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.     

Mechanical deconstruction of lignocellulose cell walls and their enzymatic saccharification

Laboratory mechanical softwood pulps (MSP) and commercial bleached softwood kraft pulps (BSKP) were mechanically fibrillated by stone grinding with a SuperMassColloider^®. The extent of fibrillation was evaluated by SEM imaging, water retention value (WRV) and cellulase adsorption. Both lignin content and mechanical treatment significantly affected deconstruction and enzymatic saccharification of fibrillated MSP and BSKP. Fibrillation of MSP and BSKP cell walls occurs rapidly and then levels off; further fibrillation has only limited effect on cell wall breakdown as measured by water retention value and cellulase adsorption. Complete (100 %) saccharification can be achieved at cellulase loading of 5 FPU/g glucan for BSKP after only 15 min fibrillation with energy input of 0.69 MJ/kg. However, the presence of lignin in MSP affects the extent of fibrillation producing fibrils mainly above 1 μm. Lignin binds nonproductively to cellulases and blocks cellulose thereby reducing its accessibility. As a result, the cellulose saccharification efficiency of MSP fibrils (6 h of fibrillation, energy input of 13.33 MJ/kg) was only 55 % at same cellulase loading of 5 FPU/g glucan.     

A comparative study of different cellulase preparations in the enzymatic treatment of cotton fabrics

Twenty-nine cellulase preparations from different sources were compared interms of their abrasive activities (the ability to remove Indigo from denim) and their ability tosaccharify cellulose. Nodirectrelationship could be found between these two abilities. The preparations were divided into three groups: (1) with a high yield of reducing sugars after 24 h hydrolysis of Avicel cellulose but low abrasive activity; (2) universal cellulases that could both effectively hydrolyze cellulose and remove Indigo from denim; and (3) cellulase samples with high abrasive activity but low saccharification ability. Cellobiohydrolase (CBH) and xylanase were purified from different fungi by chromatofocusing on a Mono P column and subjected to limited proteolysis with papain yielding cellulose-binding and core (catalytic) domains. The adsorption ability and backstaining index of both CBH and xylanase core proteins were notably lower than the respective parameters for the in itial nondigested enzymes indicating that protein adsorption on the surface of cotton fibers is a crucial factor causing Indigo backstaining during the enzymatic denim washing procedure.     

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 129.49 mg/g lignin, which was lower than that on MWLa (160.25 mg/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.     

Mixtures of Thermostable Enzymes Show High Performance in Biomass Saccharification

Optimal enzyme mixtures of six Trichoderma reesei enzymes and five thermostable enzyme components were developed for the hydrolysis of hydrothermally pretreated wheat straw, alkaline oxidised sugar cane bagasse and steam-exploded bagasse by statistically designed experiments. Preliminary studies to narrow down the optimization parameters showed that a cellobiohydrolase/endoglucanase (CBH/EG) ratio of 4:1 or higher of thermostable enzymes gave the maximal CBH-EG synergy in the hydrolysis of hydrothermally pretreated wheat straw. The composition of optimal enzyme mixtures depended clearly on the substrate and on the enzyme system studied. The optimal enzyme mixture of thermostable enzymes was dominated by Cel7A and required a relatively high amount of xylanase, whereas with T. reesei enzymes, the high proportion of Cel7B appeared to provide the required xylanase activity. The main effect of the pretreatment method was that the required proportion of xylanase was higher and the proportion of Cel7A lower in the optimized mixture for hydrolysis of alkaline oxidised bagasse than steam-exploded bagasse. In prolonged hydrolyses, less Cel7A was generally required in the optimal mixture. Five-component mixtures of thermostable enzymes showed comparable hydrolysis yields to those of commercial enzyme mixtures.     

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20–25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including β-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.     

Effect of sodium hydroxide treatment of bacterial cellulose on cellulase activity

The synergistic action between Thermobifida fusca exocellulase Cel6B and endocellulase Cel5A on sodium hydroxide pretreated bacterial cellulose (BC) was determined. The activities of Cel6B and Cel5A were tested singly and both activities were dramatically increased on pretreated BC, especially in the early stage of hydrolysis. Cel5A, which attacks the cellulose chain randomly, showed a larger increase on NaOH treated BC than Cel6B. Mixtures of the two enzymes were also able to degrade NaOH treated BC faster than BC and the kinetics of the mixture differed from that of the individual enzymes. The degree of synergistic effect (DSE) on BC decreased dramatically with time of hydrolysis. However, the DSE on NaOH treated BC was almost constant throughout the incubation, with a smaller effect at higher NaOH concentrations. The change caused by NaOH did not increase the DSE, although each individual cellulase activity increased. This showed that synergistic activity was more effective on recalcitrant cellulose, which requires effective cooperation between the cellulase components for hydrolysis.     

Can delignification decrease cellulose digestibility in acid pretreated corn stover?

It has previously been shown that the improved digestibility of dilute acid pretreated corn stover is at least partially due to the removal of xylan and the consequent increase in accessibility of the cellulose to cellobiohydrolase enzymes. We now report on the impact that lignin removal has on the accessibility and digestibility of dilute acid pretreated corn stover. Samples of corn stover were subjected to dilute sulfuric acid pretreatment with and without simultaneous (partial) lignin removal. In addition, some samples were completely delignified after the pretreatment step using acidified sodium chlorite. The accessibility and digestibility of the samples were tested using a fluorescence-labeled cellobiohydrolase (Trichoderma reesei Cel7A) purified from a commercial cellulase preparation. Partial delignification of corn stover during dilute acid pretreatment was shown to improve cellulose digestibility by T. reesei Cel7A; however, decreasing the lignin content below 5% (g g⁻¹) by treatment with acidified sodium chlorite resulted in a dramatic reduction in cellulose digestibility. Importantly, this effect was found to be enhanced in samples with lower xylan contents suggesting that the near complete removal of xylan and lignin may cause aggregation of the cellulose microfibrils resulting in decreased cellulase accessibility.     

The effects of lignin removal and drying on the porosity and enzymatic hydrolysis of sugarcane bagasse

The porosity of lignocellulosic materials is a key feature for the enzymatic hydrolysis of the constituent polysaccharides, being affected by its drying and lignin content. Here we evaluated the influence of both parameters in the porosity of sugarcane bagasse, using raw and chlorite-delignified samples. A fraction of these samples was air dried at room temperature and the remainder one was kept wet. All the samples were subjected to porometry (solute exclusion technique), determination of water retention value and assessment of enzymatic saccharification of polysaccharides. Samples with higher lignin contents had lower porosities and exhibited worse enzymatic conversions of polysaccharides. Mild drying collapsed only the smaller pores, which already were inaccessible to enzymes, and therefore did not affect the efficiencies of saccharification. Our results show that the lignin content plays an important role in the porosity of the material and that its removal improves the enzymatic saccharification of the constituent polysaccharides.     

Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBH I (T.r. Cel 7A) in enantioseparations

Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.     

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.     

Response of Cellulase Activity in pH-Controlled Cultures of the Filamentous Fungus <Emphasis Type="Italic">Acremonium cellulolyticus</Emphasis>

Cellulase production was investigated in pH-controlled cultures of Acremonium cellulolyticus. The response to culture pH was investigated for three cellulolytic enzymes, carbomethyl cellulase (CMCase), avicelase, and β-glucosidase. Avicelase and β-glucosidase showed similar profiles, with maximum activity in cultures at pH 5.5–6. The CMCase activity was highest in a pH 4 culture. At an acidic pH, the ratios of CMCase and avicelase activity to cellulase activity defined by filter paper unit were high, but at a neutral pH, the β-glucosidase ratio was high. The pH 6.0 culture showed the highest cellulase activity within the range of pH 3.5–6.5 cultures. The saccharification activity from A. cellulolyticus was compared to those of the cellulolytic enzymes from other species. The A. cellulolyticus culture broth had a saccharification yield comparable to those of the Trichoderma enzymes GC220 and Cellulosin T2, under conditions with the same cellulase activity. The saccharification yields from Solka floc, Avicel, and waste paper, measured as the percent of released reducing sugar to dried substrate, were greater than 80% after 96 h of reaction. The yields were 16% from carboxymethylcellulose and 26% from wood chip refiner. Thus, the A. cellulolyticus enzymes were suitable for converting cellulolytic biomass to reducing sugars for biomass ethanol production. This study is a step toward the establishment of an efficient system to reutilize cellulolytic biomass.     

Evidence Showing Duplication and Recombination of cel Genes in Tandem from Hyperthermophilic Thermotoga sp.

This study was conducted to assess the gene duplication and diversification of tandem cellulase genes in thermophilic bacteria. The tandem cellulase genes cel5C and cel5D were cloned from Thermotoga maritima MSB8, and a survey of the thermophilic bacterial genome for tandem cel genes from the databases was carried out. A clone having 2.3?kb fragment from T. maritima MSB8 showed cellulase activity, which had two open reading frames in tandem (cel5C and cel5D). The cel5C gene has 954?bp, which encodes a protein of 317 amino acid residues with a signal peptide of 23 amino acids, and the other gene cel5D consisting of 990?bp encoding a protein of 329 amino acid residues. These two proteins have similarity with the enzymes of glycosyl hydrolase family 5. From the enzyme assay, it was observed that Cel5C was extracellular and Cel5D was intracellular cellulase. Phylogenetic and homology matrix analyses of DNA and protein sequences revealed that family 12 cellulase enzymes Cel12A and Cel12B displayed higher homology (>50?%), but Cel5C and Cel5D enzymes belong to family 5 displayed lower homology (<30?%). In addition, repeated and mirror sequences in tandem genes are supposed to show the existence of gene duplication and recombination.     

Quantitative ^31p NMR Analysis of Lignocellulosic Materials in Ionic Liquids

In an effort to utilize NMR technique for the analysis of the structural components of wood and biomasses such as lignin, we made a comparative study of lignin contents of several types of lignocellulosic materials both before and after enzymatic hydrolyses. To this end, Eucalyptus Globulus, Norway spruce thermomechanical pulp(TMP) corn stover and Eucalyptus kraft pulp were ball-milled and treated with cellulase to prepare materials with high lignin contents. These materials were analyzed via 31P NMR in ionic liquids. The results show that the contents of various functional groups for the lignocellulosic materials after enzymatic hydrolyses are generally in agreement with those from their corresponding enzymatic mild acidolysis liginins(EMALs), indicating that 31P NMR analysis of lignocellulosic materials in ionic liquids is a promising method for quantitative characterization of lignocellulosic materials.     

The Effect of Lignin Removal by Alkaline Peroxide Pretreatment on the Susceptibility of Corn Stover to Purified Cellulolytic and Xylanolytic Enzymes

Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.     

Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B,Cel9A,and Cel5A on BMCC and Avicel

In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonospora fusca E₃, E₄, and E₅), it was observed that binding capacities for these three cellulases were 18–30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate, accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding The processive endoglucanase, Cel9A, had four times the extent of conversion of the end endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.     

Kinetics of the enzymatic saccharification of pretreated tapioca waste (Manihot esculenta) and water hyacinth (Eichhornia crassipes)

Studies were carried out on saccharification of pretreated tapioca waste and water hyacinth under two different conditions: using microbial enzymes (cellulase fromMyrothecium verrucaria, Coprinus comatus,Pleurotus florida, andCellulomonas sp.) and solid-state fermentation. The rate of saccharification was determined at different temperatures, pH, substrate concentration, and incubation period. It was found that as the source of the enzyme varies, the optimal temperature and pH for the saccharification varies. Among the two different treatments, enzymatic saccharification was found to be the most efficient. Among the various cellulase sources tested, M.verrucaria cellulase was found to be the most efficient one followed byC. comatus, P. florida, and finallyCellulomonas sp.     

Thermoascus aurantiacus CBHI/Cel7A production in Trichoderma reesei on alternative carbon sources

To develop functional enzymes in cellulose hydrolysis at or above 70 degrees C the cellobiohydrolase (CBHI/Cel7A) of Thermoascus aurantiacus was cloned and expressed in Trichoderma reesei Rut-C30 under the strong cbh1 promoter. Cellulase production of the parental strain and the novel strain (RF6026) was examined in submerged fermentation experiments using various carbon sources, which were lactose, Solka Floc 200 cellulose powder, and steam pretreated corn stover. An industrially feasible production medium was used containing only distiller's spent grain, KH(2)PO(4), and (NH(4))(2)SO(4). Enzyme production was followed by measurements of protein concentration, total cellulase enzyme activity (filter paper activity), beta-glucosidase activity, CBHI activity, and endogenase I (EGI) activity. The Thermoascus CBHI/Cel7A activity was taken as an indication of the heterologous gene expression under the cbh1 promoter.     

A boronate-modified renewable nanointerface for ultrasensitive electrochemical assay of cellulase activity

The saccharification of cellulosic biomass to produce biofuels and chemicals is one of the most promising industries for green-power production and sustainable development. Cellulase is the core component in the saccharification process. Simple and efficient assay method to determine cellulase activity in saccharification is thus highly required. In this work, a boronate-affinity surface based renewable and ultrasensitive electrochemical sensor for cellulase activity determination has been fabricated. Through boronate-sugar interaction, celluloses are attached to the electrode surface, forming the cellulose nano-network at the sensing interface. Cellulase degradation can lead to the variation of electrochemical impedance. Thus, electrochemical impedance signal can reflect the cellulase activity. Importantly, via fully utilizing the boronate-affinity chemistry that enables reversible fabrication of cellulose nano-network, a renewable sensing surface has been firstly constructed for cellulase activity assay. Thanks to interfacial diffusion process of electrochemical sensor, the product inhibitory effect in the cellulase activity assays can be circumvented. The proposed electrochemical sensor is ultrasensitive for label-free cellulase activity detection with a very simple fabrication process, showing great potential for activity screen of new enzymes in saccharification conversion.     

Understanding the effects of lignosulfonate on enzymatic saccharification of pure cellulose

The effects of lignosulfonate (LS) on enzymatic saccharification of pure cellulose were studied. Four fractions of LS with different molecular weight (MW) prepared by ultrafiltration of a commercial LS were applied at different loadings to enzymatic hydrolysis of Whatman paper under different pH. Using LS fractions with low MW and high degree of sulfonation can enhance enzymatic cellulose saccharification despite LS can bind to cellulase nonproductively. The enhancing effect varies with LS properties, its loading, and hydrolysis pH. Inhibitive effect on cellulose saccharification was also observed using LS with large MW and low degree of sulfonation. The concept of “LS-cellulase aggregate stabilized and enhanced cellulase binding” was proposed to explain the observed enhancement of cellulose saccharification. The concept was demonstrated by the linear correlation between the measured amount of bound cellulase and saccharification efficiency with and without LS of different MW in a range of pH.