Controllable Display of Sequential Enzymes on Yeast Surface with Enhanced Biocatalytic Activity toward Efficient Enzymatic Biofuel Cells

A precisely localized enzyme cascade was constructed by integrating two sequential enzymes, glucoamylase (GA) and glucose oxidase (GOx), on a yeast cell surface through an a-agglutinin receptor as the anchoring motif with cohesin-dockerin interaction. The overall catalytic activities of the combinant strains were significantly dependent on the assembly method, enzyme molecular size, enzyme order, and enzyme stoichiometry. The combinant strain with GA-DocC initially bound scaffoldin prior to GOx-DocT exhibited a higher overall reaction rate. The highest overall reaction rate (29.28 ± 1.15 nmol H₂O₂ min^-1mL^-1) was achieved when GA/GOx ratio was 2:1 with enzyme order: Yeast-GA-GOx-GA, 4-fold enhancement compared to free enzyme mixture. Further, the first example of starch/O₂ enzymatic biofuel cells (EBFCs) using codisplayed GA/GOx based bioanodes were assembled, demonstrating excellent direct biomass-to-electricity conversion. The optimized EBFC registered an open-circuit voltage of 0.78 V and maximum power density (P_max) of 36.1 ± 2.5 μW cm^-2, significantly higher than the P_max for other starch/O₂ EBFCs reported so far. Therefore, this work highlights rational organization of sequential enzymes for enhanced biocatalytic activity and stability, which would find applications in biocatalysis, enzymatic biofuel cells, biosensing, and bioelectro-synthesis.