Characterization of Amycolatopsis 75iv2 dye-decolorizing peroxidase on O-glycosides.

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive. IMPORTANCE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.

AA16 Oxidoreductases Boost Cellulose-Active AA9 Lytic Polysaccharide Monooxygenases from Myceliophthora thermophila.

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that MtAA16A from Myceliophthora thermophila and AnAA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the MtAA16A crystal structure showed a fairly LPMO-typical histidine brace active site, but the cellulose-acting LPMO-typical flat aromatic surface parallel to the histidine brace region was lacking. Further, we showed that both AA16 proteins are able to oxidize low-molecular-weight reductants to produce H2O2. The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila (MtLPMO9s) but not by three AA9 LPMOs from Neurospora crassa (NcLPMO9s). The interplay with MtLPMO9s is explained by the H2O2-producing capability of the AA16s, which, in the presence of cellulose, allows the MtLPMO9s to optimally drive their peroxygenase activity. Replacement of MtAA16A by glucose oxidase (AnGOX) with the same H2O2-producing activity could only achieve less than 50% of the boosting effect achieved by MtAA16A, and earlier MtLPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H2O2 to the MtLPMO9s is facilitated by protein-protein interaction. Our findings provide new insights into the functions of copper-dependent enzymes and contribute to a further understanding of the interplay of oxidative enzymes within fungal systems to degrade lignocellulose.

Extending the diversity of Myceliophthora thermophila LPMOs: Two different xyloglucan cleavage profiles.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic conversion of plant cell wall polysaccharides. Continuous discovery and functional characterization of LPMOs highly contribute to the tailor-made design and improvement of hydrolytic-activity based enzyme cocktails. In this context, a new MtLPMO9F was characterized for its substrate (xyloglucan) specificity, and MtLPMO9H was further delineated. Aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis, we found that both MtLPMOs released predominately C4-oxidized, and C4/C6-double oxidized xylogluco-oligosaccharides. Further characterization showed that MtLPMO9F, having a short active site segment 1 and a long active site segment 2 (-Seg1+Seg2), followed a "substitution-intolerant" xyloglucan cleavage profile, while for MtLPMO9H (+Seg1-Seg2) a "substitution-tolerant" profile was found. The here characterized xyloglucan specificity and substitution (in)tolerance of MtLPMO9F and MtLPMO9H were as predicted according to our previously published phylogenetic grouping of AA9 LPMOs based on structural active site segment configurations.

Strategy to identify reduced arabinoxylo-oligosaccharides by HILIC-MSn.

Identification of arabinoxylo-oligosaccharides (AXOS) within complex mixtures is an ongoing analytical challenge. Here, we established a strategy based on hydrophilic interaction chromatography coupled to collision induced dissociation-mass spectrometry (HILIC-MSn) to identify a variety of enzyme-derived AXOS structures. Oligosaccharide reduction with sodium borohydride remarkably improved chromatographic separation of isomers, and improved the recognition of oligosaccharide ends in MS-fragmentation patterns. Localization of arabinosyl substituents was facilitated by decreased intensity of Z ions relative to corresponding Y ions, when fragmentation occurred in the vicinity of substituents. Interestingly, the same B fragment ions (MS2) from HILIC-separated AXOS isomers showed distinct MS3 spectral fingerprints, being diagnostic for the linkage type of arabinosyl substituents. HILIC-MSn identification of AXOS was strengthened by using specific and well-characterized arabinofuranosidases. The detailed characterization of AXOS isomers currently achieved can be applied for studying AXOS functionality in complex (biological) matrices. Overall, the present strategy contributes to the comprehensive carbohydrate sequencing.

Fungal glycoside hydrolase family 44 xyloglucanases are restricted to the phylum Basidiomycota and show a distinct xyloglucan cleavage pattern.

Xyloglucan is a prominent matrix heteropolysaccharide binding to cellulose microfibrils in primary plant cell walls. Hence, the hydrolysis of xyloglucan facilitates the overall lignocellulosic biomass degradation. Xyloglucanases (XEGs) are key enzymes classified in several glycoside hydrolase (GH) families. So far, family GH44 has been shown to contain bacterial XEGs only. Detailed genome analysis revealed GH44 members in fungal species from the phylum Basidiomycota, but not in other fungi, which we hypothesized to also be XEGs. Two GH44 enzymes from Dichomitus squalens and Pleurotus ostreatus were heterologously produced and characterized. They exhibited XEG activity and displayed a hydrolytic cleavage pattern different from that observed in fungal XEGs from other GH families. Specifically, the fungal GH44 XEGs were not hindered by substitution of neighboring glucosyl units and generated various "XXXG-type," "GXXX(G)-type," and "XXX-type" oligosaccharides. Overall, these fungal GH44 XEGs represent a novel class of enzymes for plant biomass conversion and valorization.

Novel Two-Step Process in Cellulose Depolymerization: Hematite-Mediated Photocatalysis by Lytic Polysaccharide Monooxygenase and Fenton Reaction.

To transform cellulose from biomass into fermentable sugars for biofuel production requires efficient enzymatic degradation of cellulosic feedstocks. The recently discovered family of oxidative enzymes, lytic polysaccharide monooxygenase (LPMO), has a high potential for industrial biorefinery, but its energy efficiency and scalability still have room for improvement. Hematite (α-Fe2O3) can act as a photocatalyst by providing electrons to LPMO-catalyzed reactions, is low cost, and is found abundantly on the Earth's surface. Here, we designed a composite enzymatic photocatalysis-Fenton reaction system based on nano-α-Fe2O3. The feasibility of using α-Fe2O3 nanoparticles as a composite catalyst to facilitate LPMO-catalyzed cellulose oxidative degradation in water was tested. Furthermore, a light-induced Fenton reaction was integrated to increase the liquefaction yield of cellulose. The innovative approach finalized the cellulose degradation process with a total liquefaction yield of 93%. Nevertheless, the complex chemical reactions and products involved in this system require further investigation.

Regioselective C4 and C6 Double Oxidation of Cellulose by Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard-to-convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello-oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H2 18 O, it was confirmed that the C6 gem-diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem-diol, could not be excluded. These findings can be extended to how the reactive LPMO-cosubstrate complex is positioned towards the substrate.

Oxidized Product Profiles of AA9 Lytic Polysaccharide Monooxygenases Depend on the Type of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC). We observed that attachment of a carbohydrate-binding module 1 (CBM1) did not change the substrate specificity of LPMO9B from Myceliophthora thermophila C1 (MtLPMO9B) but stimulated the degradation of all three types of cellulose. A detailed quantification of oxidized ends in both soluble and insoluble fractions, as well as characterization of oxidized cello-oligosaccharide patterns, suggested that MtLPMO9B generates mainly oxidized cellobiose from BC, while producing oxidized cello-oligosaccharides from AVI and RAC ranged more randomly from DP2-8. Comparable product profiles, resulting from BC, AVI, and RAC oxidation, were found for three other AA9 LPMOs. These distinct cleavage profiles highlight cellulose specificity rather than an LPMO-dependent mechanism and may further reflect that the product profiles of AA9 LPMOs are modulated by different cellulose types.

Mass spectrometric fragmentation patterns discriminate C1- and C4-oxidised cello-oligosaccharides from their non-oxidised and reduced forms.

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that degrade recalcitrant polysaccharides, such as cellulose. However, the identification of LPMO-generated C1- and/or C4-oxidised oligosaccharides is far from straightforward. In particular, their fragmentation patterns have not been well established when using mass spectrometry. Hence, we studied the fragmentation behaviours of non-, C1- and C4-oxidised cello-oligosaccharides, including their sodium borodeuteride-reduced forms, by using hydrophilic interaction chromatography and negative ion mode collision induced dissociation - mass spectrometry. Non-oxidised cello-oligosaccharides showed predominantly C- and A-type cleavages. In comparison, C4-oxidised ones underwent B-/Y- and X-cleavage close to the oxidised non-reducing end, while closer to the reducing end C-/Z- and A-fragmentation predominated. C1-oxidised cello-oligosaccharides showed extensively A-cleavage. Reduced oligosaccharides showed predominant glycosidic bond cleavage, both B-/Y- and C-/Z-, close to the non-reducing end. Our findings provide signature mass spectrometric fragmentation patterns to unambiguously elucidate the catalytic behaviour and classification of LPMOs.

Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs. RESULTS: Two Neurospora crassa (Nc) LPMOs were found to represent different mode-of-action towards XG. Interestingly, the configuration of active site segments of these LPMOs differed as well, with a shorter Segment 1 (-Seg1) and a longer Segment 2 (+Seg2) present in NcLPMO9C and the opposite for NcLPMO9M (+Seg1-Seg2). We confirmed that NcLPMO9C cleaved the non-reducing end of unbranched glucosyl residues within XG via the oxidation of the C4-carbon. In contrast, we found that the oxidative cleavage of the XG backbone by NcLPMO9M occurred next to both unbranched and substituted glucosyl residues. The latter are decorated with xylosyl, xylosyl-galactosyl and xylosyl-galactosyl-fucosyl units. The relationship between active site segments and the mode-of-action of these NcLPMOs was rationalized by a structure-based phylogenetic analysis of fungal AA9 LPMOs. LPMOs with a -Seg1+Seg2 configuration clustered together and appear to have a similar XG substitution-intolerant cleavage pattern. LPMOs with the +Seg1-Seg2 configuration also clustered together and are reported to display a XG substitution-tolerant cleavage pattern. A third cluster contained LPMOs with a -Seg1-Seg2 configuration and no oxidative XG activity. CONCLUSIONS: The detailed characterization of XG degradation products released by LPMOs reveal a correlation between the configuration of active site segments and mode-of-action of LPMOs. In particular, oxidative XG-active LPMOs, which are tolerant and intolerant to XG substitutions are structurally and phylogenetically distinguished from XG-inactive LPMOs. This study contributes to a better understanding of the structure-function relationship of AA9 LPMOs.

Structural Motifs of Wheat Straw Lignin Differ in Susceptibility to Degradation by the White-Rot Fungus Ceriporiopsis subvermispora.

The white-rot fungus Ceriporiopsis subvermispora delignifies plant biomass extensively and selectively and, therefore, has great biotechnological potential. We previously demonstrated that after 7 weeks of fungal growth on wheat straw 70% w/w of lignin was removed and established the underlying degradation mechanisms via selectively extracted diagnostic substructures. In this work, we fractionated the residual (more intact) lignin and comprehensively characterized the obtained isolates to determine the susceptibility of wheat straw lignin's structural motifs to fungal degradation. Using 13C IS pyrolysis gas chromatography-mass spectrometry (py-GC-MS), heteronuclear single quantum coherence (HSQC) and 31P NMR spectroscopy, and size-exclusion chromatography (SEC) analyses, it was shown that ß-O-4' ethers and the more condensed phenylcoumarans and resinols were equally susceptible to fungal breakdown. Interestingly, for ß-O-4' ether substructures, marked cleavage preferences could be observed: ß-O-4'-syringyl substructures were degraded more frequently than their ß-O-4'-guaiacyl and ß-O-4'-tricin analogues. Furthermore, diastereochemistry (threo > erythro) and γ-acylation (γ-OH > γ-acyl) influenced cleavage susceptibility. These results indicate that electron density of the 4'-O-coupled ring and local steric hindrance are important determinants of oxidative ß-O-4' ether degradation. Our findings provide novel insight into the delignification mechanisms of C. subvermispora and contribute to improving the valorization of lignocellulosic biomass.