Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.     

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.     

Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression

The adsorption of cellulase on cellulose and a lignacious residue was examined by using cellulase from Trichoderma reesei, hardwood pretreated by dilute sulfuric acid under high pressure, and a lignacious residue prepared by a complete enzymatic hydrolysis of the pretreated wood. A significant amount of cellulase was found to adsorb on the lignacious residue during the hydrolysis of the pretreated wood. Hence, the adsorption of enzyme on the lignacious residue as well as cellulose must be taken into account in the development of the hydrolysis kinetics. It was found that the adsorption of enzyme on cellulose and on the lignacious residue could be represented by Langmuir type isotherms. The data show that the pretreatment at a higher temperature results in more enzyme adsorption on the cellulose fraction and less on the lignacious residue fraction. The relationship between the hydrolysis rate and the amount of enzyme adsorbed is discussed.     

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.     

Kinetic analysis of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase using an amperometric biosensor

An amperometric biosensor for the detection of cellobiose has been introduced to study the kinetics of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase. By use of a sensor in which pyrroloquinoline quinone-dependent glucose dehydrogenase was immobilized on the surface of electrode, direct and continuous observation of the hydrolysis can be achieved even in a thick cellulose suspension. The steady-state rate of the hydrolysis increased with increasing concentrations of the enzyme to approach a saturation value and was proportional to the amount of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism consisting of the adsorption of the free enzyme onto the surface of the substrate, the reaction of the adsorbed enzyme with the substrate, and the liberation of the product. The catalytic constant of the adsorbed enzyme was determined to be 0.044+/-0.011s(-1).     

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.     

Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose

The rates and extents of enzymatic cellulose hydrolysis of dilute acid pretreated corn stover (PCS) decline with increasing slurry concentration. However, mass transfer limitations are not apparent until insoluble solids concentrations approach 20% w/w, indicating that inhibition of enzyme hydrolysis at lower solids concentrations is primarily due to soluble components. Consequently, the inhibitory effects of pH-adjusted pretreatment liquor on the enzymatic hydrolysis of PCS were investigated. A response surface methodology (RSM) was applied to empirically model how hydrolysis performance varied as a function of enzyme loading (12-40mg protein/g cellulose) and insoluble solids concentration (5-13%) in full-slurry hydrolyzates. Factorial design and analysis of variance (ANOVA) were also used to assess the contribution of the major classes of soluble components (acetic acid, phenolics, furans, sugars) to total inhibition. High sugar concentrations (130g/L total initial background sugars) were shown to be the primary cause of performance inhibition, with acetic acid (15g/L) only slightly inhibiting enzymatic hydrolysis and phenolic compounds (9g/L total including vanillin, syringaldehyde, and 4-hydroxycinnamic acid) and furans (8g/L total of furfural and hydroxymethylfurfural, HMF) with only a minor effect on reaction kinetics. It was also demonstrated that this enzyme inhibition in high-solids PCS slurries can be approximated using a synthetic hydrolyzate composed of pure sugars supplemented with a mixture of acetic acid, furans, and phenolic compounds, which indicates that generally all of the reaction rate-determining soluble compounds for this system can be approximated synthetically.     

Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.     

Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process

Past technoeconomic modeling work has identified the relatively large contribution that enzymatic hydrolysis adds to the total cost of producing ethanol from lignocellulosic substrates. This cost was primarily due to the high concentration of enzyme and long incubation time that was required to obtain complete hydrolysis. Although enzyme and substrate concentration and end-product inhibition influenced the rate of hydrolysis, the effect was less pronounced during the initial stages of hydrolysis. During this time most of the cellulases were adsorbed onto the unhydrolyzed residue. By recycling the cellulases adsorbed to the residual substrate remaining after an initial 24 h, a high rate of hydrolysis, with low overall residence time and minimal cellulase input, could be achieved for several rounds of enzyme recycle. A comparison of the front end (pretreatment, fractionation, and hydrolysis) of a softwood/hardwood to ethanol process indicated that the lignin associated with the softwood-derived cellulose stream limited the number of times the cellulose containing residue could be recycled. (c) 1996 John Wiley & Sons, 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.     

Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step

Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.     

Continuous hydrolysis of sucrose by invertase adsorbed in a tubular reactor

Studies were made of invertase adsorption on Amberlite ion exchange resins. Up to 4000 units of adsorbed enzymatic activity (aea) were obtainedper g of IRA 93 resin; for an aea of 1600 units, the maximum ratio of aea over units of soluble enzyme used for adsorption was close to 50%. Nodesorption occurred during extensive washing at 30°C with 0.01M sodiumacetate buffer at pH 5. Progressive desorption of aea from the invertase–IRA 93 complex occurred when buffer molarity and temperature were increased. Desorption differed only slightly when the buffer pH was 3 or 5. Theoptimum pH of aea was 3.2 with IRA 93 resin, and varied between 3.2 and 5.1with other resins, depending on their anionic or cationic nature. Batch hydrolysis of sucrose by IRA 93–adsorbed invertase followed 1st order kinetics with respect to the substrate concentration, as in the case of soluble invertase. Continuous sucrose hydrolysis with IRA 93–adsorbed invertase was performed in a tubular reactor, and the percent conversion was experimentally determined as a function of the flow rate. The reaction was experimentally determined 50% (w/v) sucrose solution, at pH4 and 30°C; at the selected flow rate, the ratio of sucrose hydrolysis remained constant and close to 76%. This shows that invertase was not desorbed from the tubular reactor. Some continuous hydrolyses were performed with an industrial sucrose solution: enzymatic activity seemed to be stable for anextended period for time (1 month) at 30°C and pH 3 or 4.     

Modeling intrinsic kinetics of enzymatic cellulose hydrolysis

A multistep approach was taken to investigate the intrinsic kinetics of the cellulase enzyme complex as observed with hydrolysis of noncrystalline cellulose (NCC). In the first stage, published initial rate mechanistic models were built and critically evaluated for their performance in predicting time-course kinetics, using the data obtained from enzymatic hydrolysis experiments performed on two substrates: NCC and alpha-cellulose. In the second stage, assessment of the effect of reaction intermediates and products on intrinsic kinetics of enzymatic hydrolysis was performed using NCC hydrolysis experiments, isolating external factors such as mass transfer effects, physical properties of substrate, etc. In the final stage, a comprehensive intrinsic kinetics mechanism was proposed. From batch experiments using NCC, the time-course data on cellulose, cello-oligosaccharides (COS), cellobiose, and glucose were taken and used to estimate the parameters in the kinetic model. The model predictions of NCC, COS, cellobiose, and glucose profiles show a good agreement with experimental data generated from hydrolysis of different initial compositions of substrate (NCC supplemented with COS, cellobiose, and glucose). Finally, sensitivity analysis was performed on each model parameter; this analysis provides some insights into the yield of glucose in the enzymatic hydrolysis. The proposed intrinsic kinetic model parametrized for dilute cellulose systems forms a basis for modeling the complex enzymatic kinetics of cellulose hydrolysis in the presence of limiting factors offered by substrate and enzyme characteristics.     

Surface effects of solvents in hydrolysis of water-soluble lipids by candidal lipase

In the study of hydrolysis of tributyrin by the lipase of Candida cylindracea, it is shown that initial rates of hydrolysis are directly proportional to the amount of enzyme adsorbed at the substrate-water interface. As a consequence of understanding the role of the physical state of the substrate in aqueous reaction media, it was hypothesized that the inclusion of synthetic (nonsubstrate) surfaces into the reaction media may enhance the hydrolysis rate of simple liquid lipids which are partly soluble in water, like triacetin. Nonpolar n-hydrocarbons having 5-11 carbon atoms were used to create interfaces in the hydrolysis of triacetin in the soluble range. All of the C(5)-C(11) hydrocarbons showed an activating effect. For quantitative evaluation of the effects of n-hydrocarbons, n-heptane was chosen as the model n-hydrocarbon. Interrelations between the reaction kinetics and adsorption of the enzyme at the n-heptane-water interface were experimentally determined by the use of the same in-line filtration device used for the tributyrin-water system. At 35 degrees C and pH 6 the relative values of the rate constants for the decomposition of enzyme-interface-substrate complexes were calculated as 12 and 1 for the tributyrin and n-heptane-triacetin systems, respectively. The nature of activation at the solvent surfaces were accounted for by a kinetic model which assumes simultaneous adsorption of enzyme and triacetin molecules at the n-heptane-water interface. Making use of the proposed model, the value of a the apparent Michaelis constant for the soluble triacetin-n-heptane system at constant n-heptane concentration, 2 vol %, was calculated as 0.044 mol/L.     

Enzyme adsorption on SO2 catalyzed steam-pretreated wheat and spruce material

Lignocellulose is widely recognized as a sustainable substrate for biofuels production, and the enzymatic hydrolysis is regarded as a critical step for the development of an effective process for the conversion of cellulose into ethanol. One key factor affecting the overall conversion rate is the adsorption capacity of the cellulase enzymes to the surface of the insoluble substrate. Pretreatment has a strong impact on hydrolysis, which could be related to both chemical changes and morphological changes of the material. In the current work, the accessibility of four differently pretreated wheat straw substrates, two differently pretreated spruce materials, and Avicel cellulose was investigated. Adsorption isotherms (at 4 °C and 30 °C) for a cellulase preparation were obtained, and the rates of hydrolysis were determined for the different materials. Furthermore, the surface area and pore size distribution of the various materials were measured and compared to adsorption and hydrolysis properties, and the structures of the pretreated materials were examined using scanning electron microscopy (SEM).The results demonstrated a positive correlation between enzyme adsorption and the substrate specific surface area within each feedstock. Overall, the amount of enzyme adsorbed was higher for pretreated spruce than for the pretreated wheat straw, but this was not accompanied by a higher initial rate of hydrolysis for spruce. Also, the difference in the measured endoglucanase adsorption and overall FPU adsorption suggests that a larger fraction of the enzyme adsorbed on spruce was unproductive binding. The SEM analysis of the material illustrated the structural effects of pretreatment harshness on the materials, and suggested that increased porosity explains the higher rate of hydrolysis of more severely pretreated biomass.     

Kinetic modeling of rapid enzymatic hydrolysis of crystalline cellulose after pretreatment by NMMO

Pretreatment of cellulose with an industrial cellulosic solvent, N-methylmorpholine-N-oxide, showed promising results in increasing the rate of subsequent enzymatic hydrolysis. Cotton linter was used as high crystalline cellulose. After the pretreatment, the cellulose was almost completely hydrolyzed in less than 12 h, using low enzyme loading (15 FPU/g cellulose). The pretreatment significantly decreased the total crystallinity of cellulose from 7.1 to 3.3, and drastically increased the enzyme adsorption capacity of cellulose by approximately 42 times. A semi-mechanistic model was used to describe the relationship between the cellulose concentration and the enzyme loading. In this model, two reactions for heterogeneous reaction of cellulose to glucose and cellobiose, and a homogenous reaction for cellobiose conversion to glucose was incorporated. The Langmuir model was applied to model the adsorption of cellulase onto the treated cellulose. The competitive inhibition was also considered for the effects of sugar inhibition on the rate of enzymatic hydrolysis. The kinetic parameters of the model were estimated by experimental results and evaluated.     

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.     

The enzymatic hydrolysis rate of cellulose decreases with irreversible adsorption of cellobiohydrolase I

Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions. In this work the adsorption behavior of intact β-1, 4-glucan-cellobiohydrolase (CBH I) from Trichoderma reesei onto microcrystalline cellulose was monitored by surface plasmon resonance and UV-spectral method. It was found that there existed reversible binding and irreversible binding of CBH I during its interaction with cellulose substrate. To evaluate the influence of adsorption on cellulose enzymatic hydrolysis, the reaction dynamics on pure cellulose were determined. A plot of the hydrolysis rate against the surface density of irreversibly adsorbed CBH I, revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density. Taken together, results presented here should be useful for modifying the binding characteristics of CBH I and making them more effective in cellulose hydrolysis.     

Sorption of Talaromyces emersonii cellulase on cellulosic substrates

The sorption characteristics of the cellulase system of Talaromyces emersonii on various cellulosic substrates were examined. Analysis of reaction mixture supernatants by electrophoresis and enzyme assay showed that all components of the cellulase system were rapidly adsorbed by cellulose and then gradually returned to the liquid phase as the hydrolysis of the substrate progressed. The extent of adsorption in the rapid phase was influenced by pH, temperature, the nature of the substrate, and its concentration.     

Enzymatic hydrolysis of cellulose. Regulatory effect of the non-soluble substrate on the effectiveness of the enzymatic reaction

It was shown that one of the cellulase components, i.e. cellobiase, can be adsorbed on cellulose surface with the concomitant decrease of activity (by 10 times and more). The specific activity of the adsorbed cellobiase depends on the enzyme concentration in the adsorption layer and is increased with the increase in the surface concentration of cellobiase. It was found that variations in the amount of non-soluble cellulose and the corresponding changes in cellobiase activity in the system (as a result of the adsorption) can lead to a certain alteration in the shape of the kinetic curves for formation of intermediate cellobiose, which in its turn controls the rate of formation of the end product, i.e. glucose. Thus, the substrate surface causes a regulatory effect on the rate and kinetic mechanism of the enzymatic conversion of cellulose to glucose due to the adsorption effects.