Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system
Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system
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DOI：
10.1016/j.carbpol.2010.02.027
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发表时间：
2010-06
影响因子：
11.2
通讯作者：
R. Singh;G. Saini;J. Kennedy
中科院分区：
文献类型：
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作者：
R. Singh;G. Saini;J. Kennedy
pululanase在Duolite XAD761上共价固定后，热稳定性得到显著改善。固定化酶的最佳温度为60℃，比游离酶高10℃。普鲁兰、可溶性淀粉和葡聚糖的表观km值分别为4.4、20和50mg / l−1。Mn2+和Ca2+酶活性分别增加2.0倍和2.2倍。固定化生物催化剂活化能（Ea）为22.38kJmol−1。测定了固定化普鲁兰酶在不同温度（60-70℃）下不可逆失活的热力学参数（ΔH*， ΔG*， ΔS*）。60℃时d值最大（95.95h）， Q10为1.29。固定化生物催化剂在间歇体系中有效地水解了普鲁兰。在125rpm转速下，水解300min后达到固定相（95.70±1.36%）。在最佳条件下，普鲁兰水解得还原糖率为4.21±0.06%。固定化生物催化剂成功回收了33批，但在第25批后酶活性降低了一半。
Pullulanase showed significant improvement in thermal stability after covalent immobilization on Duolite XAD761. Optimum temperature of immobilized enzyme was 60°C, which was 10°C higher than the free enzyme. Apparent Kmvalues for pullulan, soluble starch and dextran were 4.4, 20 and 50mgmL−1, respectively. Mn2+and Ca2+showed 2.0- and 2.2-fold increase in enzyme activity. Activation energy (Ea) of immobilized biocatalyst was 22.38kJmol−1. Thermodynamic parameters (ΔH*, ΔG*, ΔS*) for irreversible inactivation of immobilized pullulanase at different temperatures (60–70°C) were also determined. D-value was maximum (95.95h) at 60°C and temperature quotient (Q10) was 1.29. Immobilized biocatalyst was effectively used for pullulan hydrolysis in a batch system. Stationary phase in hydrolysis (95.70±1.36%) was reached after 300min at 125rpm. Pullulan hydrolysis yielded 4.21±0.06% reducing sugars under optimal conditions. Immobilized biocatalyst was successfully recycled for 33 batches, but the enzyme activity was reduced to half after 25th cycle.