Investigation of an “alternate water supply system” in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation

Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross‐correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water‐supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β‐sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond.     

密粘褶菌胞外低分子量多肽在纤维素降解中作用的研究

从褐腐真菌中能强烈降解纤维素的代表菌株密粘褶菌(Gloeophyllum trabeum)的胞外酶液中首次分离纯化得到一低分子量的活性多肽组分(称作Gt因子),此组分能在有O.2和Fe³⁺存在时产生羟基自由基HO·;对纤维素降解的研究表明,Gt因子不同于纤维素酶对纤维素的β1.4糖苷键的水解作用,而以HO·氧化的途径作用于纤维素,导致纤维素中氢键的断裂,降低纤维素的结晶度,使其暴露出更多的末端,从而有利于纤维素的进一步降解。     

Computer simulation studies of microcrystalline cellulose Ibeta

Molecular mechanics (MM) simulations have been used to model two small crystals of cellulose Ibeta surrounded by water. These small crystals contained six different extended surfaces: (110), (11 0), two types of (100), and two types of (010). Significant changes took place in the crystal structures. In both crystals there was an expansion of the unit cell, and a change in the gamma angle to almost orthogonal. Both microcrystals developed a right-hand twist of about 1.5 degrees per cellobiose unit, similar to the twisting of beta-sheets in proteins. In addition, in every other layer, made up of the unit cell center chains, a tilt of the sugar rings of 14.8 degrees developed relative to the crystal plane as a result of a transition of the primary alcohol groups in these layers away from the starting TG conformation to GG. In this conformation, these groups made interlayer hydrogen bonds to the origin chains above and below. No change in the primary alcohol conformations or hydrogen-bonding patterns in the origin chain layers was observed. Strong localization of the adjacent water was found for molecules in the first hydration layer of the surfaces, due to both hydrogen bonding to the hydroxyl groups of the sugar molecules and also due to hydrophobic hydration of the extensive regions of nonpolar surface resulting from the axial aliphatic hydrogen atoms of the 'tops' of the glucose monomers. Significant structuring of the water was found to extend far out into the solution. It is hypothesized that the structured layers of water might present a barrier to the approach of cellulase enzymes toward the cellulose surfaces in enzyme-catalyzed hydrolysis, and might inhibit the escape of soluble products, contributing to the slow rates of hydrolysis observed experimentally. Since the water structuring is different for the different surfaces, this might result in slower hydrolysis rates for some surfaces compared to others.     

Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum in biodegradation of cellulose

A special low-molecular-weight peptide named Gt factor, was isolated and purified via HPLC from the culture extract of the brown-rot fungus Gloeophyllum trabeum. It had high-affinity Fe(3+)-chelating ability and could reduce Fe(3+) to Fe(2+). In the presence of O(2), it could produce hydroxyl radicals HO*. The effects of Gt factor on cellulose degradation suggested that Gt factor could disrupt inter- and intra- hydrogen bonds in cellulose chains by a HO*-involved mechanism. This resulted in depolymerization of cellulose chains, which produced more reducing and non-reducing ends, thus making cellulose accessible for further degradation. This pathway was quite different from the hydrolytic processes driven by cellulases, and Gt factor might play an important role in the early stage of cellulose depolymerization by brown-rot fungi.     

Real‐time detection of the morphological change in cellulose by a nanomechanical sensor

Up to now, experimental limitations have prevented researchers from achieving the molecular‐level understanding for the initial steps of the enzymatic hydrolysis of cellulose, where cellulase breaks down the crystal structure on the surface region of cellulose and exposes cellulose chains for the subsequent hydrolysis by cellulase. Because one of these non‐hydrolytic enzymatic steps could be the rate‐limiting step for the entire enzymatic hydrolysis of crystalline cellulose by cellulase, being able to analyze and understand these steps is instrumental in uncovering novel leads for improving the efficiency of cellulase. In this communication, we report an innovative application of the microcantilever technique for a real‐time assessment of the morphological change of cellulose induced by a treatment of sodium chloride. This sensitive nanomechanical approach to define changes in surface structure of cellulose has the potential to permit a real‐time assessment of the effect of the non‐hydrolytic activities of cellulase on cellulose and thereby to provide a comprehensive understanding of the initial steps of the enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2010;107: 190–194. © 2010 Wiley Periodicals, Inc.     

Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase

An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with trypto-phan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cell     

Molecular directionality in cellulose polymorphs

The recently developed technique of reductive amination, followed by gold labeling, was applied to visualize the reducing ends of cellulose microcrystals from cellulose I, cellulose II, and cellulose III(I). In these crystals, which were also characterized by electron diffraction, the labeling proved that the chains were organized in a parallel fashion in cellulose I from ramie and Valonia and also in cellulose III(I) from Valonia. In microcrystals of cellulose II from mercerized ramie, the labeling method showed that the chains were packed into an antiparallel mode. These results are discussed in terms of the fine structure of cellulose I where neighboring microfibrils of opposite polarity are visualized. The mercerization process whereby cellulose I is converted into cellulose II is therefore best described in terms of an intermingling of the cellulose chains from neighboring microfibrils of opposite polarity. As opposed to the case of mercerization the conversion of cellulose I into cellulose III(I) does not require the participation of neighboring microfibrils since the crystalline domains are converted individually.     

Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase

An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with tryptophan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cellulose can hardly proceed.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by steam pretreatment impregnated with hydrogen peroxide

Sugarcane bagasse was subjected to steam pretreatment impregnated with hydrogen peroxide. Analyses were performed using 2³ factorial designs and enzymatic hydrolysis was performed at two different solid concentrations and with washed and unwashed material to evaluate the importance of this step for obtaining high cellulose conversion. Similar cellulose conversion were obtained at different conditions of pretreatment and hydrolysis. When the cellulose was hydrolyzed using the pretreated material in the most severe conditions of the experimental design (210°C, 15 min and 1.0% hydrogen peroxide), and using 2% (w/w) water‐insoluble solids (WIS), and 15 FPU/g WIS, the cellulose conversion was 86.9%. In contrast, at a milder pretreatment condition (190°C, 15 min and 0.2% hydrogen peroxide) and industrially more realistic conditions of hydrolysis (10% WIS and 10 FPU/g WIS), the cellulose conversion reached 82.2%. The step of washing the pretreated material was very important to obtain high concentrations of fermentable sugars. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The mechanism of dextransucrase action: Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modified dextran and the nonexistence of the primer requirement for the synthesis of dextran

Streptococcus mutans OMZ 176 was grown in a sucrose-free medium containing fructose as a carbohydrate source. Dextransucrase was precipitated from the culture supernatant with 40% saturated ammonium sulfate. The activity of dextransucrase was shown to be stimulated by exogenous dextran. Maximum activity was reached when the concentration of exogenous dextran was 2 mg/ml. Dextrans modified at the nonreducing ends by reaction with tripsyl chloride and/or by hydrolysis with an exodextranase also activated dextransucrase four to six times over that of a control. The exodextranasemodified dextrans have nonreducing chains that are very short in comparison with unmodified dextran and the tripsyl-modified dextrans have chains that are blocked at the nonreducing ends with a triisopropylbenzenesulfonyl group on the C₆ hydroxyl group. Because the nonreducing ends of the modified dextrans are not available for reaction, the activation of dextransucrase by these modified dextrans cannot be due to primer reactions with the nonreducing ends. The activation of dextransucrase, thus, must be by an alternate mechanism. Two alternative mechanisms discussed are an allosteric effect and nucleophilic displacement reactions by the added dextran. It was also found that the addition of increasing amounts of dextran shifted the synthesis from an insoluble dextran to a soluble dextran.     

Mechanism of substrate inhibition in cellulose synergistic degradation.

A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.     

Solid-state hydrolysis of cellulose and methyl alpha- and beta-D-glucopyranosides in presence of magnesium chloride

Solid-state hydrolysis proceeded with cellulose and methyl alpha- and beta-D-glucopyranosides in the presence of hydrated magnesium chloride. This reaction was effective even at >100 degrees C since the hydrated water, which is held by MgCl(2) up to >200 degrees C, is utilized as a nucleophile. Excess water made this reaction ineffective due to the competition between water and sugar oxygen atoms in coordinating with Mg(2+), a Lewis acid. Consequently, this hydrolysis reaction is characteristic of solid-state reactions.     

Structure of a cellulose I–ethylenediamine complex

The structure of a crystalline cellulose I–ethylenediamine complex has been determined by x-ray diffraction methods as part of an investigation of cellulose–solvent interaction. The complex studied is that formed when native ramie fibers are swollen in ethylenediamine and then vacuum-dried. The unit cell is monoclinic with dimensions a = 12.87 Å, b = 9.52 Å, c = 10.35 Å, and γ = 118.8°, and it contains disaccharide segments of two chains, with one ethylenediamine per glucose residue. The refined model contains parallel cellulose chains that are linked by hydrogen-bonded ethylenediamine molecules. The chains along the b-axis are packed in register, leading to stacks of chains analogous to those in chitin. All the hydroxyl groups are satisfactorily hydrogen-bonded and each ethylenediamine forms four donor and two acceptor hydrogen bonds. From this work it can be seen that the interaction of cellulose I with ethylenediamine involves scission of the intermolecular hydrogen bonds followed by disruption of the stacks of quarter-staggered chains.     

Structure of a cellulose II-hydrazine complex

The structure of a crystalline cellulose II-hydrazine complex has been determined by x-ray diffraction methods as part of an investigation of cellulose-solvent interaction. The complex studied was that formed when Fortisan fibers were swollen in hydrazine and then vacuumdried. The unit cell is monoclinic with dimensions a = 9.37 Å, b = 19.88 Å, c = 10.39 Å, and γ = 120.0° and contains disaccharide segments of four chains, with one hydrazine per glucose residue. In view of the limited x-ray intensity data, the structure has been determined based on an approximate unit cell containing two chain segments, with a = 4.69 Å, using the linked-atom least-squares refinement procedures. The refined model contains antiparallel cellulose chains that are linked by both intermolecular hydrogen bonds and hydrogen-bonded hydrazine molecules. The parallel chains in the 020 planes are packed in register, leading to stacks of chains analogous to those in chitin. All the hydroxyl groups are satisfactorily hydrogen-bonded, and each hydrazine forms four donor and two acceptor hydrogen bonds, including an N? H…N bond between hydrazines. From this work it can be seen that the interaction of cellulose II with hydrazine involves scission of the intermolecular hydrogen bonds followed by disruption of the stacks of quarter-staggered chains. The latter effect is probably necessary for hydrazine to act as a cellulose solvent.     

Studies on Chrysanthemic Acid

(±) -trans-2,2-Dimethyl-3- (2′-methyl-2′-propenyl) cyclopropan-l-carboxylic acid (VII) was obtained by the treatment of (±) -pyrocin (IV) with thionyl chloride and absolute ethanol saturated with dry hydrogen chloride followed by the cyclization action of sodium tert-amylate in dry benzene and alkaline hydrolysis. This was converted into (±) -trans-chrysanthemic acid (VIII) by the catalytic action of p-toluenesulfonic acid.     

Conformational features of crystal-surface cellulose from higher plants

Native cellulose in higher plants forms crystalline fibrils a few nm across, with a substantial fraction of their glucan chains at the surface. The accepted crystal structures feature a flat-ribbon 21 helical chain conformation with every glucose residue locked to the next by hydrogen bonds from O-3' to O-5 and from O-2 to O-6'. Using solid-state NMR spectroscopy we show that the surface chains have a different C-6 conformation so that O-6 is not in the correct position for the hydrogen bond from O-2. We also present evidence consistent with a model in which alternate glucosyl residues are transiently or permanently twisted away from the flat-ribbon conformation of the chain, weakening the O-3' - 0-5 hydrogen bond. Previous molecular modelling and the modelling studies reported here indicate that this 'translational' chain conformation is energetically feasible and does not preclude binding of the surface chains to the interior chains, because the surface chains share the axial repeat distance of the 21 helix. Reduced intramolecular hydrogen bonding allows the surface chains to form more hydrogen bonds to external molecules in textiles, wood, paper and the living plant.     

Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step

Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.