Enzymatic Reaction System Research Articles

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

Enhancing nonspecific enzymatic hydrolysis of chitin to oligosaccharides pretreated by acid and green solvents under simultaneous microwave-radiation

It is hard to degrade untreated highly crystalline chitin. In this study, two solvents pretreatment chitin (acid swollen chitin (AC), super fine chitin (FC)) and microwave-heating method were used to enhance nonspecific enzymatic hydrolysis (lysozyme and pepsin), which obviously improved the enzymolysis rates by at least 1.31 times. Characterizations of chitin substrates (Mv, SEM, XRD) showed that calcium solvent pretreatment (obtained FC) was milder but effective than phosphoric acid pretreatment (obtained AC). The highest yield of chitin oligosaccharides (37.58 mg/g) were obtained after hydrolyzing AC under five-hour simultaneous microwave radiation by pepsin, among them, the content of N-acetylglucosamine was 13.76 mg/g. While, more chitin oligosaccharides with DP (degree of polymerization) 3–4 and lower DA (degree of acetylation) were obtained when using lysozyme than pepsin. Significantly, the conversion rate of chitin to oligosaccharides went best only when microwave and enzymes acting together (simultaneous strategy), which were at least 35.59% higher than separately pretreatment enzymes and substrates by microwave. The damages of microwave radiation on lysozyme and chitin substrates were revealed, and the operating principle of the whole enzyme reaction system heated by microwave was preliminatively explored.

Immobilization on magnetic PVA/SA@Fe3O4 hydrogel beads enhances the activity and stability of neutral protease

Enzyme immobilization technology has a key role in improving the stability of enzyme reaction systems and biocatalyst utilization rates. In this study, polyvinyl alcohol/sodium alginate@Fe3O4 (PVA/SA@Fe3O4) magnetic immobilized-enzyme hydrogel beads were prepared. Their structure and morphology were characterized by scanning electron microscopy, surface area and porosity analyses, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and a vibrating sample magnetometer. The capability of PVA/SA@Fe3O4 to adsorb neutral protease was investigated with variations in composition, temperature, pH, stirring speed, enzyme concentration, and crosslinking concentration. The optimal parameters of the immobilization process were determined by response surface methodology (3% neutral protease, 4% crosslinking at 200 rpm, 45 °C and pH 7.2), under which an immobilization rate of 41.98 mg/g was obtained. The thermal stability, acid-base stability, and reusability of the immobilized enzyme were improved significantly. After seven cycles, the immobilized enzyme activity remained at 30.8% that of the initial enzyme activity. The results indicate that the immobilization of NP onto magnetic PVA/SA@Fe3O4 hydrogel beads improves enzyme efficiency, giving this process potential industrial applications.

Visible light-driven photocatalytic and enzyme-like properties of novel AgBr/Ag2MoO4 for degradation of pollutants and improved antibacterial application

The manufacture of multiple catalytic materials is an important matter to solve environmental pollution and bacterial infection. AgBr/Ag2MoO4 nanoparticles with different molar ratios were prepared by a simple one-step precipitation method. The morphology and component of AgBr/Ag2MoO4 were analyzed by several characterizations means including XRD, XPS, and SEM et al., which showed that the loaded content of AgBr was different in different molar ratios. 1:1AgBr/Ag2MoO4 displayed better enzyme-like activity than that of the pure AgBr and Ag2MoO4. It can depict that 1:1AgBr/Ag2MoO4 possessed excellent photocatalytic activity with a 100% degradation rate of rhodamine B, and nearly 80% degradation rate of tetracycline hydrochloride. ∙O2– can be produced from the enzyme reaction system and the photocatalytic system, and ∙OH can be generated under visible light, which can successfully kill Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The sterilization rate of AgBr/Ag2MoO4 with visible light and H2O2 achieved 100% in a short time toward E. coli and S. aureus. The AgBr/Ag2MoO4 composite, as the bactericidal catalyst, with dual catalytic activities is a promising material in the field of environmental pollution and bacterial infection.

Non-coding RNA in alcohol use disorder by affecting synaptic plasticity.

Alcohol use disorder (AUD) is one of the most serious public health problems worldwide. AUD is a complex disorder, and there is ample evidence that genetic predisposition is critical to its development. Recent studies have shown that genetic predisposition leads to the onset of AUD, and alcohol metabolism can affect epigenetic inheritance, which in turn affects synaptic plasticity, alters brain function, and leads to more severe addictive behaviors. Non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play an important role in alcohol addiction. This paper reviews the regulatory role of ncRNAs. ncRNAs are involved in enzyme and neurotransmitter reaction systems during alcohol use disorder. Alcohol consumption regulates the expression of ncRNAs that mediate epigenetic modification and synaptic plasticity, which play an important role in the development of chronic AUD. ncRNAs may be used not only as predictors of therapeutic responses but also as therapeutic targets of AUD. Chronic alcoholism is more likely to lead to neuroimmune disorders, including permanent brain dysfunction. AUD induced by long-term alcoholism greatly alters the expression of genes in the human genome, especially the expression of ncRNAs. Alcohol can cause a series of pathological changes by interfering with gene expression, such as through disordered miRNA-mRNA expression networks, epigenetic modifications, disordered metabolism, and even synaptic remodeling. ncRNAs are involved in the transition from moderate drinking to alcohol dependence.

Expanded substrate specificity supported by P1′ and P2′ residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1′ and P2′ residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

A Highly Efficient Three-Liquid-Phase-Based Enzymatic One-Pot Multistep Reaction System with Recoverable Enzymes for the Synthesis of Biodiesel.

A three-liquid-phase system (TLPS) was developed and used as a novel enzymatic one-pot multistep reaction (EOMR) system. In this system, lipase and phospholipase were enriched in a single liquid phase with a high recovery (ca. 98%) and then used for the simultaneous catalysis of mutually inhibiting and interfering reactions (hydrolysis of phospholipids and glyceride in crude oil). A novel emulsion containing the two dispersed droplets (W2/O/W2 and W1/W2 emulsion structures) could be the key reason for this phenomenon because the emulsion system not only provided a new catalytic interface but also relieved the product inhibition. As a result, the content of free fatty acid (main hydrolysate of the glyceride) and the removal of phospholipid from the crude oil could be increased to 96 and 95%, respectively, within 1 h. The product obtained from the EOMR was directly used in the production of biodiesel via enzymatic esterification, and the content of fatty acid methanol ester could be increased to 93% within 2 h. Furthermore, the enzymes in the middle phase could also be reused, at least for eight rounds without significant loss in catalytic efficiency. Therefore, the TLPS could be considered as an ideal catalytic platform for the EOMR.

Noise-enhanced stability and resonance effect of a termolecular enzymatic reaction

Here, we investigate an enzymatic reaction system subjected to additive and multiplicative noises and a weak periodic signal. By means of numerical simulations, we find that structure of stationary probability distribution function changes from one peak to two peaks by regulating not only the noise intensities (i.e., noise-induced transition), but also system parameters. The mean first passage time as a function of the multiplicative noise intensity exhibits a maximum, showing that the noise can enhance stability of the system. Moreover, by using the two-states theory, analytically expression of signal-to-noise ratio (SNR) of the system in the adiabatic limit is derived. The results indicate that: (1) The SNR as a function of noise intensity exhibits a maximal value, i.e., a stochastic resonance (SR) phenomenon; (2) as the noise intensities remain unchanged, the curve of the SNR with respect to the parameters of the system displays a peak, i.e., a SR-like phenomenon. Because the noise and the parameters reflect, respectively, temperature and rates of the enzymatic reactions, choosing optimal temperature and rates can enhance response of the system. This findings will be beneficial for controlling enzymatic reaction systems.

High Capacity Lactate Biofuel Cell Using Enzyme Cascade without NAD

We describe a modification of the lactate biofuel cell to increase its capacity. A new multi-step enzyme reaction system without NAD was prepared using a bioanode modified with lactate oxidase, pyruvate decarboxylase, and pyrroloquinoline quinone-dependent aldehyde dehydrogenase. Its operating time was 6 times longer than that of the biofuel cell using the lactate oxidase-modified bioanode at constant current measurement of 1 mA cm−2 because of suppression of the diffusion overpotential due to the decrease in the fuel concentration.

A highly efficient and recoverable enzymatic method for removing phospholipids from soybean oil via an ionic liquid-based three-liquid-phase

A three-liquid-phase system (TLPS) with the ionic liquid (IL) constituting one of the phases was developed and used as a novel enzymatic reaction and separation system to remove phospholipids from crude soybean oils. Among the TLPSs tested, the oil/[BMIM]BF4/sodium citrate TLPS was considered to be the optimal system. In this system, a novel emulsion containing two different emulsion structures (W1/W2 and W2/O/W2 emulsion, W1 and W2 represent the IL-enriched and salt-enriched phase, respectively) could be formed, giving rise to its high catalytic efficiency. In this emulsion, not only could the phospholipids at the top/middle interface be hydrolyzed, but the hydrolysis of phospholipids also occurred at a higher efficiency at the middle/bottom interface where the interface area was much larger than the top/middle interface. As a result, the enzymatic treatment time in the TLPS (3 min) could be shortened by 60-fold relative to the conventional O/W system (3 h). Furthermore, the phospholipase in the salt-enriched phase could be reused for at least 8 batches without a significant loss in catalytic efficiency. This TLPS could offer a cost-effective, easily scaled up, and low-energy cost system for the reaction of weak polar substrate catalyzed by an interfacial enzyme.

Preparation and Characteristics of Enzymatic Reaction Systems Using Lipid Membranes and Fine Droplets of Liposomes

Preparation and Characteristics of Enzymatic Reaction Systems Using Lipid Membranes and Fine Droplets of Liposomes

Enhancing the Long-Term Stability of a Polymer Dot Glucose Transducer by Using an Enzymatic Cascade Reaction System.

Impaired glucose metabolism in diabetes causes severe acute and long-term complications, making real-time detection of blood glucose indispensable for diabetic patients. Existing continuous glucose monitoring systems are unsuitable for long-term clinical glycemic management due to poor long-term stability. Polymer dot (Pdot) glucose transducers are implantable optical nanosensors that exhibit excellent brightness, sensitivity, selectivity, and biocompatibility. Here, it is shown that hydrogen peroxide-a product of glucose oxidation in Pdot glucose sensors-degrades sensor performance via photobleaching, reduces glucose oxidase activity, and generates cytotoxicity. By adding catalase to a glucose oxidase-based Pdot sensor to create an enzymatic cascade, the hydrogen peroxide product of glucose oxidation is rapidly decomposed by catalase, preventing its accumulation and improving the sensor's photostability, enzymatic activity, and biocompatibility. Thus, a next-generation Pdot glucose transducer with a multienzyme reaction system (Pdot-GOx/CAT) that provides excellent sensing characteristics as well as greater detection system stability is presented. Pdot glucose transducers that incorporate this enzymatic cascade to eliminate hydrogen peroxide will possess greater long-term stability for improved continuous glucose monitoring in diabetic patients.

Designing a biocatalytic process involving deep eutectic solvents

AbstractDuring the last decade, deep eutectic solvents (DESs) have emerged as a promising alternative to traditional organic solvents, from both environmental and technological perspectives. The number of structural combinations encompassed by DESs is tremendous; thus, it is possible to design an optimal DES for each specific enzymatic reaction system. In (bio)catalytic processes, a DES can serve as solvent/co‐solvent, as an extractive reagent for an enzymatic product, and as a pretreatment solvent of enzymatic biomass. To date, hydrolases are the most studied enzymes in DESs, which is not surprising given that lipases are the most important industrial enzymes. At the same time, there are a limited number of papers dealing with synthetic reactions in DESs involving other hydrolytic enzymes (epoxide hydrolases, phospholipase, proteases and haloalkane dehalogenases), lyases, and dehydrogenases (as a part of the whole Saccharomyces cerevisae and Escherichia coli cell biocatalysis). When designing efficient biocatalytic processes involving DESs, independent of the reaction type and enzyme used, the following steps should be included: (i) preparation and characterisation of the DES, (ii) screening of the DES for optimal enzyme performance, (iii) selection and optimisation of the biocatalytic protocol, and (iv) recovery of the product/DES and DES recycling with possible scale‐up. In this paper, we will present some practical aspects that we experienced while working with these solvents, together with some major observations that are available in the literature. © 2020 Society of Chemical Industry

Reactive liquid‐liquid extraction as means for shifting the equilibrium in enzymatic aminase reaction systems

Reactive liquid‐liquid extraction as means for shifting the equilibrium in enzymatic aminase reaction systems

From Glucose to Polymers: A Continuous Chemoenzymatic Process

AbstractEfforts to synthesize degradable polymers from renewable resources are deterred by technical and economic challenges; especially, the conversion of natural building blocks into polymerizable monomers is inefficient, requiring multistep synthesis and chromatographic purification. Herein we report a chemoenzymatic process to address these challenges. An enzymatic reaction system was designed that allows for regioselective functional group transformation, efficiently converting glucose into a polymerizable monomer in quantitative yield, thus removing the need for chromatographic purification. With this key success, we further designed a continuous, three‐step process, which enabled the synthesis of a sugar polymer, sugar poly(orthoester), directly from glucose in high yield (73 % from glucose). This work may provide a proof‐of‐concept in developing technically and economically viable approaches to address the many issues associated with current petroleum‐based polymers.

From Glucose to Polymers: A Continuous Chemoenzymatic Process.

Efforts to synthesize degradable polymers from renewable resources are deterred by technical and economic challenges; especially, the conversion of natural building blocks into polymerizable monomers is inefficient, requiring multistep synthesis and chromatographic purification. Herein we report a chemoenzymatic process to address these challenges. An enzymatic reaction system was designed that allows for regioselective functional group transformation, efficiently converting glucose into a polymerizable monomer in quantitative yield, thus removing the need for chromatographic purification. With this key success, we further designed a continuous, three-step process, which enabled the synthesis of a sugar polymer, sugar poly(orthoester), directly from glucose in high yield (73 % from glucose). This work may provide a proof-of-concept in developing technically and economically viable approaches to address the many issues associated with current petroleum-based polymers.

Modeling Temperature-Dependent Sex Determination in Oviparous Species Using a Dynamical Systems Approach.

In many oviparous species, the incubation temperature of the egg determines the sex of the offspring. This is known as temperature-dependent sex determination (TSD). The probability of the hatched offspring being male or female varies across the incubation temperature range. This leads to the appearance of different TSD patterns in species such as FM pattern where females are predominately born at lower temperature and males at higher temperature, FMF pattern where the probability of female being born is higher at extreme temperatures and of the male being born is high at intermediate temperatures. We analyze an enzymatic reaction system proposed in the literature involving sex hormones with positive feedback effect to understand the emergence of different TSD patterns. The nonlinearity in the model is accounted through temperature sensitivity of the reaction rates affecting the catalytic mechanism in the reaction system. We employ a dynamical systems approach of singularity theory and bifurcation analysis to divide the parameter plane of temperature sensitivities into different regions where different TSD patterns are observed. Bifurcation analysis in association with the delineation of the parameter space for different TSD pattern has led to the identification of a subspace where all the TSD patterns observed in nature can be realized. We also show how modulation of the sex hormone in the species can be used to change the probability of occurrence of a specific sex, thereby preventing the extinction of endangered species.

Salt tolerance of nitrate reductase in Halomonas sp. B01

A systematic study on the lack of dissimilatory nitrate reductase (NAR) properties in Halomonas strains had been reported so far. The effects of different factors on Halomonas sp. B01 NAR activity were investigated. The salt tolerance of NAR was characterized. The denitrification process under high salt conditions was reported. Halomonas sp. B01 expressed membrane-bound NAR under induced culture by nitrate. The optimum pH of the enzyme reaction system was 8, and the optimum temperature was 30°C. The mRNA expression abundance of narH in NAR encoding gene was highest in the 60g/L NaCl inducing matrix. The NaCl concentration of optimum growth and induction of NAR were both 60g/L. The ectoine added to the NAR vitro enzyme reaction system could maintain NAR activity under high NaCl concentration. In the range of 0-60g/L NaCl, the NAR activity was stable at 17.7 (± 0.3) U/mg. The denitrification was performed by Halomonas sp. B01 at 60g/L NaCl, and the denitrification rate reached 97.1% at 24h. This study reveals for the first time the NAR properties of Halomonas strains, which provides a theoretical and technical basis for the nitrogen removal of high-salt nitrogenous wastewater using this strain.

Immobilization of Cellulase from Trichoderma Reesei on Multiwall Carbon Nanotubes (MWCNTs)

Cellulase is an enzyme commonly used to break down cellulose to beta-glucose. The demand of cellulase has been arisen due to its extensive range of applications in the industries such as detergents, foods and biofuel production. In present study, cellulase from Trichoderma reesei was immobilized on a functionalized multiwall carbon nanotube (MWCNTs) by physical adsorption method. Fourier Transform Infrared (FTIR) was used to confirm the successful functionalization of MWCNTs and immobilization of cellulase. In this approach, effects of pH and temperature for both free and immobilized cellulase were evaluated. Practically, the immobilized cellulase showed an improvement in pH and thermal stability compared to free cellulase at optimum condition of 50°C and pH 5 with 12% and 8% of increments respectively. For reusability study on CMC analysis, the bionanoconjugate retained 60% of its initial activity even after 3rd cycle of analysis. This feature is beneficial to the industrial applications because of its potential to be easily separated from the end product at the end of the reaction, reuse for several times and helps in development of multiple enzyme reaction systems.

Purification of viral neuraminidase from inclusion bodies produced by recombinant Escherichia coli

Neuraminidase (NA) is one of the targets for the development of new antivirals against the influenza virus. The recombinant Escherichia coli cells, namely the strains BL21(DE3)pLysS and ArcticExpress(DE3) were used to produce the influenza virus neuraminidase. Although the different conditions of induction were tested, the accumulation of over-expressed NA in insoluble fraction occurred independently of these conditions. The level of over-expressed protein represents 26.15 % of the total cellular proteins. Therefore, the aim of these study was to design the procedure for isolation of recombinant neuraminidase from IBs and subsequently its solubilization and refolding to its native and active form. The highest purity of IBs (86 %) was achieved after repeatedly washing for at least five times with 2 M urea. The best solubilizing agent for releasing NA from IBs was the solution of 8 M urea at pH 8.0 with 94.8 ± 0.4 mg/L released proteins. The most appropriate buffer for refolding of solubilized NA was found to be 50 mM Tris-HCl at pH 7.5 (102 ± 24.2 mg proteins) and the addition of glycerol or arginine had no stimulating effect on protein recovery. The determination of non-glycosylated activity of refolded NA monomer (Km = 0.51 g/L; Vmax = 9.73 U/mg; kcat = 8.76 s−1) using fetuin as a substrate in the coupled enzyme reaction system was the highlight of this work. This procedure provides a way to produce active form of NA monomer by recombinant E. coli cells.