Direct immobilization of a recombinant invertase to Avicel by E. coli overexpression of a fusion protein containing the extracellular invertase from Zymomonas mobilis and the carbohydrate-binding domain CBDCex from Cellulomonas fimi

The Zymomonas mobilis gene invB, encoding an extracellular invertase, was translationally fused to the cellulose binding domain Cex (CBDCex) from Cellulomonas fimi and expressed in Escherichia coli. The fusion protein obtained, termed INVB-CBD, was purified and immobilized to Avicel (crystalline cellulose) in a single step. INVB-CBD showed an invertase activity of 20.8 IU/mL and was able to bind Avicel at a ratio of 5.3 mg of protein per gram of Avicel in 5 min. Both free and immobilized INVB-CBD showed the same optimal pH (5.5) than native INVB. However, an optimal temperature shift of −10 °C was observed for both free and immobilized INVB-CBD (45 °C) compared with the native INVB (55 °C). The Km value for the free and immobilized INVB-CBD was 193 and 81 mM of sucrose, respectively; whereas, the Km value for the native INVB was 208 mM of sucrose. Thus, suggesting an improvement in the substrate affinity of INVB after the addition of the CBDCex tag. Additionally, a decrease in the Vm value was found for the free and immobilized INVB-CBD compared with the native INVB.