The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers.

Background: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM "immobilizes" the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface.

Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section.

Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers.

Oxidation of cellulose fibers using LPMOs with varying allomorphic substrate preferences, oxidative regioselectivities, and domain structures.

Lytic polysaccharide monooxygenases (LPMOs) are excellent candidates for enzymatic functionalization of natural polysaccharides, such as cellulose or chitin, and are gaining relevance in the search for renewable biomaterials. Here, we assessed the cellulose fiber modification potential and catalytic performance of eleven cellulose-active fungal AA9-type LPMOs, including C1-, C4-, and C1/C4-oxidizing LPMOs with and without CBM1 carbohydrate-binding modules, on cellulosic substrates with different degrees of crystallinity and polymer chain arrangement, namely, Cellulose I, Cellulose II, and amorphous cellulose. The potential of LPMOs for cellulose fiber modification varied among the LPMOs and depended primarily on operational stability and substrate binding, and, to some extent, also on regioselectivity and domain structure.

A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres.

Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications.

Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases.

Background: Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied.

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

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 HO may be a more efficient co-substrate for LPMOs than O.

Cloning of novel bacterial xylanases from lignocellulose-enriched compost metagenomic libraries.

Xylanases are in important class of industrial enzymes that are essential for the complete hydrolysis of lignocellulosic biomass into fermentable sugars. In the present study, we report the cloning of novel xylanases with interesting properties from compost metagenomics libraries. Controlled composting of lignocellulosic materials was used to enrich the microbial population in lignocellulolytic organisms.

Effect of hydrothermal pretreatment severity on lignin inhibition in enzymatic hydrolysis.

Hydrothermal pretreatment is commonly used for enhancing enzymatic hydrolysis of lignocellulosics. Spruce and wheat straw were pretreated with increasing severity and lignin characteristics were analysed. The effect of enzymatically isolated lignin on the hydrolysis of Avicel and the adsorption of a cellobiohydrolase onto lignin was measured.

Cellulases adsorb reversibly on biomass lignin.

Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin.

Enzyme biotechnology in degradation and modification of plant cell wall polymers.

Lignocelluloses are abundant raw materials for production of fuels, chemicals and materials. The purpose of this paper is to review the enzyme-types and enzyme-technologies studied and applied in the processing of the lignocelluloses into different products. The enzymes here are mostly glycoside hydrolases, esterases and different redox enzymes.

A new crystal form of Aspergillus oryzae catechol oxidase and evaluation of copper site structures in coupled binuclear copper enzymes.

Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.

Characterization of sulfhydryl oxidase from Aspergillus tubingensis.

Background: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.

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 (RaOx; EC 1.

Lignin Films from Spruce, Eucalyptus, and Wheat Straw Studied with Electroacoustic and Optical Sensors: Effect of Composition and Electrostatic Screening on Enzyme Binding.

Lignins were isolated from spruce, wheat straw, and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and P NMR to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy, and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption.

Lignin-derived inhibition of monocomponent cellulases and a xylanase in the hydrolysis of lignocellulosics.

Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a β-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw.

Chemical characterization of hydrothermally pretreated and enzyme-digested wheat straw: An evaluation of recalcitrance.

There is great interest in understanding changes that occur to cell wall constituents during saccharification of pretreated lignocellulose, particularly in relation to recalcitrance of the residues. This paper reports the effects of hydrothermal pretreatment followed by enzyme hydrolysis on the extractability and properties of recalcitrant wheat straw polymers. The results show that the undigested residue had lost much of its archestructure.

Impact of steam explosion on the wheat straw lignin structure studied by solution-state nuclear magnetic resonance and density functional methods.

Chemical changes of lignin induced by the steam explosion (SE) process were elucidated. Wheat straw was studied as the raw material, and lignins were isolated by the enzymatic mild acidolysis lignin (EMAL) procedure before and after the SE treatment for analyses mainly by two-dimensional (2D) [heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)] and (31)P nuclear magnetic resonance (NMR). The β-O-4 structures were found to be homolytically cleaved, followed by recoupling to β-5 linkages.

Cellulose hydrolysis and binding with Trichoderma reesei Cel5A and Cel7A and their core domains in ionic liquid solutions.

Ionic liquids (ILs) dissolve lignocellulosic biomass and have a high potential as pretreatment prior to total enzymatic hydrolysis. ILs are, however, known to inactivate cellulases. In this article, enzymatic hydrolysis of microcrystalline cellulose (MCC) and enzyme binding onto the cellulosic substrate were studied in the presence of cellulose-dissolving ILs.

The crystal structure of an extracellular catechol oxidase from the ascomycete fungus Aspergillus oryzae.

Catechol oxidases (EC 1.10.3.

Cellulase-lignin interactions-the role of carbohydrate-binding module and pH in non-productive binding.

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme-lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase-lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A.

Effect of temperature on lignin-derived inhibition studied with three structurally different cellobiohydrolases.

Non-productive enzyme adsorption onto lignin inhibits enzymatic hydrolysis of lignocellulosic biomass. Three cellobiohydrolases, Trichoderma reesei Cel7A (TrCel7A) and two engineered fusion enzymes, with distinctive modular structures and temperature stabilities were employed to study the effect of temperature on inhibition arising from non-productive cellulase adsorption. The fusion enzymes, TeCel7A-CBM1 and TeCel7A-CBM3, were composed of a thermostable Talaromyces emersonii Cel7A (TeCel7A) catalytic domain fused to a carbohydrate-binding module (CBM) either from family 1 or from family 3.

On the factors affecting product distribution in laccase-catalyzed oxidation of a lignin model compound vanillyl alcohol: experimental and computational evaluation.

Laccases (EC 1.10.3.

Topochemical pretreatment of wood biomass to enhance enzymatic hydrolysis of polysaccharides to sugars.

The surface chemistry of milled birch and pine wood pretreated by ionic liquid, hydrothermal and hydrotropic methods, followed by enzymatic hydrolysis was studied in this work. Surface coverage by lignin was measured by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to describe the surface chemical composition after pretreatment in detail, and the morphology after pretreatment was investigated by FE-SEM. Ionic liquid (1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride) pretreatment at room temperature made the samples swell but did not dissolve the wood.