Optimized Enzymatic Assay for LPMO Turnover

The assay conditions to measure
turnover numbers (TN) were optimized in a series of preliminary experiments.
We used the following standard assay conditions unless otherwise noted:
the optimized assays were performed by operating the amperometric
H₂O₂ sensor at an applied potential of 100 mV
vs SHE and an angular velocity of the RDE of 50 s^–1. Every second, 12.5 data points were collected. The H₂O₂ sensor was operated in 4 mL of 30 mM sodium acetate
buffer, pH 6.0, containing 100 mM KCl for improved conductivity in
the temperature-controlled (30 °C) electrochemical cell. Substrate
concentrations were 4 g L^–1 for hemicelluloses,
cellopentaose, or PASC and 100 g L^–1 for CNC and
MCC, and the initial concentration of H₂O₂ was
100 μM. LPMO concentrations varied according to the studied
substrate and therefore will be indicated in figures and table legends.
Typically, the following concentrations were used: 50 nM LPMO for
hemicellulosic substrates, 100 nM LPMO for cellopentaose and PASC,
and 200 nM LPMO for PASC, CNC, and MCC.
Reaction rates also
depended on the concentration of the reductant, and thus it was important
to determine saturating reductant concentrations. We, therefore, determined
the amount of ascorbate required to achieve saturation for all studied
LPMOs at pH 6.0 (Table S4). Figure S5 shows that saturation is achieved for
all LPMOs acting on xyloglucan or PASC with 500 μM ascorbate
but not for NcAA9F and HjAA9B acting
on PASC and xyloglucan, respectively. For these two LPMOs, 2 mM ascorbate
was used to achieve full saturation. Any deviation from these conditions
is indicated in the figure and table legends. A control experiment
with saturating amounts of cellobiose dehydrogenase from N. crassa (NcCDHIIA) (Figure S6) to reduce NcAA9C
acting on 4 g L^–1 of xyloglucan showed that the
measured maximum catalytic rate of 31.9 ± 0.5 s^–1 equals the rate obtained when using ascorbate as a reductant (31.6
± 1.5 s^–1; averaged over 3 different experiments
using 50–200 nM NcAA9C) (Table S3). This supports the notion that the experimentally
obtained reaction rates are independent of the reductant.
To
study the effect of different substrates, the cosubstrate H₂O₂, and the reductant ascorbate on catalysis, their
concentrations were varied. The final substrate concentrations in
experiments shown in Figure 2 were 0.125–8 g L^–1 (DP5, hemicelluloses,
PASC) and 10–100 g L^–1 (CNC). The ascorbate
concentration was varied between 0.005 and 12 mM to determine the
pH-dependent concentration needed to achieve full saturation at pH
4.0–7.0 (Figure 4). To study the influence of pH (5.0–8.0) on catalysis by NcAA9C at varying xyloglucan and H₂O₂ concentrations, shown in Figure 4, the ascorbate concentration was 3 mM, which is at
least 10 times higher than the highest half-saturating concentration
of ascorbate in the pH 5.0–8.0 range (at pH 5.0; Table S10). The H₂O₂ concentration
was varied between 25 and 300 μM, while the xyloglucan concentration
was kept constant at 4 g L^–1. All measurements were
performed in independent triplicate. The displayed H₂O₂ time traces were averaged over three independent measurements.