Abstract
The filamentous fungus Trichoderma sp. GL2 produces an extracellular glucuronan lyase (GL) when grown on glucuronan as the sole carbon source. In this paper, we report the purification to electrophoretical homogeneity of this polysaccharide lyase by size exclusion chromatography and anion exchange chromatography. The purified GL, classified as an endopolyglucuronate lyase, is a monomer with an apparent molecular weight of 27 kDa and an isoelectric point of 6.95. Despite an inhibition of the activity when polysaccharide substrates were substituted by acetates, the enzyme was active toward glucuronans (acetylated or not) and ulvan, leading to various (4,5)-unsaturated products as oligoglucuronans (acetylated or deacetylated), highly acetylated low-molecular-weight (LMW) glucuronans, and LMW ulvans.
Similar content being viewed by others
References
Askew DJ, Laing MD (1993) An adapted selective medium for the quantitative isolation of Trichoderma species. Plant Pathol 42:686–690
Brimmer TA, Boland G (2003) A review of the non-target effects of fungi used to biologically control plant diseases. Agric Ecosyst Environ 100:3–16
Courtois J, Seguin JP, Declomesnil S, Heyraud A, Colin-Morel P, Dantas L, Barbotin JN, Courtois B (1993) A (1→4)-β-d-glucuronan excreted by a mutant of Rhizobium meliloti M5N1 strain. J Carbohydr Chem 12:441–448
Da Costa A, Michaud P, Petit E, Heyraud A, Colin-Morel P, Courtois B, Courtois J (2001) Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472). Appl Environ Microbiol 67:5197–5203
Da Costa A, Michaud P, Heyraud A, Colin-Morel P, Courtois B, Courtois J (2003) Acetyl substitution of glucuronan influences glucuronan cleavage by glya from Sinorhizobium meliloti M5N1CS (NCIMB 40472). Carbohydr Polym 51:223–228
Dantas L, Courtois J, Courtois B, Séguin JP, Gey C, Heyraud A (1994) NMR spectroscopic investigation of oligoglucuronates prepared by enzymatic hydrolysis of a (1→4)-β-d-glucuronan. Carbohydr Res 265:303–310
Deane EE, Whipps JM, Lynch JM, Peberdy JF (1998) The purification and characterization of a Trichoderma harzanium exochitinase. Biochim Biophys Acta 1383:101–110
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 harzanium. J Bacteriol 177(23):6937–6945
De Ruiter GA, Josso SL, Colquhoun IJ, Voragen AGJ, Rombouts FM (1992) Isolation and characterization of β(1–4)-d-glucuronans from extracellular polysaccharides of moulds belonging to Mucorales. Carbohydr Polym 18:1–7
Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56
Hjeljord L, Tronsmo A (1998) Trichoderma and Gliocladium in biological control: an overview. In: Harman GE, Kubicek C (eds) Trichoderma and Gliocladium: enzymes, biological control and commercial applications, vol 2. Taylor and Francis, London, UK, pp 131–151
Kubicek CP (1992) The cellulase proteins of Trichoderma reesei: structure, multiplicity, mode of action and regulation of formation. Adv Biochem Eng Biotechnol 45:1–27
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lahaye M (1998) NMR spectroscopic characterisation of oligosaccharides from two Ulva rigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase. Carbohydr Res 314:1–12
Lahaye M, Brunel M, Bonnin E (1997) Fine chemical structure analysis of oligosaccharides produced by an ulvan–lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp. (Ulvales, Chlorophyta). Carbohydr Res 304:325–333
Michaud P, Roblot C, Courtois J, Séguin JP, Heyraud A, Barbotin JN, Courtois B (1995) Effect of Mg2+ on production and O-acetylation of glucuronan excreted by the Rhizobium meliloti M5N1CS strain during fermentation. Lett Appl Microbiol 20:110–112
Michaud P, Da Costa A, Courtois B, Courtois J (2003) Polysaccharide lyases—recent development as biotechnological tools. Crit Rev Biotechnol 23(4):233–266
Pecina A, Paneque A (1994) Detection of alginate lyase by activity staining after SDS PAGE and subsequent renaturation. Anal Biochem 217:124–127
Ray B, Lahaye M (1995) Cell-wall polysaccharides from the marine green algaeUlva “rigida” (ulvales, chlorophyta). Extraction and chemical composition. Carbohydr Res 274:251–261
Schoffelmeer EAM, Klis FM, Sietsma JH, Cornelissen BJC (1999) The cell wall of Fusarium oxysporum. Fungal Genet Biol 27:275–282
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MDE, Fujimoto K, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Acknowledgements
This work was supported by the European Social Fund and by Conseil Régional de Picardie (France).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Delattre, C., Michaud, P., Keller, C. et al. Purification and characterization of a novel glucuronan lyase from Trichoderma sp. GL2. Appl Microbiol Biotechnol 70, 437–443 (2006). https://doi.org/10.1007/s00253-005-0077-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-005-0077-8