Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose

The production of sugars by the enzymatic hydrolysis of cellulose is a two-step process that includes conversion of the intermediate cellobiose to glucose by beta-glucosidase. The hydrolysis was followed by analyzing the two sugar products (cellobiose and glucose). The enzyme showed maximum activity at pH 4.8. Thermal deactivation was significant at temperatures above 45 degrees C. At 50 degrees C (optimum temperature) thermal deactivation was found to follow first-order kinetics. Several models were tested by modeling the kinetics of the reaction. Their parameter values were determined by numerical optimization, including temperature dependence. The best fitting model was a competitive product inhibition for the two reactions in the operational range.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.     

Cellobiose versus glucose utilization by the ruminal bacterium Ruminococcus albus.

Cellulose degradation and metabolism in the rumen can be adversely affected by the presence of soluble sugars, but relatively little information is available on substrate preferences of cellulolytic bacteria. When the ruminal bacterium Ruminococcus albus was incubated with a combination of cellobiose and glucose, the organism preferentially utilized the disaccharide. This preference appeared to be related to repression of glucose uptake systems in cellobiose-grown cells. Glucose transport kinetics exhibited low- and high-affinity uptake, and high-affinity transport was apparently driven by ATP hydrolysis. Bacterial yield in continuous culture was as much as 38% greater when the organism was grown on cellobiose versus glucose, and the increased yield could be partially attributed to constitutive cellobiose phosphorylase activity. The maintenance coefficient of glucose-grown cells was significantly greater than that of cells provided with cellobiose (0.225 g of glucose per g of protein per h versus 0.042 g of cellobiose per g of protein per h), and this result suggested that more energy was devoted to glucose uptake. Substrate affinities were examined in carbon-excess continuous culture, and affinities for glucose and cellobiose were relatively low (0.97 and 3.16 mM, respectively). Although R. albus maintained a proton motive force of approximately 60 mV from pH 6.7 to 5.5, growth ceased below pH 6.0, and this inhibition of growth may have been caused by a depletion of ATP at low pH.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and glucose on enzyme-catalyzed cellulose hydrolysis reactions impose significant limitations on the efficiency of lignocellulose conversion — especially at high-biomass dry matter conditions. To provide the base for selecting the optimal reactor conditions, this paper reviews the reaction kinetics, mechanisms, and significance of this product inhibition, notably the cellobiose and glucose inhibition, on enzymatic cellulose hydrolysis. Particular emphasis is put on the distinct complexity of cellulose as a substrate, the multi-enzymatic nature of the cellulolytic degradation, and the particular features of cellulase inhibition mechanisms and kinetics. The data show that new strategies that place the bioreactor design at the center stage are required to alleviate the product inhibition and in turn to enhance the efficiency of enzymatic cellulose hydrolysis. Accomplishment of the enzymatic hydrolysis at medium substrate concentration in separate hydrolysis reactors that allow continuous glucose removal is proposed to be the way forward for obtaining feasible enzymatic degradation in lignocellulose processing.     

Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase

Soluble cellodextrins (linear β-1,4-d -gluco-oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom-up synthesis from glucose is promising for bulk production, but to ensure a completely water-soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α-d -glucose 1-phosphate (αGlc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1-P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg²⁺. Second, the product DP was controlled by the molar ratio of glucose/αGlc1-P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1-P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1–10 mM; 200 mM αGlc1-P), the reaction was shifted completely towards insoluble product formation (DP ∼9–10). In summary, this study provides the basis for an efficient and product DP-controlled biocatalytic synthesis of cellodextrins from expedient substrates.     

The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

Hydrolysis of cellobiose using β-glucosidase from Penicillium funiculosum. Kinetic analysis

The kinetics of cellobiose hydrolysis was studied using β-glucosidase from Penicillium funiculosum, both free and immobilized on nylon powder, at different temperatures, pH values, enzymatic activities and initial cellobiose and glucose concentrations. The experimental results were fitted to a kinetic model by considering the substrate and product inhibitions as well as the thermal deactivation of β-glucosidase with a mean deviation of less than 10%. The immobilization of β-glucosidase led to an increase in the stability of the enzyme against changes in the pH value.     

Components of cellulase from the higher termite,Nasutitermes walkeri

The cellulase from the termite Nasutitermes walkeri consists of two enzymes. Each has broad specificity with predominantly one activity. One enzyme is an endo-gb-1,4-glucanase (EC 3.2.1.4) which predominantly cleaves cellulose randomly to glucose, cellobiose and cellotriose. It hydrolyses cellotetraose to cellobiose but will not hydrolyse cellobiose or cellotriose. The second enzyme component is a β-1,4-glucosidase (EC 3.2.1.21) as its major activity is to hydrolyse cellobiose, cellotriose and cellotetraose to glucose; it has some exoglucosidase activity as glucose is the only product produced from cellulose. Its cellobiase activity is inhibited by glucono-δ-lactone.     

Characterization of a cellodextrin glucohydrolase with soluble oligomeric substrates: experimental results and modeling of concentration-time-course data

A previously isolated cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei QM 9414 is characterized using beta-1,4-glucose oligomers with defined degrees of polymerization as soluble substrates. The enzyme splits off glucose units from the nonreducing chain ends of cellooligomers. Besides this hydrolytic activity there is also evidence for transfer activity depending on the concentration and degree of polymerization of substrates. Concentration-time-course data have been gathered for the degradation of cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose covering a wide range of enzyme and substrate concentrations. A Michaelis-Menten type kinetic model has been developed, which is able to satisfactorily describe the complex system of parallel and series reactions during the conversion of oligomers to glucose. The only kind of inhibition considered is competitive inhibition by the final product glucose. The model takes into account the formation of multiple enzyme-substrate complexes and is limited to those conditions, in which no transglucosylation products are observed. Cellodextrins with higher degrees of polymerization are found to be better substrates for this enzyme than is the dimer cellobiose.     

Product inhibition of the recombinant CelS,an exoglucanase component of the Clostridium thermocellum cellulosome

CelS is the most abundant subunit and an exoglucanase component of the Clostridium thermocellum cellulosome, multicomponent cellulase complex. The product inhibition pattern of CelS was examined using purified recombinant CelS (rCelS) produced in Escherichia coli. The rCelS activity on cellopentaose was strongly inhibited by cellobiose. The rCelS activity was also inhibited by lactose. Glucose was only marginally inhibitory. Cellobiose appeared to inhibit the rCelS activity through a competitive mechanism. The inhibition was relieved when -glucosidase was added, presumably because of the conversion of cellobiose into glucose. These hydrolysis product inhibition patterns are consistent with those of the crude enzyme (cellulosome), suggesting that CelS is a rate-limiting factor in the activity of the cellulosome.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. Quantification of inhibition and suitability of membrane reactors

Product inhibition of cellulolytic enzymes affects the efficiency of the biocatalytic conversion of lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are required for alleviation of glucose product inhibition. Supported by numerous calculations this review assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation rates. The significance of glucose product inhibition on dimensioning of different ideal reactor types, i.e. batch, continuous stirred, and plug-flow, is illustrated quantitatively by modeling different extents of cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up, dilution rate, glucose output profile, and the problem of cellobiose are examined to illustrate the quantitative significance of the glucose product inhibition and the total glucose concentration on the cellulolytic conversion rate. Comprehensive overviews of the available literature data for glucose removal by membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efficiency (kg_product/kg_enzyme). Current membrane reactor designs are however not feasible for large scale operations. The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to achieve efficient enzyme-catalyzed cellulose degradation.     

Enzymatic hydrolysis of lignocellulosic biomass from Onopordum nervosum

Some properties of the cellulolytic complex obtained from Trichoderma reesei QM 9414 grown on Solka floc as carbon source and its ability to hydrolyze the lignocellulosic biomass of Onopordum nervosum Boiss were studied. The optimum enzyme activity was found at temperatures between 50 and 55 degrees C and pH ranging from 4.3 to 4.8. Hydrolysis of 4-nitropnenyl-beta-D-glucopyranoside (4-NPG) and cellobiose by the beta-glucosidase of the complex, showed competitive inhibition by glucose with a K(i) value of 0.8 mM for 4-NPG and 2. 56 mM for cellobiose. Enzymatic hydrolysis yield of Onopordum nervosum, evaluated as glucose production after 48 h, showed a threefold increase by pretreating the lignocellulosic substrate with alkali. When the loss of glucose incurred by de pretreatment was taken into account, a 160% increase in the final cellulose to glucose conversion was found to be due to the pretreatment.     

Simultaneous saccharification and fermentation of cellulose to lactic acid

Recent interest in the industrial manufacture of ethanol and other organic chemicals from biomass has led to the utilization of surplus grain and cane juice as a fermentation feedstock. Since those starting materials are also foods, they are expensive. As an alternative, cellulosic substances-the most abundant renewable resources on earth(1)-have long been considered for conversion to readily utilizable hydrolyzates.(2, 3)For the production of ethanol from cellulose, we have proposed the simultaneous saccharification and fermentation (SSF) process.(4) In SSF, enzymatic cellulose hydrolysis and glucose fermentation to ethanol by yeast proceed simultaneously within one vessel. The process advantages-reduced reactor volume and faster saccharification rates-have been confirmed by many researchers.(5-8) During SSF, the faster saccharification rates result because the glucose product is immediately removed, considerably diminishing its inhibitory effect on the cellulase system.(9)To effectively apply the SSF method to produce substances fermented from glucose, several conditions should be satisfied. One is coincident enzymatic hydrolysis and fermentation conditions, such as pH and temperature. The other is that cellulase inhibition by the final product is less than that by glucose and/or cellobiose. One of us has reported that acetic acid, citric acid, itaconic acid, alpha-ketoglutaric acid, lactic acid, and succinic acid scarcely inhibit cellulase.(10) This suggests that if the microorganisms which produce these organic acids were compatible with cellulase reaction conditions, the organic acids could be produced efficiently from cellulosic substrates by SSF.In this article, the successful application of SSF to lactic acid production from cellulose is reported. Though there have been several reports of direct cellulose conversion to organic acids by anaerobes such as Clostridium, only trace amounts of lactic acid were detected in the fermentation medium among the low-molecular-weight fatty acid components.(11-13) Lactic acid is one of the most important organic acids and has a wide range of food-related and industrial applications.     

Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

The rate of celluose degradation, limited due to the inhibition by cellobiose, can be increased by the hydrolysis of cellobiose to glucose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. A study of the immobilization of beta-glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. A retention of 96.5% of initial activity after ten sequential batch uses of the immobilized preparation was observed. The pH and temperature optima for free and immobilized cells were the same, i.e., 6.5 (0.05M Maleate buffer) and 50 degrees C, respectively. Even though the temperature optimum was found to be 50 degrees C, the enzyme exhibits a better thermal stability at 45 degrees C. Beads stored at 4 degrees C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells shown that glucose is a noncompetitive product inhibitor.The immobilized preparation was found to be limited by pore diffusion but exhibited no film-diffusion resistance during packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half-life of over seven days was obtained at the operating temperature of 45 degrees C in continuous operation of the packed bed reactor. However, the half-life in the column was found to be greatly affected by temperature, increasing to over seventeen days at a temperature of 40 degrees C and decreasing to less than two days at 50 degrees C.     

A product inhibition study of cellulases from Trichoderma longibrachiatum using dyed cellulose

Amorphous acid-swollen cellulose dyed with Reactive Orange was used to determine the relevant inhibition constants of cellulases from Trichoderma longibrachiatum by cellulose hydrolysis products (glucose and cellobiose). The method is based on the initial rate of increasing the hydrolysate absorbance (A_490mn) in the presence of added product. On adding glucose, the initial rate of glucose formation from cellulose and the rate of dye release were lower than the relevant rates in the absence of added product; however, the rate of cellobiose formation did not change. On the other hand, added cellobiose inhibited the rate of cellobiose formation from dyed cellulose and the rate of increase of the hydrolysate absorbance but did not affect the glucose formation. The constants of competitive inhibition of cellulases by glucose and cellobiose were 0.072 and 0.012 M, respectively. These inhibition parameters differed from those obtained from the analysis of the progress kinetics for extended reaction times.     

Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods

Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, -14.4 kcal/mol using SMD and -11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are -13.1, -6.0, -11.5, -7.5, and -8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion.     

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobia converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.     

Products of enzymatic hydrolysis of cellulose and its derivatives

Cellobiose and glucose were determined in a mixture of the two carbohydrates by methods involving the use of glucose oxidase and of β-glucosidase.Paper-partition chromatography is used as a confirmatory method in the identification of the hydrolysis products and in the detection of the various constituents.The cellulolytic organisms studied produce large amounts of the enzyme Cx, which diffuses into the medium. Only small amounts of β-glucosidase are found outside the cell. Cellobiose resulting from Cx activity can enter the cells as rapidly as can glucose.The role of cellobiose as a principal product in the hydrolysis of cellulose is confirmed. It is hypothesized that the principal final product of Cx activity is cellobiose, and that the presence of cellobiase in the medium is not a prerequisite to utilization of cellobiose by the organism. This is a correction of the hypothesis previously published stating that glucose appeared to be the final product of Cx activity.     

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.     

Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside by cellobiase of Trichoderma viride.

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol-buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 X 10(-3) M to 9 X 10(-2) M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate -pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol-1 over the temperature range 5-56 degrees C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol. The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-beta-D-glucoside was examined by pre-steady-state methods in which [enzyme]0 greater than [substrate]0, and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 S-1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism (formula: see text), (ii) a log rate -pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5-6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol-1 at pH 5.7 over the temperature range 10-60 degrees C.