Production of thermostable beta-mannosidase by a strain of Thermoascus aurantiacus:Isolation,partial purification and characterization of the enzyme

摘要

The production of an extracellular thermostable P-mannosidase by the thermophilic fungus Thermoascus aurantiacus Miehe was investigated in shake flask cultures.Among five different carbon sources and inducers tested,locust bean gum at 15 gl~(-1)induced the highest(4nkatml~(-1))P-mannosidase activity.Although the fungus grew very well on mannose at an initial concentration of 20 gl~(-1)there was practically no enzyme synthesis.Low level of enzyme synthesis occurred only when mannose had dropped to about 1 g l~(-1).The p-mannosidase was purified to apparent homogeneity by ethanol precipitation,differential solubility at pH 4.0 and cation ion exchange chromatography.The enzyme had a molecular mass of 99.9 kDa and a pI value of 4.8.The p-mannosidase showed maximum activity at pH 2.5-3.0 and 76 °C.It exhibited highest pH stability at 5.9 after 5 h incubation at 50 °C.It had a half-life of 10 min at 76 °C.The K_m and V_(max)values for p-nitrophenyl-beta-D-mannopyranoside(p-NP-beta-MP)were 1.1 mM and 61 nkat mg~(-1),respectively.Low transglycosylation activity was observed when the enzyme was incubated with p-NP-beta-MP as glycosyl donor and methyl-alpha-D-mannopyranoside as acceptor at 50 °C and pH 5.0 and small amounts of P-configured methylmannobioside was formed.