Applied Microbiology and Biotechnology

, Volume 89, Issue 3, pp 685–696

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii

Biotechnologically Relevant Enzymes and Proteins

Abstract

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent Km 1.66 mg mL−1; Vmax 7.69 IU mL−1) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (Ki 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. ‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.

Keywords

Exo-1,3-β-glucanase Laminaran Talaromyces emersonii Glucono-δ-lactone Purification 

References

  1. Bara MT, Lima AL, Ulhoa CJ (2003) Purification and characterization of an exo-β-1,3-glucanase produced by Trichoderma asperellum. FEMS Microbiol Lett 219:81–85. doi:10.1016/s0378-1097(02)01191-6 CrossRefGoogle Scholar
  2. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3 CrossRefGoogle Scholar
  3. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active enZymes database (CAZy): an expert resource for Glycogenomics. Nucl Acids Res 37:D233–D238. doi:10.1093/nar/gkn663 CrossRefGoogle Scholar
  4. Carlberg GE, Percival E, Anisur Rhaman M (1978) Carbohydrates of the seaweeds Desmarestia ligulata and D. firma. Phytochem 17:1289–1292. doi:10.1016/S0031-9422(00)94576-X CrossRefGoogle Scholar
  5. Cid VJ, Duran A, Del Rey F, Snyder MP, Nombela C, Sanchez M (1995) Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev 59:345–386Google Scholar
  6. de la Cruz J, Pintor-Toro JA, Benitez T, Llobell A, Romero LC (1995) A novel endo-β-1, 3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 177:6937–6945Google Scholar
  7. El-Katatny MH, Gudelj M, Robra KH, Elnaghy MA, Gübitz GM (2001) Characterization of a chitinase and an endo-β-1, 3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii. Appl Microbiol Biotechnol 56:137–143. doi:10.1007/s002530100646 CrossRefGoogle Scholar
  8. Elving P, Markowitz J, Rosenthal I (1956) Preparation of buffer systems of constant ionic strength. Anal Chem 28:1179–1180. doi:10.1021/ac60115a034 CrossRefGoogle Scholar
  9. Fontaine T, Hartland RP, Diaquin M, Simenel C, Latge JP (1997) Differential patterns of activity displayed by two exo-β-1, 3-glucanases associated with the Aspergillus fumigatus cell wall. J Bacteriol 179:3154–3163Google Scholar
  10. Galan B, Garcia-Mendoza C, Colonje M, Novices-Ledieu M (1999) Production, purification, and properties of an endo-1, 3-β-glucanase from the basidiomycete Agaricus bisporus. Curr Microbiol 38:190–193. doi:10.1007/PL00006785 CrossRefGoogle Scholar
  11. Gilleran C (2004) An environmentally-benign biotechnological strategy to utilize carbohydrate rich waste., Ph.D. dissertation, National University of Ireland, Galway, IrelandGoogle Scholar
  12. İzgü F, Altınbay D, Acun T (2006) Killer toxin of Pichia anomala NCYC 432; purification, characterization and its exo-β-1,3-glucanase activity. Enzyme Microb Technol 39:669–676. doi:10.1016/j.enzmictec.2005.11.024 CrossRefGoogle Scholar
  13. Kim Y-T, Kim E-H, Cheong C, Williams DL, Kim C-W, Lim S-T (2000) Structural characterization of β-D-(1,3;1,6)-linked glucans using NMR spectroscopy. Carbohydr Res 328:331–341. doi:10.1016/S0008-6215(00)00105-1 CrossRefGoogle Scholar
  14. Kitamoto Y, Kono R, Shimotori A, Mori N, Ichikawa Y (1987) Purification and some properties of an exo-β-1,3-glucanase from Trichoderma harzianum. Agric Biol Chem 51:3385–3386Google Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685. doi:10.1038/227680a0 CrossRefGoogle Scholar
  16. Li H, Chen J, Li A, Li D (2007) Purification and partial characterization of β-1,3-glucanase from Chaetomium thermophilum. World J Microbiol Biotechnol 23:1297–1303. doi:10.1007/s11274-007-9366-y CrossRefGoogle Scholar
  17. MacKenzie CR, Williams RE (1984) Detection of cellulose and xylanase activity in isoelectric focused gels using agar substrate gels supported on plastic films. Can J Microbiol 30:1522–1525. doi:10.1139/m84-241 CrossRefGoogle Scholar
  18. McCarthy T, Hanniffy O, Savage AV, Tuohy MG (2003) Catalytic properties and mode of action of three endo-β-glucanases from Talaromyces emersonii on soluble β-1,4 and β-1,3;1,4-linked glucans. Int J Biol Macromol 33:141–148. doi:10.1016/S0141-8130(03)00080-1 CrossRefGoogle Scholar
  19. McHale A, Coughlan MP (1982) Properties of the β-glucosidases of Talaromyces emersonii. J Gen Microbiol 128:2327–2331. doi:10.1099/00221287-128-10-2327 Google Scholar
  20. Miller GL (1959) Use of dinitrosalicyclic acid (DNS) for the determination of reducing sugars. Anal Chem 31:426–428. doi:10.1021/ac60147a030 CrossRefGoogle Scholar
  21. Moloney AP, Hackett TJ, Considine PJ, Coughlan MP (1983) Isolation of mutants of Talaromyces emersonii CBS 814.70 with enhanced cellulose activity. Enzyme Microb Technol 5:260–264. doi:10.1016/0141-0229(83)90074-1 CrossRefGoogle Scholar
  22. Moloney AP, McCrae SI, Wood TM, Coughlan MP (1985) Isolation and characterization of the 1,4-β-D-glucan glucanohydrolases of Talaromyces emersonii. Biochem J 225:365–374Google Scholar
  23. Mueller A, Raptis J, Rice PJ, Kalbfleisch JH, Stout RD, Ensley HF, Browder W, Williams DL (2000) The influence of glucan polymer structure and solution conformation on binding to (1,3)-β-D-glucan receptors in a human monocyte-like cell line. Glycobiol 10:339–346. doi:10.1093/glycob/10.4.339 CrossRefGoogle Scholar
  24. Murray PG, Grassick A, Laffey CD, Cuffe MM, Higgins T, Savage AV, Tuohy MG (2001) Isolation and characterization of a thermostable endo-β-glucanase active on 1,3-1,4-β-D-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70. Enzyme Microb Technol 29:90–98. doi:10.1016/S0141-0229(01)00354-4 CrossRefGoogle Scholar
  25. Murray PG, Aro N, Collins C, Grassick A, Penttilä M, Saloheimo M, Tuohy M (2004) Expression in Trichoderma reesei and characterisation of a thermostable family 3-glucosidase from the moderately thermophilic fungus Talaromyces emersonii. Prot Purif Expr 38:248–257. doi:10.1016/j.pep.2004.08.006 CrossRefGoogle Scholar
  26. Neves-Monteiro V, Ulhoa CJ (2006) Biochemical characterization of a β-1,3-glucanase from Trichoderma koningii induced by cell wall of Rhizoctonia solani. Curr Microbiol 52:92–96. doi:10.1007/s00284-005-0090-2 CrossRefGoogle Scholar
  27. Noronha EF, Ulhoa CJ (1996) Purification and characterization of an endo-β-1,3-glucanase from Trichoderma harzianum. Can J Microbiol 42:1039–1044. doi:10.1139/m96-133 CrossRefGoogle Scholar
  28. Noronha EF, Ulhoa CJ (2000) Characterization of a 29-kDa β-1,3-glucanase from Trichoderma harzianum. FEMS Microbiol Lett 183:119–123. doi:10.1016/S0378-1097(99)00648-5 Google Scholar
  29. O’Connell EA, Murray P, Piggott C, Hennequart F, Tuohy M (2008) Purification and characterization of a N-acetylglucosaminidase produced by Talaromyces emersonii during growth on algal fucoidan. J Appl Phycol 20:557–565. doi:10.1007/s10811-007-9291-3 CrossRefGoogle Scholar
  30. Ohno N, Nono I, Yadomae T (1989) Enzymatic and physicochemical properties of an exo-(1-3)-β-D-glucanase from Rhizoctonia solani. Carbohydr Res 194:261–271. doi:10.1016/0008-6215(89)85024-4 CrossRefGoogle Scholar
  31. Pitson SM, Seviour RJ, McDougall BM (1993) Noncellulolytic fungal β-glucanases: their physiology and regulation. Enzyme Microb Technol 15:178–192. doi:10.1016/0141-0229(93)90136-P CrossRefGoogle Scholar
  32. Raguz S, Yague E, Wood DA, Thurston CF (1992) Isolation and characterization of a cellulose-growth-specific gene from Agaricus bisporus. Gene 119:183–190. doi:10.1016/0378-1119(92)90270-Y CrossRefGoogle Scholar
  33. Ramot O, Cohen-Kupiec R, Chet I (2000) Regulation of β-1,3-glucanase by carbon starvation in the mycoparasite Trichoderma harzianum. Mycol Res 104:415–420. doi:10.1017/S0953756299001471 CrossRefGoogle Scholar
  34. Rapp P (1989) 1,3-β-Glucanase, 1,6-β-glucanase and β-glucosidase activities of Sclerotium glucanicum: synthesis and properties. J Gen Microbiol 135:2847–2858. doi:10.1099/00221287-135-11-2847 Google Scholar
  35. Santos T, Villanueva JR, Nombela C (1978) Regulation of β-1,3-glucanase synthesis in Penicillium italicum. J Bacteriol 133:542–548Google Scholar
  36. Segel IH (1976) Biochemical calculations. Wiley, New YorkGoogle Scholar
  37. Stahmann KP, Schimz KL, Sahm H (1993) Purification and characterization of four extracellular 1,3-β-glucanases of Botrytis cinerea. J Gen Microbiol 139:2833–2840. doi:10.1099/00221287-139-11-2833 Google Scholar
  38. Stone BA, Clarke AE (1983) Chemistry and biology of 1, 3-β-glucans. La Trobe University, BundooraGoogle Scholar
  39. Tuohy MG, Coughlan MP (1992) Production of thermostable xylan-degrading enzymes by Talaromyces emersonii. Bioresour Technol 39:131–137. doi:10.1016/0960-8524(92)90131-G CrossRefGoogle Scholar
  40. Wood TM, Bhat KM (1988) Methods for measuring cellulose activities. Meth Enzymol 160:87–112. doi:10.1016/0076-6879(88)60109-1 CrossRefGoogle Scholar
  41. Xue Z, Shibh M-C, Poulton JE (2006) An extracellular exo-β-(1,3)-glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression. Protein Expr Purif 47:118–127. doi:10.1016/j.pep.2005.11.025 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Elaine O’Connell
    • 1
  • Charles Piggott
    • 2
  • Maria Tuohy
    • 1
  1. 1.Molecular Glycobiotechnology Group, Biochemistry, School of Natural SciencesNational University of IrelandGalwayIreland
  2. 2.Department of Life SciencesUniversity of LimerickLimerickIreland

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