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

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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 K m 1.66 mg mL−1; V max 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 (K i 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.

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Acknowledgements

This work was funded by a Department of Agriculture Food and Fisheries Food Institutional Research Measure (FIRM) award (05R&DNUIG374) to MGT, under the National Development Plan 2000–2006 (Ireland). The authors would like to acknowledge Dr Susan Liddell at the University of Nottingham UK for her assistance and expertise in protein sequence analysis.

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  1. Charles Piggott

    Present address: Department of Life Sciences, University of Limerick, Limerick, Ireland

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  1. Molecular Glycobiotechnology Group, Biochemistry, School of Natural Sciences, National University of Ireland, Galway, Ireland

    Elaine O’Connell & Maria Tuohy

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  1. Elaine O’Connell
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Correspondence to Maria Tuohy.

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O’Connell, E., Piggott, C. & Tuohy, M. Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii . Appl Microbiol Biotechnol 89, 685–696 (2011). https://doi.org/10.1007/s00253-010-2883-x

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