During the work described in Paper I, the activity of NcLPMO9C was tested on several other soluble substrates including oligosaccharides of mannan, xylan, chitin and maltodextrin, but no activity was detected. The use of glycan microarray substrate screening system enabled a broad search for possible activities of NcLPMO9C on various polymeric substrates (Figure 1 in Paper II). Interestingly, and totally novel at the time, activity was detected on various hemicelluloses with (1,4)-linked glucan backbones, including xyloglucan, which is a β-(1,4)-linked glucan substituted with xylose and other sugars, as described in section 1.2.2.1.
Other substrates that were found to be cleaved by NcLPMO9C were mixed-linked β-glucans of oat, barley and lichens, and glucomannan.
Because of the heteropolymeric nature of xyloglucan and its various substitution patterns, the identification of xyloglucan-derived products is more challenging than identification of products generated from cellulose. However, based on known xyloglucan substitution patterns and by using MS-analyses to compare NcLPMO9C-generated product profile to product profiles generated by two endoglucanases, we managed to determine that NcLPMO9C is able to handle a variety of substitutions as long as there is an unsubstituted backbone glucose unit for the LPMO to access. This became even clearer after analyzing the degradation of XXXGXXXGOH (XG14OH, i.e. a reduced 14-mer of xyloglucan where six of the eight backbone glucose-units are substituted with xylose, indicated by X) by NcLPMO9C.
The generated product profiles showed that the enzyme primarily targets the non-substituted internal glucosyl unit that is oxidized in the non-reducing end, meaning that the only observed native product corresponded to XXX (Figure 3 in Paper II). Analyses of products generated from tamarind xyloglucan (Paper II, Figure 2) and from hemicellulose in cell wall material from Arabidopsis thaliana and tomato stem (Solanum lycopersicum) (Figure 4 in Paper II) confirmed the capability of this LPMO to accept various substitution patterns and its preference for cleaving at an unsubstituted glucose.
Products generated from konjac glucomannan, a linear β-(1,4)-linked mannan interspersed with randomly distributed glucose, generated characteristic product clusters containing the keto-, gemdiol and native form of hexose oligosaccharides, as well as acetylated species (Paper II; Fig. 2B). Since products were rather long, it seems that NcLPMO9C cannot cleave at any position in the chain and this is likely due to the occurrence of consecutive mannoses, since Figure 1 shows that galactomannan is not cleaved by this enzyme. Products generated
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from barley β-glucan showed dominance of products increasing by three sugars in size (DP4, DP7, DP10; Paper II, Figure 2C). This product profile fits well with the repetitive structure of barley β-glucan, in which every third linkage is a β-(1,3)-linkage (Ajithkumar et al., 2006).
Products generated from lichenan did not show this repetitive pattern, which may be taken to indicate that the structure of lichenan (i.e. β-glucan from moss) is less repetitive.
All in all, the data presented in Figures 2-4 of Paper II show that activity of NcLPMO9C depends on the presence of a β-(1,4)-linked glucose in the substrate backbone and that the enzyme is quite flexible when it comes to accepting substitutions near this single glucose.
Importantly, Paper II also shows a comparison of the LPMO-generated product profiles with the product profiles generated by endo-glucanases acting on the same substrates. These comparisons show clear differences, suggesting that the two enzyme types have different, and perhaps complementary, or even synergistic, roles in Nature.
In attempt to assess what would be the preferred substrates of NcLPMO9C and the natural function of the enzyme, we also assessed the initial degradation rates for cellopentaose, XG14OH, polymeric xyloglucan and PASC. The results, shown in Supplementary Figure 5 of Paper II, indicated that the enzyme has a similar rate on all substrates, varying from 0.11 s-1 for the two polymeric substrates to 0.06 s-1 and 0.03 s-1 for XG14OH and cellopentaose, respectively. Importantly, it is likely that these rates reflect the rate of the generation of H2O2
in the reaction mixtures rather than the intrinsic catalytic rate of the LPMO (Bissaro et al., 2017). It would be truly interesting to re-determine these rates in an experimental set-up where the LPMO reaction is driven by externally provided H2O2.
As described in section 1.2.2 of this thesis, a main function of hemicellulose in plant cell walls is to strengthen the cell wall by interlinking cellulose microfibrils and, in the case of xyloglucan, even weaving into the cellulose microfibrils (Hayashi, 1989). It has also been proposed that coating of cellulose by xyloglucan or xylan could provide protection against cell wall deconstruction by microorganisms (Vincken et al., 1995). While hemicelluloses are generally considered to be more easily degradable than cellulose, this may change if they interact strongly with a cellulose fibril. Furthermore, the varying substitution patterns of xyloglucan may be a challenge for xyloglucanases, i.e. hydrolytic enzymes acting on xyloglucan. Interestingly, as outlined below, some xyloglucan-active LPMOs described in the literature after the discovery of the xyloglucan activity of NcLPMO9C, are even less sensitive to substitutons (Kojima et al., 2016; Nekiunaite et al., 2016). The genomes of some biomass
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degrading microorganism contain more than 40 LPMOs (Horn et al., 2012; Lenfant et al., 2017), which are upregulated to various extents in response to growth on various substrates (Tian et al., 2009). It is likely that certain LPMOs may be targeted to certain recalcitrant sub-structures in plant cell walls. Interestingly, expression levels of the gene encoding NcLPMO9C are five times higher when the N. crassa is grown on (xyloglucan-containing) Miscanthus stems compared to pure crystalline cellulose (Avicel) (Tian et al., 2009).
The study described in Paper II has contributed to a broadening of LPMO research, including also activity on soluble hemicellulosic substrates. Following the discovery of activity on xyloglucan, LPMO activity towards other substrates has been described, in particular xylan (Frommhagen et al., 2015) and starch (Lo Leggio et al., 2015; Vu et al., 2014b). These discoveries are not only important for our understanding of the complex enzymatic systems utilized by microorganisms for biomass degradation, but are also potentially of great importance for industrial applications in enzymatic saccharification of biomasses. One of the limiting factors of enzymatic degradation of lignocellulose is the insufficient removal of hemicelluloses (Saha et al., 2013), and perhaps certain hemicellulose-active LPMOs contribute to overall process efficiency. Notably, whereas cellulose is homogeneous and relatively similar in all types of plants, hemicelluloses vary a lot in composition and amount, and optimal degradation of hemicellulose could thus require substrate-specific enzyme mixtures. In this regard, the ability of NcLPMO9C to tolerate several substitutions of its β-glucan substrate could make this enzyme particularly attractive for industrial applications.
Subsequent to the work described in Paper II, an LPMO from Podospora anserina (PaLPMO9H) was described, showing activity towards both soluble cello-oligosaccharides with DP>4 and β-(1,4)-linked hemicellulose polysaccharides like xyloglucan, lichenan and glucomannan (Bennati-Granier et al., 2015). When cleaving cello-oligosaccharides, PaLPMO9H generated both C1- and C4-oxidized products, however, the product profiles upon cleavage of xylogucan was inconclusive in regard of regioselectivity. Regardless of the regioselectivity, the analogous substrate specificity to NcLPMO9C enabled a sequence comparison. The three asparagine residues (Asn25-Asn26-Asn27) in NcLPMO9C that were discussed above (Paper I) are substituted with Ser25-Asn26-Phe27 in PaLPMO9H. Thus, only the more generally conserved Asn26 is, indeed, conserved. Bennati-Granier et al. discuss whether the presence of a phenylalanine at position 27, possibly binding stronger to a sugar compared to an asparagine, could explain the higher activity of PaLPMO9H on cellotetraose compared to NcLPMO9C (Bennati-Granier et al., 2015).
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Interestingly, NcLPMO9A (NCU02240) and NcLPMO9D (NCU01050), two C4-oxidizing LPMOs for which we could not detect activity on xyloglucan, contains the sequence Tyr25-Asp26-Gly27 and Tyr25-Asn26-Gly27, respectively. The structures of NcLPMO9A and NcLPMO9D indicate that mutation of Asn27 to glycine is needed to avoid steric hindrance of the tyrosine at position 25. We have generated and produced the N25Y-N27G mutant of NcLPMO9C and a preliminary characterization of the double mutant showed wild-type like characteristics in terms of substrate preference and activity towards shorter oligosaccharides (Isaksen & Eijsink, unpublished observations).