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Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger
- Zhu, F. -M., Du, B., Gao, H. -S., Liu, C. -J., Li, J.
- Applied biochemistry and microbiology 2010 v.46 no.6 pp. 626-632
- Aspergillus niger, Aspergillus oryzae, EDTA (chelating agent), ammonium sulfate, beta-glucosidase, calcium, calcium chloride, cellulose, enzyme activity, glucose, hydrolysis, ion exchange, iron, magnesium, molecular weight, pH, polyacrylamide gel electrophoresis, polyethylene glycol, potassium, protoplast fusion, protoplasts, size exclusion chromatography, snails, sodium, sodium chloride, temperature
- Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4,000 with 0.01 mM CaCl₂. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular (β-glucosidase (EC 126.96.36.199) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na⁺, K⁺, Ca²⁺, Mg²⁺ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe³⁺. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K m and V max values against salicin as substrate were 0.035 mM and 1.7215 μmol min⁻¹, respectively.