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.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Effect of glucose and other sugars on the beta-1,4-glucosidase activity of Thermomonospora fusca

The cell-associated beta-glucosidase activity of Thermomonospora fusca, strain YX, showed both PNPGase and cellobiase activities. The cellobiase activity was found by HPLC assay to have very low product inhibition, whereas the PNPGase activity was more significantly inhibited. Of the various sugars and sugar analogs tested for inhibition of the PNPGase activity, gluconolactone had the greatest effect. The low product inhibition of the cellobiase activity was further demonstrated by the production of glucose syrups to 20% concentration from both cellobiose and swollen cellulose (Avicel). This characteristic is of practical importance in the development of a commercial process for the production of glucose syrups from cellulose. Growth experiments gave further evidence for the probability of separate enzymes for the PNPGase and cellobiase activities.     

Surface density of cellobiohydrolase on crystalline celluloses. A critical parameter to evaluate enzymatic kinetics at a solid-liquid interface

The enzymatic kinetics of glycoside hydrolase family 7 cellobiohydrolase (Cel7A) towards highly crystalline celluloses at the solid-liquid interface was evaluated by applying the novel concept of surface density (rho) of the enzyme, which is defined as the amount of adsorbed enzyme divided by the maximum amount of adsorbed enzyme. When the adsorption levels of Trichoderma viride Cel7A on cellulose I(alpha) from Cladophora and cellulose I(beta) from Halocynthia were compared, the maximum adsorption of the enzyme on cellulose I(beta) was approximately 1.5 times higher than that on cellulose I(alpha), although the rate of cellobiose production from cellulose I(beta) was lower than that from cellulose I(alpha). This indicates that the specific activity (k) of Cel7A adsorbed on cellulose I(alpha) is higher than that of Cel7A adsorbed on cellulose I(beta). When k was plotted versus rho, a dramatic decrease of the specific activity was observed with the increase of surface density (rho-value), suggesting that overcrowding of enzyme molecules on a cellulose surface lowers their activity. An apparent difference of the specific activity was observed between crystalline polymorphs, i.e. the specific activity for cellulose I(alpha) was almost twice that for cellulose I(beta). When cellulose I(alpha) was converted to cellulose I(beta) by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose I(beta) at the same rho-value. These results indicate that the hydrolytic activity (rate) of bound Cel7A depends on the nature of the crystalline cellulose polymorph, and an analysis that takes surface density into account is an effective means to evaluate cellulase kinetics at a solid-liquid interface.     

Kinetics of growth and elemental sulfur oxidation in batch culture of thiobacillus ferrooxidans

The kinetics of oxidation of elemental sulfur by Thiobacillus ferrooxidans in a batch reactor was followed by measuring the concentration of adsorbed cells on the sulfur surface, the concentration of free cells in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate, the liquid-phase concentration of free cells continued to increase with time, whereas the surface concentration of adsorbed cells per unit weight of sulfur approached a limiting value, i.e., the maximum adsorption capacity. During sulfur oxidation, there was a close correlation between the concentrations of adsorbed and free cells, and these data were well correlated with the Langmuir isotherm. The observed rates of batch growth and sulfur oxidation were consistent with a kinetic model, assuming that the growth rate of batch growth and sulfur oxidation were consistent with a kinetic model. Assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration of adsorbed cells and the fraction of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were evaluated using the experimental data and were compared with parameters determined previously for a few metal sulfides. (c) 1994 John Wiley & Sons, Inc.     

Rapid method for determining cellobiase activity

A simple and rapid method for determining the cellobiase activity in purified enzyme preparations was developed. It is based on a series of consecutive enzymatic reactions, i. e. hydrolysis of cellobiose by cellobiase, oxidation of the forming glucose by glucose oxidase, and formation of a dyed product under peroxidase action in the same reaction system. The dyed product is recorded spectrophotometrically at 460 nm. One measurement takes from 2-3 to 7-10 min depending on a particular method of the activity determining. The reagent which is used for the activity determining can be obtained in the yophylized form and used repeatedly. The relative deviation of the method is 5-7%.     

On the mode of action of fungal cellulases

Summary The mode of action of the cellulolytic enzymes of two strong cellulose decomposing fungi, Penicillium oxalicum Curie et Thom and Helminthosporium cyclops Drechsler, was studied. The culture filtrates and enzyme preparations obtained from them showed high cellulase activity and very weak cellobiase activity. The cellulolytic system of both experimental organisms seems to be multicomponent. The cellulase component showed its activity mainly extracellulary and the cellobiase component, mainly intracellulary. It seems, therefore, that during growth of both fungi on a cellulose medium, the extracellular cellulase acts hydrolytically on the cellulose substrate forming cellobiose which is further acted upon by intracellular cellobiase to form glucose. Paper chromatographic assay of the products of the enzymatic reaction sub-stantiated this conclusion.     

Characterization of production of free gluconic acid by Gluconobacter suboxydans adsorbed on ceramic honeycomb monolith

Gluconobacter suboxydans IFO 3290 was immobilized by adsorption on ceramic honeycomb monolith and continuous production of free gluconic acid from glucose was performed in an aerated reactor. The effects of reactor residence time, aeration rate, and glucose concentration were investigated on the gluconic acid yield. Observation of SEM photographs revealed that the cells were adsorbed with a high density not only on the outer surface of the support but also on the inner surface of large pores. From measurement of the number of the adsorbed cells, it was elucidated that the biofilm comprised a monolayer or bilayer of the cells. Maximum specific rate of growth was estimated for the free and adsorbed cells, and the adsorbed cells were found to grow at a fast rate compared with the free cells. In the continuous fermentation performed for one month at the glucose concentration of 100 kg/m(3), reactor residence time of 3.5 h and aeration rate of 900 cm(3)/min, the activity of the adsorbed cells was appreciably stable. The high productivity of 26.3 kg/(m(3)-reactor . h) was attained with the gluconic acid yield of 84.6% and glucose conversion of 94%.     

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.     

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).     

Enzymatic hydrolysis of cellulosic materials by Sclerotium rolfsii culture filtrate for sugar production.

The hydrolysis of purified celluloses (cotton, Avicel, Cellulose-123, Solka Floc SW40) and cellulosic wastes (rice straw, sugarcane bagasse, wood powders, paper factory effluents) by Sclerotium rolfsii CPC 142 culture filtrate was studied. Factors which effect saccharification such as pH, temperature, enzyme concentration, substrate concentration, produce inhibition, adsorption, and inactivation of enzyme and particle size were studied. Virtually no inhibition (less than 3%) of cellulose hydrolysis by the culture filtrate was observed by cellobiose and glucose up to 100 mg/mL. Filter paper degrading enzyme(s) (but neither carboxymethylcellulase nor beta-glucosidase) was adsorbed on cellulose. The n value in the S. rolfsii system was calculated to be 0.32 for Avicel P.H. 101 and 0.53 for alkali-treated (AT) rice straw indicating penetration of cellulase into AT rice straw. In batch experiments at 10% substrate level, solutions containing 6 to 7%, 3.8 to 4.7%, 4.0 to 5.1%, and 4.2 to 4.9% reducing sugars were produced in 24 to 48 from AT rice straw. AT bagasse, alkali - peracetic acid treated mesta wood and paper factory sedimented sludge effluent, respectively. The main constituent in the hydrolysate from cellulose was glucose with little or no cellobiose, probably due to the high cellobiase content in the culture filtrate.     

Saccharification of cellulose by the cellulolytic enzyme system of Thermomonospora sp. II. Hydrolysis of cellulosic substrates

A saccharification of cellulosic material using culture filtrate from the stationary phase of a culture of Thermomonospora sp. produced primarily cellobiose up to levels inhibitory to further saccharification, while the use of whole broth resulted in the production of glucose as well. Glucose production was enhanced and continued throughout the saccharification (24–36 hr) by several additions of cellobiase activity in the form of culture solids. Using Solka-Floc as substrate, the “difference sugar” level (total soluble sugar minus glucose) rapidly rose to the same relatively stable concentration under various hydrolysis conditions, which was independent of the total sugar and glucose concentrations. A rapid hydrolusis rate was observed initially during saccharification, followed by a much slower rate of sugar production. Repeated centrifugation of the reaction mixture and replacement of the supernatant with fresh enzyme solution resulted each time in the reinitiation of a rapid hydrolysis rate. Saccharifications using A vicel microcrystalline cellulose, acid-swollen cellulose, and cotton as substrates were also studied. A modified method of making phosphoric-acid swollen cellulose is described. Saccharification of this substrate by culture filtrate and sequential additions of culture solids resulted in an inverse relationship between the attained glucose concentration and cellobiose-cellotriose concentrations.     

Kinetic studies of protein-surface interactions: A two-stage model of surface-induced protein transitions in adsorbed biofilms

The irreversible adsorption of proteins on artificial surfaces plays an important role in a wide variety of practical problems. The simple analytical models based on definite concepts regarding the mechanisms of interfacial evolution can be used efficiently for characterization of protein-surface interactions by analyzing the intrinsic kinetics of the process. In this article, analytical expressions are derived for the adsorption kinetics that take into account the presence of more than one adsorbed state for proteins in biofilms. It is shown that the experimentally observed dependence of the adsorbed mass on the concentration of protein in solution can be reproduced with this model, and the approach provides a rapid method for obtaining quantitative parameters for the adsorption process. It is shown by analytical approximation of the kinetic curves for fibrinogen adsorption onto an unmodified gold surface studied by a surface plasmon resonance biosensor that this model is in good quantitative agreement with experiments. It is found that the rate of adsorption, controlled mainly by the mass flow from the solution, determines the contribution both to self-assembling and spreading, resulting in variations of adsorbed fibrinogen interfacial structures.     

Effect of diolein on hydrolysis of phosphatidylcholine by phospholipase C from Clostridium perfringens.

The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.     

固定化纤维二糖酶的研究

黑曲霉 (AspergillusnigerLORRE 0 12 )的孢子中富含纤维二糖酶 ,将这些孢子用海藻酸钙凝胶包埋后 ,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定 ,半衰期为 38d ,耐热性和适宜的pH范围均比固定化前有所增加 ,其Km 和Vmax值分别为 6 .0 1mmol L和 7.0 6mmol (min·L)。利用固定化纤维二糖酶重复分批酶解10g L的纤维二糖 ,连续 10批的酶解得率均可保持在 97%以上 ;采用连续酶解工艺 ,当稀释率为 0 .4h- 1 ,酶解得率可达 98.5 %。玉米芯经稀酸预处理后 ,其纤维残渣用里氏木霉 (Trichodermareesei)纤维素酶降解 ,酶解得率为6 9.5 % ;通过固定化纤维二糖酶的进一步作用 ,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除 ,酶解得率提高到 84.2 % ,还原糖中葡萄糖的比例由 5 3 .6 %升至 89.5 % ,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

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.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

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.     

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.     

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.