Purification and Enzymatic Characterization of a Novel Acid-resistant and Thermostable Pullulanase

This study reports on the purification and characterization of the pullulanase from a newly isolated Aspergillus awamori XF (PulXF). The enzyme was purified to apparent electrophoretic homogeneity through ammonium sulphate precipitation, anion exchange chromatography, gel chromatography and hydrophobicity chromatography. The crude enzyme was purified 8.16 times with 309.28 U/mg specific activity. SDS-PAGE revealed that the enzyme is monomeric, with a molecular weight of 63.7 kDa. PulXF was highly active in the temperature range of 50~80℃, with an optimum activity at 60℃. It was highly active in the pH range of 4.5~8.0, with optimum pH at 5.0, and retained more than 80% of its original activity in a broad pH range of 3~8 for 24 h at 28℃. The enzyme displayed K_m and V_max values, for pullulan of 0.92 mg/mL and 11.90 μmol/min, respectively, demonstrate its greater affinity. PulXF hydrolyzed pullulan, producing maltotriose, and low hydrolytic activities were also detected with amylopectin and soluable starch, but almost not with amylose, α-cyclodextrine, β-cyclodextrine and glycogen. This proved that the purified PulXF from Aspergillus awamori XF was classified under pullulanase type I. The activity of PulXF could be promoted by Ca²⁺, Zn²⁺ and Mg²⁺. Among them, Ca²⁺ had the most significant effect. The enzyme had a tolerance for surfactants, which was relatively stable in the presence of 5% SDS, 5% CTAB, 5% Tween 80 and 30% ethanol. The excellent physical and chemical properties make PulXF suitable in the fields of starch processing, food and beverage and biotechnological industries that require high activity at high temperature and acid conditions.