Enzymatic Hydrolysis Reaction Research Articles

Efektifitas Reaksi Transesterifikasi Minyak Kelapa dan Sereh untuk Pembuatan Perisa Alami dengan Biokatalisator Lipase serta Penggunaan Ultrasonik

In general, flavor are synthesis through enzymatic hydrolysis and esterification reactions (up to 20 hours) using commercial ingredients based on free fatty acids and alcohol. Efficient endeavors utilizing enzymatic transesterification to expedite reactions are necessary to acquire flavored products. Substrates may originate from commercial or natural sources abundant in fatty acids and alcohol. Commercial fatty acids and alcohol (geraniol) are readily available in pure forms. Alternatively, fatty acids can be sourced from coconut oil, while geraniol can be derived from lemongrass oil, which is more cost-effective compared to commercial ingredients. Ultrasonics have emerged as a means to expedite enzymatic. The objective of this study is to investigate the impact of ultrasonic power and transesterification reaction time between coconut oil and geraniol on geraniol conversion. Utilizing lipase (415.99 U/g), coconut oil was subjected to transesterification with commercially obtained pure geraniol in a 1:3 weight ratio, conducted in an ultrasonic tank filled with water. Reaction durations spanned 30, 60, 90, 120, and 150 minutes, with ultrasonic powers set at 50, 70, and 90 watts. The study findings elucidated that the highest geraniol conversion rate of 50.59% was achieved with 70 watts of ultrasonic power over a 90-minute period. These optimal conditions were subsequently applied to transesterify coconut oil with geraniol from lemongrass oil, yielding a conversion rate of 90.29%. This finding demonsttrate the posibility of employing coconut oil and lemongrass oil in a one-stage transesterification process to produce natural flavors via enzymatic lipase catalysis expedited by ultrasonic technology, facilitating swift reaction times.

Hydrolysis of Phospholipids in Presence of Phospholipase D:Thrmodynamic and Kinetic Studies of Hydrolysis in Water and Alcoholic Environments

Phospholipase D(PLD)catalyzes the hydrolysis of phospholipids to produce phosphatidic acid and hydroxyl compounds. Phosphatidylcholine (PC) is the most abundant phospholipid in animals and plants, often constituting nearly 50% of total complex lipids in animals and plants. However, Phosphatidylserine (PS) makes up around 5–10% of all phospholipids. This work includes the monitoring thermodynamic and kinetic of the enzymatic hydrolysis of PC and PS in both water and ethanol. All experiments were performedusing the Schlink line technique inN2present as an inert gas to prevent the oxidative stress. UV/Vis. spectrophotometer was used to observe the kinetic of all enzymatic reactions. In addition, the Origin 2019 software was used to analyze and find the kinetic parameters of the enzymatic hydrolysis reactions. The results show that the enzymatic hydrolys is reactions of PC in water and in ethanol occurredat the same conditions, enzymatic activity 1.752 U/mg, temperature 37 ᵒC, and pH =7. However, the enzymatic hydrolysis reactions of PS in water occurred at different conditions than in ethanol. The enzymatic substitution reaction of PSinethanol was thermodynamic favorable reaction due to the value of the ∆G =_164.868 J, but in water was thermodynamically unfavorable ΔG = 65.048 J. However, the enzymatic hydrolysis reaction of PC in water was thermodynamically unfavorable ∆G = 345.319 Jaswellas in ethanol ∆G = 74.433 J. The study shows that there is clear impact of present nitrogen bases of PC and the environment of the hydrolysis on the activity of the enzymatic catalyzing.

The Effect of Enzymatic Hydrolysis and Maillard Reaction on the Flavor of Chicken Osteopontin.

To reveal the changes in the flavor quality of chicken osteopontin (CO) before and after enzymatic hydrolysis and a thermal reaction, the present study was carried out to evaluate the volatile compounds and non-volatile compounds in CO. The results show that the chicken boneset enzymatic solution (CBES) presented a notably richer aroma after the enzymatic hydrolysis treatment. At the same time, the concentrations of the total free amino acids (FAAs) and 5'-nucleotides in the CBES increased dramatically. The ERP (enzymatic reaction paste) scores and the ORC (osteopontin reactive cream) scores were exceptionally high in terms of the umami and salty flavor profiles. As precursors, FAAs and 5'-nucleotides also boosted the Maillard reaction, leading to the generation of wide volatile compounds. Compared to CO, CBES, and ORC, the sensory evaluation showed that ERP scored the highest. In summary, the enzymatic hydrolysis treatment coupled with the Maillard reaction significantly enhanced the flavor profile of CO. These findings offer valuable insights into the high-value utilization of bone by-products, making a significant advancement in the field.

Quantitative at-line monitoring of enzymatic hydrolysis using benchtop diffusion nuclear magnetic resonance spectroscopy.

Benchtop diffusion nuclear magnetic resonance (NMR) spectroscopy was used to perform quantitative monitoring of enzymatic hydrolysis. The study aimed to test the feasibility of the technology to characterize enzymatic hydrolysis processes in real time. Diffusion ordered spectroscopy (DOSY) was used to measure the signal intensity and apparent self-diffusion constant of solubilized protein in hydrolysate. The NMR technique was tested on an enzymatic hydrolysis reaction of red cod, a lean white fish, by the endopeptidase alcalase at 50°C. Hydrolysate samples were manually transferred from the reaction vessel to the NMR equipment. Measurement time was approximately 3 min per time point. The signal intensity from the DOSY experiment was used to measure protein concentration and the apparent self-diffusion constant was converted into an average molecular weight and an estimated degree of hydrolysis. These values were plotted as a function of time and both the rate of solubilization and the rate of protein breakdown could be calculated. In addition to being rapid and noninvasive, DOSY using benchtop NMR spectroscopy has an advantage compared with other enzymatic hydrolysis characterization methods as it gives a direct measure of average protein size; many functional properties of proteins are strongly influenced by protein size. Therefore, a method to give protein concentration and average size in real time will allow operators to more tightly control production from enzymatic hydrolysis. Although only one type of material was tested, it is anticipated that the method should be applicable to a broad variety of enzymatic hydrolysis feedstocks.

Mass transfer studies on the solvent-free lipase-catalyzed partial hydrolysis of palm oil in packed-bed reactor

Packed-bed reactor (PBR) is widely employed for enzyme-mediated reactions at an industrial scale because the reactor configuration enables reusability of the biocatalyst with ease, thereby reducing the overall operating expenses. However, mass transfer limitation remains a key challenge in fixed-bed column systems, especially at large scale. Therefore, the present study investigated the external mass transfer effects on solvent-free lipase-catalyzed partial hydrolysis reaction and aimed to develop a mass transfer model for the operation. The influence of volumetric flow rate, one of the crucial operating parameters affecting the external mass transfer resistance, was examined. Results demonstrated increasing the flow rate led to an increase in hydrolysis reaction with the highest reaction rate observed at 5 ml min-1 . A dimensionless mathematical mass transfer model of Colburn factor, JD = K(Re)n-1 which is a function of Reynolds and Schmidt numbers, was then proposed to simulate the enzymatic partial hydrolysis reaction of palm oil in PBR. The mass transfer model was analyzed with different n values and results revealed that the mass transfer correlation of JD=0.92(Re)-0.2 was able to predict the experimental data accurately. The model developed could describe the dominance of mass transfer effects besides providing useful information for the scale-up design of industrial reactor.

Process control of rice protein enzymolysis by field monitoring

By devising a novel near-infrared (NIR) in-situ online monitoring system, this study proposed a new process control method for rice protein (RP) enzymatic hydrolysis in order to address the challenge of monitoring certain chemical indices in real-time. The endpoint of the enzymatic hydrolysis reaction and the flexible switching point of dual-enzyme hydrolysis were determined through real-time spectrum collection. Correction and prediction models were constructed to predict the angiotensin I-converting enzyme (ACE) inhibitory activity of RP hydrolysis as well as the endpoint and the dual-enzyme flexible switching point at different substrate concentrations in an attempt to establish the new control system. At the RP substrate concentrations of 35, 40, and 45 g/L, the R2 of predicted and measured ACE inhibitory activity values under hydrolysis with a single alcalase were 0.8852, 0.8360, and 0.8613, respectively. In addition, the ACE inhibitory activity of dual-enzyme hydrolysis showed a high growth trend before and after the flexible switching point, and the hydrolysis time was significantly shortened at the same endpoint threshold as that of single alcalase hydrolysis. The results indicated that this method is capable of accurately determining and regulating the endpoint and adaptable transition point of RP hydrolysis. Consequently, this approach holds promise for the regulation of food basic materials during large-scale processing operations.

Production and purification of xylooligossaccharides from sugarcane bagasse and antioxidant potential assessment for functional ingredient application in the food industry

Xylooligosaccharides (XOS) are a type of prebiotic non-digestible carbohydrate obtained from lignocellulosic biomass through the enzymatic hydrolysis of hemicellulose; a raw material that is generated as a by-product of activity in the agro-industrial sector. As the most representative of lignocellulosic materials, sugarcane bagasse (SB) is a source for XOS production. Owing to the high impurity XOS content produced by direct enzymatic hydrolysis of xylan extracted from SB, subsequent refining steps (clarification and purification) are essential for food-related applications. This study aimed to investigate the XOS production process using enzymatic hydrolysis of xylan. Additionally, a refining step using active carbon during the XOS process was also applied; and the evaluation of the impacts the process may have on XOS antioxidant activity for potential application in food products. Xylan extraction was evaluated with potassium hydroxide (KOH) in the concentration range of 5–18 %, achieving a xylan extraction efficiency (E) up to 85 %, under hydrothermal conditions, and using 12 % hydroxide concentration for 45 min. The enzymatic hydrolysis reaction was performed by applying an endo-1,4-xylanase (NS50030-Novozymes®) cocktail, and 4.5 g/L of substrate with prebiotic fractions (xylobiose and xylotriose) as the major products, with a conversion yield of 260.82 g XOS/Kg xylan. The desorption was systematically investigated after evaluating of the ethanol elution concentration, and achieved up to 45 % of XOS recuperation in the translucent solution from 40 % ethanol eluate (v/v); demonstrating a high potential for use as a functional ingredient in food applications.

Experimental and kinetic analyses of delignification of lignocellulosic grass with minimal holocellulose loss during pretreatment

This work aims to develop an experimental and theoretical framework for converting the holocellulose present in an agro-residue to bioethanol without separating the cellulose and the hemicellulose from each other. Three delignification methods are employed to reduce holocellulose loss, and achieve a high yield of fermentable sugars through enzymatic hydrolysis: sodium chlorite-acetic acid (SC-AA), sodium chlorite-acetone (SC-AC), and separate SC-AA. The experimental methodology and theoretical analyses suggest that the separate SC-AA method is an effective delignification technology that preserves the maximum possible holocellulose content (11.7 % loss) while simultaneously removing lignin from agro-residues. Furthermore, this process also increases the accessibility of the holocellulose pores by restricting the adsorption of delignified products in the pores of the substrate, thus increasing the pore volume available for enzymatic hydrolysis reactions. The multi-stage modeling approach predicts the reaction time for delignifying agro-residues and quantifies the impact of reaction parameters on holocellulose retention.

Improvement of hydrolysis efficiency and interfacial properties of zein using nanoemulsions prepared by a low energy emulsification method

Alcalase hydrolysis can improve the interfacial properties and functional features of proteins. It usually reacts in water. However, zein is difficult to dissolve in water due to its amino acid composition, which greatly limits its application in food industry. To achieve heterogeneous hydrolysis of zein, a new and stable reaction system which called bicontinuous microemulsion was constructed. It consisted of Tween-80, absolute ethanol, n-octanol and distilled water. The average particle size of the nanoemulsion system was 18.23 nm. In this dispersion system, alcalase hydrolysis expanded the protein structure, exposed hydrophobic groups and significantly improved the emulsifying activity, foaming capacity and other interface properties. The scavenging activity of DPPH free radicals and ABTS free radicals were greatly promoted, reached 59.66% and 82.91% respectively. This study provides a new idea for enzymatic hydrolysis reaction medium of zein and is conducive to the application of zein in future.

Target Enzyme-Triggered Click Chemistry and Hybridization Chain Reaction for Fluorescence Nonculture Homogeneous Analysis of E. coli in Bloodstream Infections.

Escherichia coli is the major pathogen that causes bloodstream infections (BSI). It is critical to develop nonculture identification methods which can meet the urgent need of clinical diagnosis and treatment. In this study, we reported a homogeneous fluorescence E. coli analysis system using β-galactosidase (β-Gal) as the biomarker and double-stranded DNA-templated copper nanoparticles (dsDNA-Cu NPs) as the signal output. The product of the enzymatic hydrolysis reaction, p-aminophenol (PAP), could reduce Cu2+ to Cu+, triggering the alkyne-azido cycloaddition reaction (CuAAC). Subsequently, the hybrid chain reaction (HCR) was initiated, producing the dsDNA template used to generate Cu NPs in situ. The system achieved a wide linear range for β-Gal and E. coli 1-104 mU/L and 10-2-10 colony-forming unit (CFU)/mL, and a detection limit of 0.3 mU/L and 0.003 CFU/mL, respectively. 65 samples (45 blood and 20 urine) were collected to evaluate the clinical practicality. The results demonstrated remarkable area under the curve (AUC) values of 0.95 and 0.916 from uncultured urine and blood, respectively. It had 100% specificity and 83.3% sensitivity. The whole duration of the strategy was 3.5 h, which significantly reduced the turnaround time (TAT) and facilitated early BSI diagnosis to improve patients' prognosis. Our work had the potential to be an alternative to culture-based methods in clinics.

Critical study on the extraction of collagen from eggshell membrane by enzymatic hydrolysis reaction

Eggshells and their membranes are classified as refuse, which is costly to be disposed of in landfills. But many people do not realise that they can actually offer valuable collagen, which is obtained through the enzymatic hydrolysis process. This study aims to address the effects of enzyme type, enzyme-to-membrane ratio and water-to-material ratio on collagen extraction rate. It is found that the processing condition with a pepsin-to-membrane ratio of 0.5 and a water-to-material ratio of 1:200 can achieve an extraction rate of 2.44%. This research has created an opportunity to turn eggshell membrane waste into one of the potential sources of extracting collagen, relieving the pressure on waste treatment.

Biovalorization of fruit waste to gallic acid synthesis through the application of Bacillus haynesii SSRY4 MN031245 tannase

Present research investigation reports the detection and quantitative estimation of gallic acid synthesis by bacterial tannase catalysed hydrolysis of fruit waste tannins. Partially purified Tannase from Bacillus haynesiiSSRY4 MN031245 was employed for the enzymatic hydrolysis reaction. Tannase could successfully carry out hydrolysis reactions for synthesis of gallic acid from fruit waste. Gallic acid synthesis was detected and confirmed by High-Performance Thin Layer Chromatography (HPTLC) analysis. Partially purified tannase was able to synthesize 20.61 mg/mL gallic acid from the crude tannin extract of black plum seed powder after 24 h of incubation at 40 °C. The results suggest that bacterial tannase can be cost effective for the synthesis of gallic acid from the fruit waste.

Study on Thermodynamics and Kinetics of Cephalexin Enzymatic Hydrolysis and Its Process Development to Prepare 7-ADCA

Cephalosporin enzymatic hydrolysis technology is a green technology for recovering 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) from cephalosporin mother liquor. Solubility is critical for the production and purification of 7-ADCA. In this paper, the solubility of 7-ADCA and phenylglycine was measured. Solubility-temperature correlation model and solubility-pH correlation model were investigated, and Akaike information criterion (AIC) analysis was performed. The kinetic parameters of the enzymatic hydrolysis reaction of cephalexin, cefradine, and cefadroxil were determined, and the reaction rates under different substrate concentrations were measured, and the Lineweaver–Burk double-reciprocal equation was used to draw a graph. The Michaelis constants Km/(mg/mL) were 73.98, 583.84, 38.66, Vmax/(mg/mL·min) 4.20, 16.00, 1.96, respectively. The experimental results show that amphoteric compounds and buffers can prompt the reaction, low concentration of methanol promotes the reaction, while high concentration of methanol inhibits the reaction, and ethanol, isopropanol, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,4-dioxane all have different degrees of inhibition on the reaction speed. Finally, based on thermodynamic and kinetic studies, a process technology for the preparation of 7-ADCA by hydrolysis catalyzed of cephalexin was developed. It was confirmed that the proposed process route of preferential removal of phenylglycine by elution and/or cooling crystallization was reasonable and effective. The 7-ADCA crystal products obtained by crystallization were characterized by PXRD, thermal analysis, infrared, electron microscope, and high-performance liquid chromatography (HPLC).

Additives Enhancing Enzymatic Hydrolysis of Wheat Straw to Obtain Fermentable Sugar.

In order to explore the effect of additives on enzymatic hydrolysis of lignocellulose biomass, the effect of two different additives, Triton X-100 (TX-100) and Bovine serum albumin (BSA), enzyme dosages, and additive concentrations on enzymatic hydrolysis to obtain fermentable sugar using cellulose extracted from wheat straw (WS) as the substrate was investigated in this study. An enzymatic hydrolysis kinetic model was used to successfully describe the enzymatic hydrolysis in a heterogeneous system. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) were used to determine the effect of extraction and enzymatic hydrolysis on the composition and structure of the samples. The results showed that the total reducing sugar concentration of the raw was 1.535g/L at 120h, but that of the extracted cellulose (EC) increased to 5.087g/L at 120h, indicating that EC from WS is more conducive to enzymatic hydrolysis compared with the raw. The total reducing sugar concentration with the addition of the TX-100 was 6.737g/L at 120h, which was greater than that with the addition of the BSA (5.728g/L at 120h), indicating that the addition of two additives improved the enzymatic hydrolysis efficiency, especially TX-100. The kinetic studies showed that the initial enzymatic hydrolysis reaction rate (Km) of the EC was more than four times greater than that of the raw. The Km of the EC added with TX-100 and BSA were increased by 29.50% and 22.89% compared with that of the EC without the addition of additive. The addition of additives is an effective method for enhancing enzymatic hydrolysis efficiency and fermentable sugar production from lignocellulosic biomass.

Highly selective kinetic resolution of D/L-syn-p-sulfone phenylserine catalyzed by d-threonine aldolase in two-phase ionic solvent

In the chemical synthesis of L-syn-p-methylsulfoxide phenylserine ethyl ester (D-ethyl ester), l-tartaric acid or enzymatic resolution is employed to resolve the racemate, and thus obtain the target compound, and the remaining isomer can be recycled to obtain the raw material. In this study, high-purity L-syn-p-methylsulfoxide phenylserine (L-syn-MPS) was obtained. The kinetics of the d-threonine aldolase enzymatic hydrolysis reaction reveals that D-syn-p-sulfoxylphenylserine resolves well in [BMIM][BF4] ionic solvents. The D/L-syn-MPS racemate was resolved using a two-phase ionic solvent [BMIM][NTf2] to afford L-syn-MPS (ee (enantiomeric excess) > 99%) and a white solid in 41.7% yield. Therefore, this system is suitable for the separation of insoluble aldehydes and successfully avoids the condensation of hydroxyl aldehydes to form D-anti-MPS.

How the type of interface can alter the behavior of an aprotic ionic liquid-water mixture entrapped in different reverse micelles: An exploratory study using an enzymatic reaction as a sensor

In this work, we decipher how an aprotic Ionic Liquid (IL)-water mixture structure can be altered upon confinement in different reverse micelles (RMs) media. Thus, the effect of the polar pseudophase composition in RMs formulated by anionic or cationic surfactants was monitored using the well-known enzymatic hydrolysis reaction of 1-naphthyl phosphate (1-NP) with alkaline phosphatase (AP). Water and an aqueous mixture formed by the IL 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4C1im, TfO]) as the polar pseudophase were used. As surfactants, the anionic sodium diethylhexyl sulfosuccinate (AOT) and the cationic benzyl-n-hexadecyldimethylammonium chloride (BHDC) were employed. The effect of the nanoconfinement, as well as the impact of the polar solvent content and the type of interface, were analyzed by determining the Michaelis-Menten parameters. In homogeneous media, the catalytic efficiency of the hydrolysis reaction is strongly dependent on the amount of water in the mixtures evaluated. In RMs the catalytic efficiency is dependent on both, the polar content and the type of surfactant used. Thus, by using a model reaction with an enzyme entrapped inside different RMs it was possible to learn that an aprotic IL:water mixture can be strongly disturbed when it is confined in a cationic or anionic RMs. This particular behavior can produce a deep impact on the technological applications of these organized systems.

Conventional and Novel Technologies in the Production of Dairy Bioactive Peptides.

BackgroundIn recent years, researchers have focused on functional ingredients, functional foods, and nutraceuticals due to the rapidly increasing interest in bioactive components, especially in bioactive peptides. Dairy proteins are a rich and balanced source of amino acids and their derived bioactive peptides, which possess biological and physiological properties. In the dairy industry, microbial fermentation and enzymatic hydrolysis are promising methods for producing bioactive peptides because of their rapid efficiency, and mild reaction conditions. However, these methods utilize less raw material, take long reaction time, result in low yields, and low activity products when used alone, which pose industry to seek for novel methods as pretreatments to increase the yield of bioactive peptides.Scope and ApproachThis review emphasizes the production of peptides from the dairy proteins and discusses the potential use of novel technologies as pretreatments to conventional methods of bioactive peptides production from dairy proteins, including the mechanisms of novel technologies along with respective examples of use, advantages, limitations, and challenges to each technology.Key Findings and ConclusionNoteworthily, hydrolysis of dairy proteins liberate wide-range of peptides that possess remarkable biological functions to maintain human health. Novel technologies in the dairy industry such as ultrasound-assisted processing (UAP), microwave-assisted processing (MAP), and high pressure processing (HPP) are innovative and environmentally friendly. Generally, novel technologies are less effectual compared to conventional methods, therefore used in combination with fermentation and enzymatic hydrolysis, and are promising pretreatments to modify peptides’ profile, improve the yields, and high liberation of bioactive peptides as compared to conventional technologies. UAP is an innovative and most efficient technology as its mechanical effects and cavitation change the protein conformation, increase the biological activities of enzymes, and enhance enzymatic hydrolysis reaction rate.

Application of the enzymatic method for the quantitative determination of dequalinium chloride in lozenges

Aim. To develop a new kinetic spectrophotometric enzymatic method suitable for the quantitative determination of dequalinium chloride in lozenges.Materials and methods. An enzymatic kinetic spectrophotometric method for the quantitative determination of dequalinium chloride in lozenges has been proposed. It is based on the ability of dequalinium chloride to inhibit the enzymatic hydrolysis reaction of acetylcholine by cholinesterase in the presence of the acetylcholine (AСh) excess and H2O2. The degree of inhibition was determined by the kinetic method using two conjugated reactions: ACh perhydrolysis (interaction with an excess of hydrogen peroxide) followed by oxidation with the peroxyacid formed. Peracetic acid formed in situ by the reaction between unreacted ACh and H2O2 interacts with p-phenetidine forming a product, which absorbs at λmax = 354 nm, in the phosphate buffer solution with pH 8.3 at room temperature.Results and discussion. The linear dependence of the calibration graph for the quantitative determination of dequalinium chloride was in the concentration range of 0.2 – 0.8 μg mL–1 (r = 0.999). LOD and LOQ were 0.01 × 10–6 and 0.03 × 10–6 mol L–1, respectively. For the quantitative determination of dequalinium chloride in lozenges, RSD ≤ 2.65 % (accuracy, δ = -1.10…+1.78 %).Conclusions. A new enzymatic kinetic spectrophotometric method has been developed, and its applicability to the quantitative determination of dequalinium chloride in lozenges has been shown. It does not require a complicated treatment of the analyte and a tedious extraction procedure. The method proposed is sensitive enough to determine a small amount of the active pharmaceutical ingredient. These advantages encourage the application of the method proposed in routine quality control of the drugs studied in analytical laboratories.

Structure and Mechanism of a Cold-Adapted Bacterial Lipase.

The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic–psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.

A Portable Fluorescent Hydrogel-Based Device for On-Site Quantitation of Organophosphorus Pesticides as Low as the Sub-ppb Level.

Portable devices possess powerful application prospects in on-site sensing without the limitation of bulky instruments. Given the relevance of pesticides to food safety, we herein fabricated a robust gold nanocluster (AuNC)-based hydrogel test kit for precisely quantified chlorpyrifos by using a three-dimensional (3D) printed subsidiary device. In this work, the fluorescence of AuNC-based hydrogel could be efficiently quenched by cobalt oxyhydroxide nanoflakes (CoOOH NFs) through the Förster resonance energy transfer effect. Chlorpyrifos as an acetylcholinesterase inhibitor controls the enzymatic hydrolysis reaction and further regulates the production of thiocholine that could decompose CoOOH nanoflakes into Co2+, resulting in the fluorescence response of AuNC-based hydrogel. By using a homemade subsidiary device and smartphone, the fluorescence color was transformed into digital information, achieving the on-site quantitative detection of chlorpyrifos with the limit of detection of 0.59 ng ml−1. Owing to specific AuNC signatures and hydrogel encapsulation, the proposed fluorescence hydrogel test kit displayed high sensitivity, good selectivity, and anti-interference capability in a real sample analysis, providing great potential in on-site applications.