Facile and green fabrication of biocatalytic chitosan beads by one-step genipin-mediated β-glucosidase immobilization for production of bioactive genistein

Abstract

While glutaraldehyde is a commonly used cross-linker for enzyme immobilization, it is unsafe and requires the multi-step cross-linking procedure. Here, we report a facile, fast and cost-effective fabrication of immobilized β-glucosidase by mixing chitosan beads with the commercial enzyme in the presence of 0.1% w/v genipin as a greener cross-linker in one step at 40 °C for 6 h. The resulting beads exhibited improved resistance to acid and had a satisfactory β-glucosidase activity of 3.85 U/g beads. In contrast, the immobilized enzyme prepared by using 1% w/v glutaraldehyde required 3-h pre-activation of beads and 12-h incubation with the enzyme to achieve the maximum activity of 2.89 U/g beads. Compared with the soluble enzyme and immobilized enzyme fabricated by two-step cross-linking using glutaraldehyde or genipin, the immobilized enzyme prepared by one-step genipin cross-linking showed the greater stability as more than 80% of the maximum activity was retained over a broad range of pH 4–8 and temperature of 40–50 °C. It efficiently hydrolyzed genistin to genistein and could be repeatedly used by exhibiting 85% of the initial substrate conversion performance after 5 cycles. After 30 days of storage at 4 °C, more than 80% of its initial activity was retained. From these results, one-step genipin-mediated enzyme immobilization is a feasible and effective route to the fabrication of biocatalyst applied to pharmaceutical synthesis