Stability of WSCP-Chla versus free Chl α
One of the central aims of photobiocatalysis is to use light to drive enzymes that catalyze reactions of interest such as the degradation of recalcitrant substrates like cellulose. For application of light-driven systems, the lifetime of the photosensitizer is, therefore, vital to prolong the catalytic lifetime.
The photostability of free Chl α and Chl α bound to the WSCP (WSCP-Chl α) was measured over time in an LPMO light-driven system which includes TtAA9 and a reductant (ascorbic acid, Asc). Photostability is in this context defined as the loss of Chl fluorescence over time relative to initial fluorescence (F/F0).
As expected, we observed that, in the light-driven LPMO system, the WSCP-Chl αcomplex is more stable than free Chl αin all conditions (Fig. 1a). When combined with TtAA9 and Asc, WSCP-Chl αshowed 76 ± 4 % fluorescence after 1 h compared to Chl α where only 5 ± 0.2 % remained. In the partial assay systems, ascorbic acid enhances the photostability of both the WSCP-Chl α complex and Chl α, whereas the presence of the TtAA9 decreases the apparent photostability. However, when combining the TtAA9 and Asc the negative effects caused by the enzyme seem to be counteracted for both WSCP-Chl αand Chl α. The final fluorescence ratio (F/F0 at 60 min) was analyzed with single factor ANOVA. All WSCP-Chl α samples were significantly different from each other (p < 0.001).
Effect of light and temperature on the stability of WSCP-Chl α versus free Chl α
For application of photobiocatalytic LPMO reactions, the photosensitizer should ideally be stable under a broad range of temperatures. For example, several fungal LPMOs (AA9) have been shown to have the highest activity levels at temperatures ranging between 40-50 °C30. Therefore, photostability was tested at 25 and 50 °C. Together with temperature, light intensity was also varied to investigate which of the two factors has a larger influence on the photostability of the complex. Both photosensitizers were subjected to 50 and 200 µmol m-2 s-1 for 1 h at 25 and 50 °C (Fig. 1b). As expected, lower light conditions (50 µmol m-2 s-1) were beneficial for both WSCP-Chl α and free Chl α. When the light was increased to 200 µmol m-2 s-1, a considerable loss in photostability was observed. The rise in temperature causes an extra 15 % loss of fluorescence in free Chl αcompared to only 5 % in WSCP-Chl αcomplex.
Effect of pH on stability of WSCP-Chl α versus free Chl α
Many enzymatic reactions require rather acidic or basic environments. For example, LPMO containing enzyme cocktails have been shown to achieve a maximum depolymerization at pH 531,32. Therefore, it is important that a photosensitizer remains stable across a broad range of pH-values. In order to determine the pH stability of the WSCP-Chl αcomplex and free Chl α, both were incubated in different buffers, ranging from pH 5-8, and their photostability was measured over time (Fig. 1c).
The photostability of WSCP-Chl α is unaffected by the changes in pH as there is no significant difference between all four samples with single factor ANOVA (p > 0.05). The stability of free Chl αis expected to be favored by high pH as these pigments are known to lose their central Mg ion in acidic conditions33,34. Although this effect is not seen under our experimental conditions, Chl α remains unstable with between 4-12 % fluorescence remaining after 60 min under all conditions. WSCP-Chl α retained between 61-63 % At pH 5-8.
Photostability of WSCP-Chl α and Chl α after 24 hours
A 24-hour assay was done at low light (50 µmol m-2 s-1) to demonstrate the long-term stability of WSCP-Chl α (Fig. 1d). One phase decay model was used to approximate the half-life of both pigments with a confidence interval over 95%. Chl α shows a half-life of approximately 1.5 hours while WSCP-Chl α is estimated at approximately 24 hours.
Effect of light intensity on H2O2 production
In light of the recent publications suggesting H2O2 is a key factor in light-driven LPMO9 we proceeded to investigate the light-driven formation/generation of H2O2 from WSCP-Chl αand free Chl α under varying light intensities (0, 50, 100, 200, and 500 µmol m-2 s-1) (Fig. 2a). With WSCP-Chl α, higher light intensities lead to a faster rate of H2O2 formation, as measured by the Ampliflu™ assay, with a maximum value of 298 µM after 30 min in 500 µmol m-2 s-1 light. The highest H2O2 formation seen in free Chl α is at 200 µmol m-2 s-1 with a total of 60 µM H2O2 after 30 min. In the absence of light, no formation of H2O2 is observed. Interestingly, Chl α exposed to a light intensity of 500 µmol m-2 s-1 also showed greatly reduced formation of H2O2 compared to the same experiment with light intensity of 200 µmol m-2 s-1. This is likely a result of rapid to fast photobleaching of Chl αin the first minutes of the reaction (Fig. 2a). To establish the correlation between light intensity and H2O2, the time traces in Fig. 2a were each fitted with a linear function. The resulting slopes correspond to the rate of H2O2 measured per minute (Fig. 2b).
Effect of reductant concentration and light on H2O2 formation
After having determined the correlation between light intensity and H2O2 formation, we investigated the effects of the reductant (Asc) concentration on H2O2 formation. The assay was set up with WSCP-Chl α and four different concentrations of Asc (0, 250, 500, and 1000 µM) (Fig. 2c). Higher concentrations of Asc led to greater endpoint H2O2 formation, however, 1000 µM Asc forms similar concentrations of H2O2 as 250 µM Asc after 10 min. H2O2 levels were below the detection limit in the absence of Asc, indicating the necessity of reductant in the light-driven mechanism. With 1000 µM Asc, the addition of TtAA9 reduces amount of detected H2O2 to that in the absence of Asc, suggesting that either TtAA9 prevents the formation of H2O2, or, more likely, that TtAA9 degrades H2O2 that is formed before it can react with Ampliflu™. In order to demonstrate the tight control of H2O2 production by WSCP-Chl α, a light-dark alternating assay was performed. In this assay, WSCP-Chl α was placed in 50 µmol m-2 s-1 for 5 min followed by 5 min of fluorescence measurements in the dark (Fig 2d). This assay clearly demonstrates the light-dependence of H2O2 production by WSCP-Chl α. Once again, this assay also shows the importance of Asc in the system for H2O2 production.
Light-driven TtAA9 assays
To assess whether the higher photostability of the WSCP-Chl α would lead to higher TtAA9 product formation, light-driven assays were performed with TtAA9 using varying concentrations of reductant. Since high concentrations of H2O2 can be detrimental to LPMO activity19,20,35, and the WSCP was shown to be more stable at lower light intensities (Fig. 1b), the light intensity was reduced to 100 µmol m-2 s-1 for TtAA9 experiments. Subsequently, the optimization process was focused on the “feed rate” of Asc to control the H2O2 production in the assays. The feed rate is defined as the concentration (mM) of Asc added at certain time intervals (min). It is difficult to determine the necessary reductant concentration since there are many factors involved. To demonstrate the importance of reductant concentrations, three assays with varying Asc feed rates: 2 mM Asc (Fig. 3a), 1 mM Asc every 60 min (Fig. 3b), and 500 µM every 20 min (Fig. 3c), with gluconic acid concentrations measured every 20 min for 2 hours.
In Fig. 3a, in the presence of 2 mM Asc, all samples show high productivity in the first 20 min, after which productivity halts. Final concentrations were 61, 39, and 56 (mg L-1) for samples WSCP-Chl α, free Chl α, and no pigment, respectively. Upon halving the concentration of Asc, and adding it every hour (1 mM/h), there is a noticeable increase in productivity of all samples (Fig. 3b); however, there is still no significant increase in TtAA9 productivity upon the addition of WSCP-Chl α. In the final assay (Fig. 3c), the concentration of Asc was changed to 500 µM, added every 20 min, which led to significantly increased productivity (p < 0.05; determined for t = 120 min) for both assays containing photosensitizers. TtAA9 with WSCP-Chl αresulted in a final gluconic acid concentration of 110 mg L-1. Chl α also boosted TtAA9 productivity significantly with 75 mg L-1 compared to TtAA9 alone at 59 mg L-1.
High-Performance Anion-Exchange Chromatography (HPAEC) was performed to confirm the results of the gluconic acid measurements. The slightly acidic nature of carbohydrates allows for highly selective separations using anion exchange at high pH. The C1-oxidized products are easily characterized as seven distinct singular peaks (Fig. 4). For this study, the chromatograms can be used to compare the relative signal intensities of the oxidized products in the different samples. TtAA9 productivity after 3 hours with WSCP-Chl α shows a max signal intensity of 168.8 (nC) at 22 min corresponding to cellotetraonic acid (Glc3Glc1A). TtAA9 + Chl α and TtAA9 on its own also demonstrate max intensities with Glc3Glc1A at 115.31 and 81.2 (nC), respectively. A control containing WSCP-Chl α with no TtAA9 shows no C1 oxidations peaks the visible cellobiose (Glc2) is background from the substrate. The area under the C1 oxidation peaks was used to estimate the photobiocatalytic enhancement. The area for TtAA9 + Chl α was 1.88x than TtAA9 alone, while TtAA9 + WSCP-Chl α was 3.4x greater compared to TtAA9 alone.