Enzymatically Oxidized Carbohydrates As Dicarbonyl Biobased Cross-Linkers for Polyamines.

Carbonyl cross-linkers are used to modify textiles and form resins, and are produced annually in megatonne volumes. Due to their toxicity toward the environment and human health, however, less harmful biobased alternatives are needed. This study introduces carbonyl groups to lactose and galactose using galactose oxidase from Fusarium graminearum (FgrGalOx) and pyranose dehydrogenase from Agaricus bisporus (AbPDH1) to produce four cross-linkers. Differential scanning calorimetry was used to compare cross-linker reactivity, most notably resulting in a 34 °C decrease in reaction peak temperature (72 °C) for FgrGalOx-oxidized galactose compared to unmodified galactose. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and proton nuclear magnetic resonance (1H NMR) spectroscopy were used to verify imine formation and amine and aldehyde depletion. Cross-linkers were shown to form gels when mixed with polyallylamine, with FgrGalOx-oxidized lactose forming gels more effectively than all other cross-linkers, including glutaraldehyde. Further development of carbohydrate cross-linker technologies could lead to their adoption in various applications, including in adhesives, resins, and textiles.

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks.

Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing. IMPORTANCE The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.

Chitin-Active Lytic Polysaccharide Monooxygenases Are Rare in Cellulomonas Species.

Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member (CflaLPMO10D) as a chitin-active LPMO. Both the full-length CflaLPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that CflaLPMO10D is strictly active on the ß-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. IMPORTANCE Species from the genus Cellulomonas have a long history of study due to their roles in biomass recycling in nature and corresponding potential as sources of enzymes for biotechnological applications. Although Cellulomonas species are more commonly associated with the cleavage and utilization of plant cell wall polysaccharides, here, we show that C. flavigena produces a unique lytic polysaccharide monooxygenase with activity on ß-chitin, which is found, for example, in arthropods. The limited distribution of orthologous chitinolytic LPMOs suggests adaptation of individual cellulomonads to specific nutrient niches present in soil ecosystems. This research provides new insight into the biochemical specificity of LPMOs in Cellulomonas species and related bacteria, and it raises new questions about the physiological function of these enzymes.

Enzymatic synthesis of kraft lignin-acrylate copolymers using an alkaline tolerant laccase.

Softwood kraft lignin is a major bioresource relevant to the production of sustainable bio-based products. Continued challenges to lignin valorization, however, include poor solubility in organic solvents and in aqueous solutions at neutral pH. Herein, an alkaline tolerant laccase was used to graft acrylate functionalities onto softwood kraft lignin, which is expected to enhance the reactivity of lignin with isocyanate when producing bio-based polyurethanes. Proton nuclear magnetic resonance, Fourier-transform infrared spectroscopy, and high-performance liquid chromatography were used to confirm successful grafting of the acrylate monomer onto lignin and verify the importance of including tert-butyl hydroperoxide as an initiator in the grafting reaction. Laccase-mediated grafting of softwood kraft lignin under alkaline conditions produced lignin products with approximately 30% higher hydroxyl value and higher reactivity toward isocyanate. The reported enzymatic and aqueous process presents an opportunity for the sustainable valorization of softwood kraft lignin. KEY POINTS: • Softwood kraft lignin displayed high phenolic hydroxyl content, polydispersity index and average molecular weight • Grafting hydroxyethyl acrylate (HEA) monomer onto kraft lignin by laccase was successful at 60 °C and alkaline conditions • Lignin-HEA grafted copolymer showed an increase in total OH value and an increase in average molecular weight.

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans.

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a ß-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.

Retracted and Republished from: "Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola"

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species­aspen, pine, and spruce­under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Atypical lignification in eastern leatherwood (Dirca palustris).

Lignin is a complex phenolic biopolymer found mainly in the secondary cell walls of vascular plants, where it contributes to mechanical strength, water conduction, and plant defence. We studied the lignin of eastern leatherwood (Dirca palustris) because this slow-growing woody shrub is known for its flexible stems. Various analytical techniques and microscopy methods were employed to examine the composition and distribution of lignin and structural polysaccharides in leatherwood xylem in comparison with trembling aspen (Populus tremuloides) and white spruce (Picea glauca). We found that leatherwood has low overall levels of lignin, a high syringyl lignin content, and a unique distribution of lignin. Most remarkably, the cell corners and middle lamellae remain unlignified in mature xylem. These findings help explain the flexibility of leatherwood and also call into question the classical model of lignification, which purports that lignin polymerization begins in the cell corners and middle lamellae. This atypical lignification regime vividly illustrates the diversity in plant secondary cell wall formation that abounds in nature and casts leatherwood as a new model for the study of lignin biogenesis.

Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed the gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that enable fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species, aspen, pine, and spruce, under various culture conditions. We examined both gene expression (transcription levels) and RNA editing (posttranscriptional modification of RNA, which can potentially yield different proteins from the same gene). We found that F. pinicola is able to modify both gene expression and RNA editing profiles across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This work provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Constructing arabinofuranosidases for dual arabinoxylan debranching activity.

Enzymatic conversion of arabinoxylan requires α-L-arabinofuranosidases able to remove α-L-arabinofuranosyl residues (α-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 α-L-arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 α-L-arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A_BadAbf43A and BadAbf43A_SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity toward native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly α-(1 → 3)-L-Araf and α-(1 → 2)-L-Araf substituents attached to disubstituted Xylp (d-2,3-WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A_BadAbf43A fusion removed 83% of all α-L-Araf from WAX after a 20 hr treatment. 1 H NMR analyses further revealed differences in SthAbf62A_BadAbf43 rate of removal of specific α-L-Araf substituents from WAX, where 9.4 times higher activity was observed toward d-α-(1 → 3)-L-Araf compared to m-α-(1 → 3)-L-Araf positions.

Chemo-enzymatic Synthesis of Clickable Xylo-oligosaccharide Monomers from Hardwood 4-O-Methylglucuronoxylan.

A chemo-enzymatic pathway was developed to transform 4-O-methylglucuronic acid (MeGlcpA) containing xylo-oligosaccharides from beechwood into clickable monomers capable of polymerizing at room temperature and in aqueous conditions to form unique polytriazoles. While the gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum was used to oxidize C6-propargylated oligosaccharides, the acid-amine coupling reagents 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDAC) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) were employed and compared for their ability to append click functionalities to carboxylic acid groups of enzyme-treated oligosaccharides. While DMT-MM was a superior coupling reagent for this application, a triazine side product was observed during C-1 amidation. Resulting bifunctional xylo-oligosaccharide monomers were polymerized using a Cu(I) catalyst, forming a soft gel which was characterized by 1H NMR, confirming the triazole product.

Direct analysis by time-of-flight secondary ion mass spectrometry reveals action of bacterial laccase-mediator systems on both hardwood and softwood samples.

The modification and degradation of lignin play a vital role in carbon cycling as well as production of biofuels and bioproducts. The possibility of using bacterial laccases for the oxidation of lignin offers a route to utilize existing industrial protein expression techniques. However, bacterial laccases are most frequently studied on small model compounds that do not capture the complexity of lignocellulosic materials. This work studied the action of laccases from Bacillus subtilis and Salmonella typhimurium (EC 1.10.3.2) on ground wood samples from yellow birch (Betula alleghaniensis) and red spruce (Picea rubens). The ability of bacterial laccases to modify wood can be facilitated by small molecule mediators. Herein, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), gallic acid and sinapic acid mediators were tested. Direct analysis of the wood samples was achieved by time-of-flight secondary ion mass spectrometry (ToF-SIMS), a surface sensitive mass spectrometry technique that has characteristic peaks for H, G and S lignin. The action of the bacterial laccases on both wood samples was demonstrated and revealed a strong mediator influence. The ABTS mediator led to delignification, evident in an overall increase of polysaccharide peaks in the residual solid, along with equal loss of G and S-lignin peaks. The gallic acid mediator demonstrated minimal laccase activity. Meanwhile, the sinapic acid mediator altered the S/G peak ratio consistent with mediator attaching to the wood solids. The current investigation demonstrates the action of bacterial laccase-mediator systems directly on woody materials, and the potential of using ToF-SIMS to uncover the fundamental and applied role of bacterial enzymes in lignocellulose conversion.

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted ß-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40T.

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40(T), SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C(+) domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C(+) in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.

Influence of a family 29 carbohydrate binding module on the activity of galactose oxidase from Fusarium graminearum.

BACKGROUND: Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO. RESULTS: Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29. MAJOR CONCLUSIONS: Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs. GENERAL SIGNIFICANCE: This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family.

We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.

Enzymatically Debranched Xylans in Graft Copolymerization.

Wheat arabinoxylan was treated with two α-arabinofuranosidases exhibiting different mode of action to create three different polymeric substrates. These three substrate preparations were characterized by xylopyranose backbone sugars that are (1) singly substituted by arabinose at C2 or C3, (2) doubly substituted by arabinose at C2 and C3, and (3) largely unsubstituted. All xylan preparations were grafted with glycidyl methacrylate using cerium ammonium nitrate and then evaluated in terms of graft yield and adsorption to cellulose surfaces. The highest graft yield was observed for the xylan preparation characterized by a largely unsubstituted xylopyranose backbone. Furthermore, QCM-D analyses revealed that grafted xylans exhibited a two-stage desorption pattern, which was not seen with the ungrafted xylans and was consistent with increased water sorption. Accordingly, this study demonstrates the potential of arabinofuranosidases to increase the yield and influence the viscoelastic properties of grafted xylans used as biobased cellulose coatings.

Microplate-Based Detection of Lytic Polysaccharide Monooxygenase Activity by Fluorescence-Labeling of Insoluble Oxidized Products.

Most existing methods for screening the activity of lytic polysaccharide mono-oxygenases (LPMOs) on polysaccharides are based on the detection of soluble oxidized sugars. This approach might underestimate the total performance of LPMOs since oxidation events that do not lead to oligosaccharide release are not detected. Using PcLPMO9D as a model enzyme, a microplate-based method has been developed to detect C1-oxidizing LPMO activity by covalently linking a water-soluble fluorophore to oxidized positions within the cellulose fiber. This fluorescence method was validated using X-ray photoelectron spectroscopy and then combined with high-performance anion-exchange chromatography to track total PcLPMO9D activity.

Functional comparison of versatile carbohydrate esterases from families CE1, CE6 and CE16 on acetyl-4-O-methylglucuronoxylan and acetyl-galactoglucomannan.

BACKGROUND: The backbone structure of many hemicelluloses is acetylated, which presents a challenge when the objective is to convert corresponding polysaccharides to fermentable sugars or else recover hemicelluloses for biomaterial applications. Carbohydrate esterases (CE) can be harnessed to overcome these challenges. METHODS: Enzymes from different CE families, AnAcXE (CE1), OsAcXE (CE6), and MtAcE (CE16) were compared based on action and position preference towards acetyl-4-O-methylglucuronoxylan (MGX) and acetyl-galactoglucomannan (GGM). To determine corresponding positional preferences, the relative rate of acetyl group released by each enzyme was analyzed by real time 1H NMR. RESULTS: AnAcXE (CE1) showed lowest specific activity towards MGX, where OsAcXE (CE6) and MtAcE were approximately four times more active than AnAcXE (CE1). MtAcE (CE16) was further distinguished by demonstrating 100 times higher activity on GGM compared to AnAcXE (CE1) and OsAcXE (CE6), and five times higher activity on GGM than MGX. Following 24h incubation, all enzymes removed between 78 and 93% of total acetyl content from MGX and GGM, where MtAcE performed best on both substrates. MAJOR CONCLUSIONS: Considering action on MGX, all esterases showed preference for doubly substituted xylopyranosyl residues (2,3-O-acetyl-Xylp). Considering action on GGM, OsAcXE (CE6) preferentially targeted 2-O-acetyl-mannopyranosyl residues (2-O-acetyl-Manp) whereas AnAcXE (CE1) demonstrated highest activity towards 3-O-acetyl-Manp positions; regiopreference of MtAcE (CE16) on GGM was less clear. GENERAL SIGNIFICANCE: The current comparative analysis identifies options to control the position of acetyl group release at initial stages of reaction, and enzyme combinations likely to accelerate deacetylation of major hemicellulose sources.

Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.