AbstractBACKGROUNDThis study developed a feasible catalytic method for d‐allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers.RESULTSd‐Allulose 3‐epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo‐inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L−1 Mn2+, the CSCA‐linkerE‐DAE fusion enzyme obtained through high cell‐density cultivation displayed a maximal exo‐inulinase activity of 21.8 U mg−1 and resulted in a yield of 6.3 g L−1 d‐allulose and 39.2 g L−1 d‐fructose using 60 g L−1 inulin as the raw material. Catechol‐modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA‐immobilized CSCA‐linkerE‐DAE (8 U g−1) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L−1 d‐allulose and 32.7 g L−1 D‐fructose from 60 g L−1 inulin.CONCLUSIONThis study shed light on a feasible and cost‐effective approach for the production of syrup containing d‐allulose and D‐fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry