Isolation and Properties of Cellobiase from Penicillium verruculosum

Cellobiase (-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i. e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose--lactone competitively inhibited cellobiase (K _i0.19 mM and 17 M, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.     

Two beta-glucosidase activities in Fibrobacter succinogenes S85.

Few bacteria are capable of degrading crystalline cellulose but there is considerable interest in the properties of enzyme systems with this capability. In the bovine and ovine rumen the principal cellulolytic bacterium is Fibrobacter (formerly Bacteroides) succinogenes. The cellulase system of this organism is composed of multiple enzyme components, including a constitutive and cell-associated beta-glucosidase active against cellobiose. The properties of the beta-glucosidase activity have been investigated with the chromogenic substrate p-nitrophenyl beta-D-glucoside (pNPG). Hydrolytic activity against pNPG was located primarily in the cytoplasm and the cytoplasmic membrane but showed a gradual migration to the periplasm during growth on either glucose or cellobiose. Activity against cellobiose was found in the periplasm in significant amounts in all growth phases. Of the beta-glucosides tested, only cellobiose and pNPG were hydrolysed by crude cell extracts. In the presence of cellobiose, however, the rate of hydrolysis of pNPG was stimulated up to 10-fold, and extracts hydrolysed methylumbelliferyl beta-D-glucoside, 5-bromo-4-chloro-3-indolyl beta-D-glucoside, arbutin and aesculin. Activities against pNPG in the presence and absence of cellobiose displayed similar instability in the presence of oxygen; both were stabilized by dithiothreitol and the temperature and pH optima were identical. A significant proportion of the membrane-associated beta-glucosidase was released by treatment with 0.3 mol/1 KCl, and fractionation by chromatography on CM-cellulose showed the presence of two activities against pNPG, only one of which was stimulated by cellobiose.     

Two β-glucosidase activities in Fibrobacter succinogenes S85

Few bacteria are capable of degrading crystalline cellulose but there is considerable interest in the properties of enzyme systems with this capability. In the bovine and ovine rumen the principal cellulolytic bacterium is Fibrobacter (formerly Bacteroides ) succinogenes. The cellulase system of this organism is composed of multiple enzyme components, including a constitutive and cell-associated β -glucosidase active against cellobiose. The properties of the β -glucosidase activity have been investigated with the chromogenic substrate β -nitrophenyl β -D-glucoside (pNPG). Hydrolytic activity against pNPG was located primarily in the cytoplasm and the cytoplasmic membrane but showed a gradual migration to the periplasm during growth on either glucose or cellobiose. Activity against cellobiose was found in the periplasm in significant amounts in all growth phases. Of the β -glucosides tested, only cellobiose and pNPG were hydrolysed by crude cell extracts. In the presence of cellobiose, however, the rate of hydrolysis of pNPG was stimulated up to 10-fold, and extracts hydrolysed methylumbelliferyl β -D-glucoside, 5-bromo-4-chloro-3-indolyl β -D-glucoside, arbutin and aesculin. Activities against pNPG in the presence and absence of cellobiose displayed similar instability in the presence of oxygen; both were stabilized by dithiothreitol and the temperature and pH optima were identical. A significant proportion of the membrane-associated β -glucosidase was released by treatment with 0.3 mol/1 KCl, and fractionation by chromatography on CM-cellulose showed the presence of two activities against pNPG, only one of which was stimulated by cellobiose.     

Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides.

Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation.     

Kinetics and mathematical model of hydrolysis and transglycosylation catalysed by cellobiase

The kinetics of hydrolysis and transglycosylation reactions catalysed by cellobiase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus foetidus in the cellobiose-d-glucose reaction system have been studied. The formation of transglycosylation products was observed at cellobiose concentrations $\text{>10}{}^{\mathrm{?2}}\text{m}$, whereas at lower substrate concentrations the only reaction product was d-glucose. In the cellobiase-catalysed transglycosylation a (1→6)-β-linkage was formed after the transfer of a d-glucose residue to acceptor molecule. The basic transglycosylation products were isocellotriose and gentiobiose. A small amount of oligosaccharides with a higher degree of polymerization was also formed. The maximum content of transglycosylation products amounted to 25–30% of the total saccharide content in the system at the initial cellobiose concentration (0.1–0.3 m). The processes in the reaction system were inhibited by the substrate and product (d-glucose). A general scheme for cellobiose hydrolysis has been proposed and validated, allowing for the inhibition and transglycosylation effects. Based on this scheme, a mathematical model for cellobiose hydrolysis has been suggested to describe the kinetics of substrate consumption and product (d-glucose) accumulation, as well as the kinetics of formation and consumption of transglycosylation products throughout the course of enzymatic reaction with various initial amounts of cellobiose, starting from low concentrations up to 0.2–0.3 m (7–11% bv weight).     

Isolation and purification of isoenzymes of cellobiohydrolase I and II of trichoderma reesei using LPLC methods

Five cellulases were fractionated from a commercial cellulase preparation (Celluclast^TM) Two isoenzymes of cellobiohydrolase I (CBHI)(pI = 4.1) could be proved to be real exo-glucanases due to their activity towards MU (=methylumbelliferyl)-lactoside being inhibited by cellobiose (5 mM) and due to production of cellobiose from carboxymethylcellulose (CMC) as the sole final product.Two isoenzymes of CBHII (pI=6.15, 6.0) were shown to act as endo-glucanases because they produced glucose, cellobiose and cellotetraose from CMC and because they were not inhibited by cellobiose when decomposing MU-lactoside. Results confirm recent reports in the literature classifying CBHI and CBHII as exo-type and endo-type cellulases, respectively. Both the CBHI and the CBHII isoenzymes were shown to be active towards CMC and amorphous cellulose.CBHI and CBHII reactions could be differentiated from one another by the velocities of decomposition of CMC: CBHI acts slowly and linearly whereas CBHII acts strongly and exponentially.The fifth of the purified enzymes must be classed as a conventional endoglucanase which exhibits activity towards CMC but fails to be active towards MU-lactoside and amorphous cellulose.     

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.     

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.     

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.     

Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of the genes is starch hydrolysis, esculin hydrolysis, cellobiose utilization, chitin hydrolysis, carboxymethyl cellulose hydrolysis, and polygalacturonic acid hydrolysis. A restriction endonuclease cleavage map was constructed, and the genes for starch, carboxymethyl cellulose, cellobiose, chitin, and pectin hydrolysis were subcloned.     

Kinetics of solka floc cellulose hydrolysis by trichoderma viride cellulase

A product inhibition model is developed to describe the hydrolysis of cellulose by the Trichoderma viride enzyme system. It is assumed that noncompetitive inhibition by cellobiose dominates the reaction kinetics. Experiments show that this is indeed a reasonable assumption for initial cellulose concentrations of up to 15 g/liter and at hydrolysis extents up to 65′. Kinetic parameters were determined for the noncompetitive inhibitionmodel in batch experiments with durations of up to 1.5 hr. These parameterswere then used in predicting reaction progress for up to 10 hr. Cellobiose was added to the reaction mixture at the onset of some runs and againreliable predictions were obtained for up to 8 hr of hydrolysis. Finally reaction was carried out in a membrane reactor whereby the product cellobiose was being continuously removed and again reasonable predictability was obtained with a higher net reaction rate.     

Kinetics of cellobiose hydrolysis using cellobiase composites from Ttrichoderma reesei and Aspergillus niger

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary(1) to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.     

Development and validation of a kinetic model for enzymatic saccharification of lignocellulosic biomass

A multireaction kinetic model was developed for closed-system enzymatic hydrolysis of lignocellulosic biomass such as corn stover. Three hydrolysis reactions were modeled, two heterogeneous reactions for cellulose breakdown to cellobiose and glucose and one homogeneous reaction for hydrolyzing cellobiose to glucose. Cellulase adsorption onto pretreated lignocellulose was modeled via a Langmuir-type isotherm. The sugar products of cellulose hydrolysis, cellobiose and glucose, as well as xylose, the dominant sugar prevalent in most hemicellulose hydrolyzates, were assumed to competitively inhibit the enzymatic hydrolysis reactions. Model parameters were estimated from experimental data generated using dilute acid pretreated corn stover as the substrate. The model performed well in predicting cellulose hydrolysis trends at experimental conditions both inside and outside the design space used for parameter estimation and can be used for in silico process optimization.     

Modelling of the enzymatic hydrolysis of cellobiose and cellulose by a complex enzyme mixture of Trichoderma reesei QM 9414

Summary The enzymatic hydrolysis of cellobiose and cellulose by the cell-free culture filtrate of Trichoderma reesei QM 9414 was investigated. The concentrations of cellobiose and glucose were measured as a function of time for different initial concentrations of cellobiose. It was not possible to describe these concentration variations by a model which considers only the cellobiase hydrolysis with competitive and noncompetitive substrate and product inhibition; it is necessary that the endo--1.4-glucanase with competitive product inhibition is also taken into account.The enzymatic hydrolysis of cellulose (Avicel) was described with a mathematical model by using the results of the decomposition of cellobiose by the same enzyme mixture.the identified model parameters are presented. A sensitivity analysis of the parameter was carried out also.     

Effect of hydrogen peroxide pretreatment on the structural features and the enzymatic hydrolysis of rice straw

Assessment was made to evaluate the effect of hydrogen peroxide pretreatment on the change of the structural features and the enzymatic hydrolysis of rice straw. Changes in the lignin content, weight loss, accessibility for Cadoxen, water holding capacity, and crystallinity of straw were measured during pretreatment to express the modification of the lignocellulosic structure of straw. The rates and the extents of enzymatic hydrolysis, cellulase adsorption, and cellobiose accumulation in the initial stage of hydrolysis were determined to study the pretreatment effect on hydrolysis. Pretreatment at 60 degrees C for 5 h in a solution with 1% (w/w) H(2)O(2) and NaOH resulted in 60% delignification, 40% weight loss, a fivefold increase in the accessibility for Cadoxen, an one times increase in the water-holding capacity, and only a slight decrease in crystallinity as compared with that of the untreated straw. Improvement on the pretreatment effect could be made by increasing the initial alkalinity and the pretreatment temperature of hydrogen peroxide solution. A saturated improvement on the structural features was found when the weight ratio of hydrogen peroxide to straw was above 0.25 g H(2)O(2)/g straw in an alkaline H(2)O(2) solution with 1% (w/w) NaOH at 32 degrees C. The initial rates and extents of hydrolysis, cellulase adsorption, and cellobiose accumulation in hydrolysis were enhanced in accordance with the improved structural features of straw pretreated. A four times increase in the extent of the enzymatic hydrolysis of straw for 24 h was attributed to the alkaline hydrogen peroxide pretreatment.     

Cellobiose chemotaxis by the cellulolytic bacterium Cellulomonas gelida.

In the course of a study on the bacterial degradation of plant cell wall polysaccharides, we observed that growing cells of motile cellulolytic bacteria accumulated, without attachment, near cellulose fibers present in the cultures. Because it seemed likely that the accumulation was due to chemotactic behavior, we investigated the chemotactic responses of one of the above-mentioned bacteria (Cellulomonas gelida ATCC 488). We studied primarily the responses toward cellobiose, which is the major product of cellulose hydrolysis by microorganisms, and toward hemicellulose hydrolysis products. We found that cellobiose, cellotriose, D-glucose, xylobiose, and D-xylose, as well as other sugars that are hemicellulose components, served as chemoattractants for C. gelida, as determined by a modification of Adler's capillary assay. Competition and inducibility experiments indicated that C. gelida possesses at least two types of separately regulated cellobiose chemoreceptors (Cb1 and cellobiose, cellotriose, xylobiose, and D-glucose, and it is constitutively synthesized. The presence in C. gelida of a constitutive response toward cellobiose and of at least two distinct cellobiose chemoreceptors has implications for the survival of this cellulolytic bacterium in nature. A possible mechanism for cellobiose-mediated bacterial chemotaxis toward cellulose is proposed. We suggest that, in natural environments, motile cellulolytic bacteria migrate toward plant materials that contain cellulose and hemicellulose by swimming up cellobiose concentration gradients and/or concentration gradients of other sugars (e.g., xylobiose, D-xylose, and D-glucose) formed by enzymatic hydrolysis of plant cell wall polysaccharides.     

Kinetic studies of cellodextrins hydrolyses by exocellulase from Trichoderma reesei

The kinetics of the hydrolyses of cellotriose and of cellotetraose by cellobiohydrolase were studied using a convenient integral technique. Reaction mechanisms and mathematical models were postulated to describe the reactions. The end-products of the reaction were found to be inhibitory toward hydrolysis in a competitive mode. Hydrolysis of cellotetraose produces cellobiose and hydrolysis of cellotriose produces cellobiose and glucose. Both sugars inhibit the enzyme with cellobiose being a stronger inhibitor.     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Cellobiose hydrolysis by endoglucanase (glucan glucanhydrolase) from Trichoderma reesie: Kinetics and mechanism

Glucanohydrolase from Trichoderma reesei, having a molecular weight of 52,000, was evaluated for kinetic properties with respect to cellobiose. Results from this work include: (1) initial rate studies that show that glucanohydrolase hydrolyzes cellobiose by a competitive mechanism and that the product, glucose, inhibits the enzyme; (2) low-pressure aqueous liquid chromatography that shows that formation of a reversion product, cellobiose, is minor and occurs in detectable amounts only a very high (90mM) cellobiose concentrations; (3) development of an equation based on the mechanism of glucanohydrolase action as determined by initial rate kinetics, which accurately predicts the time course of cellobiose hydrolysis; (4) derivation of an initial rate expression for the combined activity of cellobiase and glucanohydrolase on cellobiose. Based on data in this paper it is shown that the difference in inhibition pattern of the two enzymes could be used for determining the contamination of one enzyme by small quantities of the other.     

Ultrasonic analysis of kinetic mechanism of hydrolysis of cellobiose by β-glucosidase

High-resolution ultrasonic spectroscopy (HR–US) was applied for real-time analysis of enzymatic hydrolysis of cellobiose by a β-glucosidase from Aspergillus niger (Novozyme 188) at 50 °C and pH 4.9. This technique is noninvasive, it does not require optical transparency and is suitable to continuously monitor the time dependence of the reaction progress in a broad range of experimental conditions. The time profiles of the amount of glucose released and the reaction rate were obtained from the time profile of ultrasonic velocity. The results are in good agreement with a discontinuous glucose assay (hexokinase method). The kinetic parameters of the reaction were estimated by fitting the ultrasonic time profiles of the reaction rates to several inhibition models. In addition, the equilibrium constant for the reaction of hydrolysis of cellobiose and the molar Gibbs free energy of hydrolysis were determined from the ultrasonic time profiles of concentration of glucose in the reverse reaction (glucose condensation). The results suggest the existence of more complex mechanisms regulating the activity of cellobiase than the combination of simple inhibitions. An extended kinetic model based on two sites for the competitive inhibitor (glucose) is proposed.