Fig. 4: Lignin-driven ScAA10C-catalyzed solubilization of cellulose and reduction kinetics for SmAA10A.
a The figure shows time courses for the formation of solubilized oxidized products by ScAA10C (0.5 µM) in reactions with native (KLN; circles) or dialyzed (KLD; diamonds) kraft lignin (0.9 g L−1) and Avicel (10 g L−1) in sodium phosphate buffer (50 mM, pH 7.0) at 40 °C with magnetic stirring, in the dark (darker color) or when irradiated by white light (lighter color; I = 10% Imax, ~16.8 W cm−2). Before quantification of soluble oxidized products, solubilized cello-oligosaccharides were hydrolyzed by TfCel6A to convert LPMO products with varying degree of polymerization (DP) to a mixture of DP 2 and 3 [GlcGlc1A, (Glc)2Glc1A], the amounts of which were summed up to yield the concentration of oxidized sites. b The figure shows the observed pseudo-first-order constants, kobs, for reduction of SmAA10A-Cu(II) as a function the kraft lignin concentration, derived from the fluorescence traces shown in Supplementary Fig. 3a, b. Kraft lignin concentrations were calculated based on an average molecular mass (provided by the supplier) of 10,000 g/mol for both lignin preparations; since the average mass of the dialyzed lignin is expected to be somewhat higher, compared to the native lignin, the second order rate constant for the dialyzed lignin is underestimated. SmAA10A-Cu(II) (10 µM) was anaerobically mixed with varying concentrations of native (KLN; circles) and dialyzed (KLD; diamonds) kraft lignin, and the change in fluorescence was monitored as a function of time. The reactions were carried out in sodium phosphate buffer (50 mM, pH 7.0) at 25 °C. Data were fit to single exponential functions to give observed rate constants (kobs) at each lignin concentration. The apparent second order rate constant k1applignin was determined from linear regression using the reported data points and displayed an R2 > 0.99. The data in a and b are reported as mean values from three independent experiments and the error bars show ± s.d. (n = 3).
