Materials and enzymes
All chemicals were of the highest purity grade available and purchased from Sigma-Aldrich (Saint Louis, USA), unless otherwise stated. Glacial acetic acid, hydrogen peroxide and fructose were purchased from Merck (Darmstadt, Germany).
LPMOs from Thermoascus aurantiacus (TaAA9A), UniProtKB: G3XAP7, Thermothielavioides terrestris (TtAA9), UniProtKB: D0VWZ9, Lentinus similis (LsAA9A), UniProtKB: A0A0S2GKZ1, Aspergillus nidulans (AnAA13), UniProtKB: Q5B1W7, as well as the laccase from Myceliophthora thermophila (MtL), UniProtKB: G2QFD0 were kind gifts from Novozymes A/S (Bagsværd, Denmark). LPMOs from Bacillus atrophaeus (BatAA10), UniProtKB: A0A0H3E2X6, Serratia marcescens (SmAA10), UniProtKB: O83009, Thermobifida fusca (TfAA10A), UniprotKB: Q47QG3 were expressed using the “LyGo” platform as previously described . Copper chaperone from Pseudomonas fluorescens (PfCopC), UniProtKB: A0A0D0TME7  and laccase from Bacillus subtilis (CotA), UniProtKB: H8WGE7  were expressed heterologously in Escherichia coli as previously described. All enzymes were purified to homogeneity before use. Horseradish peroxidase (HrP) was purchased from Sigma-Aldrich and used without further purification. LPMO concentrations were quantified after acid hydrolysis and separation of individual amino acids by ion-exchange chromatography .
A stock solution of rPHP was created by reducing PHP with zinc dust. For this, 1 g PHP, 4 g zinc dust, and 2.6 g NaOH were suspended in 200 mL water and boiled for 20 h with reflux. The rPHP solution was neutralized with 4 mL glacial acetic acid and boiled for another 2 h. The solution was brought to room temperature and zinc oxide was removed by decanting the rPHP solution into a new flask. Some fresh zinc dust was added to the rPHP and it was stored at 4 °C for over a year without loss of activity. The rPHP concentration was determined to be 16 mM by first oxidising a 1.25% solution to completion by HrP and comparing absorption at 552 nm to a standard curve for PHP (Fig. S6).
rPHP is commercially available under the trivial name phenolphthalin and we purchased a vial from Glentham Life Sciences (Corsham, UK). Activity of this commercial rPHP was similar to what we observed for the in-house prepared compound, although the assay response was lower.
LPMO activity assay protocol
The rPHP assay went through several stages of optimization. However, during the preparation of this manuscript, the experiments were repeated using the optimized assay conditions: triplicates of 200 µL of 0.3 µM Cu-TaAA9A, 200 µM rPHP, 25 mM citrate–phosphate buffer at pH 7.25 and 100 µM DHA were incubated in a microtiter plate well for 30 min at 40 °C with shaking at 450 rpm. The assay colour was developed by adding 50 µL 1 M Na2CO3 at pH 10.3, and the absorption measured at a wavelength of 552 nm on a BioTek Synergy H1 plate reader. The components were combined in parts of 50 µL from fourfold concentrated stocks. Prior to the experiments the LPMOs stocks were incubated over night at 4 °C with stoichiometric amounts of CuCl2. This optimized assay condition was used as the core setup on which further characterisation of the assay was carried out by altering one or two parameters as described in the text. Some of the conditions needs further elaboration.
The rPHP assay is discontinuous and the progress curves were measured by starting a series of technical replicate reactions and stopping them at different timepoints. These samples were not incubated in a thermomixer but rather inside the plate reader (preheated to 40 °C). At pre-programmed timepoints, the plate reader measured the ascorbate absorption at 265 nm before injection of 50 µL 1 M Na2CO3 to specified wells, shook the plate for 4 s and read the PHP absorption at 552 nm.
Lower limit of detection
The lower limit of assay sensitivity was determined by analysis of 16 independently prepared samples. In practice, four assay plates were set up over the course of 2 days, each plate with four samples containing 0, 10 or 15 nM TaAA9A. Each sample was copper loaded individually and all pipetting carried out using a single channel pipette. Fresh stock of DHA was used for each plate. Standard deviations at σ = 1.645 were calculated using the STDEV function in Microsoft Excel 2016.
rPHP concentrations were varied and the resulting assay measurement used in kinetic analysis using the Michaelis–Menten model: v = E0 × kcat [rPHP]/(KM + [rPHP]), where v is the rate of PHP formation and E0 the enzyme concentration. rPHP oxidation was assumed to be linear with time for all concentrations of rPHP and the conversion factor v = A552/(1.6 × 10–2 µM−1 × 30 min) was used (Fig. S6).
Temperature optimum and enzyme stability
For the determination of the temperature profile of TaAA9A in the assay, the assay was scaled down to 100 µL and mixed in a PCR tube. The reactions were incubated in a thermocycler with temperature gradient capability in three runs: 40–60 °C, 50–70 °C, and 60–80 °C. After 10 min incubation, the tubes were put on ice and the reaction stopped with 25 µL 1 M Na2CO3. The samples were withdrawn from the PCR tubes and transferred to a low-volume microtiter plate for absorption measurements. Data showed that there was a significant edge effect from the thermocycler gradient and the outer data points were omitted giving a total of 30 data points in each series.
Duplicate mixtures of 0.75 µM Cu-TaAA9A, 0.4% PASC, 25 mM citrate phosphate buffer at pH 7.25 and various co-substrates (total 200 µL) were incubated at 50 °C with shaking at 850 rpm. The co-substrates ascorbate, DHA, glutathione, and fructose were added at concentrations of 1 mM, and the reaction mixtures were incubated for 1 h or 23 h. After incubation, the reactions were stopped by the addition of 50 µL 0.5 M NaOH and then passed through a 0.45 µm filter. The released saccharides were analysed by HPAEC on an ICS 5000 equipped with a PAD detector (Dionex, Sunnyvale, CA, USA) and a CarboPac PA1 column. Chromatography was carried out following the method of Westereng et al. . Briefly, the saccharides were eluted in 0.1 M NaOH with a non-linearly increasing amount NaOAc.
rPHP and cellulose assays were investigated in anaerobic environment. In preparation of the experiments, buffer, substrates, enzyme and stop solutions were placed with mild agitation in a rigid acrylic glovebox (Belle Technology UK Ltd) and the box was purged with N2 gas for at least 1 day. On the day of experiment, DHA and ascorbic acid were dissolved in the already purged buffer. During the experiments, the build-in oxygen meter showed values of 20–40 ppm O2.
Detailed rPHP assay protocol
Step 1: Prepare an assay buffer: Dissolve 6 g Na2HPO4-7⋅H2O in 200 mL milliQ water and add citric acid to pH 7.25. Prepare a 800 µM rPHP solution. Prepare a 40 mM DHA solution (43.5 mg in 5 mL milliQ water) and dilute to 400 µM. Remaining DHA can be aliquoted and stored frozen. Prepare a stop solution: Dissolve 5.25 g Na2CO3 and 4.2 g NaHCO3 in 100 mL milliQ water and check that the pH is 10.3.
Step 2: Prepare enzyme solution: Equal volumes of 2.4 µM apo-enzyme and 2.4 µM CuCl2 are mixed and incubated overnight at 4 °C.
Step 3: Prepare an assay mix by combining equal volumes of assay buffer, rPHP solution, and DHA solution. In a microtiter plate, add 50 µL of LPMO sample to each well in a microtiter plate, and initiate the assay with 150 µL of the assay mix. Incubate the plate at 40 °C for 30 min while shaking at 450 rpm.
Step 4: Add 50 µL of stop buffer to each well and a pink colour will appear. Measure the absorption at 552 nm shortly after.