Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii.
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.
O'Connell
Elaine
E
National University of Ireland, Galway, Ireland.
Piggott
Charles
C
Tuohy
Maria
M
D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005936:Glucans; D005942:Gluconates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology*; D013696:Temperature
D004791:Enzyme Inhibitors; D005936:Glucans; D005942:Gluconates; D007783:Lactones; D011134:Polysaccharides; C008247:laminaran; D043326:Glucan 1,3-beta-Glucosidase; C010730:beta-glucono-1,5-lactone