Saccharification Of Lignocellulosic Biomass Research Articles

Multi enzyme production from mixed untreated agricultural residues applied in enzymatic saccharification of lignocellulosic biomass for biofuel

Lignocellulose recalcitrance essentially dictates high cost and low efficiency of its enzymatic saccharification. The complex lignocellulosic structure is a major obstacle to enzymatic saccharification and green ionic liquid pretreatment methods face difficulty of water washing prior enzymatic hydrolysis. Ionic liquid tolerant lignocellulolytic enzymes overcome the inhibition of enzymes during enzymatic saccharification and eliminated water washing step after pretreatment of lignocellulosic biomass. The cell wall composition cellulose, hemicellulose, and lignin, three main wall polymers of various agricultural residues and food waste lignocellulose substrates also affected the enzyme activities secreted by the B. subtilis strain. This study explores the potential of the halophilic, alkalophilic, and ionic liquid (IL)-tolerant strain Bacillus subtilis BC-001 for the simultaneous production of hydrolytic enzymes essential for lignocellulosic bio-refinery processes. BC-001 produced cellulase, amylase, xylanase, pectinase and protease are 70.41, 87.14, 65.50, 122.55 and 66.48 U/mL, respectively under optimized fermentation conditions. Cellulase produced by BC-001 retained more than 80% activity after 72 hours in 20% w/w of different ILs tested and enzymes retained more than 68% activities after 12 h in 50% w/w ILs. Employing such IL-stable cellulase, enzymatic saccharification is conducted without water washing on rice straw (RS) that has been pretreated with various ILs, including 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium chloride, and choline chloride. This research is novel as the study marks the first instance of multi-hydrolytic enzyme production from a novel alkalophilic, halophilic, thermophilic, and IL-tolerant bacterial strain using a mixture of untreated agricultural residues.

Coupling chemical modification and immobilization of cellulase improves thermal stability and low-melting mixture solvent tolerance for in situ saccharification of bagasse

In situ saccharification of lignocellulosic biomass in low-melting mixture solvents (LoMMSs) is a promising approach for biorefinery. In this study, chemical modification of cellulase coupled with immobilization was designed to improve thermal stability and LoMMS tolerance of cellulase for in situ saccharification. Cellulase was firstly chemically modified with succinic anhydride and ethylenediamine respectively to form succinylated cellulase (S-Ce) and aminated cellulase (A-Ce), then the modified cellulases were immobilized with amino-functionalized metal-organic frameworks to establish ZIF-8/A immobilized A-Ce and S-Ce. Circular dichroism, fluorescence measurements, X-ray diffraction, fourier transform infrared spectra and scanning electron microscopy were applied to characterize the modified cellulase or immobilization carrier. ZIF-8/A immobilized A-Ce presented desired thermal stability and LoMMS tolerance. Reducing sugar yields via ZIF-8/A immobilized A-Ce during in situ saccharification of bagasse maintained stably till Bet/AA concentration reached 50%, and declined relatively slowly compared with free or other immobilized cellulase when Bet/AA concentration further increased to 90%. In situ saccharification of bagasse via ZIF-8/A immobilized A-Ce in concentrated Bet/AA (50%) can be operated at a wide temperature range (50–60 °C). The corresponding reducing sugar yield with ZIF-8/A immobilized A-Ce was 58% higher than that with free cellulase in 50% Bet/AA at 60 °C.

A detailed sensitivity analysis identifies the key factors influencing the enzymatic saccharification of lignocellulosic biomass

Corn stover is the most abundant form of crop residue that can serve as a source of lignocellulosic biomass in biorefinery approaches, for instance for the production of bioethanol. In such biorefinery processes, the constituent polysaccharide biopolymers are typically broken down into simple monomeric sugars by enzymatic saccharification, for further downstream fermentation into bioethanol. However, the recalcitrance of this material to enzymatic saccharification invokes the need for innovative pre-treatment methods to increase sugar conversion yield. Here, we focus on experimental glucose conversion time-courses for corn stover lignocellulose that has been pre-treated with different acid-catalysed processes and intensities. We identify the key parameters that determine enzymatic saccharification dynamics by performing a Sobol's sensitivity analysis on the comparison between the simulation results from our complex stochastic biophysical model, and the experimental data that we accurately reproduce. We find that the parameters relating to cellulose crystallinity and those associated with the cellobiohydrolase activity are predominantly driving the enzymatic saccharification dynamics. We confirm our computational results using mathematical calculations for a purely cellulosic substrate. On the one hand, having identified that only five parameters drastically influence the saccharification dynamics allows us to reduce the dimensionality of the parameter space (from nineteen to five parameters), which we expect will significantly speed up our fitting algorithm for comparison of experimental and simulated saccharification time-courses. On the other hand, these parameters directly highlight key targets for experimental endeavours in the optimisation of pre-treatment and saccharification conditions. Finally, this systematic and two-fold theoretical study, based on both mathematical and computational approaches, provides experimentalists with key insights that will support them in rationalising their complex experimental results.

Enhanced Xylanolytic enzyme production from Parthenium hysterophorus through assessment of the RSM tool and their application in saccharification of lignocellulosic biomass

Enhanced Xylanolytic enzyme production from Parthenium hysterophorus through assessment of the RSM tool and their application in saccharification of lignocellulosic biomass

Exploring the cellulolytic and hemicellulolytic activities of manganese peroxidase for lignocellulose deconstruction

BackgroundA cost-effective pretreatment and saccharification process is a necessary prerequisite for utilizing lignocellulosic biomass (LCB) in biofuel and biomaterials production. Utilizing a multifunctional enzyme with both pretreatment and saccharification functions in a single step for simultaneous biological pretreatment and saccharification process (SPS) will be a green method of low cost and high efficiency. Manganese peroxidase (MnP, EC 1.11.1.13), a well-known lignin-degrading peroxidase, is generally preferred for the biological pretreatment of biomass. However, exploring the role and performance of MnP in LCB conversion will promote the application of MnP for lignocellulose-based biorefineries.ResultsIn this study, we explored the ability of an MnP from Moniliophthora roreri, MrMnP, in LCB degradation. With Mn2+ and H2O2, MrMnP decomposed 5.0 g/L carboxymethyl cellulose to 0.14 mM of reducing sugar with a conversion yield of 5.0 mg/g, including 40 μM cellobiose, 70 μM cellotriose, 20 μM cellotetraose, and 10 μM cellohexaose, and degraded 1.0 g/L mannohexaose to 0.33 μM mannose, 4.08 μM mannotriose, and 4.35 μM mannopentaose. Meanwhile, MrMnP decomposed 5.0 g/L lichenan to 0.85 mM of reducing sugar with a conversion yield of 30.6 mg/g, including 10 μM cellotriose, 20 μM cellotetraose, and 80 μM cellohexose independently of Mn2+ and H2O2. Moreover, the versatility of MrMnP in LCB deconstruction was further verified by decomposing locust bean gum and wheat bran into reducing sugars with a conversion yield of 54.4 mg/g and 29.5 mg/g, respectively, including oligosaccharides such as di- and tri-saccharides. The catalytic mechanism underlying MrMnP degraded lignocellulose was proposed as that with H2O2, MrMnP oxidizes Mn2+ to Mn3+. Subsequently, it forms a complex with malonate, facilitating the degradation of CMC and mannohexaose into reducing sugars. Without H2O2, MrMnP directly oxidizes malonate to hydroperoxyl acetic acid radical to form compound I, which then attacks the glucosidic bond of lichenan.ConclusionThis study identified a new function of MrMnP in the hydrolysis of cellulose and hemicellulose, suggesting that MrMnP exhibits its versatility in the pretreatment and saccharification of LCB. The results will lead to an in-depth understanding of biocatalytic saccharification and contribute to forming new enzymatic systems for using lignocellulose resources to produce sustainable and economically viable products and the long-term development of biorefinery, thereby increasing the productivity of LCB as a green resource.

Effective hydrothermal-enzymatic sequential process of agave bagasse enhances saccharification and methane production

The efficiency of enzymatic saccharification of lignocellulosic biomass is a key step for biofuels production in the biochemical biorefineries. A novel sequential process, hydrothermal pretreatment followed by enzymatic hydrolysis, of agave bagasse was investigated to improve the release of sugars and the methane production. After optimization of the hydrothermal pretreatment by surface response methodology, a hemicellulose removal of 93.6% was obtained at 180 °C/50 min. Hemicellulose removal enhanced cellulose depolymerization during the enzymatic hydrolysis of the pretreated bagasse by 2.3 times as compared to the untreated bagasse. The single-stage batch methane production of the hydrothermal and enzymatic hydrolysates were 0.223 ± 0.02 NL CH4/ g CODadd and 0.305 ± 0.03 NL CH4/g CODadd respectively, indicating a higher biodegradability of the enzymatic hydrolysate. The combined energy recovered from the hydrothermal and enzymatic hydrolysates was 3.4 times greater than that recovered from untreated agave bagasse hydrolysate. Also, the combined energy recovery was higher than the energy recovery reported in the literature for agave bagasse pretreated with other chemical and hydrothermal pretreatments. Overall, this study enhanced the saccharification efficiency and biodegradability of polysaccharides present in agave bagasse through efficient saccharification of cellulose and hemicellulose. This was reflected in a high value of combined energy recovery efficiency (54.1%) in the form of methane.

Molecular dynamics-based structural insights of the first putative endoglucanase, PsGH5A of glycoside hydrolase family 5 from Pseudopedobacter saltans.

The putative endoglucanase, PsGH5A, from Pseudopedobacter saltans of family GH5 contains a catalytic module, PsGH5 (β/α)8-TIM barrel), at N-terminal followed by a family 6 carbohydrate-binding module (CBM6, β-sandwich). Superposition of PsGH5A with PDB homologs revealed Glu220 and Glu318 as evolutionarily conserved and catalytic residues performing the hydrolysis through retaining-type mechanism, a canonical property of GH5 family. PsGH5A showed higher affinity for longer cellooligosaccharides, as long as cellodecaose with binding free energy (∆G) of - 13.72kcal/mol upon the molecular docking, thereby indicating the endo-mode of hydrolysis. The radius of gyration, Rg (2.7nm), and solvent accessible surface area, SASA (229.6 nm2), of PsGH5A-Cellotetraose complex were determined by MD simulation which was lower than that of PsGH5A (Rg, 2.8nm, SASA, 267 nm2) demonstrating the compactness and affinity of PsGH5A with the cellulosic ligands. Cellulose compatibility of PsGH5A was further confirmed by MMPBSA and per-residue decomposition analysis, where notable ∆G of - 54.38kcal/mol for PsGH5A-Cellotetraose complex was observed. Thus, PsGH5A could be potentially an efficient endoglucanase as it accommodated larger cellooligosaccharides at its active-site. PsGH5A is the first putative endoglucanase studied here from P. saltans which could be genome-mined for lignocellulosic biomass saccharification in the renewable energy sector. The 3-D structure of PsGH5A generated by AlphaFold2, RaptorX, SwissModel, Phyre2 and Robetta tool; YASARA was used for energy minimization of built models. UCLA SAVES-v6 was used for quality assessment of models. Molecular Docking was performed using SWISS-DOCK server and Chimera software. Molecular Dynamics simulations and MMPBSA analysis of PsGH5A and PsGH5A-Cellotetraose complex were performed on GROMACS 2019.6.

An overview on the factors affecting enzymatic saccharification of lignocellulosic biomass into fermentable sugars

Abstract Lignocellulosic biomass (LCB) is a widely available and sustainable energy resource that can be directly or indirectly converted to biofuels and value-added bioproducts. In such LCB conversion, enzymatic saccharification is commonly regarded as a green alternative to chemical hydrolysis due to less energy-intensive, less toxic, and more environment-benign for efficient fermentable sugar recovery. However, enzymatic saccharification faces substantial challenges, since the complex polymeric matrices of LCB necessitates a variety of enzymes for complete and adequate saccharification. Empirical evidence on enzymatic saccharification has paved the way for optimizing the processes and design for enhancing the performance in LCB. This review examines the enzymatic saccharification of LCB, focusing on the important parameters affecting the process, such as pH, temperature, agitation, enzyme/substrate loading, residence time, and the enzymes required to degrade various LCB components. Various strategies have been reported to improve the performance in saccharification and to address the non-productive adsorption of enzymes. A preliminary economic competency valuation of enzyme-derived fermentable sugars is proposed. Wheat straw, sugarcane bagasse and corn stalk appear, in this case, to be the most economic competent LCBs for commercial enzyme-derived fermentable sugar production. Lastly, practical challenges and future research directions on the enzymatic saccharification of LCB are discussed.

Cellulose esterification with carboxylic acid in deep eutectic solvent pretreatment inhibits enzymatic hydrolysis

Avicel cellulose was pretreated using two commonly used carboxylic acid-based deep eutectic solvents, i.e., choline chloride-lactic acid and choline chloride-formic acid. The pretreatment process resulted in the formation of cellulose esters with lactic acid and formic acid, which was confirmed by infrared and nuclear magnetic resonance spectra. Surprisingly, the esterified cellulose led to a significant decrease in the 48-h enzymatic glucose yield (≥75%) compared to raw Avicel cellulose. Analysis of changes in cellulose properties caused by pretreatment, including crystallinity, degree of polymerization, particle size and cellulose accessibility, contradicted the observed decline in enzymatic cellulose hydrolysis. However, removing the ester groups through saponification largely recovered the reduction in cellulose conversion. The decreased enzymatic cellulose hydrolysis by esterification may be attributed to changes in the interaction between cellulose-binding domain of cellulase and cellulose. These findings provide valuable insights into improving the saccharification of lignocellulosic biomass pretreated by carboxylic acid-based DESs.

Efficient bioconversion of lignocellulosic waste by a novel computationally screened hyperthermostable enzyme from a specialized microbiota

A large amount of lignocellulosic waste is generated every day in the world, and their accumulation in the agroecosystems, integration in soil compositions, or incineration for energy production has severe environmental pollution effects. Using enzymes as biocatalysts for the biodegradation of lignocellulosic materials, especially in harsh processing conditions, is a practical step towards green energy and environmental biosafety. Hence, the current study focuses on enzyme computationally screened from camel rumen metagenomics data as specialized microbiota that have the capacity to degrade lignocellulosic-rich and recalcitrant materials. The novel hyperthermostable xylanase named PersiXyn10 with the performance at extreme conditions was proper activity within a broad temperature (30–100 ℃) and pH range (4.0–11.0) but showed the maximum xylanolytic activity in severe alkaline and temperature conditions, pH 8.0 and temperature 90 ℃. Also, the enzyme had highly resistant to metals, surfactants, and organic solvents in optimal conditions. The introduced xylanase had unique properties in terms of thermal stability by maintaining over 82% of its activity after 15 days of incubation at 90 ℃. Considering the crucial role of hyperthermostable xylanases in the paper industry, the PersiXyn10 was subjected to biodegradation of paper pulp. The proper performance of hyperthermostable PersiXyn10 on the paper pulp was confirmed by structural analysis (SEM and FTIR) and produced 31.64 g/L of reducing sugar after 144 h hydrolysis. These results proved the applicability of the hyperthermostable xylanase in biobleaching and saccharification of lignocellulosic biomass for declining the environmental hazards.

Natural lignin modulators improve lignocellulose saccharification of field-grown sugarcane, soybean, and brachiaria

Saccharification of lignocellulosic biomass might contribute to the bioethanol production, positively impacting energy security and sustainability. Lignin reduces polysaccharide saccharification and needs to be depolymerized and separated to improve fermentable sugar yield. Here, we report a substantial increase in lignocellulose saccharification after treating plants with the natural compounds: 3,4-(methylenedioxy)cinnamic acid (MDCA), a competitive inhibitor of 4-coenzyme A ligase (4CL); piperonylic acid (PIP), a quasi-irreversible inhibitor of cinnamyl 4-hydroxylase (C4H); and daidzin (DZN) a competitive inhibitor of coniferaldehyde dehydrogenase (CALDH). A single treatment with these inhibitors on different young developmental stages significantly increased lignocellulosic saccharification of crops at maturity stage. Field trial experiments with sugarcane (Saccharum spp), soybean (Glycine max), and brachiaria (Urochloa decumbens), showed that 12-month-old sugarcane bagasse increased saccharification up to 120%, 90-days-old soybean residues increased saccharification up to 36%, and 40-days-old brachiaria straw increased saccharification up to 21%. Treatments did not affect biomass productivity, lignin content or its monomeric composition. In contrast, anatomic assessment by UV microscopy revealed that MDCA affected tissue formation, increasing the lignification in fibers and vessels, but not in parenchyma. The tissue-specific and developmental persistent phenomena allowed a substantial increase in saccharification, while preserving the mechanical strength necessary for plant growth and development, suggesting that natural compounds are opportune for bioenergy applications.

Microbial saccharification – Biorefinery platform for lignocellulose

Lignocellulosic biomass has gained increasing attention as it can be bio-transformed into biofuel, such as bioethanol , biobutanol, biohydrogen, biogas and biodiesel . The conventional strategy includes the biotransformation of lignocellulose into soluble and fermentable sugars through enzymatic hydrolysis for bioenergy production. However, the exogenous cellulase reagent preparation leads to increased complexity and a high saccharification cost. Microbial saccharification (MS) can also realize in-situ simultaneous enzymatic production and hydrolysis to obtain value-added products in a single step. However, its application is limited due to the high temperature of enzymatic hydrolysis than the optimum growth and enzyme synthesis temperature of most natural microorganisms. Nevertheless, some microbial strains and consortiums with excellent potential for microbial saccharification have been discovered and developed. Therefore, this review comprehensively summarizes different microorganisms with MS strategies, including natural and modified strains, the limitations of different strains, and possibilities for industrial application. • Compare the microbial saccharification of lignocellulosic biomass by different microorganisms. • Most bacteria mainly degrade hemicellulose while fungi degrade cellulose. • Mix enzymes/fungi promote microbial saccharification of lignocellulosic biomass. • Industrial application of microbial saccharification for biofuels production.

Multi-efficient endoglucanase from Aspergillus niger MPS25 and its potential applications in saccharification of wheat straw and waste paper deinking

The deinking in the paper industry is in great demand, and microbial enzymes are key players. In the present study, the endoglucanase production from newly isolated fungi Aspergillus niger MPS25 is reported. The optimization of endoglucanase production was carried out using one factor at a time approach resulting in endoglucanase activity (2.37 IU/ml) at 45 °C and pH 8 in submerged fermentation (SmF), which shows optimum enzyme activity at 60 °C. Interestingly, the metal ions viz. Co2+ stimulated the endoglucanase activity, whereas Mn2+ reduced the enzyme activity, which shows that this enzyme can be used for effluent treatment released through deinking. The enzymatic hydrolysis of wheat straw produced 26.96 ± 0.108 mg/g of reducing sugars, indicating its potential in saccharification and the biofuel industry. Furthermore, the validation of the deinking efficiency of this enzyme resulted in improved deinking of mixed office waste and old newspapers by 31.5% and 20.4%, respectively. The strength properties, viz. burst factor and tear index, breaking length, and tensile index of the handmade paper sheets, were also improved which were analyzed by the scanning electron micrographs. The FTIR and XRD analysis of pulp provided insights into the changes in functional groups and cellulose crystallinity, respectively. These results indicate that multi-efficient endoglucanase from Aspergillus niger MPS25 is suitable for enzyme-based eco-friendly deinking for waste paper recycling and lignocellulosic biomass saccharification.

Biochemical characterization of the β-glucosidase Glu1B from Coptotermes formosanus produced in Pichia pastoris

β-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze β-1,4-glycosidic bonds from non-reducing terminal residues in β-D-glucosides, with the release of glucose. β-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the β-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated β-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431–597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant β-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the β-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass will promote the use of this renewable material. Here, we have studied several process parameters (reaction temperature, type of enzyme blend, type of substrate, type of reductant, and in situ supply of hydrogen peroxide) to better understand how saccharification could be optimized, focusing on the role of lytic polysaccharide monooxygenases (LPMOs). Comparison of a simple, LPMO-rich cellulolytic secretome from the thermophilic fungus Thermoascus aurantiacus with the commercial cellulase preparation Cellic CTec2 showed that saccharification of (lignin-poor) sulfite-pulped spruce at 60 °C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 °C. Quantification of LPMO products showed that while LPMO activity contributed to saccharification efficiency, high levels of LPMO activity were not necessarily beneficial. Reactions with steam-exploded birch, rich in redox-active lignin, highlighted a strong impact of the feedstock on enzyme performance. In this case, the reaction with Cellic CTec2 at 50 °C was clearly most efficient. At 60 °C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H2O2-generating glucose oxidase to reactions with Cellic CTec2 at 50 °C led to strongly increased LPMO activity and, only for reactions with the lignin-poor substrate, improved saccharification yields. These results underpin the potential of the T. aurantiacus secretome for hydrolysis of lignin-poor substrates, and the usefulness of glucose oxidase for optimizing their saccharification. They also show that the efficiency of LPMO-containing cellulase preparations is highly dependent on the nature of the reductant and the substrate.

Transxylosylation of stevioside by a novel GH39 β-xylosidase, and simultaneous valorization of agroindustrial byproducts

Stevia rebaudiana Bertoni is becoming increasingly popular as a sweetener, due to its high steviol glycoside content. However, the bitter aftertaste of those compounds is an issue for consumers, which can be alleviated with the addition of glycosyl substituents to the molecule. β-Xylosidases are known to transxylosylate various acceptors, mostly alcohols and sugars. In this work, a putative novel β-xylosidase gene from Ruminiclostridium cellulolyticum was heterologously expressed in E. coli, purified and characterized. The recombinant enzyme RcXyl39A, belonging to CAZy family GH39, showed satisfactory thermostability and tolerance towards product inhibition, while it was active in a wide pH and temperature range. Surprisingly, RcXyl39A was shown to possess a relaxed substrate specificity, since it was able to cleave various synthetic substrates, but also polymeric xylans. RcXyl39A was applied to the transxylosylation of stevioside, using enzymatically hydrolyzed xylan as a mixture of donor sugars. After optimization of several process parameters, the final yield obtained for stevioside conversion reached 27.4 ± 0.6 %. The results of the present study indicate that RcXyl39A could be applied to the saccharification of lignocellulosic biomass, but also to the synthesis of novel compounds with desirable properties.

Surfactant-assisted alkaline pretreatment and enzymatic hydrolysis of Miscanthus sinensis for enhancing sugar recovery with a reduced enzyme loading.

Surfactants play a vital role in the delignification and saccharification of lignocellulosic biomass. A strategy for coupling surfactant-assisted alkaline pretreatment (SAP) with surfactant-assisted enzymatic hydrolysis (SEH) has been proposed for improving sugar recovery from a potential energy crop, Miscanthus sinensis. Poly (ethylene glycol) 2000 (PEG 2000) was found to be more efficient in SAP than in other tested surfactants. Compositional and structural analysis revealed that the SAP process with 1% of PEG 2000 produced more efficient lignin removal and microstructure disruption of the pretreated sample, thus indicating much higher reducing sugar yields of 544.4–601.2 mg/g compared to the samples that were untreated or pretreated by alkali alone. Moreover, SEH with 1% Tween 80, which could block the lignin-enzyme interactions, produced a substantial reduction of 33.3% in the enzyme loading to achieve a higher sugar recovery from the SAP sample.

Investigation of biochemical and biotechnological potential of a thermo-halo-alkali-tolerant endo-xylanase (GH11) from Humicola brevis var. thermoidea for lignocellulosic valorization of sugarcane biomass

Hemicellulose is an important source of pentose sugars, which are precursors of products of high economic value, such as prebiotic oligomers, sugar alcohols, organic acids, and biofuels. Enzymatic hydrolysis is the most efficient method of converting hemicellulose into sugars, where efficient thermotolerant fungal xylanases hydrolyze the xylan fractions present in the hemicellulose into xylose and xylooligomers. Xylanases are potential candidates for improved enzyme cocktails that increase saccharification yields in synergy with other cellulolytic and hemicellulolytic enzymes complexes. In this context, endo-xylanase from the thermophilic fungus Humicola brevis var. thermoidea (XylHb) was purified, biochemically and biophysically characterized, and its biotechnological potential was evaluated. XylHb exhibited a molecular weight of 26 kDa and biophysical analyzes revealed that XylHb is mainly formed by β sheets, and exhibits maximum exposure of its tryptophan residues at high temperatures. The characterization of the biochemical properties of XylHb indicated greater tolerance to wide pH (5.0–10.0), temperatures (50–70 °C), and NaCl concentrations (0.1–2.0 mol L−1), as well as better kinetic efficiency than the Cellic®HTec2 cocktail for birchwood xylan hydrolysis. Additionally, saccharification tests of pre-hydrothermally treated sugarcane bagasse after 72 h showed that the complementation of Cellic®CTec2 with XylHb (1:1; w/w) caused a significant increase in glucose released (7.52 ± 0.01%) and total reducing sugars (14.07 ± 0.02%) about commercial enzymatic control. Thus, the addition of XylHb to commercial cellulase suggests a potential for diverse industrial applications improving the saccharification of lignocellulosic biomass.

An Insight into Enzymatic Immobilization Techniques on the Saccharification of Lignocellulosic Biomass

An Insight into Enzymatic Immobilization Techniques on the Saccharification of Lignocellulosic Biomass

Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

BackgroundFungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases.ResultsHere we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity.ConclusionUsing small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.