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 inv B, encoding an extracellular invertase, was translationally fused to the cellulose binding domain Cex (CBD Cex ) 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 K m value for the free and immobilized INVB-CBD was 193 and 81 mM of sucrose, respectively; whereas, the K m 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 CBD Cex tag. Additionally, a decrease in the V m value was found for the free and immobilized INVB-CBD compared with the native INVB.