Exopolysaccharide production by a marine cyanobacterium Cyanothece sp.

The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBHI. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.     

Reactivities of cellulases from fungi towards ribbon-type bacterial cellulose and band-shaped bacterial cellulose

We have investigated the reactivities of various cellulases onribbon-type bacterial cellulose (BC I) and band-shaped bacterial cellulose (BCII) so as to clarify the properties of different cellulases. BC I waseffectively hydrolyzed by exo-type cellulases from different fungi from twicetofour times as much as BC II, but endo-type cellulases showed little differencein reactivity on those substrates. One of the endo-type cellulases, EG II fromTrichoderma reesei, degraded BC II more rapidly thanexo-type cellulases even in the production of reducing sugars. The degree ofpolymerization (DP) of BC II was rapidly decreased by endo-type cellulases atanearly stage, while exo-type cellulases did not cause the decrease of DP atthe initial stage, though the decrease of DP was observed after an incubation of24 h. All exo-type cellulases adsorbed on BC I and BC II,whileendo-type cellulases except for EG II adsorbed slightly on both substrates. Itwas interesting to observe EG II adsorbed on BC I but not on BC II. It issuggested that the adsorption of enzyme on cellulose is important for thedegradation of BC I, but not for BC II. It is proposed that the ratio of aspecific activity of each enzyme between BC I and BC II represents thedifference in the mode of action of cellulase. Furthermore, the K _RW value, which we can calculate from thedecrease of DP/reducing sugar produced, is effective for discriminating themode of action of cellulase, especially the evaluation of randomness in thehydrolysis of cellulose by endo- and exo-type cellulases.     

Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.     

The effect of Trichoderma reesei cellulases and hemicellulases on the paper technical properties of never-dried bleached kraft pulp

Four purified cellulases, a xylanase and mannanase from Trichoderma reesei were used to treat never-dried bleached pine kraft pulp prior to refining, and the effects on pulp properties were evaluated. The enzymatic treatments hydrolysed up to 0.8% of pulp dry weight. The results demonstrated that the individual cellulases have profoundly different modes of action in modifying pulp carbohydrates. This is especially clear when comparing their effects at the same level of hydrolysis. Pretreatment with cellobiohydrolases I (CBH I) and II (CBH II) had virtually no effect on the development of pulp properties during refining, except for a slight decrease in strength properties. On the contrary, endoglucanase I (EG I) and endoglucanase II (EG II) improved the beatability of the pulp as measured by Schopper--Riegler value, sheet density and Gurley air resistance. Of the endoglucanases, EG II was most effective in improving the beating response. The combinations of CBH I with EG I and EG II had similar effects on the pulp properties as the endoglucanases alone, although the amount of hydrolysed cellulose was increased. Pretreatments with xylanase or mannanase did not appear to modify the pulp properties. The same enzyme treatments which improved the beatability, however, slightly impaired the pulp strength, especially tear index at the enzyme dosages used. When compared at a given level of cellulose hydrolysis, the negative effect of EG II on strength properties was more pronounced compared with EG I. Thus, the exploitation of cellulases for fibre treatments requires careful optimization of both enzyme composition and dosage. Since the endoglucanases had no positive effect on the development of tensile strength, it is suggested that the explanation for the increased beating response is increased fibre breakage and formation of fines, rather than improved flexibilization. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hydrolysis of steam-pretreated lignocellulose

The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose. Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures. Adsorption isotherms were determined at 4 and 40 degrees C during 90-min reaction times. Both CBH I and EG II adsorbed stronger at 40 than at 4 degrees C. The time course of adsorption and hydrolysis, 3 min to 48 h, was studied at 40 degrees C. About 90% of the cellulases were adsorbed within 2 h. The hydrolysis rate was high in the beginning but decreased during the time course. Based on adsorption data, the hydrolysis and synergism were analyzed as function of adsorbed enzyme. CBH I showed a linear correlation between hydrolysis and adsorbed enzyme, whereas for EG II the corresponding curve leveled off at both 4 and 40 degrees C. At low conversion, below 1%, EG II produced as much soluble sugars as CBH I. At higher conversion, CBH I was more efficient than EG II. The synergism as function of adsorbed enzyme increased with bound enzyme before reaching a stable value of about 2. The effect of varying the ratio of CBH I:EG II was studied at fixed total enzyme loading and by changing the ratio between the enzymes. Only a small addition (5%) of EG II to a CBH I solution was shown to be sufficient for nearly maximal synergism. The ratio between EG II and CBH I was not critical. The ratio 40% EG II:60% CBH I showed similar conversion to 5% EG II:95% CBH I. Modifications of the conventional endo-exo synergism model are proposed.     

Isotherms for adsorption of cellobiohydrolase I and II fromtrichoderma reesei on microcrystalline cellulose

Adsorption to microcrystalline cellulose (Avicel) of pure cellobiohydrolase I and II (CBH I and CBH II) fromTrichoderma reesei has been studied. Adsorption isotherms of the enzymes were measured at 4‡C using CBH I and CBH II alone and in reconstituted equimolar mixtures. Several models (Langmuir, Freundlich, Temkin, Jovanovic) were tested to describe the experimental adsorption isotherms. The isotherms did not follow the basic (one site) Langmuir equation that has often been used to describe adsorption isotherms of cellulases; correlation coefficients (R²) were only 0.926 and 0.947, for CBH I and II, respectively. The experimental isotherms were best described by a model of Langmuir type with two adsorption sites and by a combined Langmuir-Freundlich model (analogous to the Hill equation); using these models the correlation coefficients were in most cases higher than 0.995. Apparent binding parameters derived from the two sites Langmuir model indicated stronger binding of CBH II compared to CBH I; the distribution coefficients were 20.7 and 3.7 L/g for the two enzymes, respectively. The binding capacity, on the other hand, was higher for CBH I, 1.0 Μmol (67 mg) per gram Avicel, compared to 0.57 Μmol/g (30 mg/g) for CBH II. The isotherms when analyzed with the combined Langmuir-Freundlich model indicated presence of unequal binding sites on cellulose and/or negative cooperativity in the binding of the enzyme molecules.     

A new thermostable endoglucanase,Acidothermus cellulolyticus E1

A new thermostable endoglucanase,Acidothermus cellulolyticus E1, and another bacterial endoglucanase, E₅ fromThermomonospora fusca, each exhibit striking synergism with a fungal cellobiohydrolase (Trichoderma reesei CBH I) in the saccharification of microcrystalline cellulose. In neither case did the ratio of endoglucanase to exoglucanase that demonstrated maximum synergism coincide exactly with the ratio that actually released the maximum quantity of soluble sugar for a given total cellulase loading. The difference between the two ratios, after significant hydrolysis of the substrate, was considerably larger in the case ofA. cellulolyticus E1. For both endoglucanase pairings with CBH I, the offset between the ratio for maximum synergism and the ratio for maximal soluble sugar production was found to be a function of digestion time.     

Determination of the <Superscript>13</Superscript>C chemical shift anisotropies of cellulose I and cellulose II

The chemical shift anisotropies (CSAs) of cellulose I and I, the two crystalline constituents of bacterial cellulose produced by Acetobacter xylinum (DSM 14666), and regenerated cellulose II are reported for each of the spectroscopically resolved carbon resonances using the phase adjusted spinning sideband (PASS) experiment. The data are compared with experimental results using the recoupling of anisotropy information (RAI) technique and with theoretical calculations of the structure of cellulose, including the hydrogen bonding systems.     

TEM study of band-like cellulose assemblies produced by Acetobacter xylinum at 4 °C

Two kinds of band-like cellulose assemblies, coarse and densestructures, are produced in Hestrin–Schramm medium at 4 °Cusing smooth colonies isolated from Acetobacter xylinumATCC23769, whereas ribbon cellulose assemblies are produced by the samecoloniesat 28 °C. The dense and the coarse band-like assembliesconsist of many strand-like cellulose entities (strands) and areextruded perpendicularly to the long axis of the bacterial cell. In an earlystage of incubation at 4 °C, the dense band-like assembly isproduced and the number of strands decreases gradually with increasingincubation time at 4 °C, probably because the number of activeTC subunits decreases as a result of the low-temperature shock for thebacteria.In contrast, the coarse band-like assembly is clearly observed after about 1h of incubation at 4 °C. The number of strands inthe coarse band-like assembly is about one third of that of the dense band-likeassembly and does not change during the incubation time of about 6h. In the selected-area electron diffraction (ED) experiment, thedense band-like assembly gives crystalline reflections corresponding to thecellulose II type crystal, while the coarse band-like assembly does not giveanycrystalline reflections under the same ED conditions.     

Thermal denaturation ofTrichoderma reesei cellulases studied by differential scanning calorimetry and tryptophan fluorescence

The thermal denaturation of four purified Trichoderma reesei cellulase components, cellobiohydrolase (CBH) I, CBH II, endoglucanase (EG) I, and EG II, has been monitored using a combination of classical temperature/activity profiles, differential scanning calorimetry (DSC), and thermal scanning fluorescence emission spectrometry. Significant correlations were found between the results of enzyme activity studies and the results obtained through the more direct physical approaches, in that both DSC and the activity studies showed EG II (T_m = 75°C) to be much more thermostable (by 10–11 °C) than the other three enzymes, all three of which were shown by both activity profiles and DSC to be very similar in thermal stability. The temperature dependence of the wavelength of maximum tryptophan emission showed a parallel result, with the three enzymes exhibiting less thermostable activity being grouped together in this regard, and EG II differing from the other three in maintaining a less-exposed tryptophan microenvironment at temperatures as high as 73 °C. The DSC results suggested that at least two transitions are involved in the unfolding of each of the cellulase components, the first (lower-temperature) of which may be the one correlated with activity loss.

     

Interaction of cellulose with amine oxide solvents

Cellulose I, mainly as ramie or as Avicel microcrystalline cellulose, has been monitored by optical microscopy and by ¹³C CPMAS NMR, over the course of its dissolution in hot N-methylmorpholine N-oxide solvent. Its interaction with the near-solvent N-ethylmorpholine N-oxide and related non-solvents has also been investigated. NMR shows that N-methylmorpholine N-oxide partly converts crystalline cellulose I into amorphous solid cellulose. The changes in chemical shift imply increased flexibility at the glycosidic bonds. In contrast, N-ethylmorpholine N-oxide converts cellulose I to cellulose III_I, without dissolution. Microscopy shows that the ramie fibres swell laterally, and at least some also shorten longitudinally, during dissolution. Model studies using methyl--d-glucopyranose show no evidence from ¹³C chemical shifts for different modes of binding with different solvents. However, N-methylmorpholine N-oxide binds more strongly to methyl--d-glucopyranose in DMSO than does N-ethylmorpholine N-oxide, whereas N-ethylmorpholine N-oxide binds better to H₂O. Also, ¹³C T ₁ values for aqueous cellobioside show increasing rotational freedom of the –CH₂OH sidechains as N-methylmorpholine N-oxide is added. Together, these observations imply the initial penetration of solvents and near-solvents between the molecular cellulose sheets. Subsequently, N-methylmorpholine N-oxide breaks H-bonds, particularly to O-6, just sufficiently to loosen individual chains and then dissolve the sheets.     

Conversions between ordered and disordered cellulose. Effects of mechanical treatment followed by cyclic wetting and drying

An investigation into the effects of mechanical treatment and hydration on the order of cellulose substrates (microcrystalline cellulose and Cladophora cellulose) was performed by the use of ball milling followed by cyclic wetting and drying. The results, monitored by¹³C-CP/MAS NMR-spectroscopy, were evaluated by calculation of the crystallinity indices and principal component analysis of the NMR data acquired. The results showed that a large part of the disorder induced by the mechanical treatment of cellulose by ball milling is reversible and reordering upon hydration leads to the cellulose I form initially present. The C4 signals corresponding to the reversibly disordered cellulose chains are observed in the amorphous region between 79 and 86 ppm in the¹³C-CP/MAS NMR-spectra together with signals from cellulose chains on the surface of ordered regions. The peak cluster which contains the C2, C3 and C5 ring carbons can be divided into two specific spectral regions; one between 74 and 77 ppm largely originates from ring carbons within disordered cellulose structures, and one between 70 and 74 ppm contains larger contributions from ordered cellulose. The behaviour of the celluloses upon milling is in accordance with a concept of ordered cellulose fibrils containing amorphous cellulose mainly as surface layers and induced reversible lattice distortions.     

A multivariate characterization of crystal transformations of cellulose

A spectroscopic study of cellulose transformation processes, such as alkali treatment and annealing, showed that, in combination with multivariate data analysis techniques, a detailed understanding of the crystalline transformation processes could be reached.¹³C cross-polarization magic-angle spinning (CPMAS) NMR and near-infrared (NIR) spectroscopy of cotton linters and softwood pulps analysed during the processing revealed information, after data reduction using principal components data analysis, that could be connected to structural changes of the cellulose polymorphs. The data showed that alkali treatment of cotton linters led to a cellulose conversion from cellulose I to II, while annealing, both for linters and pulps, yielded a transformation from I to I.     

Ultrastructural aspects of the acetylation of cellulose

An ultrastructural study of the acetylation of cellulose was achieved by subjecting well characterized cellulose samples fromValonia cell wall and tunicin tests to homogeneous and heterogeneous acetylation. The study involved transmission electron microscopy observations on negatively stained microcrystals as well as diffraction contrast images of the cross sections of wall fragments at various stages of the reaction. These observations showed that the acetylation of crystalline cellulose proceeds by a reduction of the diameters of the crystals while their lengths are reduced to a lower extent. These results were corroborated by electron and X-ray diffraction experiments that showed that during the reaction there was a rapid decrease in the intensities of the equatorial diffraction spots of cellulose, whereas those located on the meridian or close to the meridian stayed constant. A model of acetylation of the cellulose crystal is presented. It is based on a non swelling reaction mechanism that affects only the cellulose chains located at the crystal surface. In the case of homogeneous acetylation, the partially acetylated molecules are sucked into the acetylating medium as soon as they are sufficiently soluble. In heterogeneous conditions the cellulose acetate remains insoluble and surrounds the crystalline core of unreacted cellulose.     

Visualization of cellulase interactions with cellulose microfibril by transmission electron microscopy

In this study, we developed a method to observe interactions between cellulase and cellulose microfibril by transmission electron microscopy. Although negative staining and low-angle metal shadowing increase image contrast, neither method is sufficient to view enzyme interactions with microfibril. However, we found that the combination of negative staining and low-angle metal shadowing provided better contrast for enzyme-like particles on the microfibril. The lengths of the particles interacting with microfibrils were 7.03 and 5.05 nm, parallel and perpendicular to the fiber direction, respectively. Accounting for the additional thickness owing to metal shadowing, the particle sizes were consistent with that of CBH I from Trichoderma reesei based on a crystalline structural analysis. The combination of these electron staining techniques successfully visualized morphological changes in microfibril as well as enzymes adsorbed on it, thus demonstrating cellulase in action. These results indicate that appropriate staining techniques can be applied to extend the applications of transmission electron microscopy, which may be particularly beneficial for studies on enzymatic behavior.     

The structural changes in crystalline cellulose and effects on enzymatic digestibility

The enzymatic hydrolysis of cellulose I achieves almost complete digestion when sufficient enzyme loading as much as 20 mg/g-substrate is applied. However, the yield of digestion reaches the limit when the enzyme dosage is decreased to 2 mg/g-substrate. Therefore, we have performed three pretreatments such as mercerization, dissolution into phosphoric acid and EDA treatment. Transformation into cellulose II hydrate by mercerization and dissolution into phosphoric acid were not sufficient because substrate changed to highly crystalline structure during saccharification. On the other hand, in the case of crystalline conversion of cellulose I to III_I by EDA, almost perfect digestion was achieved even in enzyme loading as small as 0.5 mg/g-substrate, furthermore, hydrolyzed residue was typical cellulose I. The structural analysis of substrate after saccharification provides an insight into relationships between cellulose crystalline property and cellulase toward better enzymatic digestion.     

Phase diagrams and phase structures of identical and mixed chain lithium di-n-alkyl phosphate-water binary systems. Asymmetric molecular shape effect

Lithium salts of di-n-pentyl (DPP),n-butyl(n-hexyl) (BHP),n-propyl(n-hexyl) (PHP) and ethyl(n-octyl) (EOP) phosphates were synthesized and the phase diagrams of the lithium phosphate-water binary systems were determined. The phase diagrams of the DPP-, BHP- and PHP-water systems contain three regions (I, II and III) in common, which correspond to a homogeneous transparent one-phase solution, and lyotropic liquid crystalline and coagel phases, respectively. However, the EOP-H₂O system contains an additional hard gel phase (region IV). ³¹P NMR spectra suggest that region I is a monomermicelle equilibrium phase and region II is a lamellar phase. X-ray diffraction results show that for the DPP-, BHP-and PHP-water systems the twon-alkyl chains are closely packed in the lamellar phase in a manner which alternatively combines short and long chains, while in EOP-water system the two long chains are loosely packed. Furthermore, it may be assumed from³¹P NMR spectra and x-ray diffraction results that region IV in the EOP-water system is a cubic phase.Thermotropic properties for these DAP-water systems were also investigated by DSC temperature profile curves. From the H variation upon the III thermal transition, we assumed that stability of the aggregate structure in the liquid crystalline state increases in the order EOP相似文献   

Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose

This paper investigates the enzymatic hydrolysis of three main allomorphic forms of microcrystalline cellulose using different cellulases, from Trichoderma reesei and from Aspergillus niger, respectively. It was demonstrated that both the morphological and crystalline structures are important parameters that have a great influence on the course of the hydrolysis process. The efficiency of the enzymatic hydrolysis of cellulosic substrates was estimated by the amounts of reducing sugar and by the yield of the reaction. Changes in the average particle sizes of the cellulose allomorphs were determined during enzymatic hydrolysis. The accumulation of soluble sugar within the supernatant was used as a measure of the biodegradation process’s efficiency, and was established by HPLC-SEC analysis. Any modifications in the supramolecular structure of the cellulosic residues resulting from the enzymatic hydrolysis were determined by X-ray diffraction. The action of each cellulase was demonstrated by a reduction in the crystalline index and the crystallite dimensions of the corresponding allomorphic forms. The crystalline structure of allomorphic forms I and II did not suffer significant modifications, while cellulose III recorded a partial return to the crystalline structure of cellulose I. The microstructures of cellulose allomorph residues were presented using optical microscopy and scanning electron microscopy.     

Enzymatic hydrolysis of cellulose I is greatly accelerated via its conversion to the cellulose II hydrate form

Cellulose II hydrate was prepared from microcrystalline cellulose (cellulose I) via its mercerization with 5 N NaOH solution over 1 h at room temperature followed by washing with water. The structure of cellulose II hydrate changed to that of cellulose II after drying. Compared with cellulose II, cellulose II hydrate exhibited a slightly (8.5%) expanded structure only along the direction. The hydrophobic stacking sheets of the cellulose II were conserved in the cellulose II hydrate, and water molecules could be incorporated in the inflated two-chain unit cell of cellulose II hydrate. Enzymatic hydrolysis of cellulose I, cellulose II hydrate, and cellulose II was carried out at 37 °C using solutions comprising a mixture of cellulase and β-glucosidase. The hydrolysis of cellulose II hydrate proceeded much faster than the hydrolysis of the other two substrates, while the saccharification ratio of cellulose II was only slightly higher than that of cellulose I. The alkaline mercerization treatment was also applied to sugarcane bagasse. After its direct mercerization, the cellulose in bagasse was converted from cellulose I to cellulose II hydrate, and then to cellulose II after drying. Similar to in the case of microcrystalline cellulose, the rate of the enzymatic hydrolysis of the mercerized bagasse without drying (cellulose II hydrate) was much faster than the enzymatic hydrolysis of the other two substrates. Thus, the wet forms of cellulose and cellulosic biomass after mercerization, and after hydrolysis with cellulolytic enzymes, afforded superior products with extremely high degradability.