Saccharification Of Cellulose Research Articles

Microwave Effect in Hydrolysis of Levoglucosan with a Solid Acid Catalyst for Pyrolysis-Based Cellulose Saccharification.

Pyrolysis-based saccharification consisting of fast pyrolysis followed by hydrolysis of the resulting anhydrosugars such as levoglucosan is a promising method for converting cellulosic biomass into glucose that can be used for producing biofuels and biochemicals. In the present study, hydrolysis of levoglucosan was evaluated in water with a polystyrene sulfonic acid resin (a solid acid catalyst) by heating under microwave irradiation or in an oil bath at 95 °C-120 °C. When the equilibrium temperature of the solution was the same, the conversion rate of levoglucosan was greater under microwave irradiation than in an oil bath. Model experiments indicate that the sulfonyl groups of the solid acid catalyst were selectively heated by microwave irradiation. The temperature of the reaction solution in the vicinity of the catalyst was locally higher than the equilibrium temperature of the solution, which enabled hydrolysis to proceed efficiently.

Dual Role of Gluconic Acid in the Cascading Saccharification of Hemicellulose and Cellulose from Various Lignocellulosic Stuff

Dual Role of Gluconic Acid in the Cascading Saccharification of Hemicellulose and Cellulose from Various Lignocellulosic Stuff

Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part I. Characterization of Thermotolerant Mutant from the Basidiomycete Phanerochaete chrysosporium.

Cellobiohydrolase (CBH), belonging to glycoside hydrolase family 6 (GH6), plays an essential role in cellulose saccharification, but its low thermotolerance presents a challenge in improving the reaction efficiency. Based on a report that chimeric CBH II (GH6) engineered to remove non-disulfide-bonded free Cys shows increased thermotolerance, we previously mutated the two free Cys residues to Ser in GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCel6A) and obtained a thermotolerant double mutant, C240S/C393S (Yamaguchi et al., J. Appl. Glycosci. 2020; 67: 79-86). Here, characterization of the double mutant revealed that its activity towards both amorphous and crystalline cellulose was higher than that of the wild-type enzyme at elevated temperature, suggesting that the catalytic domain is the major contributor to the increased thermotolerance. To investigate the role of each free Cys residue, we prepared both single mutants, C240S and C393S, of the catalytic domain of PcCel6A and examined their residual activity at high temperature and the temperature-dependent changes of folding by means of circular dichroism measurements and thermal shift assay. The results indicate that the C393S mutation is the main contributor to both the increased thermotolerance of C240S/C393S and the increased activity of the catalytic domain at high temperature.

Investigation of the alkaline hydrogen peroxide pretreatment: From cellulose saccharification to lignin isolation

Alkaline hydrogen peroxide (AHP) pretreatment, a green and effective technology for biomass fractionation, was studied to overcome the recalcitrance of bamboo for lignin isolation and facilitating enzymatic saccharification. In order to explore the impact of AHP pretreatment on the cellulose saccharification of bamboo, the glucan/xylan recovery, lignin removal, and structural properties of solid substrate were investigated in this research. AHP pretreatment at 90 ℃ for 60 min with 3.0 % H2O2 resulted in the maximum glucan/xylan hydrolysis yield reaching 90.62 % and 88.30 %, respectively. Specially, for the first time, the recovered lignin from the AHP liquid possesses up to 59.84/100Ar of β-O-4 linkages, abundant ferulic acid (FA), and p-coumarate (pCE) structures, as well as a homogeneous molecular structure with high purity (84.04%-96.20%) and preferable antioxidant activities (IC50: 0.015–0.018 mg/mL). These results provided theoretical support for the comprehensive valorization of lignocellulose, which is favorable for achieving viable biorefineries.

A novel Ruminiclostridium thermocellum cellulase system enhances cellulosic saccharification by elimination of cellobiose feedback inhibition

The Ruminiclostridium thermocellum cellulosome system is one of the most efficient cellulase systems in nature. The main hydrolysis product of cellulosic saccharification by R. thermocellum was cellobiose, which served as a strong feedback inhibitor to the cellulosome. In this study, R. thermocellum M3, which directly hydrolyzes cellulosic biomass into monosaccharides, was used to degrade cellobiose to investigate the kinetic characteristics of cellobiose degradation. Moreover, the transcriptomic characteristics of strain M3 under cellobiose and Avicel culture conditions were compared. The results indicated that strain M3 was able to grow and hydrolyze cellobiose under cellobiose concentrations higher than 20 g/L. The best growth performance and glucose production were obtained under 12 g/L cellobiose. Compared with that of Avicel, cellobiose had greater β-glucosidase activity under cellobiose culture, with a maximum glucose yield of 767.97±19.13 mg/g cellobiose. The transcription results indicated that R. thermocellum M3 resisted the feedback inhibition of cellobiose by regulating genes encoding β-glucosidase, cellobiose phosphorylase and ABC transporters. The high glucose yield of strain M3 was closely related to the expression of the bglX gene. In addition, the gene related to the chemotactic pathway of strain M3 also increased its perception and adaptation to cellobiose.

Optimisation of sugar and solid biofuel co-production from almond tree prunings by acid pretreatment and enzymatic hydrolysis.

Almond pruning biomass is an important agricultural residue that has been scarcely studied for the co-production of sugars and solid biofuels. In this work, the production of monosaccharides from almond prunings was optimised by a two-step process scheme: pretreatment with dilute sulphuric acid (0.025M, at 185.9-214.1℃ for 0.8-9.2min) followed by enzyme saccharification of the pretreated cellulose. The application of a response surface methodology enabled the mathematical modelling of the process, establishing pretreatment conditions to maximise both the amount of sugar in the acid prehydrolysate (23.4kg/100kg raw material, at 195.7℃ for 3.5min) and the enzymatic digestibility of the pretreated cellulose (45.4%, at 210.0℃ for 8.0min). The highest overall sugar yield (36.8kg/100kg raw material, equivalent to 64.3% of all sugars in the feedstock) was obtained with a pretreatment carried out at 197.0℃ for 4.0min. Under these conditions, moreover, the final solids showed better properties for thermochemical utilisation (22.0MJ/kg heating value, 0.87% ash content, and 72.1mg/g moisture adsorption capacity) compared to those of the original prunings.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities

BackgroundThe polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear.ResultsAn LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid).ConclusionsThis study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Characterization of a Novel Hyperthermophilic GH1 β-Glucosidase from Acidilobus sp. and Its Application in the Hydrolysis of Soybean Isoflavone Glycosides.

A putative β-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of β-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, Km, Vmax, and Kcat/Km of BglAc toward pNPG were 357.62 U mg-1, 3.41 mM, 474.0 μmol min-1·mg-1, and 122.7 s-1mM-1. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new β-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation.

Efficient in situ saccharification of microcrystalline cellulose over immobilized cellulase on magnetic biochar in ionic liquid media

Saccharification serves as the entry point for the biomass into biorefinery schemes and plays a vital role in lignocellulose transformation towards bio-fuels and platform molecules. Herein, an efficient in situ saccharification of lignocellulose under the mediation of ionic liquid of [DMIM]DMP is reported, using a series of magnetically-separable biochars as catalyst supports with immobilized endoglucanase Cel5A. The physicochemical properties of these magnetic biochars with immobilized endoglucanase (abbreviated as MBIE) were thoroughly characterized by FTIR, TG, SEM/EDX, XRD, XPS, N2 physisorption, UV–vis and VSM. The results revealed that the endoglucanase, which functions as the catalytically active species, was immobilized on the biochar via simultaneous physical adsorption and covalent attachment. The extremely high surface area of biochar endows a high loading of endoglucanase and easy diffusion of reactant within the catalyst’s framework. The zeta potential of the catalyst was − 7.06 mV, indicating its excellent stability. MBIE with immobilizing endoglucanase of 484.36 mg/g exhibited the superior performance under a biphasic system (IL/water v/v 5:1), affording a glucose yield of 932.5 mg/g. The activity of MBIE remained constant after 10 consecutive cycles via magnetic recycling. This work demonstrates an efficient in situ saccharification process catalyzed by the developed MBIE with high activity and excellent regeneration ability.

Chemical composition and structural features of cellolignin from steam explosion followed by enzymatic hydrolysis of Eucalyptus globulus bark

Bark is one of the main wastes of the chemical and mechanical processing of Eucalyptus globulus wood. The proposed biochemical processing of bark via saccharification pathway involves steam explosion (SE) pretreatment (severity factor log R0 of 4.22) followed by enzymatic hydrolysis using an enzymatic cocktail composed of cellulolytic and xylanolytic enzymes. Almost 70% cellulose saccharification was achieved. The remaining cellolignin residue (CLEZ) was analysed for its chemical composition and structural features by conventional wet chemistry methods and a series of spectroscopic tools (FTIR-ATR, solid-state CP/MAS 13C NMR spectroscopy and wide-angle X-ray scattering (WAXS)). The main CLEZ component (about 51%) is acid-insoluble lignin, the chemical composition of which in terms of the ratio of syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H) units (70:28:2) is very close to that in the initial bark. This lignin is highly condensed and structurally associated with condensed tannins, which makes CLEZ recalcitrant to delignification by common methods. About one third of cellulose in eucalyptus bark after SE was inaccessible to enzymatic hydrolysis and remained in the CLEZ. This cellulose, structurally similar to microcrystalline cellulose, is imbedded into the lignin-tannins condensed matrix and extremely difficult to purify. In contrast to cellulose, bark hemicelluloses were effectively removed in enzymatic hydrolysis, with only small amounts (<2%) remaining in CLEZ. Among other CLEZ ingredients, proteins and inorganic/organic salts were the most abundant. The latter includes noticeable amounts of calcium oxalate phytoliths (up to 9%), Fe and Si salts. The eventual application areas of CLEZ are discussed.

Integrated Nondestructive Spectroscopic Technology to Reveal the Influence Mechanism of Lignins from Pretreated Corn Stover on Cellulose Saccharification

Integrated Nondestructive Spectroscopic Technology to Reveal the Influence Mechanism of Lignins from Pretreated Corn Stover on Cellulose Saccharification

Co-immobilized multi-enzyme biocatalytic system on reversible and soluble carrier for saccharification of corn straw cellulose

Herein, three enzymes (cellulase, β-glucosidase, and pectinase) with synergistic effects were co-immobilized on the Eudragit L-100, and the recovery of co-immobilized enzymes from solid substrates were achieved through the reversible and soluble property of the carrier. The optimization of enzyme ratio overcomed the problem of inappropriate enzyme activity ratio caused by different immobilization efficiencies among enzymes during the preparation process of co-immobilized enzymes. The co-immobilized enzymes were utilized to catalytically hydrolyze cellulose from corn straw into glucose, achieving a cellulose conversion rate of 74.45% under conditions optimized for their enzymatic characteristics and hydrolytic reaction conditions. As a result of the reversibility and solubility of the carrier, the co-immobilized enzymes were recovered from the solid substrate after five cycles, retaining 54.67% of the enzyme activity. The aim of this study is to investigate the potential of co-immobilizing multiple enzymes onto the Eudragit L-100 carrier for the synergistic degradation of straw cellulose.

Unveiling the potential of novel recyclable deep eutectic solvent pretreatment: Effective separation of lignin from poplar hydrolyzed residue

To effectively convert the fermentable sugars present in lignocellulosic biomass into biofuels and additional value-added products, it is crucial to remove lignin from the biomass. With the intention of expeditiously remove lignin from poplar wood and improve cellulose saccharification, an innovative ternary deep eutectic solvent (DES) benzyl triethyl ammonium chloride-ethylene glycol-FeCl3 (T-EG-F) was studied for the pretreatment of poplar hydrolyzed residue (PHR). The results revealed that following T-EG-F DES pretreatment at 130 °C for 4 h, the lignin removal rate reached 91.88 %. The effect of DES on PHR and regenerated lignin was comprehensively investigated using X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), Thermogravimetric (TG) and other characterization methods, providing valuable insights into the mechanism of this innovative biomass pretreatment. Moreover, there was a significant improvement in the enzyme digestibility of the DES pretreatment residue. At 48 h, the enzyme load of 30 FPU/g cellulose achieved a remarkable enzyme digestibility of 97.31 %, and this value exhibited a notable increase of 6.56 times compared to the untreated poplar sample. In addition, the T-EG-F could be recycled and reused. This study demonstrates that the potential of T-EG-F DES pretreatment as a green and efficient method for lignin dissociation from lignocellulosic biomass, offering a promising approach for biomass component separation.

Chemical-free saturated steam pretreatment of oil palm empty fruit bunch for improved enzymatic saccharification

Oil palm empty fruit bunch (EFB) as the main lignocellulosic residue from the oil palm industry serves as suitable feedstock for enzymatic saccharification, yielding fermentable sugars. Due to complex inter-association between hemicellulose, cellulose and lignin within EFB lignocellulosic matrix, different saturated steam conditions at 140-230 °C, 0.3-2.3 MPa was chosen as a pretreatment strategy, targeted to hydrolyze hemicellulose and reduce crystallinity of cellulose to enhance enzymatic saccharification which need no chemical requirement and potential for energy recovery. As a result, SEM analysis of EFB morphology indicated that the enhanced silica body removal from 190-230 °C of saturated steam pretreatment correspondingly led to substantial 82% removal of hemicellulose. Consequently, enzymatic saccharification yielded between 0.621-0.637 g glucose per g EFB, equivalent to 93-95% of cellulose saccharification by Acremonium cellulase on EFB treated with saturated steam at 210-230 °C. Notably, a short duration pretreatment of EFB at 210 °C for 2 min was proved sufficient for high fermentable sugar production. The potential energy estimated for employing saturated steam pretreatment of EFB in a palm oil mill was sufficient by 2.2 x 1012 kJ/year.

Saccharification of Various Wastepaper Materials by Cellulase from Brown Garden Snail (Cornu aspersum) at Different Incubation pH Values

Increased solid waste pollution and the negative effect of fossil fuel consumption on the environment are issues that would require more scientific attention and application to deal effectively with these phenomena. Wastepaper, a major component of solid waste, is classified as organic waste due to the presence of cellulose, a glucose-based biopolymer that is part of its structural composition. The saccharification of cellulose into glucose, a fermentable sugar, can be achieved with a hydrolytic enzyme known as cellulase. Although cellulase from fungal species such as Trichoderma, Aspergillus, and Penicillium are well described, knowledge about cellulase isolated from the brown garden snail is limited as it has not been the subject of many research endeavors. The waste paper has been described as a suitable resource for bio-energy development due to cellulose, a structural component of this bio-material that can be degraded into glucose, a fermentable sugar. Although paper materials such as newspaper, office paper, filter paper, Woolworths and Pick and Pay (retailers) advertising paper, as well as foolscap paper, were saccharified by different cellulases, the degradation of these paper materials by garden snail cellulase is a novel investigation from our laboratory. With the effects of temperature and incubation time on this cellulase action when degraded paper materials have already been investigated and reported, this study dealt with the garden snail cellulase action when degraded paper materials at different pH values. Most of the paper materials were degraded optimally at a pH value of 6.0, while optimum saccharification was observed at pH 4.5 when newspaper and brown envelope paper were degraded, with office paper showing maximum bioconversion at pH 7.0. The difference in the structural composition of the paper materials also affects the degree of saccharification, as the amount of sugar released from the various paper materials at optimum pH values is not similar. Together with other catalytic parameters, the pH value of this enzymatic catalysis is also to be considered when designing the development of waste paper as a bio-product resource, with limiting environmental pollution as an additional advantage of this process.

Lewis acid/base mediated deep eutectic solvents intensify lignocellulose fractionation to facilitate enzymatic hydrolysis and lignin nanosphere preparation

In this work, Lewis acids (FeCl3, AlCl3) and bases (Na2CO3, Na2SO3) were incorporated into a neutral deep eutectic solvent (DES, choline chloride/glycerin) to intensify the lignocellulose fractionation. The efficiency of fractionation in terms of the maximum delignification rate (89.7 %) and well-pleasing cellulose saccharification (100 %) could be achieved by the Lewis acid-mediated DES. An in-depth insight of the evolution of lignin structure revealed that Lewis acid-mediated DES could cleave the β-O-4 linkages efficiently to achieve a high yield lignin fragments. Meanwhile, the lignin fragments with the enhanced amphiphilic properties facilitate the preparation of lignin nanospheres (LNSs) via the self-assembly process. The resultant LNSs extracted by Lewis acid-mediated DES exhibited an excellent thermal stability, and enhanced antibacterial capacity, which were associated with the phenolic OH content. However, the extracted lignin by Lewis base-mediated DES was mainly attributed to the cleavage of lignin-carbohydrate complexes bond, especially the lignin-carbohydrate ester bond, which retained more ether bonds and a relatively complete structure. This study illuminated the different mechanisms of lignin extraction and the structural evolution of lignin from Lewis acid/base-mediated DES, and provided guidance to select suitable fractionation techniques for upgrading the downstream products.

Enhancing biomass conversion: Efficient hemicellulose removal and cellulose saccharification in poplar with FeCl3 coupled with acidic electrolyzed water pretreatment

Due to the recalcitrant structure of woody biomass such as poplar, the efficient disassembly and separation of hemicellulose component from woody biomass is crucial for green biomass processing and full component utilization. This study presented an environmentally friendly approach to utilize acidic electrolyzed water (AEW) combined with metal salts and investigated its pretreatment effects on hemicellulose removal and cellulose and lignin retention under different conditions. Meanwhile, the structural properties and enzymatic hydrolysis performance of the pretreated residues were also characterized. As a result, under the optimized pretreatment conditions (0.03 mol/L FeCl3 with AEW at 180 °C for 10 min), hemicellulose removal from poplar wood reached 98.64 %, accompanied by xylose recovery rate of 98.46 %, cellulose retention rate of 93.43 % and lignin retention rate of 94.29 %. Enzymatic hydrolysis rate of the pretreated cellulose-enriched substrate reached 97.65 %. Furthermore, comprehensive structural characterizations revealed that FeCl3 coupled with AEW pretreatment resulted in surface damage to the poplar wood, effective removal of the amorphous hemicellulose component, and partial destruction of the cellulose crystallinity. In conclusion, FeCl3 coupled with AEW pretreatment effectively separates hemicellulose, leading to significant alterations in biomass composition and structure, ultimately resulting in improved enzymatic digestion. These results provide theoretical support for targeted dissociation of hemicellulose and full component utilization of woody biomass.

In situ saccharification of metal-containing ionic liquid pretreated (ligno)cellulose via alginate encapsulated cellulase@functionalized ZIF-8

The present work aimed to develop mixed-metal salts (MMs) assisted ionic liquid (IL) pretreatment for efficient in situ saccharification of (ligno)cellulose. To accommodate MMs-IL involved in situ saccharification, a novel immobilized cellulase based on amine-functionalized ZIF-8 and calcium alginate beads (Ce@ZIF-8-A/CA) was established. XRD, FT-IR and SEM were employed to characterize the immobilization carrier and immobilized cellulase. Compared to free cellulase, Ce@ZIF-8-A/CA showed enhanced tolerance to IL, MMs and temperature. Ce@ZIF-8-A/CA preserved more than 80% of original activity at a wide temperature range (40–70 °C) in 30% 1-butyl-3-methylpyridinium chloride ([BMPy]Cl), and retained 73.0% of initial activity in 1% MMs (FeCl3/LiCl) assisted 30% [BMPy]Cl. After pretreatment with MMs assisted [BMPy]Cl, 40%-60% (ligno)cellulose was decomposed into soluble lower-molecular-weight fragments without regeneration of cellulose after adding buffer. After in situ saccharification of pretreated slurries with Ce@ZIF-8-A/CA, total reducing sugars yields of 84.7% and 71.3% from cellulose and bagasse were obtained during 12 h, saving at least 12 h compared to traditional enzymatic in situ saccharification of (ligno)cellulose in IL.

Carboxylation of sulfated cellulose nanocrystals by family AA9 lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) from the auxiliary activity 9 (AA9) family act on cellulose through an oxidative mechanism that improves cellulose saccharification in concert with other cellulolytic enzymes. Degradation and solubilization of cellulose chains are known to take place when various cellulose hierarchies, fibers, nanofibers, and cellulose nanocrystals (CNCs) are subjected to LPMOs, either alone or in combination with other cellulose acting enzymes. The use of LPMOs to modify and prepare CNCs has been proposed mostly in top-down synthesis from larger hierarchies. Here, we attempted a direct surface modification of CNCs with LPMOs with the aim of investigating the role played by the charged sulfate groups on CNCs. Sulfate half-ester groups are introduced during the preparation of CNCs from cellulose using sulfuric acid. It has been proposed that the charged sulfate groups hinder the binding of enzymes or affinity of charged reactants on the surface and hence reduce enzymatic and chemical reaction efficiency. We demonstrate the modification of commercial sulfated CNCs using a family AA9 LPMO. Conductometric titration and spectrometric characterization of the oxidized particles indicate that carboxylation of up to 10% was possible without degradation of the crystals. Unexpectedly, the carboxyl groups could only be introduced to the crystals containing sulfate groups, while desulfated crystals remained unfunctionalized. This was deemed to be due to that the sulfate groups limit the adsorption of the enzymes and hence modulate the cuts facilitated by the enzymes on the surface. This limits the release of chains from the surface and enables the carboxylation of the insoluble substrate rather than the release of the solubilized chains. This study highlights the importance of analyzing both the solid and soluble reaction products to gain insights into the oxidation mechanism. We demonstrated that 10% functionalization suffices for the use of CNCs in coupling chemistry.

Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

BackgroundMicrobial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin.ResultsTo further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern.ConclusionsThis work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.