底物印迹技术对双重固定化果胶酶的影响

Effect of substrate imprinting technique on double immobilized pectinase

用壳聚糖微球作为载体,戊二醛交联后通过底物印迹法固定化果胶酶,并对固定化条件和酶学性质进行了研究。结果表明:以柑橘果胶S(半乳糖醛酸≥74%)和0.05%的戊二醛作为底物和交联剂,在20 m L果胶酶液中加入1 m L柑橘果胶S,50℃反应8 min进行印迹后吸附,固定化果胶酶活力回收率达到49.77%。以柑橘果胶S为底物,在pH4.0和50℃条件下,底物印迹制备的固定化果胶酶表观米氏常数Km为7.854 mg/m L;最适反应温度提高为60℃;最适反应pH为3.5,在pH3.0~5.0内稳定;温度稳定性和酸碱稳定性有显著的提高;重复使用6次后,相对酶活剩余68.79%。底物印迹技术可以提高双重固定化果胶酶的底物亲和性和稳定性。

The pectinase was immobilized by substrate blotting with chitosan microspheres as a carrier and crosslinked with glutaraldehyde. The immobilization conditions and partial properties of immobilized enzyme were investigated. The results showed that pectinase activity recovery could retain up to 49.77% when crosslinked with 0.05% glutaraldehyde, adsorption after 20 mL pectinase solution and 1 mL citrus pectin S （galacturonic acid ≥74%） reaction 8 min at 50 ℃. With citrus pectin S as substrate, the kinetics properties for immobilized enzyme were assessed as follows. The Km of imprinted double immobilized pectinase in pH 4.0 and 50 ℃ conditions were detected to be 7.854 mg/mL by a double reciprocal Lineweaver-Burk plot. The optimum temperature and optimum pH of imprinted double immobilized pectinase for the hydrolysis of pectin were determined to be pH 3.5 and 60 ℃, respectively. The immobilized pectinase was stable in a pH range from 3.0 to 5.0. The pH stability and thermostability of imprinted double immobilized pectinase were obviously improved in comparison with free enzyme. When the imprinted double immobilized pectinase was cyclically used for six of reactions, it retained 68.79% of relative activity. Substrate imprinting technology could improve the substrate affinities and stability of double immobilized pectinase.