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

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.