The liquid fraction from hydrothermal pretreatment of wheat straw provides lytic polysaccharide monooxygenases with both electrons and H2O2 co-substrate

Abstract

Background Enzyme-aided valorization of lignocellulose represents a green and sustainable alternative to the traditional chemical industry. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important components of the state-of-the art enzyme cocktails for cellulose conversion. Yet, these monocopper enzymes are poorly characterized in terms of their kinetics, as exemplified by the growing evidence for that H2O2 may be a more efficient co-substrate for LPMOs than O2. LPMOs need external electron donors and one key question of relevance for bioprocess development is whether the required reducing power may be provided by the lignocellulosic substrate. Results Here, we show that the liquid fraction (LF) resulting from hydrothermal pretreatment of wheat straw supports LPMO activity on both chitin and cellulose. The initial, transient activity burst of the LPMO reaction was caused by the H2O2 present in the LF before addition of LPMO, while the steady-state rate of LPMO reaction was limited by the LPMO-independent production of H2O2 in the LF. H2O2 is an intermediate of LF oxidation as evidenced by a slow H2O2 accumulation in LF, despite high H2O2 production rates. This H2O2 scavenging ability of LF is important since high concentrations of H2O2 may lead to irreversible inactivation of LPMOs. Conclusions Our results support the growing understanding that fine-tuned control over the rates of H2O2 production and consumption in different, enzymatic and non-enzymatic reactions is essential for harnessing the full catalytic potential of LPMOs in lignocellulose valorization.

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

“…Notably, only a handful of studies included LPMOs in their enzyme mixtures [21,61,87,174]. The results of these studies indicate a correlation between the lignin content of the pretreated feedstock and the importance of LPMO in the enzyme mixture, which may be attributed to the ability of lignin to drive LPMO reaction, as discussed above (e.g., [185,381]). When assessing the optimal proportion of LPMO in the enzyme mix, process conditions will have to be taken into account, too, since the LPMO reaction requires a source of oxygen.…”

“…An explanation for these initial findings only became clear after the discovery that LPMOs need electrons, which lignin can provide [114,381]. Recent studies indicate that lignin has a dual function in LPMO activation: it is able to reduce the active site-copper of LPMOs and to produce H 2 O 2 in situ from O 2 [185,269]. Importantly, lignin-active enzymes can affect the electrondonating and H 2 O 2 -generating abilities of lignin, providing possible links between polysaccharide-and lignin-degrading enzyme systems [42,115,269].…”

“…For saccharification of cellulose-rich sulfite-pulped spruce, it has been shown that lignin-containing spent sulfite liquor can work as an electron donor [62,65]. On the other hand, accumulating data confirm that the LPMO reaction can be driven by lignin remaining in the biomass after various pretreatments, including dilute-sulfuric acid pretreatment [134], steam explosion [250] or hydrothermal pretreatment [48,185], although to varying extents [296]. Thus, while lignin may be inhibitory to cellulases due to unproductive enzyme binding [23,32,91,260,287,288,347] or shielding the polysaccharide [90,191], it may be crucial for LPMO activity in certain experimental settings.…”

“…For substrates with low lignin content, direct supply of H 2 O 2 works extremely well [248], also at demonstration scale [65]. For lignin-rich substrates, however, the benefits of direct addition of external H 2 O 2 are less clear [248], presumably due to side-reactions occurring between added H 2 O 2 and lignin [185]. In situ production of H 2 O 2 may happen close to the enzyme, perhaps even on the enzyme, which will increase the likeliness that the generated H 2 O 2 is indeed used by the LPMO rather than being consumed in side reactions between H 2 O 2 and lignin.…”

“…Importantly, one of the current targets when optimizing saccharification setups concerns how to leverage LPMO activity while keeping LPMOs from inactivation. As discussed above, LPMO inactivation may be caused by reactive oxygen species that derive from reactions between O 2 and lignin [185] or that are formed by the LPMO itself [37] or by other redox enzymes present in the enzyme mixture [39]. It has been shown for various reaction setups that too high feeding rates of externally added H 2 O 2 [200,248] or too high levels of in situ production of H 2 O 2 [185,269] lead to LPMO inactivation.…”