Study Of ?-glucosidase Immobilization And The Biotransformation Of Soybean Isoflavone

Soybean isoflavones belong to the secondary metabolites of the soy, which are used as phytoestrogens and antioxidants. Two types of active ingredient are glucoside and aglycone after hydrolyzation, while the small mount of isoflavone aglycone has more bioactivity than glucoside which is the major part of isoflavone. Preliminary studies in our lab found that the free recombinant bata-glucosidase from Agrobacterium tumefaciens could hydrolyse glucoside into aglycone effectively. For the purpose of reducing the cost of hydrolysis and providing reference for industrial aglycone production, this research studied immobilization condition and enzymatic properties of recombinant beta-glucosidase, and the hydrolysis system of soybean isoflavones via immobilized beta-glucosidase.Beta-glucosidase, purified from the engineered strain HCCB20097, was immobilized on chitosan beads cross-linked with glutaraldehyde by cross-linking and adsorption. The optimized immobilization condition was determined as follows:adding0.3g of chitosan beads to2.5%glutaraldehyde of10ml in shaker under the speed of150r/min for2hours at the crosslinking temperature of25?; and then, after chitosan-glutaraldehyde beads were washed by distilled water, adding10ml of citric acid-sodium dihydrogen phosphate buffer solution(pH=6.0) and17.69U ?-glucosidase to above solution for8hours absorbing under the same speed and4?. Under this condition, the recovery of enzyme activity reached to56.12%.The optimal reaction temperature and pH of immobilized beta-glucosidase and free beta-glucosidase were studied and the results showed that both are40?and6.0, and the thermostability of immobilized beta-glucosidase much better than that of free beta-glucosidase. The activity of immobilized beta-glucosidase remains90%even stored in solution with pH6.0, at4?for almost one month, while the activity of free enzyme only remains40%in the same condition at the third day.Using isoflavone as substrate and immobilized recombinant beta-glucosidase as biocatalyst, the different surfactants on the biotransform system was carefully investigatived to overcome the low yield of product. The results of optimized system showed as follows:1.5%of Triton X-114,1.0mg/ml of isoflavone,1.5U of immobilized enzyme. The conversion rate by this immobilized enzyme keeps about90%after11batches under the optimal condition, which is slightly better than that of commercially available enzyme under the same condition.