Extremophiles

, Volume 7, Issue 5, pp 385–390

Mode of action and antifungal properties of two cold-adapted chitinases

  • Konstantinos Mavromatis
  • Matteo Lorito
  • Sheridan L. Woo
  • Vassilis Bouriotis
Original Paper

Abstract

The mode of action of two chitinases from the Antarctic Arthrobacter sp. strain TAD20 on N-acetyl-chitooligomers and chitin polymers has been elucidated. Identification of the length of chitin oligomers following enzymatic hydrolysis was verified by using HPLC-based analysis. It was observed that the length of the oligomer is important for enzyme action. The enzymes cannot effectively hydrolyze chitin oligomers with a degree of polymerization lower than four. ArChiA is an endochitinase which hydrolyzes chitin substrates randomly, whereas ArChiB is an exochitinase which degrades chitin chains and N-acetyl-chitooligomers from the nonreducing end, releasing N-N′-diacetyl-chitobiose. ArChiB (100 μg/ml) inhibited spore germination and hyphal elongation of the phytopathogenic fungus Botrytis cinerea by 15% and 30%, respectively. A more pronounced effect was observed with ArChiA (100 μg/ml) resulting in 70% inhibition of spore germination and 60% inhibition of germ tube elongation. A slight additive effect was observed, when the two enzymes were used in combination, only on the inhibition of germ tube elongation.

Keywords

Anomer separation Botrytis cinerea Chitinase Chitooligomers Endochitinase Exochitinase 

References

  1. A/Banat BMA, Kameyama Y, Yoshioka T, Koga D (1999) Purification and characterization of a 54 kDa chitinase from Bombyx mori. Insect Biochem Mol Biol 29:537–547CrossRefGoogle Scholar
  2. Aalten DM van, Synstad B, Brurberg MB, Hough E, Riise BW, Eijsink VG, Wierenga RK (2000) Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution. Proc Natl Acad Sci USA 97:5842–5847CrossRefPubMedGoogle Scholar
  3. Aalten DM van, Komander D, Synstad B, Gaseidnes S, Peter MG, Eijsink VG (2001) Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci USA 98:8979–8984CrossRefPubMedGoogle Scholar
  4. Armand S, Tomita H, Heyraud A, Gey C, Watanabe T, Henrissat B (1994) Stereochemical course of the hydrolysis reaction catalyzed by chitinases A1 and D from Bacillus circulans WL-12. FEBS Lett 343:177–180CrossRefPubMedGoogle Scholar
  5. Boisset C, Fraschini C, Schulein M, Henrissat B, Chanzy H (2000) Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. Appl Environ Microbiol 66:1444–1452CrossRefPubMedGoogle Scholar
  6. Brurberg MB, Nes IF, Eijsink VG (1996) Comparative studies of chitinases A and B from Serratia marcescens. Microbiology 142:1581–1589PubMedGoogle Scholar
  7. Frankowski J, Lorito M, Scala F, Schmid R, Berg G, Bahl H (2001) Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48. Arch Microbiol 176:421–426CrossRefPubMedGoogle Scholar
  8. Gaudin C, Belaich A, Champ S, Belaich JP (2000) CelE, a multidomain cellulase from Clostridium cellulolyticum: a key enzyme in the cellulosome? J Bacteriol 182:1910–1915Google Scholar
  9. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D'Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107PubMedGoogle Scholar
  10. Gooday G (1994) Physiology of microbial degradation of chitin and chitosan, In: Ratledge C (ed) Biochemistry of microbial degradation. Kluwer Academic, Dordrecht, The Netherlands, pp 279–312Google Scholar
  11. Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–696PubMedGoogle Scholar
  12. 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 9:544–551PubMedGoogle Scholar
  13. Jeuniaux C (1966) Chitinases, In: Neufield EF, Ginsberg V (eds) Methods in enzymology. Academic Press, London, pp 644–650Google Scholar
  14. Koga D, Yoshioka T, Arakane Y (1998) HPLC analysis of anomeric formation and cleavage pattern by chitinolytic enzyme. Biosci Biotechnol Biochem 62:1643–1646Google Scholar
  15. Lonhienne T, Mavromatis K, Vorgias CE, Buchon L, Gerday C, Bouriotis V (2001a) Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes. J Bacteriol 183:1773–1779CrossRefPubMedGoogle Scholar
  16. Lonhienne T, Zoidakis J, Vorgias CE, Feller G, Gerday C, Bouriotis V (2001b) Modular structure, local flexibility and cold-activity of a novel chitobiase from a psychrophilic Antarctic bacterium. J Mol Biol 310:291–297CrossRefPubMedGoogle Scholar
  17. Lorito M, Harman GE, Hayes CK, Broadway RM, Tronsmo A, Woo SL, Di Pietro A (1993) Chitinolytic enzymes produced by Trichoderma harzianum: antifungal activity of purified endochitinase and chitobiosidase. Phytopathology 83:302–307Google Scholar
  18. Lorito M, Woo SL, Garcia I, Colucci G, Harman GE, Pintor-Toro JA, Filippone E, Muccifora S, Lawrence CB, Zoina A, Tuzun S, Scala F, Fernandez IG (1998) Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 95:7860–7865PubMedGoogle Scholar
  19. Papanikolau Y, Prag G, Tavlas G, Vorgias CE, Oppenheim AB, Petratos K (2001) High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis. Biochemistry 40:11338–11343CrossRefPubMedGoogle Scholar
  20. Perrakis A, Tews I, Dauter Z, Oppenheim AB, Chet I, Wilson KS, Vorgias CE (1994) Crystal structure of a bacterial chitinase at 2.3 A resolution. Structure 2:1169–1180PubMedGoogle Scholar
  21. Richer DL (1987) Synergism: a patent view. Pestic Sci 19:309–315Google Scholar
  22. Sakuda S (1996) Studies on the chitinase inhibitors, allosamidins. In: Muzzarelli RAA (ed) Chitin enzymology. European Chitin Society, Ancona, Italy, pp 203–212Google Scholar
  23. Shiro M, Ueda M, Kawaguchi T, Arai M (1996) Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24. Biochim Biophys Acta 1305:44–48PubMedGoogle Scholar
  24. Terwisscha van Scheltinga AC, Kalk KH, Beintema JJ, Dijkstra BW (1994) Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure 2:1181–1189PubMedGoogle Scholar
  25. Watanabe T, Kobori K, Yamada T, Ito Y, Uchida M, Tanaka H (1993) Domain structures and functions of bacterial chitinases. In: Muzzarelli RAA (ed) Chitin enzymology. European Chitin Society, Ancona, Italy, pp 329–336Google Scholar
  26. Watanabe T, Ishibashi A, Ariga Y, Hashimoto M, Nikaidou N, Sugiyama J, Matsumoto T, Nonaka T (2001) Trp122 and Trp134 on the surface of the catalytic domain are essential for crystalline chitin hydrolysis by Bacillus circulans chitinase A1. FEBS Lett 494:74–78CrossRefPubMedGoogle Scholar
  27. Zecchinon L, Claverie P, Collins T, D'Amico S, Delille D, Feller G, Georlette D, Gratia E, Hoyoux A, Meuwis M-A, Sonan G, Gerday C (2001) Did psychrophilic enzymes really win the challenge? Extremophiles 5:313–321Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Konstantinos Mavromatis
    • 1
    • 2
  • Matteo Lorito
    • 3
  • Sheridan L. Woo
    • 3
  • Vassilis Bouriotis
    • 1
    • 2
  1. 1.Department of Biology, Division of Applied Biology and BiotechnologyUniversity of CreteHeraklionGreece
  2. 2.Enzyme Technology DivisionInstitute of Molecular Biology and BiotechnologyHeraklionGreece
  3. 3.Dipartmento di Arboricoltura, Botanica e Patologia Vegetale, Sezione Patologia VegetaleUniversita degli Studi di Napoli Federico IIPorticiItaly

Personalised recommendations