Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013     

Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of this phenomenon, the enzymatic reaction of the nearly pure cellulose in Avicel was interrupted over the course of nearly complete hydrolysis. Then, the solids were treated with proteinase to degrade the cellulase enzymes remaining on the solid surface, followed by proteinase inhibitors to inactive the proteinase and successive washing with water, 1.0 M NaCl solution, and water. Next, fresh cellulase and buffer were added to the solids to restart hydrolysis. The rate of cellulose hydrolysis, expressed as a percent of substrate remaining at that time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate reactivity, suggesting that other effects such as enzymes getting "stuck" or otherwise slowing down may be responsible.     

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems

Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.     

Carbohydrate derived‐pseudo‐lignin can retard cellulose biological conversion

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo‐lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo‐lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT‐IR, and SEM imaging. It was found that hemicelluloses (xylan) derived‐pseudo‐lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ～ 1.94) dilute acid pretreatment of a xylan–Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo‐lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions. Biotechnol. Bioeng. 2013; 110: 737–753. © 2012 Wiley Periodicals, Inc.     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Optimal operating policy of the ultrafiltration membrane bioreactor for enzymatic hydrolysis of cellulose

The dilution rate of an ultrafiltration membrane bioreactor in the enzymatic hydrolysis of cellulose was optimized using the kinetic model developed by Fan and Lee.(4) The sequence of optimal dilution rates was found to generally consist of an initial period of a minimal value (batch period), a subsequent period of maximum dilution rate, a period of a second batch, and a final period of a singular dilution rate. The effects of operating conditions, such as beta-glucosidase activity, operating time, maximum dilution rate, substrate feeding rate, and enzyme-to-substrate ratio on both the conversion yield and the sequence of optimal dilution rates were investigated. To evaluate the validity of kinetic model employed in this work, enzymatic hydrolysis was carried out using alpha-cellulose as a substrate in the ultrafiltration membrane bioreactor. The experimental data were well consistent with the simulation results. (c) 1993 John Wiley & Sons, Inc.     

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353. © 2008 Wiley Periodicals, Inc.     

Molecular dynamics simulation of the processive endocellulase Cel48F from Clostridium cellulolyticum: a novel “water‐control mechanism” in enzymatic hydrolysis of cellulose

Glycoside hydrolase of Cel48F from Clostridium cellulolyticum is an important processive cellulose, which can hydrolyze cellulose into cellobiose. Molecular dynamics simulations were used to investigate the hydrolysis mechanism of cellulose. The two conformations of the Cel48F‐cellotetrose complex in which the cellotetroses are bound at different sites (known as the sliding conformation and the hydrolyzing conformation) were simulated. By comparing these two conformations, a water‐control mechanism is proposed, in which the hydrolysis proceeds by providing a water molecule for every other glucosidic linkage. The roles of certain key residues are determined: Glu55 and Asp230 are the most probable candidates for acid and base, respectively, in the mechanism of inverting anomeric carbon. Met414 and Trp417 constitute the water‐control system. Glu44 might keep the substrate at a certain location within the active site or help the substrate chain to move from the sliding conformation to the hydrolyzing conformation. The other hydrophobic residues around the substrate can decrease the sliding energy barrier or provide a hydrophobic environment to resist entry of the surrounding water molecules into the active site, except for those coming from a specific water channel. Copyright © 2014 John Wiley & Sons, Ltd.     

A functionally based model for hydrolysis of cellulose by fungal cellulase

A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. The model represents the actions of cellobiohydrolases I, cellobiohydrolase II, and endoglucanase I; and incorporates two measurable and physically interpretable substrate parameters: the degree of polymerization (DP) and the fraction of beta-glucosidic bonds accessible to cellulase, F(a) (Zhang and Lynd, 2004). Initial enzyme-limited reaction rates simulated by the model are consistent with several important behaviors reported in the literature, including the effects of substrate characteristics on exoglucanase and endoglucanase activities; the degree of endo/exoglucanase synergy; the endoglucanase partition coefficient on hydrolysis rates; and enzyme loading on relative reaction rates for different substrates. This is the first cellulase kinetic model involving a single set of kinetic parameters that is successfully applied to a variety of cellulosic substrates, and the first that describes more than one behavior associated with enzymatic hydrolysis. The model has potential utility for data accommodation and design of industrial processes, structuring, testing, and extending understanding of cellulase enzyme systems when experimental date are available, and providing guidance for functional design of cellulase systems at a molecular scale. Opportunities to further refine cellulase kinetic models are discussed, including parameters that would benefit from further study.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Hydrolysis of insoluble cellulose to glucose catalyzed by cellulase‐containing liposomes in an aqueous solution of 1‐butyl‐3‐methylimidazolium chloride

The liposome containing cellulase from Trichoderma viride was prepared under the condition that an appreciable amount of cellulase was incorporated in lipid membranes. The liposomal cellulase and free enzyme were examined in their hydrolytic activities to insoluble cellulose powder CC31 in the acetate buffer solution (pH 4.8) of 15 w/w% [Bmim][Cl] (1‐butyl‐3‐methylimidazolium chloride). The mean diameter and size distribution of cellulase‐containing liposome were practically unchanged under the above condition. The free cellulase was deactivated more rapidly than the liposomal cellulase in catalyzing the hydrolysis of 2.0 g/l CC31 at 45°C in the presence of [Bmim][Cl] for 48 h. The activities of liposomal and free cellulase to cellobiose as soluble substrate were less susceptible to [Bmim][Cl] than their cellulolytic activities to CC31, meaning that β‐glucosidase is relatively stable among the three enzyme components of cellulase. The rate of glucose production could be appreciably improved by the pretreatment of CC31 with [Bmim][Cl] alone at 120°C for 30 min followed by the liposomal cellulase‐catalyzed hydrolysis of the substrate at 45°C at the [Bmim][Cl] concentration of 15 w/w%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1190–1196, 2013     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

A new approach for modeling cellulase-cellulose adsorption and the kinetics of the enzymatic hydrolysis of microcrystalline cellulose

Two fractions of substrate in microcrystalline cellulose which differ in their adsorption capacities for the cellulases and their susceptibility to enzymatic attack have been identified. On the basis of a two-substrate hypothesis, mathematical models to describe enzyme adsorption and the kinetics of hydrolysis have been derived. A new nonequilibrium approach was chosen to predict cellulase-cellulose adsorption. A maximum binding capacity of 76 mg protein per gram substrate and a half-maximum saturation constant of 26 filter paper units (FPU) per gram substrate have been calculated, and a linear relationship of hydrolysis rate vs. adsorbed protein has been found. The fraction of substrate more easily hydrolyzed, as calculated from hydrolysis data, represents 19% of the total effective substrate concentration. This fraction is only slightly different from that of other celluloses and has been estimated to be 27% and 30% for NaOH- and H(3)PO(4)-swollen cellulose, respectively. The effective substrate concentration is equal to the maximum amount of the substrate which can be converted during exhaustive hydrolysis. This in turn is determined by the overall degradability of the substrate by the cellulases (85-90% for microcrystalline cellulose) and by the cellobiose concentration during hydrolysis. The kinetic model is based on a summation of two integrated first-order reactions with respect to the effective substrate concentration. Furthermore, it includes the principal factors influencing the reaction rates: the ratio of filter paper and beta-glucosidase units per gram substrate and the initial substrate concentration. (c) 1993 John Wiley & Sons, Inc.     

Interaction between the CBM of Cel9A from Thermobifida fusca and cellulose fibers

Molecular docking and molecular dynamics (MD) simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. The fiber was found to bind to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain (CD) with a polysaccharide chain. The results also expand the current known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.     

Kinetics of the enzymatic hydrolysis of cellulose: 2. A mathematical model for the process in a plug-flow column reactor

The kinetics of enzymatic cellulose hydrolysis in a plug-flow column reactor catalysed by cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma longibrachiatum adsorbed on cellulose surface have been studied. The maximum substrate conversion achieved was 90–94%. The possibility of enzyme recovery for a reactor of this type is discussed. A mathematical model for enzymatic cellulose hydrolysis in a plug-flow column reactor has been developed. The model allows for the component composition of the cellulase complex, adsorption of cellulases on the substrate surface, inhibition by reaction products, changes in cellulose reactivity and the inactivation of enzymes in the course of hydrolysis. The model affords a reliable prediction of the kinetics of d-glucose and cellobiose formation from cellulose in a column reactor as well as the degree of substrate conversion and reactor productivity with various amounts of adsorbed enzymes and at various flow rates.     

Determination of molecular size of O-(2-hydroxyethyl)cellulose (HEC) and its relationship to the mechanism of enzymatic hydrolysis by cellulases

Z-average root mean square end-to-end distance 〈r_o²〉_z^1/2 and radius of gyration 〈s_o²〉_z^1/2 for 13 samples of O-(2-hydroxyethyl)cellulose (HEC) of different molecular weights were derived from Gel Permeation Chromatography and intrinsic viscosity measurements with water as a solvent. At 40 °C and pH 4.5, contraction of chain dimensions is observed, compared with the sizes observed under neutral conditions at room temperature. The effect is lower for samples with higher molecular weights. Values of 〈r_o²〉₄₀/DP_z also indicate that chain flexibility increases at higher temperature and acidic conditions. From the analysis of molecular weight dependence of 〈s_o²〉 _z^1/2, Flory exponent υ was derived at 40 °C and pH 4.5. A value of υ = 0.70 ± 0.02 was recorded, which indicates that a relatively stiff chain is present under these conditions. Finally, different equations to calculate persistence length L_p were evaluated. Values in the range of 260-400 Å were derived for persistence length. Implications of chain conformation in the enzymatic action of cellulases are also discussed.     

A mechanistic model for enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition

The projected cost for the enzymatic hydrolysis of cellulosic biomass continues to be a barrier for the commercial production of liquid transportation fuels from renewable feedstocks. Predictive models for the kinetics of the enzymatic reactions will enable an improved understanding of current limitations, such as the slow-down of the overall conversion rate, and may point the way for more efficient utilization of the enzymes in order to achieve higher conversion yields. A mechanistically based kinetic model for the enzymatic hydrolysis of cellulose was recently reported in Griggs et al. (2011) (Part I). In this article (Part II), the enzyme system is expanded to include solution-phase kinetics, particularly cellobiose-to-glucose conversion by β-glucosidase (βG), and novel adsorption and product inhibition schemes have been incorporated, based on current structural knowledge of the component enzymes. Model results show cases of cooperative and non-cooperative hydrolysis for an enzyme system consisting of EG(I) and CBH(I). The model is used to explore various potential rate-limiting phenomena, such as substrate accessibility, product inhibition, sterically hindered enzyme adsorption, and the molecular weight of the cellulose substrate.     

Description of cellobiohydrolases Ce16A and Ce17A fromTrichoderma reesei using Langmuir-type models

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding changes during hydrolysis is still needed. The adsorption of purffied two cellobiohydrolases (Ce17A and Ce16A) fromTrichoderma reesei cellulase to microcrystalline cellulose has been studied. Cellobiohydrolase II (Ce16A) does not affect the adsorption of cellobiohydrolase I (Ce17A) significantly, and there are specific binding sites for both Ce17A and Ce16A. The adsorption affinity and tightness of the cellulase binding domain (CBD) for Ce17A are larger that those of the CBD for Ce16A. The CBD for Ce17A binds more rapidly and tightly to Avicel than the CBD for Ce16A. The decrease in adsorption observed when the two cellobihydrolases are studied together would appear to be the result of competition for binding sites on the cellulose. Ce17A competes more efficiently for binding sites than Ce16A. Competition for binding sites is the dominating factor when the two enzymes are acting together, furthermore adsorption to sites specific for Ce17A and Ce16A, also contributes to the total adsorption.