Bifunctional acid urease can not only remove urea from rice wine, but also effectively remove ethyl carbamate (EC), thereby reducing harmful substances in rice wine. Acid urease from Providencia rettgeri JN-B815 was expressed in Escherichia coli BL21(DE3) as an inclusion body. Subsequently, acid urease was refolded gradually using dilution-ultrafiltration and specific activity of 123 U mg(-1) was obtained. The acid urease thus obtained was immobilized on graphene oxide/chitosan beads. The recovery of immobilized urease was 77% for the graphene oxide/chitosan composite when coupled for 8 h with 5% glutaraldehyde. Steady-state kinetic analysis showed that the optimum temperature for urease activity was 37 degrees C. Time-dependent thermal inactivation studies showed that the stability of the immobilized urease improved at 37 degrees C and the t(1/2) (half-life) of decay in urease activity was 12 h, whereas it was 43 days for soluble and chitosan/graphene oxide immobilized urease at 4 degrees C. A significant change in Km occurred during the immobilization; the Km for urea and EC in the free state were 9.14 mM and 386 mM, respectively, whereas they were 5.28 mM and 180 mM, respectively, in the immobilized state. The values of V-max for immobilized EC and urea were 1.08 mu mol min(-1) and 1.16 mu mol min(-1), respectively, whereas they were 0.782 mu mol min(-1) and 0.931 mu mol min(-1), respectively, for soluble EC and urea. Furthermore, 90% of the original activity of graphene oxide/chitosan urease beads were retained after the beads were used 10 times, indicating excellent reusability. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.