Showing papers on "Substrate (chemistry) published in 2009"

Understanding enzyme immobilisation

Abstract: Enzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations. At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical–chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation.

Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose

Abstract: Working at high solids (substrate) concentrations is advantageous in enzymatic conversion of lignocellulosic biomass as it increases product concentrations and plant productivity while lowering energy and water input. However, for a number of lignocellulosic substrates it has been shown that at increasing substrate concentration, the corresponding yield decreases in a fashion which can not be explained by current models and knowledge of enzyme-substrate interactions. This decrease in yield is undesirable as it offsets the advantages of working at high solids levels. The cause of the 'solids effect' has so far remained unknown. The decreasing conversion at increasing solids concentrations was found to be a generic or intrinsic effect, describing a linear correlation from 5 to 30% initial total solids content (w/w). Insufficient mixing has previously been shown not to be involved in the effect. Hydrolysis experiments with filter paper showed that neither lignin content nor hemicellulose-derived inhibitors appear to be responsible for the decrease in yields. Product inhibition by glucose and in particular cellobiose (and ethanol in simultaneous saccharification and fermentation) at the increased concentrations at high solids loading plays a role but could not completely account for the decreasing conversion. Adsorption of cellulases was found to decrease at increasing solids concentrations. There was a strong correlation between the decreasing adsorption and conversion, indicating that the inhibition of cellulase adsorption to cellulose is causing the decrease in yield. Inhibition of enzyme adsorption by hydrolysis products appear to be the main cause of the decreasing yields at increasing substrate concentrations in the enzymatic decomposition of cellulosic biomass. In order to facilitate high conversions at high solids concentrations, understanding of the mechanisms involved in high-solids product inhibition and adsorption inhibition must be improved.

The influence of time, storage temperature, and substrate age on potential soil enzyme activity in acidic forest soils using MUB-linked substrates and L-DOPA.

Abstract: The purpose of this experiment was to evaluate whether soil storage and processing methods significantly influence measurements of potential in situ enzyme activity in acidic forest soils. More specifically, the objectives were to determine if: (1) duration and temperature of soil storage; (2) duration of soil slurry in buffer; and (3) age of model substrates significantly influence the activity of six commonly measured soil extracellular enzymes using methylumbelliferone (MUB)-linked substrates and l-dihydroxyphenylalanine (l-DOPA). Soil collected and analyzed for enzyme activity within 2 h was considered the best measure of potential in situ enzyme activity and the benchmark for all statistical comparisons. Sub-samples of the same soil were stored at either 4 °C or −20 °C. In addition to the temperature manipulation, soils experienced two more experimental treatments. First, enzyme activity was analyzed 2, 7, 14, and 21 days after collection. Second, MUB-linked substrate was added immediately (i.e. <20 min) or 2 h after mixing soil with buffer. Enzyme activity of soil stored at 4 °C was not significantly different from soil stored at −20 °C. The duration of soil storage was minimal for β-glucosidase, β-xylosidase, and peroxidase activity. N-acetyl-glucosaminidase (NAGase), phosphatase, and phenol oxidase activity appeared to change the most when compared to fresh soils, but the direction of change varied. Likewise, the activities of these enzymes were most sensitive to extended time in buffer. Fluorometric MUB and MUB-linked substrates generally had a 3-day shelf life before they start to significantly suppress reported activities when kept at 4 °C. These findings suggest that the manner in which acidic forest soils are stored and processed are site and enzyme specific and should not initially be trivialized when conducting enzyme assays focusing on NAGase, phosphatase, and phenol oxidase. The activities of β-glucosidase, β-xylosidase, and peroxidase are insensitive to storage and processing methods.

Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles

Abstract: A novel and efficient immobilization of β-d-galactosidase from Aspergillus oryzae has been developed by using magnetic Fe3O4–chitosan (Fe3O4–CS) nanoparticles as support. The magnetic Fe3O4–CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe3O4 nanoparticles made through co-precipitation of Fe2+ and Fe3+. The resultant material was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry and thermogravimetric analysis. β-d-Galactosidase was covalently immobilized onto the nanocomposites using glutaraldehyde as activating agent. The immobilization process was optimized by examining immobilized time, cross-linking time, enzyme concentration, glutaraldehyde concentration, the initial pH values of glutaraldehyde and the enzyme solution. As a result, the immobilized enzyme presented a higher storage, pH and thermal stability than the soluble enzyme. Galactooligosaccharide was formed with lactose as substrate by using the immobilized enzyme as biocatalyst, and a maximum yield of 15.5% (w/v) was achieved when about 50% lactose was hydrolyzed. Hence, the magnetic Fe3O4–chitosan nanoparticles are proved to be an effective support for the immobilization of β-d-galactosidase.

Computational structure-based redesign of enzyme activity.

Abstract: We report a computational, structure-based redesign of the phenylalanine adenylation domain of the nonribosomal peptide synthetase enzyme gramicidin S synthetase A (GrsA-PheA) for a set of noncognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a noncognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.

Substrate-selective supramolecular tandem assays: monitoring enzyme inhibition of arginase and diamine oxidase by fluorescent dye displacement from calixarene and cucurbituril macrocycles.

Abstract: A combination of moderately selective host−guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays (“supramolecular tandem assays”) exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products (“substrate-selective assays”). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M−1 with arginine, 550 M−1 with ornithi...

Optimization of Enzymatic Hydrolysis of Wheat Straw Pretreated by Alkaline Peroxide Using Response Surface Methodology

Abstract: To optimize the enzymatic conversion of widely available lignocellulosic biomass-wheat straw (WS), pretreatment facilitating the enzymatic saccharification process was first performed by alkaline peroxide, resulting in a substrate consisting of 60.17% cellulose, 29.53% hemicelluloses, and 4.59% lignin. Using response surface methodology, the combined effects of enzyme loading, substrate concentration, surfactant concentration, and reaction time on hydrolysis yield from enzymatic saccharification of WS were further investigated. The results showed that both enzyme loading and substrate concentration had interactions with surfactant concentration. A quadratic polynomial equation for predicting the hydrolysis yield was developed. The experimental results were in good agreement with predicted values. Therefore, the model could be successfully used to identify the effective combinations of the four factors for predicting hydrolysis yield.

Coatings and surface treatments having active enzymes and peptides

Abstract: Disclosed herein are a materials such as a coating, an elastomer, an adhesive, a sealant, a textile finish, a wax, and a filler for such a material, wherein the material includes an enzyme such as an esterase (e.g., a lipolytic enzyme, a sulfuric ester hydrolase, an organophosphorus compound degradation enzyme), an enzyme that degrades a cell wall and/or a cell membrane component (e.g., a lysozyme, a lytic transgrycosylase, a peptidase), and/or a biocidal or biostatic peptide. Also disclosed herein are methods of decontaminating a surface comprising such a material from a chemical substrate of an enzyme such as a lipid or an organophosphorus compound, as well as reducing the growth of a microorganism on or within such a material.

Impact of substrate to inoculum ratio in anaerobic digestion of swine slurry

Abstract: Batch tests using swine slurry as substrate were conducted to elucidate the impact on anaerobic digestion of different ratio of substrate (COD)/inoculum (VS). Soluble COD, volatile fatty acids (VFAs) concentration, and pH were periodically analyzed in order to completely understand possible inhibitions. In addition, ammonia concentration was also calculated at the start up and final stage of the test. The results showed that the same methane yield was achieved with the three experimental ratios (COD/VS of 1, 2 and 3), however methane production rates were clearly different. This later parameter decreased with increasing ratios. This behaviour was explained by the accumulation of VFAs (predominantly acetic (HAc) and propionic acid (HPr)), reflected on the soluble COD profile as well. The possible inhibition by free ammonia was null since the values calculated were far below the threshold concentration reported in literature.

Front end of line plasma mediated ashing processes and apparatus

Abstract: Front end of line (FEOL) plasma mediated ashing processes for removing organic material from a substrate generally includes exposing the substrate to the plasma to selectively remove photoresist, implanted photoresist, polymers and/or residues from the substrate, wherein the plasma contains a ratio of active nitrogen and active oxygen that is larger than a ratio of active nitrogen and active oxygen obtainable from plasmas of gas mixtures comprising oxygen gas and nitrogen gas. The plasma exhibits high throughput while minimizing and/or preventing substrate oxidation and dopant bleaching. Plasma apparatuses are also described.

Electrochemical Water Splitting Coupled with Organic Compound Oxidation: The Role of Active Chlorine Species

Abstract: The need for alternative energy sources with minimal to no carbon footprint is growing. A solar-powered electrochemical system that produces hydrogen via water splitting using organic pollutants as sacrificial electron donors is a possible solution. The hybridization of a BiO_x−TiO_2/Ti anode with a stainless steel cathode powered by a photovoltaic (PV) array has been shown to achieve this process. The electrochemical degradation kinetics of a variety of organic substrates is investigated as a function of a background electrolyte, NaCl versus Na_2SO_4. The observed substrate (S) degradation kinetics (k_(obs)^S) are found to correlate well with the cell current (I_(cell)) and the H_2 production energy efficiency (EE) in the presence of NaCl as the background electrolyte. In the case of Na_2SO_4, no correlation is observed and the degradation rates are greatly reduced in comparison to NaCl. This suggests that the primary chemical oxidant is electrolyte-dependent. The k_(obs)^S’s are found to be proportional to the bimolecular rate constants of Cl_2^(•−) with the substrate (k_(Cl_2^(•−) + S)) and to substrate-induced ΔEEs (EE with substrate − EE without substrate) in the presence of NaCl. The ΔEE correlation arises from the active chlorine species acting as an electron shuttle, which compete with H_2 production for cathodic electrons. In the presence of the organic substrates, the active chlorine species are quenched, increasing the fraction of electrons utilized for the H_2 production.

Temperature sensitivity and substrate quality in soil organic matter decomposition: results of an incubation study with three substrates

Abstract: Kinetic theory suggests that the temperature sensitivity of decomposition of soil organic matter should increase with increasing recalcitrance. This ‘temperature–quality hypothesis’ was tested in a laboratory experiment. Microcosms with wheat straw, spruce needle litter and mor humus were initially placed at 5, 15 and 25 1C until the same cumulative amount of CO2 had been respired. Thereafter, microcosms from each single temperature were moved to a final set of incubation temperatures of 5, 15 and 25 1C. Straw decomposed faster than needle litter at 25 and 15 1C, but slower than needle litter at 5 1C, and showed a higher temperature sensitivity (expressed as Q10) than needle litter at low temperatures. When moved to the same temperature, needle litter initially incubated at 5 and 15 1C had significantly higher respiration rates in the final incubation than litters initially placed at 25 1C. Mor humus placed at equal temperatures during the initial and final incubations had higher cumulative respiration during the final incubation than humus experiencing a shift in temperature, both upand downwards. These results indicate that other factors than substrate quality are needed to fully explain the temperature dependence. In agreement with the hypothesis, Q10 was always higher for the temperature step between 5 and 15 1C than between 15 and 25 1C. Also in agreement with the temperature–quality hypothesis, Q10 significantly increased with increasing degree of decomposition in five out of the six constant temperature treatments with needle litter and mor humus. Q10s for substrates moved between temperatures tended to be higher than for substrates remaining at the initial temperature and an upward shift in temperature increased Q10 more than a downward shift. This study largely supports the temperature– quality hypothesis. However, other factors like acclimation and synthesis of recalcitrant compounds can modify the temperature response.

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Abstract: To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier—either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies “open” and “closed” CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

Enzymatic surface modification and functionalization of PET: A water contact angle, FTIR, and fluorescence spectroscopy study

Abstract: The purpose of this study was to investigate the changes induced by a lypolytic enzyme on the surface properties of polyethylene terephthalate (PET). Changes in surface hydrophilicity were monitored by means of water contact angle (WCA) measurements. Fourier Transform Infrared spectroscopy (FTIR) in the Attenuated Total Reflectance mode (ATR) was used to investigate the structural and conformational changes of the ethylene glycol and benzene moieties of PET. Amorphous and crystalline PET membranes were used as substrate. The lipolytic enzyme displayed higher hydrolytic activity towards the amorphous PET substrate, as demonstrated by the decrease of the WCA values. Minor changes were observed on the crystalline PET membrane. The effect of enzyme adhesion was addressed by applying a protease after-treatment which was able to remove the residual enzyme protein adhering to the surface of PET, as demonstrated by the behavior of WCA values. Significant spectral changes were observed by FTIR-ATR analysis in the spectral regions characteristic of the crystalline and amorphous PET domains. The intensity of the crystalline marker bands increased while that of the amorphous ones decreased. Accordingly, the crystallinity indexes calculated as band intensity ratios (1,341/1,410 cm(-1) and 1,120/1,100 cm(-1)) increased. Finally, the free carboxyl groups formed at the surface of PET by enzyme hydrolysis were esterified with a fluorescent alkyl bromide, 2-(bromomethyl)naphthalene (BrNP). WCA measurements confirmed that the reaction proceeded effectively. The fluorescence results indicate that the enzymatically treated PET films are more reactive towards BrNP. FTIR analysis showed that the surface of BrNP-modified PET acquired a more crystalline character.

Synthesis of a metal-organic framework film by direct conversion technique for VOCs sensing.

Abstract: For the first time, a metal–organic framework Zn3(BTC)2 film has been successfully synthesized on the substrate of zinc wafer by a direct conversion technique. The obtained crystal densities and inter-growth of the film have been improved via the zinc wafer conversion method. The effect of synthesis conditions on the crystallization of Zn3(BTC)2 and activation of the substrate were determined to optimize the strategies for the synthesis of continuous and stable layers. The crystallization took place by converting the activated zinc layer on the substrate. The reaction between the substrate-generated zinc source and the H3BTC clear solution yielded the best inter-growth of crystals and formed a high-density coating. More interestingly, the fluorescence emission of the Zn3(BTC)2 film was found to be highly sensitive and selective sensing to dimethylamine among different VOCs. The fluorescence intensity decreased with increasing contents of dimethylamine in ethanol solution due to weak fluorescence quenching effect. This high-quality MOF film may find promising applications in sensors, especially in VOCs sensing.

Effect of ionic liquid properties on lipase stabilization under microwave irradiation

Abstract: Ionic liquids (ILs) as neoteric solvents and microwave irradiation as alternative energy source are becoming two important tools for many enzymatic reactions. However, it is not well understood what properties of ILs govern the enzyme stabilization, and whether the microwave irradiation could activate enzymes in ILs. To tackle these two important issues, the synthetic activities of immobilized Candida antarctica lipase B (Novozyme 435) were examined in more than twenty ILs through microwave heating. Under microwave irradiation, enhanced enzyme activities were observed when the enzyme was surrounded by a layer of water molecules. However, such enhancement diminished when the reaction system was dried. To understand the effect of IL properties, the enzyme activities under microwave irradiation were correlated with the viscosity, polarity and hydrophobicity (log P) of ILs, respectively. The initial reaction rates bear no direct relationship with the viscosity and polarity (in terms of dielectric constant and E T N ) of ILs, but have a loose correlation (a bell curve) with log P values. The enzyme stabilization by ILs was explained from aspects of hydrogen-bond basicity of anions, dissolution of the enzyme, ionic association strength of anions, and substrate ground-state stabilization by ILs.