The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

Precisely-localized enzyme cascade was constructed by integrating two sequential enzymes glucoamylase (GA) and glucose oxidase (GOx)) on yeast cell surface through a-agglutinin receptor as anchoring motif and cohesin-dockerin interactions. The overall catalytic activities of the combinant strains were significantly dependent on assembly method, enzyme molecular size, enzyme order and enzyme stoichiometry. The combinant strain with GA-DocC initially bound scaffoldin prior to GOx-DocT exhibited higher overall reaction rate. The highest overall reaction rate (29.28±1.15 nmol H2O2 min-1 mL-1) was achieved when GA/GOx ratio was 2:1 with enzyme order: yeast-GA-GOx-GA, four-fold enhancement compared to free enzyme mixture. Further, the first example of starch/O2 enzymatic biofuel cells (EBFCs) using co-displayed GA/GOx based bioanodes were assembled, demonstrating excellent direct biomass-to-electricity conversion. The optimized EBFC registered open-circuit voltage of 0.78 V and maximum power density (Pmax) of 36.1 ± 2.5 µW cm-2 , significantly higher than the Pmax 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 cell, biosensing and bioelectro-synthesis. PCR primers; Amino acid sequences of dockerin-fused proteins; The numbers of expressed enzyme units on the yeast cell surface. Supplementary figures: Immunofluorescence micrographs; Long-term stability assays; Biocathode characterization; Power density-voltage profile of the blank control cell; Polarization curves of starch/O2 EBFCs.

No abstract