Preparation Of Cross-linked β-glucosidase Aggregates And Its Application For Soybean Isoflavone Hydrolyzation

Soybean plays an important role in the prevention of diverse chronic diseases, which has attracted great attention. The soybean, especially soybean hypocotyl is enriched in health-promoting isoflavones, mainly including genistin and daidzin. Epidemiological studies showed that individuals with soybean isoflavone-rich diets have evidently lower occurrences of some cancers and coronary heart diseases as compared to individuals with soybean isoflavone-less diets. A large number of recent studies have pointed to the fact that soybean isoflavones have various physiological activities, including the prevention of some cancers, cardiovascular disease and osteoporosis. Thus, their market prospects are evidently attractive for the natural functional health foods that the efficacious ingredients are dominantly isoflavones. While the soybean isoflavone glucoside should be hydrolyzed into isoflavone aglucone before showing its bio-activities. In this research, genistein was produced by theβ-glucosidase immobilization.Enzymes can be immobilized in different ways, among them, crosslinking is a very attractive technique because no carrier is necessary. A major breakthrough in this field was the development of cross-linked enzyme crystals (CLECs), but the preparation of CLECs was by no means trial and involves high costs because the CLECs requires the crystallization of the enzyme prior to crosslinking. The cross-linked enzyme aggregates (CLEAs) technologies offers an easy,cheap and broadly applicable method for the enzyme immobilization.In this paper, 6-glucosidase from Aspergillus niger was precipitated by 90% ammonium sulfate for 30 min at 0℃, then the aggregates were cross-linked by 5% glutaraldehyde for 150 min at 30℃. The 6-glucosidase cross-linked enzyme aggregates were obtained after the cross-linked 6-glucosidase was centrifuged and washed by pH5.0 citric acid-Na₂HPO₄ buffer solution several times. The optimum reaction temperature and pH of the CLEAs were 60 ℃ and 4.0, respectively. And its K_m was 4.61×10^-3 mol/L. Compared to the free β-glucosidase, the CLEAs had stronger thermal and storage stability, and also its affinity for the substrate had improved. Incubated for 120 min at 70 ℃, the CLEAs still retained 61% of the activity, while the free β-glucosidase was only 34%. When the CLEAs were stored in pH5.0 citric acid-Na₂HPO₄ buffer solution for 90 d at 4 ℃, about 86% of the activity was still retained. And when the CLEAs was employed to produce genistein, the conversion was 66%, and 35% of conversion was kept after consecutive use of 10 times.