Cellulase Binding Research Articles

The role of glycosylation in non-productive adsorption of cellulase to lignin isolated from pretreated corn stover

Glycosylation, a general post-translational modification for fungal cellulase, has been shown to affect cellulase binding to its substrate. However, the exact impact of glycosylation on cellulase-lignin interaction remain unclear. Here, we demonstrated that the lignin isolated from tetrahydrofuran-pretreated corn stover exhibits strong adsorption capability to cellulase due to its negatively charged and porous structure. For the cellulases with varying glycosylation levels, the less-glycosylated protein showed high adsorption capability to lignin, and that trend was observed for the main cellulase components secreted by Penicillium oxilicum, including endoglucanase PoCel5B, cellobiohydrolase PoCel7A-2, and β-glucosidase PoBgl1. Additionally, N-glycan sites and motifs were examined using mass spectrometry, and protein structures with N-glycans were constructed, where PoBgl1 and PoCel7A-2 contained 13 and 1 glycosylated sites respectively. The results of molecular dynamics simulations indicated that the N-glycans impacted on the solvent-accessible surface area and secondary structure of protein, and the binding conformation of lignin fragment on cellulase, resulting in a decrease in binding energy (14 kcal/mol for PoBgl1 and 13 kcal/mol for PoCel7A-2), particularly for van der Waals and electrostatic interaction. Those findings suggested that glycosylation negatively impacted the lignin-cellulase interaction, providing a theoretical basis for the rational engineering of enzymes to reduce lignin-enzyme interaction.

The Limitation of Unproductive Binding of Cellulases to Lignin by Ozone Pretreatment

The limitation of enzymatic saccharification of lignocellulose is attributed to the nonproductive adsorption between lignin and cellulase. This study aims to investigate the effects of ozone pretreatment on the physical structure and chemical properties of milled wood lignin (MWL). The objective is to reduce the non-productive adsorption of cellulase on lignin. The structure–activity relationship between the physical structure of MWL and the occurrence of nonproductive adsorption was analysed using two-dimensional heteronuclear single quantum coherence–nuclear magnetic resonance (HSQC-NMR) and phosphorus-31 nuclear magnetic resonance spectrum (31P-NMR), etc. The results indicate that ozone pretreatment resulted in a decrease in the phenolic hydroxyl content and S/G ratio, an increase in the carboxyl content, and a negative zeta potential of MWL. The maximum adsorption capacity decreased from 25.77 mg/g to 10.09 mg/g, the Langmuir constant decreased from 13.86 mL/mg to 10.11 mL/mg, and the binding strength decreased from 357.14 mL/g to 102.04 mL/g, as determined by Langmuir isothermal adsorption. This suggests that ozone pretreatment resulted in a reduction in the hydrophobicity of lignin and a weakening of the electrostatic attraction between lignin and cellulase, thereby effectively reducing the non-productive adsorption of cellulase on lignin. This study provides an environmentally friendly pretreatment technique and comprehensively analyses the structural changes of ozone-treated MWL. These findings contribute to a deeper understanding of the interaction between lignin and cellulase.

Fungal cellulolytic enzyme complex immobilized on chitosan-functionalised magnetic nanoparticles for paddy straw saccharification

Utilization of biopolymeric functionalized nanocomposites for enzyme immobilization has been actively explored for their potential role as catalysts in sustainable agro-residue valorization models. In the present study, cellulase immobilization on control and chitosan functionalized magnetic nanoparticles (c-MNP & Ch-MNP, respectively) was investigated. A cellulase binding efficiency of 67.50% on c-MNP and 74.06% on Ch-MNP was achieved. The enzyme immobilization on nano-supports was confirmed using electron microscopic and spectroscopic techniques. The immobilized cellulase (E-Ch-MNP) exhibited carboxymethyl cellulase, cellobiase and avicelase specific activities of 48.68, 25.98 and 33.80 nmol min−1 mg−1 protein. The optimum pH for free and immobilized enzyme were reported to be pH 5 and 6, respectively. An increased thermal stability for E-Ch-MNP was observed over a broader temperature range with a peak relative enzyme activity at 60℃. The binding constant (Km= 5.00 mg ml−1) and maximal rate of reaction (Vmax= 0.12 µmol min−1 ml−1) were also determined for E-Ch-MNP. Molecular docking studies determined the flexibilities of both the receptor cellulase protein and the ligand immobilization support molecules with respect to the binding energy and inhibition constants, reflecting their desired conformation and stability. Maximum reducing sugar content of 242.14 mg g−1 of pre-treated straw was reported at 96 h of incubation for E-Ch-MNP with a saccharification efficiency of 35.78%. The retained relative activity of immobilized cellulase was 82.83, 75.17, 44.61 and 27.95% of initial activity after second, third, fourth and fifth cycle, respectively. Therefore, immobilization of cellulase on chitosan coated magnetic nanoparticles holds promise for further optimization for improved cellulose hydrolysis.

Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution

Efficient cellulose degradation by cellulase enzymes is crucial for using lignocellulosic biomass in bioenergy production. Single-molecule microscopy showed that xylan hinders the efficiency of cellulase by inhibiting its binding to cellulose and impeding the processivity of bound enzyme molecules.

In-House Extracted Soybean Protein Can Reduce the Enzyme Dosage in Biomass Saccharification

Bioconversion of the complex carbohydrates present in lignocellulosic biomass into simple sugars, in order to obtain biofuels and bio-based products, is still limited by the low performance of the enzymatic saccharification reaction and the high cost of cellulolytic enzymes. Low-cost additives such as soybean protein can reduce the unproductive adsorption of cellulases onto lignin, increasing conversion rates and reducing enzyme losses. Here, investigation was made of the effects of different soybean protein fractions, extracted in-house, on the enzymatic saccharification of hydrothermally pretreated sugarcane bagasse. The glucose released during biomass saccharification increased by up to 76% in the presence of the in-house extracted soybean protein, compared to the control (without additive). A remarkable finding was that the technique allowed the enzyme loading to be decreased four-fold. The results suggested that the alkali-extracted proteins presented high surface hydrophobicity, which enhanced their interaction with lignin and reduced the unproductive binding of cellulases. Among the main soybean protein fractions, glycinin had the best effect in improving saccharification, which could have been due to its higher hydrophobicity. Hence, in-house extracted soybean proteins seem to be interesting alternative additives capable of increasing the lignocellulosic biomass conversion efficiency in future biorefineries.

Anti-Cancer and Anti-Inflammatory Activities of a Short Molecule, PS14 Derived from the Virulent Cellulose Binding Domain of Aphanomyces invadans, on Human Laryngeal Epithelial Cells and an In Vivo Zebrafish Embryo Model.

In this study, the anti-cancer and anti-inflammatory activities of PS14, a short peptide derived from the cellulase binding domain of pathogenic fungus, Aphanomyces invadans, have been evaluated, in vitro and in vivo. Bioinformatics analysis of PS14 revealed the physicochemical properties and the web-based predictions, which indicate that PS14 is non-toxic, and it has the potential to elicit anti-cancer and anti-inflammatory activities. These in silico results were experimentally validated through in vitro (L6 or Hep-2 cells) and in vivo (zebrafish embryo or larvae) models. Experimental results showed that PS14 is non-toxic in L6 cells and the zebrafish embryo, and it elicits an antitumor effect Hep-2 cells and zebrafish embryos. Anticancer activity assays, in terms of MTT, trypan blue and LDH assays, showed a dose-dependent inhibitory effect on cell proliferation. Moreover, in the epithelial cancer cells and zebrafish embryos, the peptide challenge (i) caused significant changes in the cytomorphology and induced apoptosis; (ii) triggered ROS generation; and (iii) showed a significant up-regulation of anti-cancer genes including BAX, Caspase 3, Caspase 9 and down-regulation of Bcl-2, in vitro. The anti-inflammatory activity of PS14 was observed in the cell-free in vitro assays for the inhibition of proteinase and lipoxygenase, and heat-induced hemolysis and hypotonicity-induced hemolysis. Together, this study has identified that PS14 has anti-cancer and anti-inflammatory activities, while being non-toxic, in vitro and in vivo. Future experiments can focus on the clinical or pharmacodynamics aspects of PS14.

Combinatorial pretreatments of reed straw using liquid hot water and lactic acid for fermentable sugar production

A green combinatorial pretreatment using liquid hot water (LHW) and lactic acid (LA) was used to treat reed straw for effective lignocellulose digestibility and xylose recovery. The reeds were first treated with LHW at 180 °C for 60 min, followed by LA treatment at 170 °C for 45 min. Using 37.5 g/L lactic acid, the lignocellulose digestibility was 80.6 % and glucose yield was 56.0 %, which was much higher than that for one-step LHW pretreatment (the lignocellulose digestibility was 27.3 % and the glucose yield was 26.3 %). These data demonstrated that LHW-LA pretreatment increased lignocellulose digestibility and glucose yield by 1.95-fold and 1.13-fold, respectively. The highest total sugar yielded 39.2 g/100 g-substrate. The results revealed that LHW-LA pretreatment removed most hemicellulose and partial lignin from the reed straw and prompted cellulase binding to reed cellulose, which enhanced lignocellulose enzymatic saccharification. The high fermentable glucose yielded from the combinatorial pretreatment as presented in this study highlighted the great potential of our pretreatment technology to be applied in the biorefinery, which could lay down the cost and sustainability of current biorefinery industry.

Process strategies to reduce cellulase enzyme loading for renewable sugar production in biorefineries

Second-generation (2G) sugar is the principal building block in biorefineries producing sustainable fuels, green chemicals, and materials. Its production involves a pre-treatment strategy, which can remove either hemicellulose or lignin, followed by a cellulase cocktail-mediated hydrolysis of pre-treated biomass applying numerous modifications. The high production cost of sugar from biomass using high-solid loadings with the desired purity is the important limiting factor for their economic conversion into bio-renewables. Pre-treatment and cellulase-mediated enzymatic hydrolysis of recalcitrant biomass are among the major technical and economic impediments to the overall success of biorefineries. Critical parameters such as the high amount of cellulase required for biomass hydrolysis, high cellulase cost, unproductive binding of cellulase to lignin, inhibitive reactions, and lower substrate applicability directly influence the recovery of sugar at a competitive price. Understanding the interaction of the relationship of the enzymes and substrates with the interactions of the reducing cellulase amount and effective hydrolysis rate should be positioned for the recovery of 2G sugar at a competitive price. Thus, effective strategies are needed to minimize cellulase dosage to overcome the cost of the enzyme. Further, on-site enzyme production, the addition of pre-treated biomass in fed-batch mode, and the washing of pre-treated biomass can play a pivotal role in the economic production of 2G sugar. This review provides a comprehensive analysis of the specific strategies such as applying extraneous proteins and additives, enzyme recycling, and effect of washing pre-treated biomass on 2G sugar production at a large scales in lignocellulose biorefineries.

Exploring the promoting mechanisms of bovine serum albumin, lignosulfonate, and polyethylene glycol for lignocellulose saccharification from perspective of molecular interactions with cellulase

Bovine serum albumin (BSA), polyethylene glycol (PEG) and lignosulfonate (LS) have been extensively employed as synergistic agents in lignocellulose saccharification. Nevertheless, the promoting mechanisms have not been fully understood and there are a number of controversial opinions existed. All attention has been paid to the interactions between respective additive and substrate. However, rarely attention has been paid to the interactions between additives and enzymes (cellulase from Trichoderma reesei in this investigation). This interaction is actually more important since cellulase interacts with the additives before it contacts with substrate. Therein, Quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasma resonance (SPR) and small angel X-ray scattering (SAXS) were incorporated to study the interaction between enzyme and additives. The results showed synergistic agents have different interaction modes with cellulase. BSA and LS can form complexes with cellulase and the formed complexes prevent them from nonproductive binding by residue lignin; what’s more, the cellulase-BSA complexes improve the hydrolytic capability of pristine enzyme whereas cellulase-LS complexes reduce. PEG prevents the unproductive binding of cellulase to the residual lignin by forming a thin layer that actually acts as a steric hindrance to the residual lignin. This investigation helps us to understand the sophisticated interactions among the components in the complicated enzymatic system, especially the interactions between enzymes and synergistic agents. It will be helpful in the design and utilization of synergistic additives in the lignocellulose biorefinery process as well.

Alleviating Nonproductive Adsorption of Lignin on CBM through the Addition of Cationic Additives for Lignocellulosic Hydrolysis.

The nonproductive adsorption of cellulase onto lignin significantly inhibited the enzymatic hydrolysis of lignocellulosic biomass. In this study, we constructed a rapid fluorescence detection (RFD) system, and using this system, we demonstrated that the addition of cationic additives DTAB or polyDADMAC greatly increased the partition coefficients of cellulose/lignin, reduced nonproductive adsorption, and enhanced the hydrolysis efficiency of lignocellulose compared to those of Tweens or PEGs. Moreover, the addition of polyDADMAC and DTAB increased the glucose yield released from the mixture of Avicel and AICS-lignin (MCL) by 16.9 and 20.6%, respectively, and reduced the inhibition rate of lignin by 16.9 and 20.7%, respectively. Interestingly, polyDADMAC or DTAB treatment performed more effectively for the enzymatic hydrolysis of pretreated lignocellulosic biomass, compared with MCL. We confirmed that the reduced hydrophobicity and increased zeta potential of lignin cocontribute to the dampening nonproductive adsorption of lignin. In particular, the zeta potential values of lignin and the partition coefficients of Avicel/lignin with the addition of additives showed a good correlation, suggesting that electrostatic force also plays a crucial role in the adsorbing of cellulase on lignin. This work will be conducive to decreasing the nonproductive binding of cellulase onto lignin and enhancing cellulose conversion.

Structural Changes of Alkali Lignin under Ozone Treatment and Effect of Ozone-Oxidized Alkali Lignin on Cellulose Digestibility

In this study, the structural changes of alkali lignin induced by ozonation were investigated, and the effect of ozone-treated alkali lignin and its mechanism on Avicel enzymatic hydrolysis was examined. The physicochemical properties of alkali lignin were analyzed by FTIR, 1H-13C HSQC NMR, and GPC. It was revealed that ozone pretreatment increased the content of carboxyl and/or aldehyde groups and the negative zeta potential of alkali lignin, which enhanced the electrostatic repulsion between alkali lignin and cellulase; The S/G ratio was reduced, indicating the hydrophobic interaction was diminished. The Langmuir adsorption isotherm showed that the cellulase binding strength of ozone pretreated alkali lignin (OL-pH3, OL-pH7, and OL-pH12 were 16.67, 13.87, and 44.05 mL/g, respectively) was significantly lower than that of alkali lignin (161.29 mL/g). The 72 h hydrolysis yields of Avicel added with OL-pH3, OL-pH7, and OL-pH12 were 55.4%, 58.6%, and 54.9% respectively, which were 2.6–6.3% higher than that of Avicel added with AL (52.3%). This research aimed to reduce the non-productive adsorption between cellulase and lignin by investigating the structural changes of lignin caused by ozone treatment. For the first time, we discovered that ozone-treated alkali lignin has a further promotion effect on the enzymatic digestion of cellulose, providing a green and feasible pretreatment process for the enzymatic hydrolysis of lignocellulose and aiding in the more efficient utilization of biomass.

Bioinspired Cellulase-Mimetic Solid Acid Catalysts for Cellulose Hydrolysis

Glucose produced by catalytic hydrolysis of cellulose is an important platform molecule for producing a variety of potential biobased fuels and chemicals. Catalysts such as mineral acids and enzymes have been intensively studied for cellulose hydrolysis. However, mineral acids show serious limitations concerning equipment corrosion, wastewater treatment and recyclability while enzymes have the issues such as high cost and thermal stability. Alternatively, solid acid catalysts are receiving increasing attention due to their high potential to overcome the limitations caused by conventional mineral acid catalysts but the slow mass transfer between the solid acid catalysts and cellulose as well as the absence of ideal binding sites on the surface of the solid acid catalysts are the key barriers to efficient cellulose hydrolysis. To bridge the gap, bio-inspired or bio-mimetic solid acid catalysts bearing both catalytic and binding sites are considered futuristic materials that possess added advantages over conventional solid catalysts, given their better substrate adsorption, high-temperature stability and easy recyclability. In this review, cellulase-mimetic solid acid catalysts featuring intrinsic structural characteristics such as binding and catalytic domains of cellulase are reviewed. The mechanism of cellulase-catalyzed cellulose hydrolysis, design of cellulase-mimetic catalysts, and the issues related to these cellulase-mimetic catalysts are critically discussed. Some potential research directions for designing more efficient catalysts for cellulose hydrolysis are proposed. We expect that this review can provide insights into the design and preparation of efficient bioinspired cellulase-mimetic catalysts for cellulose hydrolysis.

Direct Evidence for Aligned Binding of Cellulase Enzymes to Cellulose Surfaces.

The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers have been designing new cellulase enzymes for the breakdown of otherwise insoluble cellulose materials. A barrier to the rational design of new enzymes has been our lack of a molecular picture of how cellulase binding occurs. A critical factor is the attachment via the enzyme's carbohydrate binding module (CBM). To elucidate the structural and mechanistic details of cellulase adsorption, we have combined experimental data from sum frequency generation spectroscopy with molecular dynamics simulations to probe the equilibrium structure and surface alignment of a 14-residue peptide mimicking the CBM. The data show that binding is driven by hydrogen bonding and that tyrosine side chains within the CBM align the cellulase with the registry of the cellulose surface. Such an alignment is favorable for the translocation and effective cellulose breakdown and is therefore likely an important parameter for the design of novel enzymes.

A sustainable biorefinery strategy: Conversion and fractionation in a facile biphasic system towards integrated lignocellulose valorizations

A sustainable and integrated biorefinery strategy was achieved by a facile biphasic system, ferric trichloride (FeCl3) solution assisted by methyl isobutyl ketone (MIBK), yielding multiple products: furfural, sugars, and lignin nano-particles simultaneously. For the highest yield of furfural, the conditions were optimized resulting in 75.55% and over 69.38% of the yields from xylose and some typical biomasses respectively. Both the catalyst and MIBK can be recycled. Eucalyptus was lucubrated on the valorizations of three major-components with 75.18% of furfural yield, 97.54% of cellulase digestibility from the residue and lignin nano-particles. Meanwhile, the regularities of biomass degradation under this biphasic system were summarized as well as high digestibility which was resulted by hemicellulose and lignin removal in cooperation with the amount of the cellulase binding sites. Based on the proposed biorefinery strategy, 65.82% of the initial mass can be valorized into high-value end-products.

Maleic acid hydrotropic fractionation of wheat straw to facilitate value‐added multi‐product biorefinery at atmospheric pressure

AbstractWheat straw was rapidly fractionated using maleic acid (MA) as an acid hydrotrope at atmospheric pressure under a range of conditions. MA hydrotropic fractionation (MAHF) was very selective in dissolving lignin and hemicelluloses resulting in a cellulose‐rich water insoluble solids (WIS), which was evaluated for producing fibers through bleaching and cellulosic nanofibrils by fibrillation, as well as glucose through enzymatic saccharification. The residual lignin (RL) within the WIS has a low degree of condensation, which eased bleaching. The RL was also carboxylated through MAHF, which facilitated nanofibrillation to produce lignin‐containing cellulose nanofibrils. The carboxylation also aided enzymatic saccharification of WIS by decreasing nonproductive cellulase binding to RL through electrostatic repulsion between carboxylated (charged) RL and the cellulase at elevated pH (>cellulase isoelectric point). The dissolved lignin (DL) from MAHF also has a low degree of condensation, making it favorable for further catalytic conversion. Carboxylation improved DL antioxidant capability. MA is an FDA‐approved indirect food additive (21CFR175‐177) and is much less corrosive than other acids typically used for cellulosic fractionation. It is also less soluble which is a distinct and very promising advantage for acid recovery. Therefore, MAHF has notable advantages and significant potential for sustainable biorefinery.

ADDITIVES FOR CELLULASE ENHANCEMENT

Cellulases have been vital for the saccharification of lignocellulosic biomass into reduced sugars to produce biofuels and other essential biochemicals. However, the sugar yields achievable for canonical cellulases (i.e. endoglucanases, exoglucanases and β-glucosidases) have not been convincing in support of the highly acclaimed prospects and end-uses heralded. The persistent pursuit of the biochemical industry to obtain high quantities of useful chemicals from lignocellulosic biomass has resulted in the supplementation of cellulose-degrading enzymes with other biologicalcomplementation. Also, chemical additives (e.g. salts, surfactants and chelating agents) have been employed to enhance the stability and improve the binding and overall functionality of cellulases to increase product titre. Herein, we report the roadmap of cellulase-additive supplementations and the associated yield performances.

Comparison of Two Acid Hydrotropes for Sustainable Fractionation of Birch Wood.

This study reports on a comparative study of acid hydrotropic fractionation (AHF) of birch wood using maleic acid (MA) and p-toluenesulfonic acid (p-TsOH). Under the same level of delignification, lignin dissolved by MA is much less condensed with a higher content of ether aryl β-O-4 linkages. Lignin depolymerization dominated in MA hydrotropic fractionation (MAHF) and resulted in a single lower molecular weight peak, in contrast to the competitive depolymerization and repolymerization in p-TsOH AHF with a bimodal distribution. The less condensed MA-dissolved lignin facilitated catalytic conversion to monophenols. Carboxylation of residual lignin in fractionated cellulosic water-insoluble solids (WISs) enhanced enzymatic saccharification by decreasing nonproductive cellulase binding to lignin. At a low cellulase loading of 10 FPU g-1 glucan, saccharification of WIS-MT120 from MAHF at 120 °C was 95 % compared with 48 % for WIS-PT85 from p-TsOH AHF at 85 °C under the same level of delignification of 63 %. Residual lignin carboxylation also facilitated nanofibrillation of WIS for producing lignin-containing cellulose nanofibrils (LCNFs) through an enhanced lignin lubrication effect, which substantially decreases fibrillation energy. LCNFs from only one pass of microfluidization of WIS-MT120 have the same morphology as those from WIS-PT85 after three passes. MA also has a lower solubility and higher minimal hydrotropic concentration, which facilitated acid recovery. MA is U.S. Food and Drug Administration (FDA)-approved as an indirect food additive, affording significant advantages compared with p-TsOH for biorefinery applications.

Enzymatic sugar production from elephant grass and reed straw through pretreatments and hydrolysis with addition of thioredoxin-His-S

BackgroundThe bioconversion of lignocellulose to fermentable C5/C6-saccharides is composed of pretreatment and enzymatic hydrolysis. Lignin, as one of the main components, resists lignocellulose to be bio-digested. Alkali and organosolv treatments were reported to be able to delignify feedstocks and loose lignocellulose structure. In addition, the use of additives was an alternative way to block lignin and reduce the binding of cellulases to lignin during hydrolysis. However, the relatively high cost of these additives limits their commercial application.ResultsThis study explored the feasibility of using elephant grass (Pennisetum purpureum) and reed straw (Phragmites australis), both of which are important fibrous plants with high biomass, no-occupation of cultivated land, and soil phytoremediation, as feedstocks for bio-saccharification. Compared with typical agricultural residues, elephant grass and reed straw contained high contents of cellulose and hemicellulose. However, lignin droplets on the surface of elephant grass and the high lignin content in reed straw limited their hydrolysis performances. High hydrolysis yield was obtained for reed straw after organosolv and alkali pretreatments via increasing cellulose content and removing lignin. However, the hydrolysis of elephant grass was only enhanced by organosolv pretreatment. Further study showed that the addition of bovine serum albumin (BSA) or thioredoxin with His- and S-Tags (Trx-His-S) improved the hydrolysis of alkali-pretreated elephant grass. In particular, Trx-His-S was first used as an additive in lignocellulose saccharification. Its structural and catalytic properties were supposed to be beneficial for enzymatic hydrolysis.ConclusionsElephant grass and reed straw could be used as feedstocks for bioconversion. Organosolv and alkali pretreatments improved their enzymatic sugar production; however, the increase in hydrolysis yield of pretreated elephant grass was not as effective as that of reed straw. During the hydrolysis of alkali-pretreated elephant grass, Trx-His-S performed well as additive, and its structural and catalytic capability was beneficial for enzymatic hydrolysis.

Enhanced Catalytic Performance of Trichoderma reesei Cellulase Immobilized on Magnetic Hierarchical Porous Carbon Nanoparticles.

Cellulase from Trichoderma reesei was immobilized by covalent or non-covalent binding onto magnetic hierarchical porous carbon (MHPC) nanomaterials. The immobilization yield and the enzyme activity were higher when covalent immobilization approach was followed. The covalent immobilization approach leads to higher immobilization yield (up to 96%) and enzyme activity (up to 1.35Umg-1) compared to the non-covalent cellulase binding. The overall results showed that the thermal, storage and operational stability of the immobilized cellulase was considerably improved compared to the free enzyme. The immobilized cellulose catalyzed the hydrolysis of microcrystalline cellulose up to 6 consecutive successive reaction cycles, with a total operation time of 144h at 50°C. The half-life time of the immobilized enzyme in deep eutectic solvents-based media was up to threefold higher compared to the soluble enzyme. The increased pH and temperature tolerance of the immobilized cellulase, as well as the increased operational stability in aqueous and deep eutectic solvents-based media indicate that the use of MHPCs as immobilization nanosupport could expand the catalytic performance of cellulolytic enzymes in various reaction conditions.

Synergy of hemicelluloses removal and bovine serum albumin blocking of lignin for enhanced enzymatic hydrolysis

A cost efficient synergistic strategy combining mild alkaline pretreatment (0.5–5% NaOH at 70 °C for 60 min) and bovine serum albumin (BSA) blocking of lignin was evaluated for effective conversion of poplar. The highest glucose yield of 69.2% was obtained for 5% alkaline pretreated sample, which was 4.4 times that of untreated sample. The enhanced enzymatic hydrolysis was attributed to significant hemicelluloses removal with limited delignification. Delignification mainly occurred in secondary wall, leading to more open cell wall structure, thus facilitating better transport of enzyme. Hemicelluloses removal helped split adjacent microfibrils, thus increased the specific sites for cellulase binding. After BSA addition in enzymatic hydrolysis, cellulose conversion further improved to 78.4% with 33% reduction of cellulase dosage due to decreased non-specific adsorption of cellulase on residual lignin. The utilization of synergistic alkaline pretreatment – BSA strategy may improve the overall economics of biomass conversion and successful commercial implementation of biorefineries.