Assembly Of Cellulases With Synthetic Protein Scaffolds

Multienzyme cascade is one of the hotspots in biocatalysis and synthetic biology fields. Cascade enzymes in metabolic pathways are often spatially organized together, facilitating substrates flow between enzymes. Construction of artificial cellulases complex by mimicking natural multienzyme assemblies is a promising way to enhance the ability of cellulases for cellulose hydrolysis.In this study, an artificial cellulases complex was constructed by tethering three cellulases to a synthetic protein scaffold, and hydrolysis of cellulose was evaluated. Four aspects were introduced:Firstly, the genes of interactional proteins were cloned to the plasmid pET21a in Escherichia coli. The binding affinity of purified proteins was detected, suggesting that they were suitable for construction of protein scaffolds. Secondly, different cellulases were cloned and overexpressed in E. coli, and the interactional proteins were fused to the C/N terminus of cellulases. The expression and ability of cellulases was compared, and IPbEG, IPaBGL, IPaA, IPbE, IPc4 were selected for assembly. Then, artificial protein scaffolds were constructed by fusion of three interactional proteins. ScafBA was selected to assemble two cellulases according to the expression. The reduction sugars from cellulose hydrolysis catalyzed by complexes with different scaffolds were assayed, and the complex assembled with ScafBAC exhibited the highest production of reduction sugar. Finally, the synergistic effect of the complex was evaluated. Compared with the corresponding free enzyme systems, the activity of confirmed bifunctional complex was increased by 35%. After a 4-h reaction, the trifunctional complex was demonstrated and showed 1.5-fold higher activity of cellulose hydrolysis than that of unassembled cellulases.Assembly with protein scaffold can facilitate the biocatalytic capacity of enzyme cascades. This assembly technology can be used in many fields, such as synthesis of high value-added chemicals, construction of biosensor, biochemistry detection and so on.