Non-productive Enzyme Adsorption Research Articles

Unveiling the role of long-range and short-range forces in the non-productive adsorption between lignin and cellulases at different temperatures

Quantitatively understanding of interaction mechanism between lignin and cellulases is essential for the efficient improvement of lignocellulose enzymatic hydrolysis. However, the individual contribution of multiple forces between lignin and cellulases to the non-productive adsorption of enzymes still remains deeply ambiguous, especially in situations of near enzymatic hydrolysis temperatures. Herein, atomic force microscopy (AFM) and computational simulations were utilized to quantitatively analyze the intermolecular forces between lignin and enzyme at 25 °C and 40 °C. Our results unveiled that an increase in temperature obviously improved adsorption capacity and total intermolecular forces between lignin and cellulases. This positive relationship mainly comes from the increase in the decay length of hydrophobic forces for lignin-cellulases when temperature increases. Different from the hydrophobic interaction which provides long-range part of attractions, van der Waals forces dominate the intermolecular force only at approaches < 2 nm. On the other hand, electrostatic forces exhibited repulsive effects, and its intensity and distance were limited due to the low surface potential of cellulases. Short-range forces including hydrogen bonding (main) and π-π stacking (minor) stabilize the non-specific binding of enzymes to lignin, but increasing temperature reduces hydrogen bond number. Therefore, the relative contribution of long-range forces increased markedly at higher temperatures, which benefits protein capture and brings lignin and cellulase close together. Finally, the structure–activity relationships between lignin physicochemical properties and its inhibitory effect to enzymes indicated that hydrophobic interactions, hydrogen bonding, and steric effects drive the final adsorption capacity and glucose yields. This work provides quantitative and basic insights into the mechanism of lignin-cellulase interfacial interactions and guides design of saccharification enhancement approaches.

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.

The pre-addition of “blocking” proteins decreases subsequent cellulase adsorption to lignin and enhances cellulose hydrolysis

The pre-adsorption of non-catalytic/blocking proteins onto the lignin component of pretreated biomass has been shown to significantly increase the effectiveness of subsequent enzyme-mediated hydrolysis of the cellulose by limiting non-productive enzyme adsorption. Layer-by-layer adsorption of non-catalytic proteins and enzymes onto lignin was monitored using Quartz Crystal Micro balancing combined with Dissipation monitoring (QCM-D) and conventional protein adsorption. These methods were used to assess the interaction between soft/hardwood lignins, cellulases and the three non-catalytic proteins BSA, lysozyme and ovalbumin. The QCM-D analysis showed higher adsorption rates for all of the non-catalytic proteins onto the lignin films as compared to cellulases. This suggested that the “blocking” proteins would preferentially adsorb to the lignin rather than the enzymes. Pre-incubation of the lignin films with blocking proteins resulted in reduced adsorption of cellulases onto the lignin, significantly enhancing cellulose hydrolysis.

Rational feeding strategies of substrate and enzymes to enzymatic hydrolysis bioreactors

Bioreactors operating in fed-batch mode improve the enzymatic hydrolysis productivity at high biomass loadings. The present work aimed to apply rational feeding strategies of substrates (pretreated sugarcane straw) and enzymes (CellicCtec2?) to achieve sugar titers at industrial levels. The instantaneous substrate concentration was kept constant at 5% (w/v) along the fed-batch. The enzyme dosage inside the bioreactor was adjusted so that the reaction rate was not less than a pre-defined value (a percentage of the initial reaction rate ? rmin). When r reached values below rmin, enzyme pulses were applied to return the reaction rate to its initial value (r0). The optimized feeding policy indicated a reaction rate maintained at a minimum of 70% of r0, based on the trade-off between glucose productivity and enzyme saving. Initially, it was possible to process a 21% (w/v) solid load, achieving 160 g/L of glucose concentration and 80% of glucose yield. It was verified that non-productive enzyme adsorption was the main reason for some reduction of hydrolysis yield regarding the theoretical cellulose-to-glucose conversion. An increment of 30 g/L in the final glucose concentration was achieved when a lignin-blocking additive (soybean protein) was used in the enzymatic hydrolysis.

Synergistic Treatment of Alkali Lignin via Fungal Coculture for Biofuel Production: Comparison of Physicochemical Properties and Adsorption of Enzymes Used As Catalysts

White-rot fungi (WRF) is capable of producing extracellular enzymes that degrade lignin structure and facilitate biofuel production from lignocellulosic biomass wastes. However, fungal monocultures are constrained by low activities of the lignin-degrading enzyme system, leading to poor treatment efficiency and a long duration, which are not advantageous for large-scale applications. To improve enzyme production and enhance lignin degradation, a novel coculture system was proposed using the white-rot fungi Phanerochaete chrysosporium and Irpex lacteus CD2. The degradation efficiency of the alkali lignin by the fungal coculture was 26.4%, which was higher than that of the fungal monocultures. It was due to the production of lignin degrading enzymes was promoted in the liquid medium. Scanning electron microscopy (SEM), fourier transform infrared (FTIR), thermogravimetric (TG) and mercury porosimeter analyses results revealed that the alkali lignin treated with the fungal coculture had the largest porosity, and the degree of destruction of the alkali lignin structure by the fungal coculture was higher than that of the fungal monocultures. Meanwhile, the nonproductive adsorption of enzymes on alkali lignin was significantly reduced by 61.0% when the biomass was treated with the fungal coculture. As a result, the nonproductive adsorption was remarkably reduced, while it significantly improved the cellulase catalysis efficiency. These results demonstrated the synergistic effects of the fungal coculture for biomass treatment and provided a new approach for increasing lignin degradation while improving enzymatic catalysis and biofuel production through fungal coculture.

Effect of stepwise lignin removal on the enzymatic hydrolysis and cellulase adsorption

Lignin can negatively impose on the enzymatic hydrolysis of pretreated lignocellulose through non-productive enzyme adsorption and/or steric hindrance. The effect of the residual lignin on enzymatic hydrolysis was evaluated with sugarcane bagasse (SCB) samples containing different lignin contents and their milled and enzymolytic lignins (MELs) retaining the intact protolignins. The results showed that the improvement of enzymatic hydrolysis of SCB would become insignificant when the lignin removal achieved above 64.0%, and the changes in chemical groups of lignin had slight impact on adsorbing enzymes. When the lignin retention in the pretreated SCB was less than 36.0%, the residual lignin would not significantly influence the enzymatic hydrolysis via enzyme adsorption and steric hindrance. The contributions of the residual lignins in pretreated SCB samples to the total enzyme adsorptions of pretreated SCB samples were only in the range of 9.6% to 15.5%, which were not predominant for the total enzyme adsorptions.

Lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier rather than by inducing nonproductive adsorption of enzymes

BackgroundLignin is known to hinder efficient enzymatic conversion of lignocellulose in biorefining processes. In particular, nonproductive adsorption of cellulases onto lignin is considered a key mechanism to explain how lignin retards enzymatic cellulose conversion in extended reactions.ResultsLignin-rich residues (LRRs) were prepared via extensive enzymatic cellulose degradation of corn stover (Zea mays subsp. mays L.), Miscanthus × giganteus stalks (MS) and wheat straw (Triticum aestivum L.) (WS) samples that each had been hydrothermally pretreated at three severity factors (log R0) of 3.65, 3.83 and 3.97. The LRRs had different residual carbohydrate levels—the highest in MS; the lowest in WS. The residual carbohydrate was not traceable at the surface of the LRRs particles by ATR-FTIR analysis. The chemical properties of the lignin in the LRRs varied across the three types of biomass, but monolignols composition was not affected by the severity factor. When pure cellulose was added to a mixture of LRRs and a commercial cellulolytic enzyme preparation, the rate and extent of glucose release were unaffected by the presence of LRRs regardless of biomass type and severity factor, despite adsorption of the enzymes to the LRRs. Since the surface of the LRRs particles were covered by lignin, the data suggest that the retardation of enzymatic cellulose degradation during extended reaction on lignocellulosic substrates is due to physical blockage of the access of enzymes to the cellulose caused by the gradual accumulation of lignin at the surface of the biomass particles rather than by nonproductive enzyme adsorption.ConclusionsThe study suggests that lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier blocking the access of enzymes to cellulose rather than by inducing retardation through nonproductive adsorption of enzymes.

Characteristics of Lignin Fractions from Dilute Acid Pretreated Switchgrass and Their Effect on Cellobiohydrolase from Trichoderma longibrachiatum

To investigate the interactions between acid pretreated grass lignin and cellobiohydrolase, three different lignin fractions were isolated from dilute acid pretreated switchgrass by (i) ethanol extraction, followed by (ii) dioxane/H2O extraction, and (iii) cellulase treatment, respectively. Structural properties of each lignin fraction were elucidated by GPC, 13C-NMR and 2D-HSQC NMR analyses. The adsorptions of cellobiohydrolase to the isolated lignin fractions were also studied by Langmuir adsorption isotherms. Ethanol-extractable lignin fraction, mainly composed of syringyl (S) and guaiacyl (G) units, had the lowest molecular weight, while dioxane/H2O-extractable lignin fraction had the lowest S/G ratio with higher content of p-coumaric acid (pCA) unit. The residual lignin fraction after enzymatic treatment had the highest S/G ratio without hydroxyphenyl (H) unit. Strong associations were found between lignin properties such as lignin composition and S/G ratio and its non-productive enzyme adsorption factors including the maximum adsorption capacity and binding strength.

Effect of residual lignins present in cholinium ionic liquid-pretreated rice straw on the enzymatic hydrolysis of cellulose

To provide deep insights into cholinium ionic liquid (IL) pretreatment effect on the subsequent enzymatic hydrolysis, residual lignins (RLs) present in rice straw pretreated by cholinium glycinate ([Ch][Gly]), acetate ([Ch][OAc]), and glycolate ([Ch][Glc]) were isolated and characterized in this work. The effects of RLs on the enzymatic hydrolysis of cellulose and cellobiose, and their non-productive adsorption to β-glucosidase and cellulase were explored. Of the three RLs examined, RL isolated from [Ch][Gly]-pretreated straw (Gly-RL) had the highest carboxylic acid content (1.27mmol/g) and the lowest phenolic hydroxyl content (0.72mmol/g), resulting in the weakest non-productive enzyme adsorption. It might account well for the highest enzymatic digestibility of cellulose in the presence of Gly-RL. However, the strongest enzyme adsorption was found in the case of Glc-RL with the lowest carboxylic acid content (0.14mmol/g) and the highest phenolic hydroxyl content (1.19mmol/g). The enzymatic hydrolysis of cellulose was significantly affected by RLs. The negative effect of RLs on the cellulose hydrolysis mainly stemmed from their inhibition to cellulase-mediated reactions, instead of inhibition to β-glucosidase-catalyzed cellobiose hydrolysis.

Understanding the Nonproductive Enzyme Adsorption and Physicochemical Properties of Residual Lignins in Moso Bamboo Pretreated with Sulfuric Acid and Kraft Pulping.

In this work, to elucidate why the acid-pretreated bamboo shows disappointingly low enzymatic digestibility comparing to the alkali-pretreated bamboo, residual lignins in acid-pretreated and kraft pulped bamboo were isolated and analyzed by adsorption isotherm to evaluate their extents of nonproductive enzyme adsorption. Meanwhile, physicochemical properties of the isolated lignins were analyzed and a relationship was established with non-productive adsorption. Results showed that the adsorption affinity and binding strength of cellulase on acid-pretreated bamboo lignin (MWLa) was significantly higher than that on residual lignin in pulped bamboo (MWLp). The maximum adsorption capacity of cellulase on MWLp was 129.49mg/g lignin, which was lower than that on MWLa (160.25mg/g lignin). When isolated lignins were added into the Avicel hydrolysis solution, the inhibitory effect on enzymatic hydrolysis efficiency of MWLa was found to be considerably stronger than that with MWLp. The cellulase adsorption on isolated lignins was correlated positively with hydrophobicity, phenolic hydroxyl group, and degree of condensation but negatively with surface charges and aliphatic hydroxyl group. These results suggest that the higher nonproductive cellulase adsorption and physicochemical properties of residual lignin in acid-pretreated bamboo may be responsible for its disappointingly low enzymatic digestibility.

Addition of metal ions to a (hemi)cellulolytic enzymatic cocktail produced in-house improves its activity, thermostability, and efficiency in the saccharification of pretreated sugarcane bagasse

High activity and stability are essential for (hemi)cellulolytic enzymes used in biomass conversion, while non-productive binding of cellulases to lignin reduces saccharification efficiency and needs to be avoided. One potential strategy is the addition of inexpensive metal ions. This paper describes the influence of divalent metal ions on the activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes produced in-house by Aspergillus niger under solid-state fermentation (SSF). The use of Mn(2+) provided the best (hemi)cellulolytic activity and stability, with an increase in endoglucanase activity of up to 57%. The use of Mn(2+) was then investigated in the saccharification of sugarcane bagasse submitted to acid, steam-explosion, and hydrothermal pretreatments. The addition of Mn(2+) ions at 10mM in the saccharification of acid-pretreated bagasse resulted in a 34% increase in glucose release. These positive effects appeared to be due to a reduction in non-productive enzyme adsorption. The findings suggest that the addition of inexpensive metal ions can help to improve activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes.

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

Non-productive enzyme adsorption onto lignin inhibits enzymatic hydrolysis of lignocellulosic biomass. Three cellobiohydrolases, Trichoderma reesei Cel7A (TrCel7A) and two engineered fusion enzymes, with distinctive modular structures and temperature stabilities were employed to study the effect of temperature on inhibition arising from non-productive cellulase adsorption. The fusion enzymes, TeCel7A-CBM1 and TeCel7A-CBM3, were composed of a thermostable Talaromyces emersonii Cel7A (TeCel7A) catalytic domain fused to a carbohydrate-binding module (CBM) either from family 1 or from family 3. With all studied enzymes, increase in temperature was found to increase the inhibitory effect of supplemented lignin in the enzymatic hydrolysis of microcrystalline cellulose. However, for the different enzymes, lignin-derived inhibition emerged at different temperatures. Low binding onto lignin and thermostable structure were characteristic for the most lignin-tolerant enzyme, TeCel7A-CBM1, whereas TrCel7A was most susceptible to lignin especially at elevated temperature (55 °C).

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.

Inhibitory effect of lignin during cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme adsorption

The effect of lignin as an inhibitory biopolymer for the enzymatic hydrolysis of lignocellulosic biomass was studied; specially addressing the role of lignin in non-productive enzyme adsorption. Botanical origin and biomass pre-treatment give rise to differences in lignin structure and the effect of these differences on enzyme binding and inhibition were elucidated. Lignin was isolated from steam explosion (SE) pre-treated and non-treated spruce and wheat straw and used for the preparation of ultrathin films for enzyme binding studies. Binding of Trichoderma reesei Cel7A (CBHI) and the corresponding Cel7A-core, lacking the linker and the cellulose-binding domain, to the lignin films was monitored using a quartz crystal microbalance (QCM). SE pre-treatment altered the lignin structure, leading to increased enzyme adsorption. Thus, the positive effect of SE pre-treatment, opening the cell wall matrix to make polysaccharides more accessible, may be compromised by the structural changes of lignin that increase non-productive enzyme adsorption.

Pretreatment of Extruded Corn Stover with Polyethylene Glycol to Enhance Enzymatic Hydrolysis: Optimization, Kinetics, and Mechanism of Action

Due to the high potential of the extrusion technique for pretreatment of lignocellulosic substrates, several attempts have been conducted in previous studies to further increase the subsequent sugar yield from extrusion pretreatment. Examples include application of chemicals along with extrusion, such as alkali-extrusion and ethylene glycol-extrusion, or before extrusion, such as hot water extraction. In this study, a new sequential technique has been developed for pretreatment of corn stover (CS), which utilizes an initial extrusion pretreatment (155 rpm screw speed and temperatures of 90°C, 180°C and 180°C corresponding to feed, barrel and die zones, respectively at a reaction time of 45–90 s) followed by pretreatment with polyethylene glycol 6,000 (PEG). In order to fully characterize the response for sugar yield over the range of surfactant treatment conditions assessed, response surface methodology was used. Treatment temperature, incubation time and PEG concentration were varied between 45–55°C, 1–4 h, 0.15–0.6 g PEG/g glucan, respectively. Statistical analysis was conducted by fitting the glucose and xylose yields to a quadratic polynomial model. PEG concentration and temperature were found to be the most significant factors in surfactant pretreatment. The optimum condition resulted in 25.4% and 10.3% increase in glucose and xylose yield, respectively. Using the combination of 10.8 FPU/g glucan of Ctec2 and 0.3 g PEG/g glucan, the glucose yield of extruded CS reached 98%. A yield was 64% resulted from application of similar amounts of Ctec and Htec. Decreased adsorption of enzyme to the lignocellulosic substrate as well as increased enzyme activity and reaction velocity indicated by kinetic parameter evaluation and nitrogen combustion analysis suggested an increased solubilization of cellulase in the presence of PEG. We propose that a non-productive adsorption of enzymes occur during hydrolysis not only due to lignin but also due to crystalline cellulose. Comparison of enzyme adsorptions and increase in sugar yields between Avicel and CS suggests the presence of other potential mechanisms of action for PEG in addition to increase of enzyme solubilization.

Eliminating inhibition of enzymatic hydrolysis by lignosulfonate in unwashed sulfite-pretreated aspen using metal salts

This study demonstrated the efficiency of Ca(II) and Mg(II) in removing inhibition of enzymatic hydrolysis by lignosulfonate through non-productive adsorption of enzymes. Adding 1 mmol/g cellulose of either metal salt restores approximately 65% of the activity lost when a pure cellulose/cellulase solution is spiked with lignosulfonate. Addition of either Ca(II) or Mg(II) is also effective in counteracting soluble inhibitors of cellulase present in unwashed aspen solid substrate produced by SPORL (sulfite pretreatment to overcome recalcitrance of lignocelluloses). Soluble inhibitors are often removed by thoroughly washing the lignocellulosic solid substrate following pretreatment. It was determined that adding 1 mmol of MgSO(4)/g substrate (oven dry) to the unwashed aspen substrate gave enzymatic substrate digestibility (SED) equivalent to that of washing for a range of enzyme loadings. These results demonstrate that applying divalent metal salts eliminates the need for washing, thereby saving considerable process water and cost for production of chemicals and biofuels from lignocellulose.

Effects of Lignin−Metal Complexation on Enzymatic Hydrolysis of Cellulose

This study investigated the inhibition of enzymatic hydrolysis by unbound lignin (soluble and insoluble) with or without the addition of metal compounds. Sulfonated, Organosolv, and Kraft lignin were added in aqueous enzyme-cellulose systems at different concentrations before hydrolysis. The measured substrate enzymatic digestibility (SED) of cellulose was decreased by 15% when SL was added to a concentration of 0.1 g/L due to nonproductive adsorption of enzymes onto lignin. Cu(II) and Fe(III) were found to inhibit enzymatic cellulose hydrolysis in the presence of lignin. Ca(II) and Mg(II) were found to reduce or eliminate nonproductive enzyme adsorption by the formation of lignin-metal complex. The addition of Ca(II) or Mg(II) to a concentration of 10 mM can almost completely eliminate the reduction in SED caused by the nonproductive enzyme adsorption onto the lignins studied (SL, OL, or KL at concentration of 0.1 g/L). Ca(II) was also found to reduce the inhibitive effect of bound lignin in pretreated wood substrate, suggesting that Ca(II) can also form complex with bound lignin on pretreated solid lignocelluloses. Significant improvement in SED of about over 27% of a eucalyptus substrate produced by sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) was achieved with the application of Ca(II).

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.