Enzyme Adsorption Research Articles

Unit-cell wide SBA-15 type mesoporous silica nanoparticles

With large and 2D hexagonally ordered mesopores, SBA-15 is the superstar among mesoporous silicas reported so far and has found many applications. To prepare SBA-15 nanoparticles is of particular interest for biomedical application yet remains challenging. In this work, rod-shaped SBA-15 nanoparticles (SBA-15NPs), with 2–3 mesopore channels in width (called unit-cell wide) and averaged length of 94.8 nm, are for the first time prepared in a boric acid catalyzed P123 templating system based on the partitioned cooperative self-assembly principle in a one-pot preparation. The BET surface area and total pore volume of typical SBA-15NPs reach 553 m2g-1 and 1.52 cm3g-1, respectively. The preparation of SBA-15NPs relies on PCSA process that bestows long and thin tubular SBA-15 pre-structures with many defects in low-temperature synthesis step, which are then cut into SBA-15NPs by a sulfuric acid enhanced Ostwald ripening process in hydrothermal treatment step. In enzyme adsorption tests, SBA-15NPs demonstrate higher capacity and faster adsorption compared with conventional rod-shaped SBA-15.

Adsorption of enzymes with hydrolytic activity on polyethylene terephthalate

Polyethylene terephthalate (PET) degrading enzymes have recently obtained an increasing interest as a means to decompose plastic waste. Here, we have studied the binding of three PET hydrolases on a suspended PET powder under conditions of both enzyme- and substrate excess. A Langmuir isotherm described the binding process reasonably and revealed a prominent affinity for the PET substrate, with dissociation constants consistently below 150 nM. The saturated substrate coverage approximately corresponded to a monolayer on the PET surface for all three enzymes. No distinct contributions from specific ligand binding in the active site could be identified, which points towards adsorption predominantly driven by non-specific interactions in contrast to enzymes naturally evolved for the breakdown of insoluble polymers. However, we observed a correlation between the progression of enzymatic hydrolysis and increased binding capacity, probably due to surface modifications of the PET polymer over time. Our results provide functional insight, suggesting that rational design should target the specific ligand interaction in the active site rather than the already high, general adsorption capacity of these enzymes.

Combination of hydrothermal and chemi-mechanical pretreatments to enhance enzymatic hydrolysis of poplar branches and insights on cellulase adsorption

An integration of different pretreatments is important to overcome recalcitrance and realize efficient bioconversion of lignocellulosic biomass. This study aims at the effects of combination of hydrothermal pretreatment and different chemi-mechanical pretreatments on enzymatic hydrolysis, and understanding the enzymes adsorption mechanism. The combination of hydrothermal and chemi-mechanical pretreatments effectively improved the enzymatic hydrolysis of poplar substrates, in which the enzymatic hydrolysis of substrates pretreated by hydrothermal pretreatment + Fenton pretreatment + mechanical refining (HFM) was the highest (92.39% of glucose conversion yield, and 20.88 g/L of glucose concentration). The substrates’ main characteristics were obviously changed after combined pretreatments, such as swelling ability and specific surface area of substrates were increased. The Langmuir adsorption model (R2 > 0.98) and pseudo second-order adsorption kinetic model (R2≈1) were well suitable to describe the adsorption of enzymes on substrates, meanwhile the adsorption mechanism was summarized.

Enhancement of Phospholipid Binding and Catalytic Efficiency of Streptomyces klenkii Phospholipase D by Increasing Hydrophobicity of the Active Site Loop.

The mechanism of active site loops of Streptomyces phospholipase D (PLD) binding to the lipid-water interface for catalytic reactions still remains elusive. A flexible loop (residues 376-382) in the active site of Streptomyces klenkii PLD (SkPLD) is conserved within PLDs in most of the Streptomyces species. The residue Ser380 was found to be essential for the enzyme's adsorption to the interface and its substrate recognition. The S380V mutant showed a 4.8 times higher catalytic efficiency and nearly seven times higher adsorption equilibrium coefficient compared to the wild-type SkPLD. The monolayer film technique has confirmed that the substitution of Ser380 with valine in the loop exhibited positive interaction between the enzyme and PCs with different acyl chain lengths. The results of the interfacial binding properties indicated that the S380V mutant might display suitable phosphatidylserine synthesis activity. The present study will be helpful to explain the role of residue 380 in the active site loops of Streptomyces PLD.

Non-productive binding of cellobiohydrolase i investigated by surface plasmon resonance spectroscopy

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers.Graphic abstract

High-Sensitivity Enzymatic Glucose Sensor Based on ZnO Urchin-like Nanostructure Modified with Fe3O4 Magnetic Particles.

A novel and efficient enzymatic glucose sensor was fabricated based on Fe3O4 magnetic nanoparticles (Fe3O4MNPs)-modified urchin-like ZnO nanoflowers (ZnONFs). ZnONFs were hydrothermally synthesizing on a flexible PET substrate. Fe3O4MNPs were deposited on the surface of the ZnONFs by the drop-coating process. The results showed that the urchin-like ZnONFs provided strong support for enzyme adsorption. For Fe3O4MNPs, it significantly promoted the redox electron transfer from the active center of GOx to the ZnO nanoflowers beneath. More importantly, it promoted the hydrolysis of H2O2, the intermediate product of glucose catalytic reaction, and thus improved the electron yield. The sensitivity of the Nafion/GOx/Fe3O4MNPs/ZnONFs/Au/PET sensor was up to 4.52 μA·mM−1·cm−2, which was improved by 7.93 times more than the Nafion/GOx/ZnONFs/Au/PET sensors (0.57 μA·mM−1·cm−2). The detection limit and linear range were also improved. Additionally, the as-fabricated glucose sensors show strong anti-interference performance in the test environment containing organic compounds (such as urea, uric acid, and ascorbic acid) and inorganic salt (for instance, NaCl and KCl). The glucose sensor’s service life was evaluated, and it can still maintain about 80% detection performance when it was reused about 20 times. Compared with other existing sensors, the as-fabricated glucose sensor exhibits an ultrahigh sensitivity and wide detection range. In addition, the introduction of Fe3O4MNPs optimized the catalytic efficiency from the perspective of the reaction mechanism and provided potential ideas for improving the performance of other enzymatic biosensors.

Development of functionalized metal–organic frameworks immobilized cellulase with enhanced tolerance of aqueous-ionic liquid media for in situ saccharification of bagasse

In this study, two functionalized ZIF-8 were used for cellulase immobilization through physical adsorption. The in situ saccharification efficiency of immobilized cellulase was evaluated in 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP) using bagasse (BG) as the substrate. The results showed that the enzyme adsorption capacity (mg/g) of chitosan@ZIF-8 (326.04 mg/g) and PEG@ZIF-8 (264.20 mg/g) was improved compared to ZIF-8 (192.40 mg/g). The ionic liquid (IL)-tolerance of functionalized ZIF-8 immobilized cellulase was also significantly increased. In 50% (v/v) [Emim]DEP, the amount of reducing sugar produced by functionalized ZIF-8 immobilized cellulase catalyzed hydrolysis was increased by 64.34% and 16.98% compared with ZIF-8 immobilized cellulase, respectively. Furthermore, the reusability of functionalized ZIF-8 immobilized cellulase was improved in 25% (v/v) [Emim]DEP. Notably, the increase in adsorption capacity and IL-tolerance of the functionalized ZIF-8 immobilized cellulase may be related to the increase in surface potential and the hydrophobic groups on the surface of the carrier.

Construction of a structural enzyme adsorption/kinetics model to elucidate additives associated lignin-cellulase interactions in complex bioconversion system.

Enzymatic hydrolysis is a rate-limiting process in lignocellulose biorefinery. The reaction involves complex enzyme-substrate and enzyme-lignin interactions in both liquid and solid phases, and has not been well characterized numerically. In this study, a kinetic model was developed to incorporate dynamic enzyme adsorption and product inhibition parameters into hydrolysis simulation. The enzyme adsorption coefficients obtained from Langmuir isotherm were fed dynamically into first-order kinetics for simulating the equilibrium enzyme adsorption in hydrolysis. A fractal and product inhibition kinetics was introduced and successfully applied to improve the simulation accuracy on adsorbed enzyme and glucose concentrations at different enzyme loadings, lignin contents, and in the presence of bovine serum albumin (BSA) and lysozyme. The model provided numerical proof quantifying the beneficial effects of both additives, which improved the hydrolysis rate by reducing the nonproductive adsorption of enzyme on lignin. The hydrolysis rate coefficient and fractal exponent both increased with increasing enzyme loadings, and lignin inhibition exhibited with increasing fractal exponent. Compared with BSA, the addition of lysozyme exhibited higher hydrolysis rates, which was reflected in the larger hydrolysis rate coefficients and smaller fractal exponents in the simulation. The model provides new insights to support process development, control, and optimization.

Fully Inkjet-Printed Biosensors Fabricated with a Highly Stable Ink Based on Carbon Nanotubes and Enzyme-Functionalized Nanoparticles.

Enzyme inks can be inkjet printed to fabricate enzymatic biosensors. However, inks containing enzymes present a low shelf life because enzymes in suspension rapidly lose their catalytic activity. Other major problems of printing these inks are the non-specific adsorption of enzymes onto the chamber walls and stability loss during printing as a result of thermal and/or mechanical stress. It is well known that the catalytic activity can be preserved for significantly longer periods of time and to harsher operational conditions when enzymes are immobilized onto adequate surfaces. Therefore, in this work, horseradish peroxidase was covalently immobilized onto silica nanoparticles. Then, the nanoparticles were mixed into an aqueous ink containing single walled carbon nanotubes. Electrodes printed with this specially formulated ink were characterized, and enzyme electrodes were printed. To test the performance of the enzyme electrodes, a complete amperometric hydrogen peroxide biosensor was fabricated by inkjet printing. The electrochemical response of the printed electrodes was evaluated by cyclic voltammetry in solutions containing redox species, such as hexacyanoferrate (III/II) ions or hydroquinone. The response of the enzyme electrodes was studied for the amperometric determination of hydrogen peroxide. Three months after the ink preparation, the printed enzyme electrodes were found to still exhibit similar sensitivity, demonstrating that catalytic activity is preserved in the proposed ink. Thus, enzyme electrodes can be successfully printed employing highly stable formulation using nanoparticles as carriers.

Flow amperometric uric acid biosensors based on different enzymatic mini-reactors: A comparative study of uricase immobilization

This is the first report of a novel strategy for uric acid (UA) determination via newly developed highly stable flow biosensors based on different enzymatic mini-reactors in combination with a four-electron reduction of the consumed oxygen. The design of the biosensors provides a spatial separation of the detection (working electrode) and biorecognition (uricase-based mini-reactor) part. To prepare the most efficient enzymatic mini-reactor, a comparative study focused on three different techniques of the covalent uricase immobilization at two kinds of mesoporous silica powders (SBA−15 and MCM−41) and the physical enzyme adsorption at four types of carbonaceous powders (glassy carbon, graphite, Starbon® 300, and Starbon® 800) has been performed. All investigations were carried out for two kinds of uricase: from Candida sp. (UOx(c)) and from Bacillus fastidiosus (UOx(b)) to estimate the influence of uricase source. The flow-through cell with tubular detector of silver solid amalgam was used for amperometric monitoring of four-electron oxygen reduction at the highly negative potential (−1100 mV vs. SCE). It was found that the developed UA biosensors responses are strongly dependent on the enzyme immobilization strategy. UOx(c) bounded to glassy carbon by physical adsorption has provided good results in terms of sensitivity, stability, and repeatability for UA determination. The biosensor with the mini-reactor based on the covalent immobilization of UOx(b) at glutaraldehyde activated NH2 functionalized MCM–41 has shown extremely high stability (more than 1 year) and reusability (at least 600 measurements) The biosensor’s practical applicability has been tested by successful determination of UA in human urine.

Mechanisms of bio-additives on boosting enzymatic hydrolysis of lignocellulosic biomass

Expensive cellulase is one of the major obstacles hinders large-scale biorefining of lignocellulosic biomass. The cheap and biodegradable additives sophorolipid and whey protein were found to boost enzymatic hydrolysis, their mechanisms were clarified firstly in this study. Results showed that the effects of these additives on enhancing enzymatic hydrolysis were positively correlated with substrate content; when the solid dosage was 20% (w/v), the presence of sophorolipid and whey protein increased glucose yield by 17.8% and 11.9%, respectively; this could be attributed to sophorolipid favor to alleviate the non-productive adsorption between undesired substrates and enzymes caused by hydrophobic and electrostatic forces, and the ability of whey protein to block the site of enzyme adsorption of lignin; high shear and temperature conditions accelerate the inactivation of cellulase, and the addition of sophorolipid and whey protein reduced the inactivation rate by 7.8% and 13.6%, respectively, under enzymatic hydrolysis conditions.

Eucalyptus lignin modification for dynamic adsorption with lignocellulose-degradation enzymes dependent on pH values

The large amount of ineffective adsorption of cellulase on lignin seriously impeded the enzymatic hydrolysis efficiency and reduced the enzyme activity. In this study, the eucalyptus lignin obtained by alkali and hydrotropic methods was used as raw materials to investigate the differences in the dynamic adsorption with different enzymes and enzyme components in detail. The lignin influenced on enzyme activity of different enzymes and enzyme components was determined as well. Furthermore, alkali lignin (AL) and sodium xylene sulfonate lignin (SL) were modified by dimethylamine (DMA) and diethylenetriamine (DETA) through Mannich reaction. By introducing a certain amount of amine groups on lignin to reduce the surface charge and hydrophobicity of lignin for greatly reduction adsorption of cellulase. Under the optimum condition (pH 4.8), the adsorption of cellulase onto DETA-AL-amine was reduced by 96.32 % from 136 mg g−1 to 5 mg g−1. This work suggests that lignin-amine has potential applications in reducing enzyme adsorption.

Near‐Linear Controllable Synthesis of Mesoporosity in Hierarchical UiO‐66 by Template‐Free Nucleation‐Competition

AbstractHierarchies design of porosity in metal‐organic frameworks (MOFs) has gained significant interest in recent years, and customization of mesoporous sizes in MOFs is still quite challenging. Herein, a template‐free method by nucleation‐competition has been developed to realize a near‐linear control of the mesopore sizes (3–13 nm) in the hierarchical MOF UiO‐66(Hf). High selectivity of enzyme adsorption, high activity of bulky‐molecular catalysis, high stability of mesostructure, and extension to other MOFs further prove the success in the potential customization synthesis and applications.

Antifouling membranes employing a 2D planar nanobiocatalyst of crosslinked glucose oxidase aggregates wrapping extra-large graphene oxide

This paper presents highly effective antimicrobial surfaces employing a 2D structured nanobiocatalyst composed of graphene oxide (GO) and glucose oxidase (GOD). Enzyme molecules are immobilized onto extra-large GO pieces with a plane dimension of approximately 100 μm via an enzyme adsorption, precipitation, and crosslinking (EAPC) approach. This enables the effective wrapping of extra-large GO pieces by a matrix of crosslinked enzyme aggregates, which improves the enzyme loading. Consequently, the measured GOD activities of the EAPC sample via 50% (w/v) ammonium sulfate precipitation are 4,940 and 3,820 times higher than those of the control samples, i.e, the enzyme adsorption (EA) and enzyme adsorption/crosslinking (EAC) samples, respectively. The preservation of the planar GO geometry with an extra-large surface also allows the effective binding of EAPC onto a commercial membrane filter via a polydopamine coating, thus yielding a biocatalytic EAPC membrane. Compared to the commercial membrane with no bound EAPC, the in situ generation of H2O2via the EAPC-catalyzed oxidation of glucose on the membrane surface demonstrated enhanced filterability against a mixed bacterial population of activated sludge obtained from a municipal sewage plant as well as two model bacteria: gram-negative Pseudomonas aeruginosa and gram-positive Staphylococcus aureus. The bacterial decontamination of the EAPC-bound membrane surface can also be activated on demand by simply adding glucose to the bulk solution. This newly proposed mechanism of antifouling surfaces employing a localized nanobiocatalytic conversion of nontoxic glucose to bactericidal H2O2 can provide insights for biofouling control via a highly effective and environment-friendly approach.

Protective effects of non‐catalytic proteins on endoglucanase activity at air and lignin interfaces

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air-liquid interface provide rationale. This work investigated root causes including effects of the air-liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air-liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%-50% of its activity due to an air-liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.

Kinetic Study on Enzymatic Hydrolysis of Cellulose in an Open, Inhibition-Free System

Due to the complexity of cellulases and the requirement of enzyme adsorption on cellulose prior to reactions, it is difficult to evaluate their reaction with a general mechanistic scheme. Nevertheless, it is of great interest to come up with an approximate analytic description of a valid model for the purpose of developing an intuitive understanding of these complex enzyme systems. Herein, we used the surface plasmonic resonance method to monitor the action of a cellobiohydrolase by itself, as well as its mixture with a synergetic endoglucanase, on the surface of a regenerated model cellulose film, under continuous flow conditions. We found a phenomenological approach by taking advantage of the long steady state of cellulose hydrolysis in the open, inhibition-free system. This provided a direct and reliable way to analyze the adsorption and reaction processes with a minimum number of fitting parameters. We investigated a generalized Langmuir-Michaelis-Menten model to describe a full set of kinetic results across a range of enzyme concentrations, compositions, and temperatures. The overall form of the equations describing the pseudo-steady-state kinetics of the flow-system shares some interesting similarities with the Michaelis-Menten equation. The use of familiar Michaelis-Menten parameters in the analysis provides a unifying framework to study cellulase kinetics. The strategy may provide a shortcut for approaching a quantitative while intuitive understanding of enzymatic degradation of cellulose from top to bottom. The open system approach and the kinetic analysis should be applicable to a variety of cellulases and reaction systems to accelerate the progress in the field.

Mitigating the negative impact of soluble and insoluble lignin in biorefineries

The presence of inhibitors is still an economic bottleneck that needs to be resolved in order to make the biorefineries feasible, requiring the development of technologies capable of improving their competitiveness in the biofuel marketplace. Soluble and insoluble lignin can impair the enzymatic hydrolysis process by inhibition, deactivation, and unproductive adsorption of enzymes. Washing the pretreated biomass or using lignin-blocking additives during saccharification could mitigate these negative effects in future biorefineries. Here, an investigation was performed of the combined mitigation processes, in terms of their technical and economic feasibility in an integrated first and second generation (1G2G) sugarcane biorefinery. Evaluation was made of the impacts of biomass washing and soybean protein addition, separately or in combination, on glucose yields for enzymatic hydrolysis in the presence of high (liquor) and low (buffer) concentrations of soluble inhibitors/deactivators. Combining washing and soybean protein addition provided the highest glucose yields, with an increase of up to 50%. The effect of the mitigation processes could be explained by a combination of catalytic mechanisms acting on both soluble and insoluble lignin. In an industrial context, biomass washing (90 °C, 15% (w/w) solids, 3 steps) followed by soybean protein addition (12% (w/v) solids) provided a cost-competitive methodology for bioethanol production, with an estimated net present value of US$ 9.16 × 107, optimizing hydrolysis process in the 1G2G sugarcane biorefinery.

Efficient deconstruction of Chinese silvergrass by FeCl3-catalyzed γ-valerolactone/water system under mild reaction condition

In this work, γ-valerolactone (GVL) pretreatment using FeCl3 as a catalyst under mild temperature (120 °C) to facilitate efficient deconstruction and saccharification of Chinese silvergrass (CS) was reported. The pretreatment resulted in significant xylan and lignin removal of CS and the highest xylan and lignin removal reaching 84.3 % and 68.7 % at FeCl3 concentration of 100 mM. No obvious change in the crystalline structure of cellulose was observed under the mild pretreatment condition. X-ray photoelectron spectroscopy analysis indicated that the FeCl3-catalyzed GVL pretreatment caused poorer lignin but richer carbohydrates on the CS surface due to the increased O/C ratio in CS (increased from 0.23 to 0.63). The results indicated that the pretreatment could facilitate the decrease of non-productive adsorption of enzyme and was beneficial for the enzymatic saccharification of pretreated CS, and a high glucose yield of 99.7 % was achieved by cellulases and Tween 80.

Adsorption of α-amylase and Starch on Porous Zinc Oxide Nanosheet: Biophysical Study

Engineered biocatalyst and its desired products using nanotechnology has intensified the research in food industries. Zinc oxide (ZnO) nanosheet is designed and prepared; the characterization studies include surface plasmon resonance peak (364 nm), X-ray diffraction pattern determined crystallite size (25 nm), and transmission electron microscopy confirms the porous surface nature. Atomic force microscopy showed substrate and enzyme adsorbed on ZnO nanosheets. The zeta potential of ZnO nanosheet (−41.9 mV) whereas α-amylase bound with ZnO nanosheets (−32.8 mV), and starch bound with ZnO nanosheets (−28.7 mV) was analyzed using dynamic light scattering. The circular dichroism spectra displayed α-helix in native amylase at optimum concentration 54.70% compared to the adsorbed α-amylase with ZnO nanosheet that showed 37%. Freundlich isotherm model revealed multilayer adsorption behavior of α-amylase onto porous ZnO nanosheet. Enzyme kinetics study presents alteration in Michaelis–Menten constant (Km) and maximum velocity (Vmax), the α-amylase bound with porous ZnO nanosheet showed a reduction in Km and Vmax. The substrate and enzyme adsorbed together on porous ZnO nanosheet exhibited increased Km (27.77 μM), whereas Vmax (2.85 μM) remains unchanged. Moreover, α-amylase once modified at optimum pH (5.8) and temperature (52 °C), produces less maltose than α-amylase adsorbed on ZnO nanosheet, which indicates higher maltose production. In this study, ZnO nanosheet enzyme catalytic system was created, wherein enzymatic reaction shifted to different pH and temperature other than optimum conditions. All these findings suggest that careful attention to the enzyme adsorption profiles can contribute to industrial applications.

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

BackgroundConventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass.ResultsIn this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation.ConclusionsEnzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.