Purification And Properties' Study On The Pullulanase From Bacillus Stearothermophilus

Pullulanase (Pullulanase, EC.3.2.1.41) is a debranching enzyme that can hydrolyze pullulan specificity and amylopectin branch points on theα-1, 6 glycosidic bonds . It has broad application prospects in starch saccharification industry.Bacillus stearothermophilus were broken using Cell crushing machine, impurity proteins were taken out using ammonium sulfate salting out method. Cell pullulanase was purified using DEAE-32 cellulose anion-exchange. The salting out conditions were optimized through orthogonal test. The results showed that: The separate condition is the best when the buffer is three times of volume than Bacteria, ammonium sulfate saturation was 40%, pH7.0. pH has an great effect on the separation, differences were considered statistically significant where F >F0.05; The specific activity of purified pullulanase was 26.50 U/mg, the purification factor was 8.01, the recovery was 73%; A zone electrophoresis was showing after detected by SDS-PAGE, the relative molecular mass was 55.4kDa, the results above showed that the purified enzyme was obtained. Study on the enzymatic properties of pullulanase which obtained from Bacillus stearothermophilus . The results showed that: the optimum temperature was 60℃, and has a strong thermal stability; About 70% of residual activity is still sustained after treatment 90min at 60℃; The half-life greater than 50 min at 70℃; Optimum pH5.6, acid, good stability. In the range of pH4.5 6.0 for 24h still has a high activity which reacted at 50℃; Km is 0.036μmol / L, Vmax is 20.16μmol/min; Mn²⁺, Mg²⁺ and Ca²⁺ on the enzyme activity, in which the strongest activation of Mn²⁺ , and Cu²⁺ and Zn²⁺ is inhibited.The pullulanase which obtained from Bacillus stearothermophilus belongs to high-temperature acidic enzymes in starch processing industry, used in conjunction with the starch enzyme in industrial applications can have a great prospect.