  A novel approach for electrochemical tuning of alcohol oxidase (AOx) and
alcohol dehydrogenase (ADH) biocatalysis towards butanol-1 oxidation by
incorporating enzymes in various designs of amperometric biosensors is
presented. The biosensors were developed by using commercial graphene
oxide-based screen-printed electrodes and varying enzyme producing strains,
encapsulation approaches (layer-by-layer (LbL) or one-step electrodeposition
(EcD)), layers composition and structure, operating conditions (applied
potential values) and introducing mediators (Meldola Blue and Prussian Blue) or
Pd-nanoparticles (Pd-NPs). Simultaneous analysis/screening of multiple crucial
system parameters during the enzyme engineering process allowed to identify
within a period of one month that four out of twelve proposed designs
demonstrated a good signal reproducibility and linear response (up to 14.6 mM
of butanol) under very low applied potentials (from -0.02 to -0.32 V). Their
mechanical stability was thoroughly investigated by multi-analytical techniques
prior to butanol determination in cell-free samples from an anaerobic butanol
fermentation. The EcD-based biosensor that incorporates ADH, NAD+, Pd-NPs and
Nafion showed no loss of enzyme activity after preparation and demonstrated
capabilities towards low potential (-0.12 V) detection of butanol-1 in
fermentation medium (4 mM) containing multiple electroactive species with
almost 15 times enhanced sensitivity (0.2282 $\mu$A/mM $\pm$ 0.05) when
compared to the LbL design. Furthermore, the ADH-Nafion bonding for the S.
cerevisiae strain was confirmed to be 3 times higher than for E. coli.
