CBM17 is used to immobilize the sucrose isomerase PalI originated from Klebsiella sp.LX3 on the surface of microcrystalline cellulose to obtain a kind of economic,environmental friendly,high stability and recyclable immo-bilized sucrose isomerase.The fusion CBM17 tagged PalI expression vector was constructed by Restriction-Free clo-ning,and the immobilized enzyme was prepared based on the property that CBM17 binds spontaneously to microcrys-talline cellulose;The enzyme activity of the enzyme was determined by 3,5-dinitrosalicylic acid colorimetric glycome-try;Comparing the enzymatic properties of enzymes in the free state with the immobilized state,a measure of the cata-lytic performance of the immobilized enzyme;The number of recyclable uses and the storage time of the immobilized enzyme were measured to evaluate its stability.CBM17 tagged PalI was successfully constructed and immobilized on the surface of microcrystalline cellulose;The immobilized and free forms of PalI showed similar optimum temperature and pH,respectively 45℃and pH 6.0,and their temperature stability and pH stability were improved compared with those of free enzymes.The highest enzyme activity was(11.04±0.02)U/mg Avicel PH101.The immobilized en-zyme showed an enhanced affinity for the substrate sucrose with Km at(79.94±9.56)mmol/L;The immobilized en-zyme showed fine operational and storage stability,the enzyme activity remained at about 80% of the initial enzyme activity for 12 consecutive reactions,decreased to less than 60% when stored until 56 days at 4℃.In this study,an environmentally benign,economically feasible,and highly stable immobilized sucrose isomerase was successfully pre-pared,and this immobilization strategy provides a new way of thinking for the application of sucrose isomerase in in-dustry.
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