File Name : figure s1__ 600 dpi.tif
Caption : figure s1. gc analysis results of standard solutions of (r)-4-phenyl-2-butanol, (rac)-4-phenyl-2-butanol and 4- phenyl-2-butanone (8 mm in 50 mm taps buffer, ph 9.0). gc protocol: inlet temperature 200 °c, detector temperature 250 °c, oven 110 °c/hold 20 mins; 10 °c/min to 200 °c. rt (4-phenyl-2-butanone) = 20.8 minutes. retention time: rt((r)-4-phenyl-2-butanol) = 23.7 minutes, rt ((s)-4-phenyl-2-butanol) = 23.6 minutes, rt(4-phenyl-2-butanone) = 21.1 minutes. the concentration of the ketone and enantiomeric alcohols determined by gc-fid are shown in section 4.
File Name : figure s2__ 600 dpi.tif
Caption : figure s2. the stereoinversion of (r)-4-phenyl-2-butanol by w110a and lk nanoconfined in the e-leaf monitored in real-time by chronoamperometry. conditions: starting cell volume 5 ml and 1 ml sample taken at potential switch as indicated; solution stirred throughout using a magnetic flea; substrates: [(r)-4-phenyl-2-butanol] = 4 mm, [nadp+] = 20 μm, [mg2+] = 5 mm; buffer: 50 mm taps ph 9.0; temperature: 25 ℃, potential held at 0.21 v vs. she to drive oxidation during stage 1 and −0.64 v vs she for reduction. experiments performed in an anaerobic glovebox to avoid any contribution to the current form the reduction of o2. enzyme loading: fnr loaded first by drop-casting, lk and w110a loaded by placing the fnr-loaded electrode in a stirred 2.5 ml solution containing 1.28 nmols of lk and 0.47 nmols of w110a in 50 mm taps buffer ph 9 at 4 °c for 18 h; electrode (ti foil) sa: 7 cm2.
File Name : figure s3__ 600 dpi.tif
Caption : figure s3: the deracemization of (±)-4-phenyl-2-butanol solution by w110a and lk nanoconfined in the e-leaf and monitored in real-time by chronoamperometry. conditions: starting cell volume 5 ml and 1 ml sample is taken at potential switch as indicated; solution stirred throughout using a magnetic flea; substrate [(±)-4-phenyl-2-butanol] = 8 mm, [nadp+] = 20 μm, [mg2+] = 5 mm buffer: 50 mm taps ph9.0; temperature: 25 ℃, potential held at +0.21 v vs. she to drive oxidation and −0.64 v for reduction. experiments performed in an anaerobic glovebox to avoid any contribution to the current from the reduction of o2. enzyme loading: fnr loaded first by drop-casting, lk and w110a loaded after fnr, by stirring in a 2.5 ml solution containing 1.28 nmols of lk and 0.24 nmols of w110a in 50 mm taps buffer ph 9 at 4 °c for 18 h; electrode (ti foil) sa: 14 cm2.
File Name : figure s4_600dpi.tif
Caption : figure s4. gc-fid analysis signal for the stereoinversion experiment shown in figure 1
File Name : figure s5__600dpi.tif
Caption : figure s5. gc-fid analysis signal for the stereoinversion experiment shown in figure 3
File Name : figure s6_600dpi_resized.tif
Caption : figure s6: the effect of 25 mm edta on the activity of adh variants, w110a and w110v, from t. ethanolicous and lk from l. kefir. the oxidation of (±)-4-phenyl-2-butanol catalysed by a: w110a, b: w110v, and c: lk with real-time addition of edta to a final concentration of 25 mm as indicated. for the experiments in a and b, an ito@ti foil electrode (3.5 cm2) was preloaded with fnr by drop-casting followed by rinsing to remove excess enzyme; for the experiment in c, fnr was preloaded in the same way, but the electrode used was an ito@graphite (0.07 cm2). the starting solution for all three experiments contained 8 mm racemic alcohol; nadp+ (20 μm for a and b and 10 μm for c) was also introduced before the addition of each variant adh to the bulk solution as shown. a buffer exchange to remove any adh enzyme remaining in the bulk solution, was carried out as indicated, replacing the solution with fresh buffer containing only racemic alcohol and nadp+ at the same initial concentrations. conditions reaction volume: 4 ml; for a and b the solution was stirred throughout using a magnetic flea, for c, the electrode was rotated at 1000 rpm; magnesium (5 mm) was included in experiment c only; buffer: 50 mm taps ph 9.0; temperature: 25 ℃; a and b: potential held at +0.21 v vs. she, c: potential held at +0.07 v vs she. experiments performed in an anaerobic glovebox to avoid any contribution to the current from the reduction of o2.