Investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques

The benefits of applying cellulose selective enzymes as analytical tools for chemical structure characterization of cellulose derivatives have been frequently addressed over the years. In a recent study the high selectivity of cellulase Cel45A from Trichoderma reesei (Tr Cel45A) was utilized for relating the chemical structure to the flow properties of carboxymethyl cellulose (CMC). However, in order to take full advantage of the enzymatic hydrolysis the enzyme selectivity on the cellulose substrate must be further investigated. Therefore, the selectivity of Tr Cel45A on CMC was studied by chemical sample preparation of the enzyme products followed by mass spectrometric chemical structure characterization. The results strongly suggest that, in accordance with recent studies, also this highly selective endoglucanase is able to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (AGUs). Furthermore, the results also indicate that substituents on the nearby AGUs will affect the hydrolysis.     

Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field

The use of the intensive mass transfer reactor (IMTR) for enzymatic saccharification of cellulose, where the reaction mixture is intensively stirred by ferromagnetic particles (FMP), enhances the process rate and productivity drastically. The most significant enhancement of the process was observed when microcrystalline cellulose was used as a substrate. A concentration of sugars up to 5% was obtained after 1 h of cellulose hydrolysis using a cellulase activity level of 2 filter paper units (FPU)/mL (20 FPU/g substrate). In the hydrolysis of two types of industrial cellulosic wastes, the enhancement effects were less pronounced. Parameters related to the IMTR design, such as the shape, dimensions, and mass of FMP, as well as the magnetic field strength, strongly affected the process of hydrolysis. Among various kinds of FMP tested, the most efficient were found to be cylindrical particles (0.25 x 4 mm). In general, the hydrolysis rate enhanced when the magnetic field strength increased from 26,000 to 64,000 A/m. An optimal FMP loading existed at each level of the field strength. Hydrolyzates obtained in the IMTR under the action ofTrichoderma reesei andPenicillium verruculosum cellulases contained glucose and cellobiose as soluble products, cellobiose being predominant (> 50%). Only when a high level of extra Β-glucosidase was added to the IMTR (10 CBU/mL), did glucose made up more than 90% of the products. Owing to extreme shear conditions in the IMTR, significant enzyme inactivation took place.     

An extension of the Harano-Ooshima rate expression for enzymatic hydrolysis of cellulose to account for changes in the amount of adsorbed cellulase

Three empirical rate expressions, Kinetics I, II, and III, for the enzymatic hydrolysis of cellulose were evaluated in an effort to develop a easy-to-use rate expression. They are based on the following equation:-dV/dX = kV, where V and X are the hydrolysis rate and the fractional conversion. In Kinetic I,k is constant. In Kinetic II, a linear relatinship betweenk and t is assumed. In Kinetic III, an exponential relationship is assumed. The three expressions were applied to enzymatic hydrolysis carried out under seven different conditions in which the kinds of substrates, enzymes, and initial concentrations were varied. All of the examined rate expressions were applied to the hydrolysis with success, but the better accuracies were obtained by Kinetic III, Kinetic II, and Kinetic I in this order. The variations ofk with time found in this study, especially the exponential relationship, were consistent with the effect of the measured changes in the concentration of adsorbed enzyme as predicted by theory developed previously by Ooshima et al. (1).     

A rapid method for determining Mycobacterium tuberculosis based on a bulk acoustic wave impedance biosensor

The bulk acoustic wave impedance biosensor was set up and used to monitor the growth of Mycobacterium tuberculosis (M.TB). This sensor is rapid, simple, sensitive (lower limit is 2×10³ cells ml⁻¹) and cheap (easy to generalize). The typical response curve was different from other bacteria's, such as Escherichia coli, Staphylococcus aureus, Proteus mirabilis. The frequency detection time was used to quantitatively determine M.TB. It was proportional to logarithm of the initial concentration of M.TB in the range of 2×10³-3×10⁷ cells ml⁻¹. The set up sensor was applied to the direct diagnosis of M.TB samples. The interference of other bacteria was eliminated by pretreatment. Our results confirmed that the use of the set up biosensor was reliable, sensitive. It gives out the potential use for determining M.TB.     

Surface acoustic wave sensor system applied to the kinetic study of urease in plant seeds

A new type of the surface acoustic wave (SAW) sensor system was delivered. Ure-ase from several kinds of plant seeds was extracted with different extracting solvents. The urease activity, Michaelis constant and other kinetic parameters were estimated for the first time by means of the new device-SAW sensor system. Some factors such as pH, temperature, activators and inhibitors are also discussed. The method can be applied to the determination of urea content in human urine and the experimental results consist with those reported.     

Enzyme-based determination of cholesterol using the quartz crystal acoustic wave sensor

We have used the AT-cut quartz crystal sensor to measure in real-time the total cholesterol concentration in buffer and serum, using the trienzyme system of cholesterol esterase (ChE), cholesterol oxidase (ChOx) and horseradish peroxidase (HRP). The hydrogen peroxide produced from the ChE-ChOx reaction oxidises diaminobenzidine (DAB), in the presence of HRP. The response of the sensor to cholesterol is optimal in the presence of 0.1% (v/v) Triton X-100 at 0.2 U/ml ChOx, and 1 U/ml ChE. A response is obtained in less than 25 min. Using the optimal concentrations of the reagents, the linear range for free cholesterol and low density lipoprotein (LDL) cholesterol determination was between 50 and 300 μM, and 25 and 400 μM, respectively. It was found that the concentration of high density lipoprotein (HDL) cholesterol could not be determined because it solubilised the oxidised DAB, leading to poor adsorption at the crystal surface. We obtained a response to the use of cholesterol in serum at 300 μM, demonstrating that this biosensor could be used for cholesterol determination in clinical samples.     

Ion chromatographic determination of salicylate in human serum with a bulk acoustic wave sensor as detector

Salicylate is a hydrolysis product of salicylate-containing drugs (such as aspirin) in patients' blood. Monitoring of this ion in blood is helpful for diagnosing of overdosage of these drugs. The present paper describes an ion chromatography (IC) method developed for determination of total salicylate in human serum, in which a hulk acoustic wave (BAW) sensor was used as detector; 0.5 mmoI/L sodium carbonate (Na₂CO₃, pH 8.5) served as mobile phase. Interference in the determination was negligible. The method is simple, rapid, accurate, and precise. Serum salicylate was analyzed using both the proposed IC-BAW method and the classical Trinder spec-trophotometric method, and the results showed that the two method agreed well.     

A nonlinear regression model applied to kinetic multi-component analysis of esters with a SAW-impedance sensor

A kinetic nonlinear regression model for multi-component assay of esters was proposed based on their different alkaline-catalysed hydrolysis rate. The reaction rate was determined by monitoring the conductance change in solution with a liquid-purpose surface acoustic wave impedance sensor(SAW). The model was tested theoretically and experimentally with the mixture of methyl acetate and n-propyl acetate. The experimental detection limit of methyl acetate and n-propyl acetate (within 10 min) was 0.5 umol/L and 1.0 umol/L respectively and the recovery of the sensor system ranged from 93% to 106% (n=6).     

Bulk acoustic wave sensor for the ion chromatographic determination of copper in human plasma

By using ion chromatography with series bulk acoustic wave detection, a method for the determination of copper in human blood plasma has been developed. The advantages of the good selectivity of ion chromatography and the highly sensitive response of SBAW have been combined to improve detection limit, accuracy and reproducibility. The detection limit (3) of the method to copper is 0.3 g/ml. The relative standard deviation for the determination of 1.0 g/ml of copper is 2.1% (n=7). For the IC analysis, the analytical column is a Shim-pack IC C1 column, and the mobile phase is 4.0 mM tartaric acid/2.0 mM ethylenediamine solution with pH 4.2. This system has been applied to the determination of Cu in blood plasma from healthy people or patients with renal failure.     

Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content

Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.     

Development of a generalized phenomenological model describing the kinetics of the enzymatic hydrolysis of NaOH-treated pine wood

Pinus pinaster wood samples were treated during 3 h with alkaline solutions (containing 1, 5.5, or 10 weight percent NaOH) at 100, 115, or 130‡C using liquor/wood ratios of 6, 8, or 10 g/g. The solid residues obtained in treatments were used as substrates for enzymatic hydrolysis. In the hydrolysis assays, the reaction time (in the range 0–48 h) was considered as an operational variable. The cellulose conversion achieved at the end of assays were highest for samples pretreated at high temperature and high alkali concentration using low liquor/wood ratios. The experimental results obtained in each hydrolysis trial were fitted to an empirical model based on the assumption that the cellulose contained in substrates was composed by two fractions having different susceptibility to hydrolysis. The kinetic parameters obtained for the various experiments performed were correlated with the operational variables by means of empirical, statistically significant equations, which provided a generalized interpretation of the process.     

2D bitmapping approach for identification and quantitation of common base flavor adulterants using surface acoustic wave arrays and artificial neural network data analysis

Arrays of polymer-coated surface acoustic wave microsensors are used in conjunction with a variety of signal-processing algorithms known as artificial neural networks (ANN). This format of data analysis has the capability to characterize complex mixtures of volatile and semi-volatile organic compounds commonly found in base flavors. The approach described, which minimizes the number of training sets while retaining the robustness of an ANN, utilizes a 2D bitmap matrix. The matrix is obtained by converting the time domain kinetics of sensor response into a bitmap. The high data throughput of this approach enables quantitation on the order of ppm of common base flavor adulterants.     

A nonlinear regression model applied to kinetic studies of ester hydrolysis with a surface acoustic wave sensor

A non-linear regression model was derived for the simultaneous determination of the rate constant in alkaline hydrolysis of esters and the initial concentration of esters based on the linear relationship between the frequency response of the surface acoustic wave sensor system and the conductivity of the solution. The model was tested theoretically and experimentally with the methyl-, ethyl-, and n-propyl-acetate systems. The corresponding rate constants estimated at 25 degrees C are 0.147, 0.103 and 0.0671 respectively.     

Solvents effects on the mechanism of cellulose hydrolysis: A QM/MM study

This article reports a combined quantum mechanics/molecular mechanics (QM/MM) investigation on the acid hydrolysis of cellulose in water using two different models, cellobiose and a 40‐unit cellulose chain. The explicitly treated solvent molecules strongly influence the conformations, intramolecular hydrogen bonds, and exoanomeric effects in these models. As these features are largely responsible for the barrier to cellulose hydrolysis, the present QM/MM results for the pathways and reaction intermediates in water are expected to be more realistic than those from a former density functional theory (DFT) study with implicit solvent (CPCM). However, in a qualitative sense, there is reasonable agreement between the DFT/CPCM and QM/MM predictions for the reaction mechanism. Differences arise mainly from specific solute–solvent hydrogen bonds that are only captured by QM/MM and not by DFT/CPCM. © 2015 Wiley Periodicals, Inc.     

Frequency response to liquid density of a piezoelectric quartz crystal sensor with longitudinal wave

Elimination of longitudinal wave effect is an important aspect in the detection cell design, although such consideration is ignored in most of references. Three detection cells were designed to investigate the influence of longitudinal wave effect on the frequency response of a piezoelectric quartz crystal (PQC) to liquid density. In the cell with horizontally mounted quartz crystal, the air/liquid interface acts the reflection surface for the longitudinal wave. The variation in liquid height by regent addition or solvent evaporation can result in fluctuation in the oscillating frequency of the PQC. The influence of the longitudinal wave is more obvious in a test liquid of lower density. In the cell with perpendicularly mounted quartz crystal, the longitudinal wave is mainly reflected back by the inner wall body. The fine structure of plotting of frequency shift (Δf) versus (ρη)^1/2 shows a wave shape, which is different from the well-known linear relationship between of Δf and (ρη)^1/2, where ρ and η are the density and viscosity of the liquid, respectively. And wave-shaped frequency-temperature curves were observed. The longitudinal wave was a kind of potential error source in the PQC measurements. The longitudinal wave effect can be efficiently eliminated by using a rough reflection surface. After eliminating the influence of reflected longitudinal wave, the stability of the sensor PQC was much improved.     

Rapid detection of Escherichia coliform with a bulk acoustic wave sensor based on the gelation of Tachypleus amebocyte lysate

A new sensing method (BAW-TAL technique), which combined the bulk acoustic wave (BAW) technique with the gelation reaction of Tachypleus amebocyte lysate (TAL), was used for viscosity and density measurement and applied to the detection of Escherichia coliform (E. coli). This method depended on the fact that the viscosity and density of the mixture increased, and as a result, the resonance frequency decreased correspondingly after TAL was mixed with the heated E. coli solution that contained endotoxin. Results showed that the frequency shift was linearly related to the logarithm of E. coli concentration in the range of 2.7x10(4)-2.7x10(8) cells/ml. The correlation coefficient was 0.996. This BAW-TAL method was compared with the standard pour plate counts (PPC) method. The proposed method was much more rapid and simpler for detection of E. coli than the traditional methods.     

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.     

A transient enzymatic inhibition as an efficient tool for the discriminating bioluminescent analysis of three adenylic nucleotides with a fiberoptic sensor based on a compartmentalized tri-enzymatic sensing layer

A fiberoptic biosensor has been developed for the specific and alternate determination of ATP, ADP and AMP. The sensing layer is arranged by compartmentalizing the tri-enzyme sequence adenylate kinase – creatine kinase – firefly luciferase. The two kinases are covalently co-immobilized on a collagen membrane, whereas firefly luciferase is bound alone on a separate one. For the specific determination of each adenylic nucleotide, three particular reaction media are needed with which flow-injection analysis can be performed in the 2.5–2500 pmol for ATP, 10–2500 pmol for ADP and 25–5000 pmol for AMP linear ranges. For ten consecutive injections of 100 pmol ATP, 100 and 250 pmol ADP, the RSD were 3.5, 2.0 and 2.2%, respectively. When the three nucleotides are present simultaneously in the same sample, the transient inhibition of adenylate-kinase activity by adenosine 5′-monosulphate enables their specific and alternate measurement.     

SAW enzyme sensor applied to the determination of enzyme kinetic constants with the aid of a non-linear regression algorithm

A general program SAWBEH is proposed for the kinetic data evaluation of enzyme-catalysed hydrolysis by using the frequency shift response of an SAW sensor device. The Marquardt-Fletcher algorithm was employed as the minimum basis. The program was validated theoretically and compared with normal linear analysis methods by using synthetic data added with noise at different levels. The standard deviations of results obtained by the latter are about 100 times those obtained by the former. The system trypsin/benzoyl-L-arginine ethyl ester (BAEE) as a test model was studied at pH 7.6–7.8 and 32 °C. The Michaelis constant was estimated to be 6.34 × 10^–5 M.