Arabinoxylans are the most abundant polysaccharides in the bran from wheat and rye kernels. Ferulic acid moieties covalently bound to arabinosyl substitutions in arabinoxylans can be oxidised and crosslinked by lac-case enzymes, forming xylan hydrogels stabilised by chemical and physical interactions. Here, we explore the use of alpha-L-arabinofuranosidases to tune the rheological properties of laccase-crosslinked feruloylated arabinoxylans from wheat (WAX) and rye (RAX) brans, proposed to be mediated via intermolecular backbone interactions. The effect of subsequent freeze-drying and regeneration of the hydrogels on their multiscale structure and viscoelastic properties was further evaluated by X-ray scattering, microscopy and rheology measurements. The combined use of alpha-L-arabinofuranosidases from glycosyl hydrolase (GH) families GH62 and GH43 with complementary specificity towards different substitution motifs in arabinoxylan resulted in synergistic arabinose removal with a 48 % and 33 % increase in arabinose removal in WAX and RAX respectively, while retaining the ferulic acid moieties in both WAX and RAX. The extent of ferulic acid oxidation in WAX and RAX seemed to be affected by substrate inaccessibility for the laccase and polysaccharide chain aggregation, which was further accentuated by enzymatic arabinose removal. Rheological investigations revealed that laccase-crosslinked WAX hydrogels pretreated with arabinofuranosidases showed a decrease of 65-95 % in the storage and loss moduli compared to the non-pretreated WAX hydrogels, whereas arabinose removal improved the viscoelastic properties of RAX hydrogels both before and after regeneration, with an increase of storage moduli of 72-100 %. Arabinofuranosidase treatments and freeze-drying/regeneration altered the hydration properties of the hydrogels and their network structure, promoting the occurrence of ordered domains. Our results show that the biophysical properties of the arabinoxylans in terms of aggregation and hydration largely influence substrate accessibility to laccase-mediated oxidation and the multiscale assembly of the hydrogels upon freeze drying and regeneration, thus impacting their overall rheological properties. These dietary fibre hydrogels from cereal side streams have large potential to be used as food hydrocolloids, contributing to the overall circularity of the food system.

Modification of the vapor phase can dynamically alter interfacial energies, wetting, and droplet motion. We investigate the influence of vapor adsorption on the interfacial properties and droplet mobility of polymer brushes, self-assembled layers, and bare silicon. Ellipsometry and quartz crystal microbalance with dissipation experiments show that polydimethylsiloxane (PDMS) brush thickness increases by ∼45% in n-hexane vapor, whereas a negligible increase is observed for all other surfaces, indicating that PDMS brushes swell in n-hexane vapor. Despite this, we find that vapor-induced changes in interfacial energies dominate the wetting response; swelling actually hinders droplet mobility. Across all nonfluorinated surfaces, alkane vapors adsorb on the solid interface to form ultrathin liquid films that also cloak water droplets. Water droplets that are pinned or move at ≤0.2 mm/s when in air reach velocities above 30 mm/s on PDMS brushes and alkylsilane coatings when in n-pentane and n-hexane vapors. In contrast, a continuous lubricating alkane film does not form on fluorinated polymer brushes, and consequently, droplet velocities remain low (<0.1 mm/s) in all vapor environments. These results identify vapor-induced interfacial energy modification, rather than polymer swelling, as the key mechanism enabling tunable droplet mobility on smooth coatings.

d-Glucosaminic acid is a valuable amino acid useful in food and medical applications. It is a highly sought after enantiopure molecule important for the synthesis of drugs and glycopeptides. Current enzymatic synthesis pathways to d-glucosaminic acid carry disadvantages such as low product yield and long reaction times. Herein, the Auxiliary Activity 7 chito-oligosaccharide oxidase from Lentinus brumalis, LbChi7A, was shown as a potent biocatalyst capable of efficiently converting d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) to their respective C1-acids. The substrate specificity of LbChi7A towards GlcN and GlcNAc enabled the conversion of at least 90

Anaerobic microbes produce multienzyme complexes known as cellulosomes to enhance the degradation of cellulosic substrates. These complexes localize diverse enzymes onto a protein scaffold, where proteins are anchored by dockerin domains. Although the cellulosomes of anaerobic fungi incorporate a broad array of cellulolytic enzymes, they remain largely unexplored. Notably, some fungal cellulosomes reportedly comprise expansin-like proteins with potential to disrupt cellulose networks. While two bacterial cellulosomal expansin-like proteins have been characterized, no fungal cellulosomal expansin-like proteins have been functionally characterized to date. Sequence analyses of expansin-like proteins from the anaerobic fungus Neocallimastix californiae revealed similar N-terminal domains among proteins with or without appended dockerins. Those without dockerins, however, consistently lacked the first conserved aromatic residue that forms the substrate binding surface of the C-terminal family 63 carbohydrate binding module. One cellulosomal expansin-like protein from N. californiae (NcaEXLX1) was recombinantly expressed with and without (NcaEXLX1tr) the dockerin domains. The adsorption characteristics of NcaEXLX1 and NcaEXLX1tr, and impact on cellulase (Cel7B) activity, were then investigated using quartz crystal microbalance with dissipation (QCM-D). NcaEXLX1 exhibited higher binding to cellulose nanofibrils (CNF) compared to NcaEXLX1tr. Despite the lower binding of NcaEXLX1tr to CNF, both NcaEXLX1 and NcaEXLX1tr enhanced the action of Cel7B to similar extents. This study reports the production and characterization of a fungal cellulosomal expansin-like protein. The corresponding NcaEXLX1 protein and truncated variant were shown to enhance the activity of an endoglucanase, similar to observations made with non-cellulosomal expansin-like proteins. Notably, the improvement in cellulase activity upon the addition of NcaEXLX1 or NcaEXLX1tr was not correlated to extent of substrate binding.

This study describes an enzymatic pathway to produce high purity 4-O-methylglucaric acid from xylan, an underutilized fraction of lignocellulosic biomass. Beechwood xylan was enzymatically hydrolysed using a commercial xylanase and an α-glucuronidase from Amphibacillus xylanus to form 4-O-methylglucuronic acid, which was then purified by anion exchange chromatography and subsequently oxidized to 4-O-methylglucaric acid using a recombinantly produced uronic acid oxidase from Citrus sinensis. Enzymatic oxidation with uronic acid oxidase afforded 95 % yield in 72 hours which is considerably higher than yields previously achieved using a glucooligosaccharide oxidase from Sarocladium strictum. 4-O-methylglucaric acid was isolated by precipitation and purified by recrystallization. Characterization by liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy confirmed product identity and high purity (97.8 % w/w). 4-O-methylglucaric acid's performance as a detergent builder was compared to commercial glucaric acid. At 10 : 1 molar ratios of detergent builder to calcium, 4-O-methylglucaric acid provided similar calcium sequestration performance to glucaric acid (less than 5 % difference) at pH 7 and pH 10 in the presence of surfactants, including sodium dodecylbenzene sulfonate. Given their similar calcium sequestration performance, 4-O-methylglucaric acid could effectively substitute for glucaric acid in detergent formulations.

The unique surface properties of grafted polydimethylsiloxane (PDMS) chains, particularly their omniphobicity and low friction, are influenced by molecular structure and tethering density. Despite molecularly smoothness and homogeneity, these surfaces exhibit significant variability in wettability and contact angle hysteresis (CAH). This work uncovers the molecular structure of grafted PDMS chains. Grafted PDMS chains synthesized using a difunctional chlorosilane initiator, which exhibits CAH <2 degrees on silicon wafers, adopt a brush-to-mushroom conformation with a molecular weight approximate to 7,800 g mol(-1), a grafting density of 0.22 +/- 0.4 chains nm(-2), and a thickness of approximate to 3 nm. Each PDMS chain terminates with a silanol group, and approximate to 96% of substrate silanols remain unreacted. The presence of these terminal silanols is confirmed with time-of-flight secondary ion mass spectroscopy, as is their removal when exchanged for trimethylsilyl groups, both on the substrate and terminating the PDMS chains. Quartz crystal microbalance with dissipation measurements show that this "capping" procedure exchanges approximate to 1.5 silanols nm(-2); capping occurs at the substrate and PDMS chain end. The findings suggest that grafted, capped PDMS chains of this molecular weight are able to achieve excellent omniphobic properties even when the majority of surface silanols remain unreacted, which may aid in the design of future omniphobic materials.

Background Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum ) and SpATA (from Silicibacter pomeroyi ) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated. Results Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure. Conclusions Using oxidase–amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme. Graphical Abstract

Polydimethylsiloxane (PDMS) brushes showcase unparalleled omniphobic properties, repelling a diverse spectrum of liquid droplets. However, PDMS brushes exhibit limited physical durability due to their unique chain conformation, especially when subjected to mechanical abrasion. Here we report the unexpected repellency of PDMS brushes towards their own PDMS chains, i.e., like-vs-like repulsion or autophobicity, and show how the interplay between PDMS brush structure and autophobic behavior can be tuned to design damage-tolerant omniphobic surfaces. A detailed examination of grafted PDMS chain growth using difunctional chlorosilanes from the vapor phase revealed autophobic PDMS chain dewetting during synthesis. Architecturally designed PDMS brushes that favor incremental chain cluster growth demonstrate tolerance towards abrasion, where fractured chains can re-bond to the surface, resulting in extended resistance to abrasion. Tribology experiments confirm a decreasing coefficient of friction and lower contact angle hysteresis after mechanical wear. The tailored autophobicity of PDMS brushes results in surfaces that durably maintain omniphobicity, repelling low surface tension liquids after one thousand cycles of abrasion.

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization.

Various enzymes can be used to modify the structure of hemicelluloses directly in vivo or following extraction from biomass sources, such as wood and agricultural residues. Generally, these enzymes can contribute to designer hemicelluloses through four main strategies: (1) enzymatic hydrolysis such as selective removal of side groups by glycoside hydrolases (GH) and carbohydrate esterases (CE), (2) enzymatic cross-linking, for instance, the selective addition of side groups by glycosyltransferases (GT) with activated sugars, (3) enzymatic polymerization by glycosynthases (GS) with activated glycosyl donors or transglycosylation, and (4) enzymatic functionalization, particularly via oxidation by carbohydrate oxidoreductases and via amination by amine transaminases. Thus, this Perspective will first highlight enzymes that play a role in regulating the degree of polymerization and side group composition of hemicelluloses, and subsequently, it will explore enzymes that enhance cross-linking capabilities and incorporate novel chemical functionalities into saccharide structures. These enzymatic routes offer a precise way to tailor the properties of hemicelluloses for specific applications in biobased materials, contributing to the development of renewable alternatives to conventional materials derived from fossil fuels.

Environmentally unsustainable and toxic chemical flocculants and dispersants from nickel (Ni) processing contribute to industrial effluents that greatly impact biodiversity and aquatic life. Despite the industry's efforts to reduce its ecological footprint-primarily due to the lack of commercially available biodegradable, environmentally benign, and nontoxic reagents- these reagents continue to harm natural ecosystems. Chemicals used during the processing operations often target a specific unit operation that negatively affects downstream operations. Herein, we discover the concentration-dependent behavior of cellulose nanocrystals (CNCs) as a dispersant and flocculant, avoiding the use of various harmful chemicals in the nickel processing stages. Electrophoretic, optical brightfield microscopy, and quartz crystal microbalance-dissipation studies detected charge neutralization behavior by renewable and biodegradable CNCs, which can greatly benefit Ni processing operations. Microflotation experiments demonstrated that CNCs enhanced Ni recovery from 62 to 77 wt % and concentrate grade from 15 to 20 wt %. Settling and turbidity studies demonstrated the dual flocculant-dispersant behavior of CNCs resulting from the alignment of CNC fibers along the octahedral brucite basal and amphoteric edge plane of serpentine. CNCs can be used as dispersants during froth flotation to improve Ni beneficiation and as postprocessing flocculants for tailings management and dewatering, which is one of the major environmental and social concerns facing the mining and mineral processing industry. In addition, this study paves the way for CNCs to be used as flocculants and dispersants in a range of industries from healthcare to pharmaceuticals to semiconductor devices.

Fouling at liquid-solid interfaces is a pernicious problem for a wide range of applications, including those that are implemented by digital microfluidics (DMF). There are several strategies that have been used to combat surface fouling in DMF, the most common being inclusion of amphiphilic surfactant additives in the droplets to be manipulated. Initial studies relied on Pluronic additives, and more recently, Tetronic additives have been used, which has allowed manipulation of complex samples like serum and whole blood. Here, we report our evaluation of 19 different Pluronic and Tetronic additives, with attempts to determine (1) the difference in antifouling performance between the two families, (2) the structural similarities that predict exceptional antifouling performance, and (3) the mechanism of the antifouling behavior. Our analysis shows that both Pluronic and Tetronic additives with modest molar mass, poly(propylene oxide) (PPO) ≥50 units, poly(ethylene oxide) (PEO) mass percentage ≤50%, and hydrophilic-lipophilic balance (HLB) ca. 13-15 allow for exceptional antifouling performance in DMF. The most promising candidates, P104, P105, and T904, were able to support continuous movement of droplets of serum for more than 2 h, a result (for devices operating in air) previously thought to be out of reach for this technique. Additional results generated using device longevity assays, intrinsic fluorescence measurements, dynamic light scattering, asymmetric flow field flow fractionation, supercritical angle fluorescence microscopy, atomic force microscopy, and quartz crystal microbalance measurements suggest that the best-performing surfactants are more likely to operate by forming a protective layer at the liquid-solid interface than by complexation with proteins. We propose that these results and their implications are an important step forward for the growing community of users of this technique, which may provide guidance in selecting surfactants for manipulating biological matrices for a wide range of applications.

Xylan is one of the most abundant, natural polysaccharides, and much recent interest focuses on upgrading heteroxylan to make use of its unique structures and chemistries. Significant progress has been made in the discovery and application of novel enzymes for debranching and modifying heteroxylans. Debranching enzymes include acetylxylan esterases, α-l-arabinofuranosidases and α-d-glucuronidases that release side groups from the xylan backbone to recover both biochemicals and less substituted xylans for polymer applications in food packaging or drug delivery systems. Besides esterases and hydrolases, many oxidoreductases including carbohydrate oxidases, lytic polysaccharide monooxygenases, laccases and peroxidases have been also applied to alter different types of xylans for improved physical and chemical properties. This review will highlight the recent discovery and application of enzymes for upgrading xylans for use as added-value chemicals and in functional polymers.

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.

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e(-8) [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm(2) to 63 dyn/cm(2)). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 +/- 0.24 mu m/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. Statement of significance This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration. (c) 2022 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

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 wooddecaying polypore Fomitopsis pinicola on three different wood species-aspen, pine, and spruce-under 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.

Natural products and their derivatives have been commonly used in our daily life, as they play important roles in boosting immune systems and fighting diseases [...]