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.     

Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulase

Enzymatic hydrolysis involves complex interaction between enzyme, substrate, and the reaction environment, and the complete mechanism is still unknown. Further, glucose release slows significantly as the reaction proceeds. A model based on Langmuir binding kinetics that incorporates inactivation of adsorbed cellulase was developed that predicts product formation within 10% of experimental results for two substrates. A key premise of the model, with experimental validation, suggests that V(max) decreases as a function of time due to loss of total available enzyme as adsorbed cellulases become inactivated. Rate constants for product formation and enzyme inactivation were comparable to values reported elsewhere. A value of k(2)/K(m) that is several orders of magnitude lower than the rate constant for the diffusion-controlled encounter of enzyme and substrate, along with similar parameter values between substrates, implies a common but undefined rate-limiting step associated with loss of enzyme activity likely exists in the pathway of cellulose hydrolysis.     

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.     

Purification and properties of alpha-ketoglutarate reductase from Micrococcus aerogenes

Micrococcus aerogenes grown in media containing glutamate has high levels of glutamate dehydrogenase and alpha-ketoglutarate reductase. The latter enzyme catalyzes the reversible reduction of alpha-ketoglutarate to alpha-hydroxyglutarate in the presence of reduced nicotinamide adenine dinucleotide (NADH). The enzyme has a high specificity for both substrates in either direction and displays Michaelis-Menten kinetics at moderate substrate concentrations. K(m) values of 0.12 to 0.17 mm alpha-ketoglutarate and 0.3 mm NADH for the forward reaction were calculated from data obtained at low substrate concentrations. At high concentrations, this reaction was inhibited by both substrates. The reverse reaction, which proceeded at 0.1 to 0.2 times the rate of the forward reactions, was inhibited by one of the products, alpha-ketoglutarate. K(m) values for the substrates of this reaction were 10 mm for alpha-hydroxyglutarate and 1 mm for nicotinamide adenine dinucleotide. alpha-Ketoglutarate reductase has a molecular weight of 7.5 x 10(4) to 8.2 x 10(4) and is composed of identical polypeptide chains with a molecular weight of 3.6 x 10(4) to 3.8 x 10(4).     

Change in Molecular Size of Cellulose during Regeneration of Cell Wall on Carrot Protoplasts

Protoplasts isolated from carrot cells were cultured in a chemically defined medium. The majority of them regenerated cell wall and underwent cell division. Cellulose synthesis started without a noticeable lag but the rate of deposition was very low during the initial stage. The degree of polymerization of cellulose was determined by viscosity measurement of the nitrated product. The cellulose formed in the early stage of the wall regeneration consisted mainly of low molecular weight glucan chains. Change in the average molecular weight of cellulose was found during the growth cycle of carrot cells in normal suspension culture.     

Structural properties of cellulose and cellulase reaction mechanism

The effects of structural properties and their changes during cellulose hydrolysis on the enzymatic hydrolysis rate have been studied from the reaction mechanism point of view. Important findings are the following: (1) The crystallinity index (CrI) of partially crystalline cellulose increases as the hydrolysis reaction proceeds, and a significant slowing down of the reaction rate during the enzymatic hydrolysis is, in large part, attributable to this structural change of cellulose substrate. (2) The crystallinity of completely disordered cellulose, like phosphoric-acid-treated cellulose, does not change significantly, and a relatively high hydrolysis rate is maintained during hydrolysis. (3) The specific surface area (SSA) of partially crystalline cellulose decreases significantly during enzymatic hydrolysis while the change in SSA of regenerated cellulose is found to be negligible. (4) The value of degree of polymerization (DP) of highly ordered crystalline cellulose remains practically constant whereas the change in DP of disordered regenerated cellulose is found to be very significant. (5) Combination of these structural effects as well as cellulase adsorption, product inhibition, and cellulase deactivation all have important influence on the rate of cellulase reaction during cellulose hydrolysis. More experimental evidence for a two-phase model, which is based on degradation of cellulose by simultaneous actions of cellulase complex on the crystalline and amorphous phases, has been obtained. Based on experimental results from this study and other results accumulated, the mode of cellulase action and a possible reaction mechanism are proposed.     

Differential recognition of natural and nonnatural substrate by molecular chaperone alpha-crystallin-A subunit exchange study

alpha-Crystallin is a molecular chaperone that recognizes proteins substrates in stress. It binds to the unstable conformer of a large variety of related or unrelated substrates and thus prevents them aggregating and holds them in a folding competent state. In this article, we have tried to critically analyze, from experimental point of view, whether alpha-crystallin has any preference for its natural substrates compared to the nonnatural one. Our results clearly show that alpha-crystallin is exceptionally active and sensitive in preventing aggregation of its natural substrates and can fully prevent such an aggregation in a substoichiometric ratio, but nonnatural substrates require a considerably higher amount of alpha-crystallin. Using suitable fluorescent-labeled alpha-crystallins and performing fluorescence resonance energy transfer experiments, we were able to determine the subunit exchange kinetics between the alpha-crystallin oligomers. It was found that while alpha-crystallin was bound to its natural substrate, the rate of subunit exchange was slightly decreased. But, when a nonnatural substrate carbonic anhydrase remained bound to the chaperone, further loss in subunit exchange rate was observed. Nonnatural substrate was found to create higher activation energy barrier for the subunit exchange reaction compared to the native substrates. Similarities in major beta-sheet structure of both alpha-crystallin and its natural substrates may be the reason for the preference in molecular recognition in comparison with the nonnatural substrate.     

Analysis and quantification of a mixed exo-acting and endo-acting polysaccharide depolymerization system

A new mathematical model has been proposed based on a model presented by Suga, van Dedem, and Moo-Young.(10) The model requires a separate differential equation for each polymeric species (differentiated by degree of polymerization) in the reaction mixture. The main contribution of this model is the incorporation of experimental molecular weight distributions as the initial conditions. These molecular weight distributional as the initial conditions were obtained using modern analytical equipment previouly unknown for this application. The equipment, SEC/LALLS, measures relative concentrations of specific molecular weight species along with the corresponding molecular weights, thus yielding (through some mathematical manipulation) the absolute concentration of each molecular weight species. The concentration at each molecular weight can then be incorporated as the initial condition for that equation. Theoretically, the system of differential equations can be solved to give a more realistic time course of reaction.Synergism between endo-acting and exo-acting enzymes was examined theoretically using the mathematical model. Through model predictions, it was found that synergy is based on two fundamental parameters: (1) each enzyme's activity relative to the sum of enzyme activities and, (2) overall substrate concentration relative to the exo-acting enzyme's Michaeiis kinetic constant K(m). Theoretically, synergism increases as a function of reaction time. Intermediate endo fractions (ratio of endo-acting enzyme activity to the sum of endo-acting and exo-acting enzyme activity) from 0.3 to 0.7 exhibit the most synergism. Values of k[log(K(m, exo)/S(0))] above about zero also exhibits the most synergism.An examination of experimental data obtained both by SEC/LALLS and by reducing sugar measurements shows that the model is inadequate for successfully predicting quantities associated with the substrate during reaction. This is especially true for synergism predictions. At short reaction times, the model predicts the data fairly well, but at longer times the predictions are inconsistent with experimental data. These inconsistencies may be due to complicating phenomena such as enzyme inhibitions.     

密粘褶菌Gloeophyllum trabeum胞外低分子量多肽的分离纯化及其对纤维素生物降解作用

通过HPLC高效液相层析由褐腐真菌中能强烈降解木质纤维素的代表菌株密粘褶菌Gloeo phyllumtrabeum的胞外培养液 ,分离纯化得到一低分子量的活性多肽组分 (Gt因子 ) .Gt因子具有较好热稳定性 ,在pH 2 5～ 6 5范围内保持稳定 .Gt因子分子量在 4 0 0 0左右 ,等电点pI 6 6 .Gt因子具有络合Fe3 + 的能力 ,且能够将Fe3 + 还原为Fe2 + .在O2 存在时 ,能以纤维素物质为电子供体形成羟基自由基HO·.利用循环伏安法 ,观察到Gt因子与纤维素底物之间的氧化还原过程 .研究表明 ,Gt因子极有可能在褐腐菌的纤维素降解初期起着重要的作用 .     

How important is secretion of exoenzymes through apical cell walls of fungi?

A hypothesis is proposed that the passage of exoenzymes through cell walls occurs more easily through the more plastic and porous nascent cell wall, e. g., the apical region of fungal hyphae. It also accounts for the occurrence of some exoenzymes in cell walls. As the porous and nascent apical wall of fungi is transformed to the less porous lateral wall during growth, some exoenzymes are trapped in transit, thus becoming bound into the wall. Enzymes with binding sites in the wall are not considered in the hypothesis. Several experimental tests performed on Neurospora crassa yield results consistent with its predictions: 1. under selected growth conditions, a group of three exoenzymes of high molecular weight has a significantly higher percent of the total cellular enzyme activity in the wall fraction than another group of three exoenzymes of low molecular weight; this complies with the prediction that larger molecules are more easily trapped in transit, 2. during germ tube outgrowth and early log phase, when the relative amount of surface area occupied by hyphal tips is larger than in older cultures, there is decreased molecular sieving of secreted exoenzymes as judged by a) a smaller proportion of the secreted invertase, comprising light invertase (mol wt=51,500) and heavy invertase (mol wt=210,000), being in the light form, and b) a larger amount of proteins with molecular weights over 40,000 than those of 20,000–40,000 in the culture filtrate. Some of the possible applications of the hypothesis to other microorganisms are discussed.     

Production of extracellular enzymes by Thermomonospora curvata during growth on protein-extracted lucerne fibres

After extraction of food protein from lucerne, the residual fibre was used as a carbon and energy source by the thermophilic actinomycete, Thermomonospora curvata. Induction of catabolic exoenzymes during growth for 7 d on the fibre at 53°C in a mineral salts minimal medium was compared with that on a variety of other inductive substrates. A fibre concentration of 1.5% (w/v) was optimal for total protein secretion. The fibre was a poor substrate for amylase production due to lack of inducer rather than to catabolite repression by soluble sugars released during degradation. β-Glucosidase release during growth on the fibre was about 10 times that observed in cultures grown on cellobiose or cellulose, but production of other cellulolytic enzymes was about one-half that produced on cellulose. Pectinolytic activity (measured as polygalacturonate lyase) was equal to that produced on pectin. Cells grown on the fibre released about eight times as much proteinase as those grown on cellulose, but proteolytic activity was transient and decreased rapidly during later growth. Xylanase appeared to be co-ordinately induced with cellulolytic enzymes; comparable maximal activities, observed during growth on either the fibre or cellulose, were three times that produced on xylan or xylose.     

Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis

In the cellulase-cellulose reaction system, the adsorption of cellulase on the solid cellulose substrate was found to be one of the important parameters that govern the enzymatic hydrolysis rate of cellulose. The adsorption of cellulase usually parallels the rate of hydrolysis of cellulose. The affinity for cellulase varies depending on the structural properties of cellulose. Adsorption parameters such as the half-saturation constant, the maximum adsorption constant, and the distribution coefficient for both the cellulase and cellulsoe have been experimentally determined for several substrates. These adsorption parameters vary with the source of cellulose and the pretreatment methods and are correlated with the crystallinity and the specific surface area of cellulose substrates. The changing pattern of adsorption profile of cellulase during the hydrolysis reaction has also been elucidated. For practical utilization of cellulosic materials, the cellulose structural properties and their effects on cellulase adsorption, and the rate of hydrolysis must be taken into consideration.     

Kinetics of enzymatic solid-to-solid peptide synthesis: intersubstrate compound, substrate ratio, and mixing effects

A systematic study of thermolysin-catalyzed solid-to-solid peptide synthesis using Z-Gln and Leu-NH2 as model substrates was carried out. The aim was to extend the kinetic knowledge of this new reaction system involving highly concentrated substrate mixtures with little water (10% to 20% w/w). Preheating of the substrates, and ultrasonic treatment, as described in the literature, had no significant effect on our system. The formation of a third compound, the salt of the two substrates, was discovered during melting point experiments. This was associated with a very strong dependence of kinetics on the exact substrate ratio (e.g., twofold higher initial rate with 60% Leu-NH2 and 40% Z-Gln than with the equimolar substrate ratio). A model was developed to show how the composition and pH of the liquid phase depends on the substrate ratio, and seemed to explain the experimental rates. In addition, the influences of different mixing and water distribution methods are described. Finally, we can now summarize the major effects of the reaction system as a starting point for further research and scale-up studies.     

Mechanism of substrate dephosphorylation in low Mr protein tyrosine phosphatase.

Substrate dephosphorylation by the low molecular weight protein tyrosine phosphatases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol(-1), which is in good agreement with experimental data. Proteins 1999;36:370-379.     

Boundaries of the butyrylcholinesterase anion site from data of a conformational analysis of substrates

By means of molecular mechanics, theoretical conformational analysis has been made of 19 substrates of butyrylcholinesterase - acetylcholine derivatives with different structure of the ammonium group. It was concluded that the anionic point is located in the cavity of the enzymic molecule. Dimensions and shape of this cavity were established which provide satisfactory correlation between its filling by substrate conformers and the rate of their enzymic hydrolysis. Some suggestions were made with respect to the mechanism of the effect of non-productive sorbtion of the substrates on the rate of their enzymic hydrolysis.     

Enzymatic bromination of barbituric acid and some of its derivatives

Barbituric acid, 1-methyl- and 1,3-dimethylbarbituric acid, some of its 5-phenyl derivatives, and 5-chlorobarbituric acid are presented as new substrates for the bromoperoxidase isolated from the brown alga Ascophyllum nodosum. This enzyme is able to convert these substrates into the corresponding 5-bromo or 5,5-dibromo derivatives in good yields. Kinetic measurements show that the structure of the examined substrates has little or no effect on the enzymatic rate of bromination. However, at low substrate concentration the reaction rate depends on both the concentration of the organic substrate and the concentration of hydrogen peroxide. A mechanism is proposed for the reactions of bromoperoxidase with its substrates. These reactions involve the formation of free hypobromous acid which can either brominate the organic halogen acceptor or produce singlet oxygen by a competing reaction with hydrogen peroxide.     

Sucrose Synthetase of Sweet Potato Roots

The effect of concentration of each substrate in the reaction catalyzed by sucrose synthetase isolated from sweet potato roots was determined. For the sucrose synthesizing reaction, UDP-glucose(ADP-glucose)+fructose→sucrose+UDP(ADP), the substrate saturation curves for UDP-glucose, ADP-glucose and fructose were hyperbolic in shape and the reaction was strongly inhibited by UDP competitively. On the other hand, the substrates for the reversal of sucrose synthetase reaction, sucrose+UDP(ADP)→UDP-glucose(ADP-glucose)+fructose, exhibited a sigmoidal shaped saturation curve which was deviated from the Michaelis-Menten equation. The plot of data according to the empirical Hill equation gives a values greater than 1.0 for every substrate examined in the latter case. In view of these experimental data, the major role of sucrose synthetase is postulated in that this enzyme is involved in the breakdown of sucrose in sweet potato root tissues instead of the sucrose synthesizing reaction. The molecular weight of the enzyme was determined to be about 540,000 by the Sephadex gel filtration chromatography.     

Molecular Modelling of Chymotrypsin-Substrate Interactions: Calculation of Enantioselectivity

A method has been developed for the calculation of the enantioselectivity of chymotrypsin catalysed hydrolytic reactions using molecular mechanics and molecular dynamics. Nine different ester substrates, which are hydrolysed by the enzyme over a wide range of reaction rates have been studied. Models of the transition state of the ester hydrolysis were built using computer aided molecular modelling. The energies of the transition state models were calculated by molecular mechanics and molecular dynamics methods. The point charges of the substrates were modelled from known force field parameters and by semiempirical methods. The difference in free energy of activation between the enantiomers of each substrate were compared with experimental values. The calculations approximated the experimental results. The calculated structure of the transition state model of the chymotrypsin catalysed hydrolysis of acetyl-phenylalanine ester was virtually the same as the published crystal structure of a chymotrypsin-trifluoromethyl ketone inhibitor complex (Brady et al., Biochemistry 29: 7600-7607, 1990).     

CTP-phosphatidic acid cytidyltransferase from Saccharomyces cerevisiae. Partial purification, characterization, and kinetic behavior.

CTP-phosphatidic acid cytidyltransferase catalyzes the formation of CDP-diglyceride from CTP and phosphatidic acid. The enzyme was solubilized from crude mitochondrial membrane by treatment with digitonin and was further purified by chromatography on DEAE-Sephadex, quaternary aminoethyl (QAE) Sephadex, and Sepharose 6B columns. At this stage the enzyme, enriched 550-fold over crude cell homogenate, still remains associated with phospholipid and has an estimated approximate molecular weight of 400,000 on the basis of gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 550-fold enriched enzyme yielded two major protein bands having molecular weights of 45,000 and 19,000. The enzyme exhibits an absolute dependence on Triton X-100, a sharp Mg2+ dependence with an optimum at 20 mM, and a pH optimum of 6.5 for activity. The product of the CTP-phosphatidic acid cytidyl-transferase reaction has been isolated and identified as CDP-diglyceride, both for the crude enzyme preparation as well as for the 550-fold enriched enzyme. CTP-phosphatidic acid cytidyltransferase is capable of catalyzing the reverse reaction in the presence of pyrophosphate, utilizing CDP-diglyceride as substrate. The product of the reverse reaction was identified as CTP. Kinetic analysis of the behavior of CTP-phosphatidic acid cytidyltransferase was performed at three different stages of its purification. Initial analysis of the data yielded biphasic behavior in double reciprocal plots with respect to both substrates. Hill plots of the data indicated the presence of negative cooperativity. A detailed analysis of the kinetic behavior was performed on the enzyme purified 550-fold. The data suggest a mechanism involving two distinct cycles of catalysis, responsive to homotropic modification, with different affinities for both substrates. Further analysis of the kinetic behavior in the presence of inhibitors (dCTP and PPi) yielded a reaction order for the entrance of substrates and departure of products from the reaction cycles. The high affinity site catalyzes the reaction via a double displacement mechanism and is the predominant form at low concentrations of substrates. At high concentrations of substrates the low affinity site starts contributing significantly to the reaction velocity with an ordered single displacement mechanism. In each case CTP is the first substrate to attach and PPi is the first product released.     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.