Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres.     

Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and β-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both β-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on β-Glucosidase activity. However, these sugars were shown to have significant inhibitory effects on cellulase activity during cellulose hydrolysis. Our study suggests that high-substrate consistency hydrolysis with supplementation of hemicellulose is likely to be a practical solution to minimizing end-product inhibition effects while producing hydrolysate with high glucose concentration.     

Enzymatic hydrolysis of steam-exploded and ethanol organosolv-pretreated douglas-fir by novel and commercial fungal cellulases

Softwood residues are the most abundant feedstock available for bioconversion in many northern countries. However, the high costs for delignification and enzymatic hydrolysis currently deter commercialization of softwood bioconversion processes. This study evaluates the abilities of two novel fungal preparations (MSUBC1 and MSUBC2) and two commercial cellulase preparations (TR1 and TR2) to hydrolyze cellulose in Douglas-fir pretreated by steam explosion or ethanol organosolv process. MSUBC1 showed significantly better performance than the other preparations on both lignocellulosic substrates. In particular, MSUBC1 achieved >76% cellulose conversion for hydrolysis of steam-exploded Douglas-fir (～44% lignin) after 72 h at low enzyme loading (10 filter paper units/g of cellulose) and without β-glucosidase supplementation.     

Utilization of waste cellulose—III. Comparative study of the activity of the cellulases of Trichoderma viride and Aspergillus niger towards different cellulosic substrates

Filter paper, carboxymethylcellulase and β-glucosidase activities have been determined and compared for cellulases originating from Trichoderma viride (TV) and Aspergillus niger (AN). The formation of glucose and of total reducing sugar has been measured as a function of time for the hydrolysis of cellulose I by the same quantity of FP units from TV, AN or a mixture of both strains. Long term efficiency is lower for AN but an important synergistic effect has been observed for the mixture of the enzymes. This synergistic action has been assigned to a better balance of endo- and exoglucanases and essentially to the addition to TV of thermally stable endoglucanases from AN. The β-glucosidases formed in large quantity by AN have been found to be thermally unstable and susceptible to product inhibition. They do not play any role in the observed synergistic action.     

Enzymatic activity of cellulase adsorbed on cellulose and its change during hydrolysis

Hydrolysis of pure cellulose Avicel has been carried out, using Meicelase from Trichoderma viride, where the enzymatic activity of cellulase adsorbed on cellulose and its changes during the hydrolysis were investigated. A rapid drop of the hydrolysis rate during the reaction, that is always observed in enzymatic hydrolysis of cellulose, could be explained by a decline of specific activity of adsorbed enzyme, and it was implied that the decline results from a loss of synergistic action between endoglucanase and exoglucanase. An empirical equation expresses the change of hydrolysis rate during the reaction and also shows that the change of the hydrolysis rate is caused by the decline of the specific enzymatic activity of adsorbed enzyme.     

Effects of dilute acid and steam explosion pretreatments on the cellulose structure and kinetics of cellulosic fraction hydrolysis by dilute acids in lignocellulosic materials

Worldwide, yellows diseases impact plants important in human nutrition, the natural environment, and the culture and commerce of humans. Since the presumed pathogens, mycoplasma-like organisms (MLOs), have not been isolated in pure culture in vitro, their study must proceed by other experimental approaches. In a study of disease affecting grapevines in Europe and North America, polymerase chain reactions (PCR) and restriction analyses of PCR-amplified DNA were used to detect and differentiate strains of MLOs associated with grapevine yellows. MLOs were detected both in naturally diseased grapevines and in experimentally inoculated host plants. The data indicated an unexpected genomic diversity among grapevine-infecting MLOs, and supported their classification with MLOs in the aster yellows, X-disease, and elm yellows groups. The presence of diverse MLOs in grapevines provokes consideration that these MLOs may be present in overlapping geographic ranges and that multiple MLO infections may occur in individual plants, increasing the complexity of grapevine yellows epidemiology and control and the significance of sensitive MLO detection in planting stock and phytosanitary-regulated germplasm.     

Determination of the nitrogen content of nitrocellulose from smokeless gunpowders and collodions by alkaline hydrolysis and ion chromatography

In this work, a method to determine the nitrogen content of nitrocellulose from gunpowders and collodions is proposed. A basic hydrolysis of nitrocellulose with 1.0% (m/v) NaOH at 150 °C during 30 min was carried out for nitrocellulose from gunpowders (after its previous isolation by a protocol optimized by our research group) and from collodion samples. The concentration of nitrate and nitrite ions in the hydrolysate was determined by ion chromatography with suppression and conductimetric detection. The nitrogen content of nitrocellulose was calculated from the values of the concentration of both ions. The quantitative method was evaluated in terms of selectivity, sensitivity, robustness, limits of detection and quantification, and precision, measured as repeatability and intermediate precision. These parameters were good enough to demonstrate the validity of the method and its applicability to the determination of the nitrogen content of nitrocellulose contained in different types of gunpowders (single- and double-base gunpowders, manufactured from 1944 to 1997) and in commercial collodion samples. For gunpowders, the nitrogen content determined with the optimized method was compared with the values reported by the official label of the ammunition (obtained by a digestion/titration method) and errors, by defect, ranging from 1% to 15.2% (m/m) were calculated. The highest errors were obtained for the oldest gunpowders and could be attributed to the loss of nitro groups in the nitrocellulose molecule during aging. For collodion samples, errors could not be calculated since the real nitrogen content for these samples was not given in the label. In addition, the analysis time (2 h for nitrocellulose isolation, 1.5 h for nitrocellulose hydrolysis, and 0.2 h for chromatographic separation) was about 10 times lower than in the digestion/titration method nowadays used for gunpowder samples.     

Effect of chemical compounds on amino acid content of some Fusarium species and its significance to fungal chemotaxonomy

The cellular amino acid profiles of nine species of Fusarium; namely, Fusarium anthophilum, Fusarium avenaceum, Fusarium cerealis, Fusarium graminearum, Fusarium graminum, Fusarium oxysporum f. sp. conglutinans, Fusarium pseudograminearum, Fusarium roseum, and Fusariumsacchari var. elongatum growing on malt extract medium were determined. The amino acid profiles of the investigated fungi were varied and could be used for identification and characterisation of certain Fusarium species. Addition of certain chemical compounds including aspartic acid, glutamic acid, methionine, selenium, and urea to the growth medium affected the amino acid profiles. However, susceptibility of amino acid content to environmental conditions increased the variation of amino acid profiles among all the investigated Fusarium species. Some amino acids were only produced when certain chemical compounds were added to the growth medium. Valine was produced by F. anthophilum only in the presence of aspartic acid or selenium, while serine was produced in the presence of aspartic acid, glutamic acid, or methionine. Also, cysteine was produced by F. avenaceum in the presence of glutamic acid or urea. F. cerealis produced tryptophan only in the presence of aspartic acid or urea, while F. graminearum produced leucine in glutamic, methionine or urea. Similarly, many different amino acids were produced by each Fusarium species only in the presence of certain chemical compounds. The results revealed that the amino acid profiles will be more useful for characterisation and identification of fungi if they are determined under different conditions.     

Comparative hydrolysis analysis of cellulose samples and aspects of its application in conservation science

Cellulose - Knowledge about the carbohydrate composition of pulp and paper samples is essential for their characterization, further processing, and understanding the properties. In this study, we...     

Rapid flow injection spectrophotometric determination of monofluorophosphates in toothpastes after on-line hydrolysis by alkaline phosphatase immobilized on a cellulose nitrate membrane

A new, rapid flow injection (FI) method is reported for the spectrophotometric determination of monofluorophosphate (MFP) ions in toothpastes. MFP ions are hydrolyzed on-line by alkalinephosphatase (APase) immobilized on a cellulose nitrate membrane, prior to injection in the FI system. The yielded orthophosphate ions are determined spectrophotometrically (lambda(max) = 690 nm) using the molybdenum blue approach. The chemical and FI variables that affected the enzymatic reaction were studied and optimized. A study of interferences was also carried out. The proposed method is very precise (s(r) = 0.7% at 1.0 x 10(-4) mol l(-1) MFP, n = 12), fast (sampling rate of 72 h(-1)) and allows the determination of MFP ions in the range of 4.0 x 10(-5) to 6.0 x 10(-4) mol l(-1) with a satisfactory 3sigma detection limit of 4.0 x 10(-6) mol l(-1). The application of the proposed FI method to toothpaste samples yielded accurate results (e(r) < 2.0%) compared with a potentiometric reference procedure.     

Kinetics of the alkaline hydrolysis of isatin and N-methylisatin in water and water-N,N-dimethylacetamide mixtures

The kinetics of amide bond cleavage of isatin and N-methylisatin in the presence of N,N-dimethylacetamide (DMA) was followed spectrophotometrically in the range of solvent composition (0–48.53 wt.%) and temperatures (40–70 °C) using piperidine as a nucleophile. The reaction was studied under pseudo-first-order kinetics. The rate of the reaction decreases largely with increasing organic solvent content. The thermodynamic activation parameters were calculated and discussed in terms of solvation of the activated complex. No linearity was observed between log rate constant and the reciprocal dielectric constant for the solvent used suggesting that there is a selective solvation by higher polar solvent (water). Finally, a mechanism for the ring opening for isatin and N-methylisatin was proposed.     

A DSC study of the effect of ascorbic acid on bound water content and distribution in chitosan-enriched bread rolls during storage

Changes of tightly and loosely bound water relative content in bread were studied using differential scanning calorimetry method. Combination of chitosan with ascorbic acid changes water redistribution between starch and gluten and in such a way can be related to bread quality and sensory properties. The decrease of the water vaporization temperatures, melting temperatures and enthalpies in fresh bread containing chitosan were detected when ascorbic acid was added in combination with chitosan. The decrease of melting peak temperature has been attributed to the increase of interaction of loosely bound water and the decrease of vaporization peak temperature has been attributed to the decrease of interaction of tightly bound water with bread components as a result of ascorbic acid addition. Freezable water amount and total water amount in crumb decrease more rapidly during first stage of staling and more slowly at the second stage of staling in the bread nutritionally fortified with chitosan.     

Computational and NMR analyses for the identification of bound water molecules in ribonuclease T1.

A structural characterization of bound water molecules in ribonuclease T1 (RNase T1) was carried out by nuclear magnetic resonance spectroscopy and molecular dynamics simulation. Amide protons of residues Trp59, Leu62, Tyr68 and Phe100 were found to cross-relax with protons of bound waters. Molecular dynamics simulations of the 120 water molecules observed in the free form of the crystal structure indicate that these amide protons donate hydrogen bonds to the less mobile water molecules. Hydrogen-bonded chains of the water molecules that are identified in the simulation study are located in the hairpin-like loop of RNase T1, comprising residues 62 to 76. The temperature factors of the observed water molecules in the crystal structure are very low, indicating that these bound waters are intrinsic components of RNase T1.     

Sensitive determination of dissolved tryptophan in freshwater by alkaline hydrolysis and HPLC.

A sensitive method involving alkaline hydrolysis and HPLC analysis has been developed to detect nanomolar levels of tryptophan dissolved in freshwater. The procedure includes 1) alkaline hydrolysis with nitrogen atmosphere in Teflon-lined Pyrex tubes with 4.2 M sodium hydroxide at 110 degrees C for 16 h, in which ascorbic acid was used as an antioxidant; 2) a determination step, in which tryptophan was separated efficiently from other amino acids by HPLC and measured by fluorescence. The mean recovery for tryptophan standard was 91.4% with an RSD of 3.3%. Using this method, dissolved tryptophan concentrations ranged from 10.91 to 41.83 nM in water samples from Lake Biwa, Japan.     

Stability and efficiency improvement of ASA in internal sizing of cellulosic paper by using cationically modified cellulose nanocrystals

Alkenyl succinic anhydride (ASA) is a reactive sizing agent that can impart good water repellence to paper by decreasing the wettability of the cellulose fibers. However, ASA can undergo hydrolysis, which is detrimental to the ASA sizing efficiency. In order to improve the ASA emulsion stability and ASA sizing efficiency, we used cationically modified cellulose nanocrystals (CNCs) to stabilize the cationic starch-emulsified ASA. Transmission electron microscope observation revealed that ASA droplets were well shielded by both the cationic CNCs and cationic starch, which may be responsible for the improved stabilization of ASA. The Hercules size test sizing degree, contact angle and particle size measurements demonstrated that cationic CNCs–ASA sized paper exhibited improved results in comparison with the control (without cationic CNCs under otherwise the same conditions). Furthermore, the resulting cationic CNCs–ASA system can improve the tensile index and burst index of the sized paper.