来源于Anoxybacillus sp. SK3-4普鲁兰酶PulASK的固定化及酶学性质研究

Immobilization of Pullulanase PulASK from Anoxybacillus sp. SK3-4 and Its Enzymatic Properties

摘要:

普鲁兰酶在淀粉加工、多糖制备以及啤酒酿造等领域有着广泛应用。然而，该酶游离下催化产物需分离纯化和本身不可再生限制了其工业应用。磁性纳米材料具有可重复使用、磁回收性质和比表面积大等特点，有助于解决游离酶的工业化应用难题。作者将源于LAnoxybacillus sp. SK3-4的普鲁兰酶PulASK固定在纳米磁性材料Fe₃O₄@Histidine上以制备固定化酶复合体：Fe₃O₄@Histidine/PulASK，测定固定化酶的酶学性质和动力学参数。结果显示，与游离酶PulASK相比，固定化酶Fe₃O₄@Histidine/PulASK的最适反应温度由60 ℃提高到65 ℃，最适pH与游离PulASK相同；在60 ℃，pH 6.0下孵育7 h，固定化酶残余酶活为62%，而游离酶的仅为30%。分析酶动力学数据可知，在60 ℃，pH 6.0的情况下，游离酶PulASK的K_m为4.7 mmol/L，为Fe₃O₄@Histidine/PulASK的1.47倍；Fe₃O₄@Histidine/PulASK的k_cat值是350 s^-1，与PulASK的k_cat值相当，Fe₃O₄@Histidine/PulASK的k_cat/K_m是PulASK的1.57倍；并且Fe₃O₄@Histidine/PulASK在重复使用9次后，活力仍保持50%以上。与PulASK相比，Fe₃O₄@Histidine/PulASK具有良好的热稳定性且可重复使用，具有潜在应用于食品工业的价值。

Abstract:

Pullulanase is of high industrial value in the fields of starch processing, functional medicine polysaccharide preparation and beer brewing. However, the high costs of the separation and purification of catalytic products and non-renewability of free pullulanase limit its industrial application. Magnetic nanoparticles possess unique features, such as reusability, magnetic recovery and specific surface area, which is helpful to solve the problems of industrial application of free enzymes. The immobilized enzyme complex Fe₃O₄@Histidine/PulASK was prepared by immobilizing PulASK from Anoxybacillus sp. SK3-4 onto the Fe₃O₄@Histidine magnetic nanoparticles. The enzymatic properties and kinetic parameters of the immobilized enzyme was investigated. In comparison with free PulASK, the optimal reaction temperature of Fe₃O₄@Histidine/PulASK increased from 60 ℃ to 65 ℃, while their optimal reaction pH was the same. The residual activity of immobilized enzyme was 62% after 7 h incubation at pH 6.0 under 60 ℃, while that of free enzyme was only 30%. The enzyme kinetics data analysis revealed that K_m of the free enzyme PulASK was 4.7 mmol/L at pH 6.0 under 60 ℃, which was 1.47 times as that of Fe₃O₄@Histidine/PulASK. The k_cat value of Fe3O4@Histidine/PulASK was 350 s^-1, which was comparable to that of PulASK. The k_cat /K_m values of Fe₃O₄@Histidine/PulASK was 1.57 times as that of PulASK. Furthermore, Fe₃O₄@Histidine/PulASK remained more than 50% active after 9 cycles of usage. Compared with PulASK, Fe₃O₄@Histidine/PulASK obtained remarkable thermal stability and reusability, exhibiting potential application value in food industry.