1887

Abstract

produces several chitinolytic enzymes, including chitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad pH optimum around pH 5.0-6.0, and a temperature optimum between 50 and 60 C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 C, pH 6.1. Analyses of the degradation of various -acetylglucosamine oligomers, fluorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc)] from (GlcNAc) and thus possess an exo--diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc) substrates, indicating that they also have an endochitinase (ChiA) or exo--triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with 4-methylumbel-β-D--diacetylchitobioside, an analogue of (GlcNAc), showed cooperative kinetics for ChiA, whereas for ChiB normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than ChiB and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in indicate possible roles for ChiA and ChiB in chitin breakdown.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-7-1581
1996-07-01
2021-04-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/7/mic-142-7-1581.html?itemId=/content/journal/micro/10.1099/13500872-142-7-1581&mimeType=html&fmt=ahah

References

  1. Blaak H., Schnellmann J., Walter S., Henrissat B., Schrempf H. 1993; Characteristics of an exochitinase from Streptomjces olivaceo- viridis, its corresponding gene, putative domains and relationship to other chitinases. Eur J Biochem 214:659–669
    [Google Scholar]
  2. Bork P., Doolittle R. F. 1992; Proposed acquisition of an animal protein domain by bacteria. Proc Natl Acad Sci USA 89:8990–8994
    [Google Scholar]
  3. Bronnenmeier K., Rücknagel K.P., Staudenbauer W. L. 1991; Purification and properties of a novel type of exo-1,4-β-glucanase (Avicellase II) from the cellulolytic Clostridium stercorarium. Eur J Biochem 200:379–385
    [Google Scholar]
  4. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
    [Google Scholar]
  5. Brurberg M. B., Eijsink V. G. H., Nes I. F. 1994; Characterization of a chi tinase gene (chi A) from Serratia marcescens strain BJL200 and one-step purification of the gene product. FEMS Microbiol Lett 124:399–404
    [Google Scholar]
  6. Brurberg M. B., Eijsink V. G. H., Haandrikman A. J., Venema G., Nes I. F. 1995; Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing. Microbiology 141:123–131
    [Google Scholar]
  7. Cleland W.W. 1963; Computer programmes for processing enzyme kinetic data. Nature 198:463–465
    [Google Scholar]
  8. Davies G., Henrissat B. 1995; Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859
    [Google Scholar]
  9. Davis B., Eveleigh D. E. 1984; Chitosanases : occurrence, production and immobilization. In Chitin, Chitosan, and Related Enzymes pp. 161–179 Edited by Zikakis J. P. London: Academic Press;
    [Google Scholar]
  10. De la Cruz J., Hidalgo-Gallego A., Lora J. M., Benitez T., Pintor-Toro J., Llobell A. 1992; Isolation and characterization of three chitinases from Trichoderma harzianum. Eur J Biochem 206:859–867
    [Google Scholar]
  11. Fuchs R. L., McPherson S. A., Drahos D. J. 1986; Cloning of a Serratia marcescens gene encoding chitinase. Appl Environ Microbiol 51:504–509
    [Google Scholar]
  12. Gooday G.W. 1990; Physiology of microbial degradation of chitin and chitosan. Biodégradation 1:177–190
    [Google Scholar]
  13. Harpster M.H., Dunsmuir P. 1989; Nucleotide sequence of the chitinase B gene of 3. marcescens QMB1466. Nucleic Acids Res 17:5395
    [Google Scholar]
  14. Imoto T., Yagishita K. 1971; A simple activity measurement of lysozyme. Agr Biol Chem 35:1154–1156
    [Google Scholar]
  15. John M., Röhrig H., Schmidt J., Wieneke U., Schell J. 1993; Rhizobium Nod B protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase. Proc Natl Acad Sci USA 90:625–629
    [Google Scholar]
  16. Jones J.D.G., Grady K. L., Suslow T. V., Bedbrook J. R. 1986; Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J 5:467–473
    [Google Scholar]
  17. Kless H., Sitrit Y., Chet H., Oppenheim A. B. 1989; Cloning of the gene coding for the chitobiase of Serratia marcescens. Mol Gen Genet 217:471–473
    [Google Scholar]
  18. Koo J. C., Lim C. O., Choi Y. J., Kim C. Y., Bahk J. D., Lee S. Y., Cho M. J. 1993; Expression of bacterial chitinase in tobacco and enhanced resistance against phytopathogenic fungi. EMBL Database Accession number LOI455
    [Google Scholar]
  19. Krishnan A., Nair P. N., Jones D. 1994; Isolation, cloning and characterization of new chitinase stored in active form in chitin- lined venom reservoir. J Biol Chem 269:20971–20976
    [Google Scholar]
  20. Kuranda M.J., Robbins P. W. 1987; Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae. Proc Natl Acad Sci USA 84:2585–2589
    [Google Scholar]
  21. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  22. Lorito M., Peterbauer C., Hayes C. K., Harman G. E. 1994; Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiology 140:623–629
    [Google Scholar]
  23. Manoil C., Beckwith J. 1986; A genetic approach to analyzing membrane protein topology. Science 233:1403–1408
    [Google Scholar]
  24. Monreal J., Reese E. 1969; The chitinase of Serratia marcescens. Can J Microbiol 15:689–696
    [Google Scholar]
  25. Perrakis A., Tews I., Dauter Z., Oppenheim A. B., Chet I., Wilson K. S., Vorgias C. E. 1994; Crystal structure of a bacterial chitinase at 2'3 A resolution. Structure 2:1169–1180
    [Google Scholar]
  26. Renkema G. H., Boot R. G., Muijsers A. O., Donker-Koopman W. E., Aerts J. M. F. G. 1995; Purification and characterization of human chitotriosidase, a novel member of the chitinase family of proteins. J Biol Chem 270:2198–2202
    [Google Scholar]
  27. Roberts R.L., Cabib E. 1982; Serratia marcescens chitinase : one- step purification and use for the determination of chitin. Anal Biochem 127:402–412
    [Google Scholar]
  28. Roberts W.K., Selitrennikoff C. P. 1988; Plant and bacterial chitinases differ in antifungal activity. J Gen Microbiol 134:169–176
    [Google Scholar]
  29. Robbins P. W., Albright C., Benfield B. 1988; Cloning and expression of a Streptomjces plicatus chitinase (chitinase-63) in Escherichia coli. J Biol Chem 263:443–447
    [Google Scholar]
  30. Schickler H., Haran S., Oppenheim A. B., Chet I. 1993; Cloned chitinases and their role in biological control of plant pathogenic fungi. In Chitin Enzymology pp. 375–382 Edited by Muzzarelli R. A. A. Ancona, Italy: European Chitin Society;
    [Google Scholar]
  31. Segel I.H. 1975 Enzyme kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems New York: John Wiley;
    [Google Scholar]
  32. Shapira R., Ordentlich A., Chet I., Oppenheim A. B. 1989; Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli.. Phytopathology 79:1246–1249
    [Google Scholar]
  33. Stoll V.S., Blanchard J. S. 1990; Buffers : principles and practice.. Methods Ensymol 182:24–38
    [Google Scholar]
  34. Sundheim L., Poplawsky A. R., Ellingboe A. H. 1988; Molecular cloning of two chitinase genes from Serratia marcescens and their expression in Pseudomonas species. Phys Mol Plant Pathol 33:483–491
    [Google Scholar]
  35. Takayanagi T., Sjisaka K., Takiguchi Y., Shimahara K. 1991; Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochim Biophys Acta 1078:404–410
    [Google Scholar]
  36. Tronsmo A., Harman G. E. 1993; Detection and quantification of N-acetyl-β-D-glucosaminidase, chitobiase and endochitinase in solutions and on gels. Anal Biochem 208:74–79
    [Google Scholar]
  37. Vessey J.C., Pegg G. F. 1973; Autolysis and chitinase production in cultures of Verticillium albo-atrum. Trans Br My col Soc 60:133–143
    [Google Scholar]
  38. Vorgias C. E., Tews I., Perrakis A., Wilson K. S., Oppenheim A. B. 1993; Purification and characterization of the recombinant chitin degrading enzymes, chitinase A and chitobiase from Serratia marcescens.. In Chitin Ensymology pp. 417–422 Edited by Muzzarelli R. A. A. Ancona, Italy: European Chitin Society;
    [Google Scholar]
  39. Walkley J.W., Tillman J. 1977; A simple thin-layer chromatographic technique for the separation of mono- and oligosaccharides. J Chromatogr 132:172–174
    [Google Scholar]
  40. Watanabe T., Oyanagi W., Suzuki K., Tanaka H. 1990a; Chitinase system of Bacillus circulons WL-12 and importance of chitinase Al in chitin degradation. J Bacteriol 172:4017–4022
    [Google Scholar]
  41. Watanabe T., Suzuki K., Oyanagi W., Ohnishi K., Tanaka H. 1990b; Gene cloning of chitinase Al from Bacillus circulons WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin.. J Biol Chem 265:15659–15665
    [Google Scholar]
  42. Watanabe T., Oyanagi W., Suzuki K., Ohnishi K., Tanaka H. 1992; Structure of the gene encoding chitinase D of Bacillus circulons WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases.. J Bacteriol 174:408–414
    [Google Scholar]
  43. Watanabe T., Kobori K., Miyashita K., Fujii T., Sakai H., Uchida M., Tanaka H. 1993; Identification of glutamic acid 204 and aspartic acid 200 in chitinase Al of Bacillus circulons WL-12 as essential residues for chitinase activity. J Biol Chem 268:18567–18572
    [Google Scholar]
  44. Watanabe T., Ito Y., Yamada T., Hashimoto M., Sekine S., Tanaka H. 1994; The roles of the C-terminal domain and type III domains of chitinase Al from Bacillus circulons WL-12 in chitin degradation. J Bacteriol 176:4465–4472
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-7-1581
Loading
/content/journal/micro/10.1099/13500872-142-7-1581
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error