Selection of the Compromised Operating Condition of a One-Pot Multi-Enzymatic Reaction Using Individually Immobilised Enzymes: Production of Lactofructose Syrup

We selected a compromised operating condition (temperature and pH) in a one-pot multi-enzyme system by considering the stability and initial reaction rate of the catalysts in a single parameter (catalytic potential). The production of lactose-fructose syrup (LFS) was used as a model of a multi-enzymatic system. The enzymes used were β-galactosidase (β-gal), extracted from Bifidobacterium bifidum, which was previously immobilised and commercially immobilised glucose isomerase (GI). The compromised catalytic potential for both enzymes was obtained at a temperature of 50 °C and pH of 6.5, with a catalytic potential of 19,034.9 µmol g⁻¹ for β-gal and 1894.7 µmol g⁻¹ for GI. Under these conditions, the initial reaction rate of β-gal was 100% and GI was 20%, whereas the half-life was 25 h and 300 h for β-gal and GI, respectively. After 20 batches (equivalent to 40 h of operation), no significant productivity losses were detected (less than 10%). The average productivity of the first five batches was 0.111 g _fructose‧h⁻¹, whereas that of the last five batches was 0.106 g _fructose‧h⁻¹. These results showed the applicability of the catalytic potential as a tool for selecting the operating conditions in a process where two or more enzymes work simultaneously.