Enhancing stability and by-product tolerance of β-glucuronidase based on magnetic cross-linked enzyme aggregates

Abstract

β-glucuronidase is an important catalyst which is highly specific for β-glucuronides. Here, we constructed magnetic cross-linking β-glucuronidase aggregates (MCLEAs) to for the production of glycyrrhetinic acid (GA). Before crosslinking via glutaraldehyde, we used carbodiimide to enhance the interaction between enzymes and carboxyl-functionalized Fe3O4, efficiently improving the activity recovery. Compared to free enzymes, both kcat and kcat/Km enhanced, indicating that crosslinking and aggregation brought higher catalytic efficiency to enzymes. MCLEAs enhanced pH and thermal stabilities and retained 63.3% of catalytic activity after 6 cycles. More importantly, it was first found that the glucuronic acid tolerance of β-glucuronidase after the formation of MCLEAs enhanced 221.5% in 10 mM of glucuronic acid. According to the Raman spectroscopy, the ordered structure of β-glucuronidase increased from 43.9% to 50.6% after immobilization, which explained the increased stability and tolerance. To sum up, MCLEAs provided an efficient strategy for immobilization of enzymes, which enhanced stability and glucuronic acid tolerance of enzymes. It might be an effective solution to the serious inhibition caused by by-products during the preparation of aglycone from natural glycosides, having a significant applied prospect in industry.