22. Kawase T, Yokokawa S, Saito A, Fujii T, Nikaidou N, Miyashita K, Watanabe T (2006) Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2). Biosci Biotechnol Biochem 70(4):988–998CrossRefPubMedGoogle Scholar
  23. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721CrossRefPubMedGoogle Scholar
  24. Kramer KJ, Muthukrishnan S (1998) Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem Mol Biol 27:887–900CrossRefGoogle Scholar
  25. Labeda DP, Goodfellow M, Brown R, Ward AC, Lanoot B, Vanncanneyt M, Swings J, Kim SB, Liu Z, Chun J, Tamura T, Oguchi A, Kikuchi T, Kikuchi H, Nishii T, Tsuji K, Yamaguchi Y, Tase A, Takahashi M, Sakane T, Suzuki KI, Hatano K (2012) Phylogenetic study of the species within the family Streptomycetaceae. Antonie Van Leeuwenhoek 101:73–104CrossRefPubMedGoogle Scholar
  26. Lehane MJ (1997) Peritrophic matrix structure and function. Annu Rev Entomol 42:525–550CrossRefPubMedGoogle Scholar
  27. Lehane MJ, Billingsley PF (1996) Biology of the insect midgut. Chapman & Hall, LondonCrossRefGoogle Scholar
  28. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39(D):225–229CrossRefGoogle Scholar
  29. Marco JL, Valadares-Inglis MC, Felix CR (2004) Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis perniciosa. Appl Microbiol Biotechnol 64:70–75CrossRefGoogle Scholar
  30. Martínez CP, Echeverri C, Florez JC, Gaitan AL, Góngora CE (2012) In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops. AMB Express 2:22 [http://www.amb-express.com/content/2/1/22]
  31. Mizuno R, Fukamizo T, Sugiyama S, Nishizawa Y, Kezuka Y, Nonaka T, Suzuki K, Watanabe T (2008) Role of the loop structure of the catalytic domain in rice class I chitinase. J Biochem 143:487–495CrossRefPubMedGoogle Scholar
  32. Nagpure A, Gupta RK (2013) Purification and characterization of an extracellular chitinase from antagonistic Streptomyces violaceusniger. J Basic Microbiol 63:871–877Google Scholar
  33. Nawani NN, Kapadnis BP (2005) Optimization of chitinase production using statistics based experimental designs. Process Biochem 40:651–660Google Scholar
  34. Oppenheim AB, Chet I (1992) Cloned chitinases in fungal plant-pathogen control strategies. Trends Biotechnol 10:392–394CrossRefGoogle Scholar
  35. Ramı́rez MG, Avelizapa LIR, Avelizapa NGR, Camarillo RC (2004) Colloidal chitin stained with Remazol Brilliant Blue R®, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. J Microbiol Methods 56(2):213–219CrossRefGoogle Scholar
  36. Rong XY, Huang Y (2010) Taxonomic evaluation of the Streptomyces griseus clade using multilocus sequence analysis and DNA–DNA hybridization, with proposal to combine 29 species and three subspecies as 11 genomic species. Int J Syst Evol Microbiol 60:696–703CrossRefPubMedGoogle Scholar
  37. Rong XY, Huang Y (2012) Taxonomic evaluation of the Streptomyces hygroscopicus clade using multilocus sequence analysis and DNA–DNA hybridization, validating the MLSA scheme for systematics of the whole genus. Syst Appl Microbiol 35(1):7–18CrossRefPubMedGoogle Scholar
  38. Rong XY, Guo YP, Huang Y (2009) Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA-DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens. Syst Appl Microbiol 32:314–322CrossRefPubMedGoogle Scholar
  39. Rosselló-Mora R, Amann R (2001) The species concept for prokaryotes. FEMS Microbiol Rev 25(1):39–67CrossRefPubMedGoogle Scholar
  40. Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:404–425Google Scholar
  41. Sambrook J, Russell DW (2001) Molecular cloning—a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  42. Shahidi F, Kamil YVAJ (2001) Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci Technol 12:435–464CrossRefGoogle Scholar
  43. Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Evol Microbiol 16:313–340Google Scholar
  44. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedCentralPubMedGoogle Scholar
  45. Terra WR, Ferreira C (2005) Biochemistry of digestion. In: Gilbert LI, Iatrov K, Gill S (eds) Comprehensive Molecular Insect Science 4:171–224. Elsevier, OxfordGoogle Scholar
  46. Terra WR, Ferreira C, Bianchi AGD (1979) Distribution of digestive enzymes among the endoperitrophic and ectoperitrophic spaces and midgut cells of Rhynchosciara and its physiological significance. J Insect Physiol 25:487–494CrossRefGoogle Scholar
  47. Tharanathan RN, Kittur FS (2003) Chitin—the undisputed biomolecule of great potential. Crit Rev Food Sci 43:61–87CrossRefGoogle Scholar
  48. Usui T, Matsui H, Isobe K (1990) Enzymic-synthesis of useful chito-oligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium-sulfate. Carbohydr Res 203:65–77CrossRefPubMedGoogle Scholar
  49. Wang F, Xiao X, Saito A, Schrempf H (2002) Streptomyces olivaceoviridis possesses a phosphotransferase system that mediates specific, phosphoenolpyruvate-dependent uptake of N-acetylglucosamine. Mol Genet Genomics 268:344–351CrossRefPubMedGoogle Scholar
  50. Watanabe T, Kanai R, Kawase T, Tanabe T, Mitsutomi M, Sakuda S, Miyashita K (1999) Family 19 chitinases of Streptomyces species: characterization and distribution. Microbiology 145:3353–3363CrossRefPubMedGoogle Scholar
  51. Williamson N, Brian P, Wellington EMH (2000) Molecular detection of bacterial and streptomycete chitinases in the environment. Antonie Van Leeuwenhoek 78:315–321CrossRefPubMedGoogle Scholar
  52. Wu Y, Liu F, Liu Y, Zhang Z, Zhou T, Liu X, Shen Q, Shen B (2011) Identification of chitinases Is-chiA and Is-chiB from Isoptericola jiangsuensis CLG and their characterization. Appl Microbiol Biotechnol 89:705–713CrossRefPubMedGoogle Scholar
  53. Zucchi TD, Almeida LG, Dossi FCA, Cônsoli FL (2010) Secondary metabolites produced by Propionicimonas sp. (ENT-18) induce histological abnormalities in the sclerotia of Sclerotinia sclerotiorum. BioControl 55:811–819CrossRefGoogle Scholar
  54. Zucchi TD, Guidolin AS, Consoli FL (2011a) Isolation and characterization of actinobacteria ectosymbionts from Acromyrmex subterraneus brunneus (Hymenoptera, Formicidae). Microbiol Res 166:68–76CrossRefPubMedGoogle Scholar
  55. Zucchi TD, Rossi GD, Cônsoli FL (2011b) Characterization of a β-amylase from Propionicimonas sp. ENT-18 ectosymbiont of Acromyrmex subterraneus brunneus. Ann Microbiol 61:985–990Google Scholar
  56. Zucchi TD, Almeida LG, Moraes LAB, Cônsoli FL (2014) Albocycline, the main bioactive compound from Propionicimonas sp. ENT-18 against Sclerotinia sclerotiorum. Ind Crops Prod 52C:264–268CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Authors and Affiliations

  • Guilherme Duarte Rossi
    • 1
    • 2
  • Tiago Domingues Zucchi
    • 1
    • 3
  • Aline Sartori Guidolin
    • 1
  • Aline Peruchi
    • 1
  • Fernando Luis Cônsoli
    • 1
  1. 1.Lab de Interações em Insetos, Depto de Entomologia & Acarologia, Escola Superior de Agricultura “Luiz de Queiroz”Univ de São Paulo (ESALQ/USP)PiracicabaBrazil
  2. 2.Depto de Fitossanidade, Faculdade de Ciências Agrárias e VeterináriasFCAV/UNESPJaboticabalBrazil
  3. 3.Lab Microbiologia AmbientalEMBRAPA Meio AmbienteJaguariúnaBrazil

Personalised recommendations