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.     

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.     

Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

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.     

Kinetic model of enzymatic hydrolysis of steam-exploded wheat straw

The hydrolysis kinetics of steam-exploded wheat straw treated with cellulase NS 50013 enzyme complex in combination with β-glucosidase NS 50010 is studied. The time dependence of the reducing sugars amount is followed at varying the temperature value and the amount of the enzyme introduced. The activation energy determined on the ground of the rate temperature dependence stays unchanged in the course of the process. The preexponential factor decreases with the increase of the degree of hydrolysis and is responsible for the process rate decrease. A new expression for the dependence of degree of hydrolysis of one of carbohydrate polymers (cellulose) in wheat straw on the time, the enzyme concentration and the temperature is obtained. It is of practical importance as well because it provides estimation of the degree of hydrolysis required at predetermined values of the temperature, the enzyme concentration and the time used. The expression can be used for control of the enzyme hydrolysis of cellulose in the wheat straw.     

Kinetics of tributyrin hydrolysis by lipase

The kinetics for the tributyrin hydrolysis using lipase (Pseudomonas fluorscenes CCRC-17015) were investigated in the liquid–liquid and liquid–solid–liquid reaction systems in a batch reactor. The lipase was covalently immobilized onto the surface of porous polymethylacrylamide (PMAA) crosslinking with N,N-methylene biacrylamide with a spacer of ethylenediamine actived by glutaraldehyde. The conditions such as tributyrin concentration, temperature, agitation, and pH value, were evaluated to achieve the optimum reaction conditions for both free lipase and immobilized lipase. The kinetic parameters in the reaction system were also obtained for two reaction systems. The turnover numbers calculated for free lipase and immobilized lipase were 29 and 5.7 s⁻¹, respectively. The parameters of k and k_m obtained using Lineweaver-Burk plot method were 26.2 mol/(mg min) and 1.35 mol/dm³ for free lipase, 5.2 mol/(mg min) and 0.2 mol/dm³ for immobilized lipase, respectively. The experimental results revealed good thermal stability, with greater stability at higher pH value for immobilized lipase in the liquid–solid–liquid reaction.     

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K_m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K_m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.     

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.     

Kinetics of p-nitrophenyl pivalate hydrolysis catalysed by cytoplasmic aldehyde dehydrogenase.

The effects of modifiers (NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol and p-nitrobenzaldehyde) on the hydrolysis of p-nitrophenyl (PNP) pivalate (PNP trimethylacetate) catalysed by cytoplasmic aldehyde dehydrogenase are reported. In each case a different inhibition pattern is obtained to that observed when the substrate is PNP acetate; for example, propionaldehyde and chloral hydrate competitively inhibit the hydrolysis of PNP acetate, but are mixed inhibitors with PNP pivalate. The kinetic results can be rationalized in terms of different rate-determining steps: acylation of the enzyme in the case of the pivalate but acyl-enzyme hydrolysis for the acetate. This is confirmed by stopped-flow studies, in which a burst of p-nitrophenoxide is observed when the substrate is PNP acetate, but not when it is the pivalate. PNP pivalate inhibits the dehydrogenase activity of the enzyme competitively with the aldehyde substrate; this is most simply explained if the esterase and dehydrogenase reactions occur at a common enzymic site.     

Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.     

Measurement of saccharification by cellulases

Techniques for screening and measurement of the cellulase complex are reviewed under qualitative or quantitative headings, with emphasis on recent methods of analysis. Appropriate substrates are considered for the constituent enzymes of the complex in the light of physical constraints on hydrolysis.     

Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells

Xyloglucan endotransglycosylases (XETs) cleave and then re-join xyloglucan chains and may thus contribute to both wall-assembly and wall-loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: "integrational" (in which a newly secreted xyloglucan reacts with a previously wall-bound one) and "restructuring" (in which one previously wall-bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in "heavy" or "light" media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml-1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2-h pulse of L-[1-3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non-radioactive walls and chased by light, non-radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall-bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall-bound non-radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall-bound [3H]xyloglucan (2-11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall-bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall-assembly and -loosening.     

Structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation

Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)₈ triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities.     

Synthesis of β-mercaptoethyl-glycosides by enzymatic reverse hydrolysis and transglycosylation

Summary The synthetic potential ofAlmond β-glucosidase andJack bean α-mannosidase in the presence of high amounts of β-mercaptoethanol as glycosyl acceptor for the synthesis of β-mercaptoethyl-glycosides was studied. The regioselectivity, O-glycosylation and/or S-glycosylation, and the stereoselectivity were analyzed with the reverse hydrolysis and the transglycosylation methods. With both enzymes, high yields of condensation are obtained without the use of chemical protective groups.     

Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis–Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37 °C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis–Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.     

Kinetic study of a thermostable beta-glycosidase of Thermus thermophilus. Effects of temperature and glucose on hydrolysis and transglycosylation reactions

A -glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttgly) has a broad substrate specificity towards -D-glucoside, -D-galactoside and -D-fucoside derivatives. The thermostability of Ttgly was exploited to study its kinetic properties within the range 25–80[emsp4 ]°C. Whatever the temperature, except around 60[emsp4 ]°C, the enzyme displayed non-Michaelian kinetic behavior. Ttgly was inhibited by high concentrations of substrate below 60[emsp4 ]°C and was activated by high concentrations of substrate above 60[emsp4 ]°C. The apparent kinetic parameters (k _cat and K _m ) were calculated at different temperatures. Both k _cat and K _m increased with an increase in temperature, but up to 75[emsp4 ]°C the values of k _cat increased much more rapidly than the values of K _m . The observed kinetics might be due to a combination of factors including inhibition by excess substrate and stimulation due to transglycosylation reactions. Our results show that the substrate could act not only as a glycosyl donor but also as a glycosyl acceptor. In addition, when the glucose was added to reaction mixtures, inhibition or activation was observed depending on both substrate concentration and temperature. A reaction model is proposed to explain the kinetic behavior of Ttgly. The scheme integrates the inhibition observed at high concentrations of substrate and the activation due to transglycosylation reactions implicating the existence of a transfer subsite.     

Production of a new sucrose derivative by transglycosylation of recombinant Sulfolobus shibatae beta-glycosidase

The gene encoding beta-glycosidase of the hyperthermophilic archaea Sulfolobus shibatae (SSG) was expressed in Escherichia coli. Recombinant SSG (referred to as rSSG hereafter) was efficiently purified, and its transglycosylation activity was tested with lactose as a donor and various sugars as acceptors. When sucrose was used as an acceptor, we found a distinct intermolecular transglycosylation product and confirmed its presence by TLC and high performance anion exchange chromatography (HPAEC). The sucrose transglycosylation product was isolated by paper chromatography, and its chemical structure was determined by 1H and 13C NMR. The sucrose transfer product was determined to be beta-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-beta-d-fructofuranoside with a galactose molecule linked to sucrose via a beta-(1-->6)-glycosidic bond.     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

纤维素酶与木质纤维素生物降解转化的研究进展

利用纤维素酶将预处理后的秸秆降解成可发酵性单糖,然后发酵生产所需的液体燃料及化工产品的技术,对于我国解决能源、环境、人口就业等难题有着巨大的积极影响。在木质纤维素生物降解转化工艺中,减少纤维素酶用量及提高酶解效率是降低木质纤维素降解成本的关键。纤维素酶系和木质纤维素酶水解技术的改进需要深入了解纤维素酶系统的组成及其协同作用、纤维素酶的结构与功能以及纤维素酶的生产技术。将就以上几个方面的研究进展进行讨论,并深入探讨了纤维素酶糖化能力的评价方法。     

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K_m (15.6260 g/L) and the rate constant of deactivation, K_de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V_max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.