Photoenzymatic reaction and analysis
This protocol is extracted from research article:
Nicotinamide adenine dinucleotide as a photocatalyst
Sci Adv, Jul 19, 2019; DOI: 10.1126/sciadv.aax0501

A TEOA-buffered solution (150 mM, pH 7.5) containing NAD+, TsOYE, CaCl2, and substrate was prepared in a microcentrifuge tube. For the control experiment, in the absence of TEOA, MOPS was used as a substitute for TEOA because it is extensively used in the OYE biocatalysis, acts as an electron donor of photoactivated NAD+, and does not produce a precipitate with Ca2+ ions; the divalent ion is required for activity of TsOYE (41). Note that MOPS and TEOA have buffering capacities. The sample was irradiated with a 450-W xenon lamp at 318.15 K. For the unit conversion of light intensity from mW cm−2 to μE cm−2 s−1, the average photon energy was ca. 2.14 eV according to the spectral irradiance of the xenon lamp. Note that μE cm−2 s−1 refers to the number of moles of photons in micromole hitting a defined surface per second. For quantitative analysis of the product using gas chromatography (GC), organic substrates and products were extracted with ethyl acetate solvent containing 1-octanol as an internal standard. The mixture was centrifuged to collect the organic phase and, after which, was dried with MgSO4 to eliminate residual water content. The organic supernatant was analyzed by GC using a 7890A gas chromatograph (Agilent Technologies, USA). The machine was equipped with a flame ionization detector and a CP-Chirasil-Dex CB column (25 m by 0.32 mm by 0.25 μm). The oven temperature program for all enzymatic substrates and products was 363.15 K held for 2 min, 4 K min−1 to 388.15 K held for 0 min, and 20 K min−1 to 453.15 K held for 1 min. The yield, enantiomeric excess (ee), TOF, and TTN were calculated according to the following equations (Eqs. 6 to 10)Yield(%)=Concentration of productInitial concentration of substrate×100(6)ee(%)=Moles of an enantiomerMoles of the other enantiomerTotal moles of product×100(7)TOFTsOYE(hour1)=Concentration of product at the given timeConcentration of TsOYE×Time(8)TTNTsOYE=Maximum concentration of productConcentration of TsOYE(9)TTNNAD+=Maximum concentration of productConcentration of NAD+(10)

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