Dual-Site Cooperative Modification Strategy Empowers Cellulase: Performance Optimization and pH-Sensitive Recovery.

The utilization of cellulose by enzymatic hydrolysis is of great significance for realizing resource recycling and carbon neutrality. The cellulase with efficient activity and recyclability has always been the focus and hotspot of research. Here, the modified cellulase Mal-Cell-FA with pH-sensitive recovery properties is constructed based on the transition state theory and dynamic amide bonds. It is achieved by using an aromatic aldehyde poly(ethylene glycol) to perform "dual-site cooperative modification" on the catalytic domain and the binding domain of the enzyme. The activity of Mal-Cell-FA was increased by 138.00% compared to that of natural cellulase. Through pH regulation, Mal-Cell-FA was easily cyclically utilized, and its relative enzyme activity remained above 80% after 5 cycles. The study indicates that Mal-Cell-FA has excellent enzymatic activity due to its effective accessibility to substrates. This strategy provides a new approach for the efficient utilization of cellulose and the improvement of enzyme performance.