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In this study, we have investigated the mathematical model of an immobilized enzyme system that follows the Michaelis–Menten (MM) kinetics for a micro-disk biosensor. The film reaction model under steady state conditions is transformed into a couple differential equations which are based on dimensionless concentration of hydrogen peroxide with enzyme reaction (H) and substrate (S) within the biosensor. The model is based on a reaction–diffusion equation which contains highly non-linear terms related to MM kinetics of the enzymatic reaction. Further, to calculate the effect of variations in parameters on the dimensionless concentration of substrate and hydrogen peroxide, we have strengthened the computational ability of neural network (NN) architecture by using a backpropagated Levenberg–Marquardt training (LMT) algorithm. NNs–LMT algorithm is a supervised machine learning for which the initial data set is generated by using MATLAB built in function known as “pdex4”. Furthermore, the data set is validated by the processing of the NNs–LMT algorithm to find the approximate solutions for different scenarios and cases of mathematical model of micro-disk biosensors. Absolute errors, curve fitting, error histograms, regression and complexity analysis further validate the accuracy and robustness of the technique.

Background Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome. Methods Structure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures. Results The glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0. Conclusion The findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

A revolution in chemical biology occurred with the introduction of click chemistry. Click chemistry plays an important role in protein chemistry modifications, providing specific, sensitive, rapid, and easy-to-handle methods. Under physiological conditions, click chemistry often overlaps with bioorthogonal chemistry, defined as reactions that occur rapidly and selectively without interfering with biological processes. Click chemistry is used for the posttranslational modification of proteins based on covalent bond formations. With the contribution of click reactions, selective modification of proteins would be developed, representing an alternative to other technologies in preparing new proteins or enzymes for studying specific protein functions in different biological processes. Click-modified proteins have potential in diverse applications such as imaging, labeling, sensing, drug design, and enzyme technology. Due to the promising role of proteins in disease diagnosis and therapy, this review aims to highlight the growing applications of click strategies in protein chemistry over the last two decades, with a special emphasis on medicinal applications.

In 2012, 7 million premature deaths were attributed to air pollution, of which 4.3 million to indoor air pollution inducing the "sick building syndrome." In particular, formaldehyde, a toxic and highly reactive chemical, has been linked to cancer. The use of soil microbial biomass and plants to remove formaldehyde from polluted air by plants has been proposed, but there are so far few quantitative reports. Here, we developed an efficient plant–microbe technology to remove formaldehyde by adding cultured microorganism into the rhizosphere of three plant species, Tradescantia zebrina Bosse (T. zebrina), Aloe vera (Haw.) Ber (A. vera) and Vigna radiata (Linn.) Wilczek (V. radiata). Shoots were exposed to 0.72 mg of formaldehyde per m3 of air for 24 h. Results show that formaldehyde removal rates were 23.1 ± 0.1 μg/h/g fresh weight with microbes versus 18.5 ± 0.21 μg/h/g without microbes for A. vera, 86.4 ± 0.7 versus 59.3 ± 0.2 μg/h/g for T. zebrine and 97.6 ± 0.9 versus 25.1 ± 4.2 μg/h/g for V. radiata. Overall, formaldehyde removal was increased by 6.7–90.5% using microbes. Formaldehyde removal was influenced by light intensity, with this effect more pronounced in plant–microbe systems. [ABSTRACT FROM AUTHOR]

A fluorometric and colorimetric method are described for the determination of hydrogen peroxide and glucose by integrating copper nanoclusters (CuNCs) into a Fenton-like reaction. The mechanism mainly depends on the fast formation of long-strand DNA-templated CuNCs with strong red fluorescence (with excitation/emission maxima at 340/640 nm) in the absence of H2O2. The DNA can be cleaved into short-oligonucleotide fragments by hydroxy radicals as formed in the Ce(III)-triggered Fenton-like reaction in the presence of H2O2. As a result, short-strand DNA loses the ability as a template for the formation of CuNCs. This leads to a decrease of fluorescence. The colorimetric assay, in turn, is based on the oxidation of colorless Ce(III) ions to the distinctly yellow Ce(IV) ions (with an absorption maximum at 400 nm) by H2O2. Compared with those assays based on the use of enzyme mimics, this method does not require any chromogenic substrates such as ABTS and TMB. Based on the dual-signal readout platform, we successfully achieved the detection of H2O2 and glucose. LODs are as low as 0.266 μM and 2.92 μM. The methods were applied to the sensitive determination of glucose by using glucose oxidase (GOx) which catalyzes the oxidization of glucose to produce H2O2. The practical application was demonstrated by determination of glucose in human serum, with apparent recoveries of 98.4–101.9% and 99.1–105.6%, respectively. The concentration of glucose ranges from 1 to 500 μM and 50 to 600 μM based on the dual-signal readout platform, respectively. This fluorometric and colorimetric dual-mode strategy will pave a new avenue for constructing effective assays for H2O2-related analytes in biochemical and clinical applications. [ABSTRACT FROM AUTHOR]

Unlabelled Image • Flavanones were produced by enzyme treatment of citrus wastes. • Enzyme treatments promoted high bioactive phenolic concentrations in citrus wastes. • Enzymes increased the bioactivity of citrus wastes. • Higher amounts of hesperetin were found after 24 h of enzymatic treatment. • Tannase and β-glucosidase seemed to have a synergistic effect on aglycon production. [ABSTRACT FROM AUTHOR]

Cephalosporins, a large group of β-lactam antibiotics, contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C, and substitutions of chemical groups or modifications of 7-ACA side-chains resulting in varying pharmacologic properties and antimicrobial activities, development of useful antibiotic agents, also. Cephalosporin C obtained by fungus fermentation can be transformed to 7-ACA by two-step or one step enzymatically conversion process. The most important step in 7-ACA downstream process is represented by its separation from enzymatically produced reaction mixture. Among the used methods new separation techniques have been developed and applied to bioseparations, like reactive extraction and pertraction which have considerable potential. [ABSTRACT FROM AUTHOR]

Determination of kinetic parameters of enzymes is important in biotechnology research. It is also one of the most challenging processes in methanol production. The activity of enzyme is determined in term of initial rates at various substrate concentrations. The enzymatic hydrolysis of methanol by pectin methyl esterase (PME) enzyme was investigated at 25 °C and pH 9 over the reaction time range from 0 to 90 min. In this study, the parameters of the enzyme's kinetic, KM and Vmax were directly determined using a modified Michaelis-Menten equation by applying the Lineweaver-Burk plots. Besides, nonlinear regression of Michaelis-Menten equation was calculated based on Euler’s and Runge-Kutta 4th order methods by using Solver supplement application. The result of kinetic constant was tested by comparing the experimental data with model predictions. It was found that Euler and Runge-Kutta method was successful in determining the kinetic parameter rather than Lineweaver-Burk plot. The application of the Michaelis-Menten equation describes the enzyme kinetic very well. From the kinetic analysis, it showed the good agreement between the result obtained and the predictions model in the production of methanol using PME enzyme. [ABSTRACT FROM AUTHOR]

In the present paper, fatty diethanolamide has been produced from Calophyllum inophyllum L (local name: nyamplung) kernel oil. The synthesis of fatty diethanolamide was performed by reacting triglycerides from the nyamplung kernel oil with diethanolamine catalysed by Thermomyces lanuginosus immobilized lipase (Lipozyme TL IM). The optimum reaction conditions were of the ratio of oil to diethanolamine at 1:5 (g:mmol), amount of enzyme at 0.05 g, reaction temperature at 40°C, and incubation time at 2 h. The percentage yield of fatty diethanolamide at optimum condition was 44%. Characterization of the products were performed using gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy. [ABSTRACT FROM AUTHOR]

Highlights • The enzymatic reaction between SBP and EUG, IEUG and VAI, respectively is studied. • Central composite design is used to optimize the different experimental variables. • MCR-ALS method is used to obtain the concentration vs time profiles. • Apparent Michaelis-Menten constants and kinetic parameters were calculated. • Antioxidant apparent acidity constants were also determined. Abstract The oxidation of eugenol, isoeugenol and vanillin natural antioxidants catalyzed by the soybean peroxidase enzyme was studied using uv–vis spectroscopy. An experimental design was used to optimize the different variables. The multivariate curve resolution method was used to obtain the profiles of antioxidant absorbance's as a function of time due to uv–vis absorption bands of both antioxidants and the enzymatic reaction product/s show a strong overlap. From these results, apparent Michaelis-Menten constants as well as the kinetic parameters k 1 and k 3 involved in the catalytic cycle of peroxidases were calculated. The antioxidant apparent acidity constants were also determined at different pH's from uv–vis spectrophotometric measurements. Values of k 1 were (0.6 ± 0.1) × 105 M−1 s−1, (2.0 ± 0.2) × 105 M−1 s−1 and (7.0 ± 0.5) × 106 M−1 s−1 and k 3 (4.0 ± 0.2) × 105 M−1 s−1, (6.0 ± 0.6) × 105 M−1 s−1 and (6.0 ± 0.9) × 106 M−1 s−1 for eugenol, isoeugenol and vanillin, respectively. [ABSTRACT FROM AUTHOR]

The schematic illustration of developing the TdT-mediated isothermal nucleic acids amplification for detecting APE1 activity. [Display omitted] • Designing the TdT-mediated isothermal G-quadruplexes amplification. • Designing a facile and template-free APE1 activity detection assay. • Achieving the high-contrast, specific, and rapid-response fluorescence towards APE1 activity. • Achieving the sensitive detection performances of APE1 activity in biological samples. Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential role in regulating physiological functions and is a critical node in tumor progression. However, conventional detection methods for discriminating APE1 activity are hampered by labor-intensive procedures, multiple operations, and limited detection sensitivity. Herein, we have developed a terminal deoxynucleotidyl transferase (TdT)-mediated randomly arrayed G-quadruplexes (G4s) biosynthesis assay (TGBA) for facile and sensitive detection of APE1 activity. In this assay, the recognition DNA hairpin probe (rDHP) with an AP-site in the stem and modification of the phosphate group in 3′-end (3′-P) has been well-designed to be specifically cleaved by APE1 and then expose the new 3′-OH for rDHP template-free elongation by TdT. Following the rational proportion of the G-rich dNTP pool, the amplified and well-formed G4s can be obtained to bind to the G4 fluorescent dye, such as Thioflavin T (ThT). The effective, specific, high-contrast, and rapid fluorescence readout of TGBA has been validated by the in vitro analysis of APE1 activity and the evaluation of APE1 inhibitor, which enables the sensitive detection of APE1 activity with a lower detection limit of 0.001 U/mL and the detection linear range from 0.005 to 1 U/mL. Inspired by these results, we seek to explore the analysis of APE1 activity in different scenarios, including human serum and cell lysates. The unambiguously distinguishing capability of TGBA is proved by both detecting endogenous APE1 activity in cell lysates and exogenous APE1 activity in human sera. Collectively, TGBA will exhibit promising potential in the diagnosis of APE1-related diseases and the evaluation of drug efficacy. [ABSTRACT FROM AUTHOR]

Enzymatic reactions during tea growth, processing, storage and post-processing conduce to the diversity in the organoleptic qualities and health benefits of tea. While chemical component transformations by enzymatic catalysis have been extensively explored, the dynamic alteration of endogenous and exogenous enzyme activities throughout manufacture chains remain in a mist. This paper discusses main enzymatic reactions involved in tea growth, processing, storage and post-processing, including the dominant enzymes of specific processes, the chemical transformation driven by enzymes and the alteration of endogenous and exogenous enzymes activities. The direct and indirect regulatory factors influencing enzyme activities and their possible applications in tea production practice are also explored. The endogenous enzyme system of tea plants regulates secondary metabolism and stress defense, contributing to the primary accumulation of volatile and non-volatile compounds in tea plants. Endogenous enzymes activities altered unceasingly and are exposed to more substrates due to cell rupture during the withering, spread, rolling and tumbling stages, establishing the intricate substance basis for distinct organoleptic characteristics. Microbial or intentionally added exogenous enzymes, such as β-glucosidase and tannase, dominate during fermentation, storage, and post-processing, transforming chemical profiles to a more radical extent. This review provides theoretical foundation for precise controlling and adequate utilization of enzymes throughout the tea manufacture chains. • The extensive existence of enzymes throughout tea manufacture chains was presented. • Enzyme-mediated quality formation is comprehensively analyzed. • Factors influencing enzymatic reactions are thoroughly summarized. • Future development trends involve the programmed regulation of enzymatic activities. [ABSTRACT FROM AUTHOR]

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO 2. Pressurization was found to lead to an increase of the binding affinity (decrease of K M , respectively) and a decrease of the turnover number, k cat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the K M -values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches. We addressed the combined effect of an aqueous two-phase system and high hydrostatic pressure on the kinetics of formate dehydrogenase-catalyzed oxidation of formate to CO 2. The experimental approach was supported using thermodynamic modeling with the ePC-SAFT equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. [Display omitted] • We studied the combined effects of an aqueous two-phase system and high hydrostatic pressure on the kinetics of a formate dehydrogenase-catalyzed reaction. • The experimental approach was supported using thermodynamic modeling with ePC-SAFT to predict the lphase separation and kinetic parameters. • ePC-SAFT was able to quantitatively predict the K M values up to a pressure of 1000 bar. • The framework presented enables significant advances in bioprocess engineering. [ABSTRACT FROM AUTHOR]

The identification and quantification of viable Escherichia coli (E. coli) are important in multiple fields including the development of antimicrobial materials, water quality, food safety and infections diagnosis. However, the standard culture-based methods of viable E. coli detection suffer from long detection times (24 h) and complex operation, leaving the unmet requirement for fast assessing the efficiency of antimicrobial materials, early alerting the contamination of water and food, and immediately treatment of infections. We present a digital β- d -glucuronidase (GUS) assay in a self-priming polydimethylsiloxane (PDMS) microfluidic chip for rapid E. coli identification and quantification. The GUS expression in viable bacteria was investigated to develop a fast GUS assay at the single-cell level. Single E. coli were stochastically discretized in picoliter chambers and identified by specific GUS activity. The digital GUS assay enabled identifying E. coli within 3 h and quantifying within 4 h for different E. coli subtypes. The specificity of our method was confirmed by using blended bacteria including E. coli, Bacillus , Shigella and Vibrio. We utilized digital GUS assay to enumerate viable E. coli after incubated with antibacterial materials for assessing the antibacterial efficiency. Moreover, the degassed chip can realize automatic sample distribution without external instruments. The results demonstrated the functionality and practicability of digital GUS assay for single E. coli identification and quantification. With air-tight packaging, the developed chip has the potential for on-site E. coli analysis and could be deployed for diagnosis of E. coli infections, antimicrobial susceptibility testing, and warning the fecal pollution of water. Digital GUS assay provides a paradigm, examining the activity of metabolic enzyme, for detecting the viable bacteria other than E. coli. [Display omitted] • A self-dispensing microwell array chip was developed. • Digital GUS assay enabled to differentiate viable E. coli. • E. coli identification and quantification was achieved in 4 h. • The bactericidal efficiency of 2D materials was assessed with digital GUS assay. [ABSTRACT FROM AUTHOR]

D-amino acids, the enantiomers of naturally abundant L-amino acids, bear unique stereochemistry properties that lead to the resistance towards most of the endogenous enzymes. Previous works have demonstrated applications of D-amino acids in therapeutic development with the aid of mirror-image phage display and retro-inverso peptide synthesis. In this review, we highlight the recent progress and challenges in the exploration of D-amino acids at the interface of chemistry and life science. First, we will introduce some progress made in traditional application of D-amino acids to enhance biostability of peptide therapeutics. Then, we discuss some works that explore the relatively underexplored interactions between the enzyme and D-amino acids and enzymatic reactions of D-amino acids. To highlight the enzymatic reactions of D-amino acids, we will describe several emerging works on the enzyme-instructed self-assembly (EISA) and their potential application in selective anti-inflammatory or anticancer therapies. At the end, we briefly mention the challenges and possible future directions.

Microalgae has received overwhelming attention worldwide as a sustainable source for energy generation. However, the production of biofuel from microalgae biomass consists of several steps, of which lipid extraction is the most important one. Because of the nature of feedstock, extraction needs special attention. Three different methods were studied to extract algal oil from two different algae variant, Chlorella sp. and Spirulina sp. The highest percentage oil yield was obtained by ultrasonication (9.4% for Chlorella sp., 6.6% for Spirulina sp.) followed by the Soxhlet and solvent extraction processes. Ultrasonication and Soxhlet extraction processes were further optimized to maximize oil extraction as solvent extraction was not effective in extracting lipid. For ultrasonication, an amplitude of 90% recorded the highest percentage yield of oil for Spirulina sp. and a 70% amplitude recorded the highest percentage yield of oil for Chlorella sp. On the other hand, for Soxhlet extraction, a combination of chloroform, hexane, and methanol at a 1:1:1 ratio resulted in the highest yield of algal oil. Afterward, the crude algae oil from the ultrasonication process was transesterified for 5 h using an immobilized lipase (Novozyme 435) at 40 °C to convert triglycerides into fatty acid methyl ester and glycerol. Thus, ultrasonic-assisted lipid extraction was successful in producing biodiesel from both the species.

The food industry requires rapid and simple detection methods for preventing harm from pathogenic bacteria. Until now, various technologies used to detect foodborne bacteria were time-consuming and laborious. Therefore, we have developed an automated immunomagnetic separation combined with a colorimetric assay for the rapid detection of E. coli O157:H7 in food samples. The colorimetric detection method using enzymatic reaction is fascinating because of its simplicity and rapidity and does not need sophisticated devices. Moreover, the proposed procedures for the detection of bacteria in food take less than 3 h including pre-enrichment, separation and detection steps. First, target-specific immunomagnetic beads were introduced to contaminated milk in a pre-enrichment step. Second, the pre-enriched sample solution containing target bacteria bound on immunomagnetic beads was injected into an automated pretreatment system. Subsequently, the immunomagnetic beads along with target bacteria were separated and concentrated into a recovery tube. Finally, released β-galactosidase from E. coli O157:H7 after lysis was reacted with chlorophenol red β-galactopyranoside (CPRG) used as a substrate and the colorimetric change of CPRG was determined by absorbance measuring or the naked eye. By the proposed approach in this study, we could detect 3 × 102 CFU/mL of E. coli O157:H7 from a milk sample within 3 h.

The estrogen-like mycotoxin zearalenone (ZEN) is one of the most widely distributed contaminants especially in maize and its commodities, such as corn oil. ZEN degrading enzymes possess the potential for counteracting the negative effect of ZEN and its associated high safety risk in corn oil. Herein, we targeted enhancing the secretion of ZEN degrading enzyme by Pichia pastoris through constructing an expression plasmid containing three optimized expression cassettes of zlhy-6 codon and signal peptides. Further, we explored various parameters of enzymatic detoxification in neutralized oil and analyzed tocopherols and sterols losses in the corn oil. In addition, the distribution of degraded products was demonstrated as well by Agilent 6510 Quadrupole Time-of-Flight mass spectrometry. P. pastoris GSZ with the glucoamylase signal was observed with the highest ZLHY-6 secretion yield of 0.39 mg/mL. During the refining of corn oil, ZEN in the crude oil was reduced from 1257.3 to 13 µg/kg (3.69% residual) after neutralization and enzymatic detoxification. Compared with the neutralized oil, no significant difference in the total tocopherols and sterols contents was detected after enzymatic detoxification. Finally, the degraded products were found to be entirely eliminated by washing. This study presents an enzymatic strategy for efficient and safe ZEN removal with relatively low nutrient loss, which provides an important basis for further application of enzymatic ZEN elimination in the industrial process of corn oil production.

The purpose of this study is to present an activity based on integrated Science, Technology, Engineering, and Mathematics [STEM] approach, its implication, steps, and necessary documents. The activity started with a daily-life problem stated in the STEM literature. Later, activity worksheets were provided. Learners were asked to design a process to prevent or slow down apple’s browning reaction. After the first designing step, groups’ preventing processes were compared by the use of a rubric prepared previously. Then, groups were given a chance to re-design preventing apple’s browning process. Finally, in light of the rubric, the most successful preventing process was determined. The activity can be used both in high school chemistry and biology courses. Teachers who implemented the activity stated that the participating learners enjoyed the lesson and came up with interesting ideas. Furthermore, they stated that the activity does not necessitate expensive materials, and can easily be applied.

In this paper, a comprehensive study has been made on the detection of free fatty acids (FFAs) in palm oil via an optical technique based on enzymatic aminolysis reactions. FFAs in crude palm oil (CPO) were converted into fatty hydroxamic acids (FHAs) in a biphasic lipid/aqueous medium in the presence of immobilized lipase. The colored compound formed after complexation between FHA and vanadium (V) ion solution was proportional to the FFA content in the CPO samples and was analyzed using a spectrophotometric method. In order to develop a rapid detection system, the parameters involved in the aminolysis process were studied. The utilization of immobilized lipase as catalyst during the aminolysis process offers simplicity in the product isolation and the possibility of conducting the process under extreme reaction conditions. A good agreement was found between the developed method using immobilized Thermomyces lanuginose lipase as catalyst for the aminolysis process and the Malaysian Palm Oil Board (MPOB) standard titration method (R2 = 0.9453).

Development of point-of-care platforms combining reliability and ease of use is a challenge for the evolution of sensing in healthcare technologies. Here, we report the development and testing of a fully integrated enzymatic colorimetric assay for the sensing of phenylalanine in blood samples from phenylketonuria patients. The platform works with a customized mobile app for data acquisition and visualization and comprises an electronic system and a disposable sensor. The sensing approach is based on specific enzymatic phenylalanine recognition, and the optical transduction method is based on in situ gold nanostructure formation. The phenylketonuria (PKU) smart sensor platform is conceived to perform self-monitoring on phenylalanine levels and real-time therapy tuning, thanks to the direct connection with clinicians. Validation of the technologies with a population of patients affected by PKU, together with the concurrent validation of the platform through centralized laboratories, has confirmed the good analytical performances in terms of sensitivity and specificity, robustness, and utility for phenylalanine sensing. The self-monitoring of phenylalanine for the daily identification of abnormal health conditions could facilitate rapid therapy tuning, improving the wellness of PKU patients.

Abstract A Generalized Integral Transform Technique (GITT) solution scheme for transient non-linear heat and mass diffusion in chemically reacting systems with Michaelis–Menten type kinetics has been developed. A model with two one-dimensional coupled PDEs was selected for illustrating the solution methodology, in which temperature and substrate concentration are the dependent variables. Although the model is one-dimensional, a generalized formulation valid for both plane and curved systems was employed. The main novelty of this investigation is the treatment of the reaction kinetics term, whose functional form is a major obstacle for GITT solutions. This term was resolved by an automatic scheme based on Taylor Series approximations for which the expansion point can be progressively updated with the numerical time-integration, using data from the previous time-step. The outcome of the solution implementation is then verified with data from a reference finite-volumes solution, exhibiting a very good agreement. Finally, illustrative results of averaged temperature and substrate concentration are presented, showing that different type of solutions are obtained within the considered range of dimensionless parameters. [ABSTRACT FROM AUTHOR]

Based on the signal transduction with a horseradish peroxidase (HRP)-functionalized gold nanoprobe, this work develops a new electrochemical aptasensing method for kanamycin (Kana) detection. The aptasensor was constructed through the hybridization of biotinylated Kana-aptamer at its complementary oligonucleotide strand-modified electrode, followed by intercalating methylene blue (MB) into the formed double-stranded DNA (dsDNA). Then, the streptavidin and high-content HRP functionalized gold nanoparticle probes were bound onto the aptasensor. As the MB-mediated HRP-catalytic reaction could produce sensitive electrochemical signal and the aptamer-biorecognition toward Kana could cause quantitative decrease of MB intercalation and nanoprobe capture, convenient electrochemical signal transduction was achieved. Both the enzymatic reaction and nanoprobe signal amplification greatly enhance the electrochemical signal, offering ultrahigh sensitivity of the method. The MB intercalation into dsDNA not only provides necessary electron mediator for the enzymatic reaction but also simplifies the electrochemical measurement. Under optimal conditions, this method showed a wide linear range over four-order of magnitude with a low detection limit of 0.88 pg/mL. In addition, the aptasensor features high specificity, excellent repeatability and stability as well as satisfactory reliability. Thus it possesses valuable application potentials. [ABSTRACT FROM AUTHOR]

Fatty hydroxamic acids (FHA) have been successfully synthesized from Terminalia catappa L. (local name: ketapang) kernel oil by a one-step lipase catalyzed reaction. The use of immobilized lipase as a catalyst to synthesize FHA has several advantages such as a simple purification of product and environmentally friendly. In addition, it also allows the reaction to be carried out under mild conditions, which reduces the reaction's side products. The optimal reaction condition obtains were 40°C temperature reaction for 25 h with the ratio of ketapang seeds oil (g) : hydroxilamin (mmol) is 1 : 2.861 mmol and the ratio of lipase enzyme (g) : ketapang kernel oil (g) is 0,015: 1. Amount of hydroxamic group in 1 gr of sample is 2.46 mol. Qualitative test of FHAs was carried out by color test and FTIR. Complex colors of the FHA with copper (II) and iron (III) are green and dark red, respectively. For FTIR analysis of FHAs group, various peak appeared such as C=O amide (1685 cm-1), O-H (3434 cm-1), N-H (3261,09 and 1568,64 cm-1) and C-N (939,91 cm-1). [ABSTRACT FROM AUTHOR]

Alkyldiethanolamides (fatty acid diethanolamides) synthesis from Terminalia catappa L. kernel oil was optimized using lypozyme as a catalyst. The result showed that the optimal reaction conditions were 2 hours reaction time, with a ratio of oil mass (g) to diethanolamine (mmol) of 1:5, a ratio of oil mass to enzyme (g) of 1: 0.075, and a temperature of 40°C. The percentage of alkyldiethanolamides at optimum condition was 56-60%. The synthesis results were also analyzed by FTIR. FTIR spectra revealed specific absorption at several wave numbers (3434 cm-1, 1655 cm-1, 1280 cm-1), indicating that amide and alcohol bonds (C=O, C-N, and O-H) were formed. GC-MS was employed to identify the types of fatty acid diethanolamides that were successfully synthesized. The fatty acid diethanolamides formed were palmitoyldiethanolamide (Rt = 32.96 min) and oleyldiethanolamide (Rt = 35.57 min). The total nitrogen content of alkyldietanolamides was 0.26%, or 0.19 mmol of the amide group in 1 g of sample. [ABSTRACT FROM AUTHOR]

The new synthetic route to α‐acyloxy amides based on Passerini multicomponent reaction followed by transformation into the corresponding five‐, six‐, and seven‐membered 3‐hydroxy‐lactams has been proposed. The influence of the reaction conditions, which includes substrate structure on the reaction course that led to desired lactams, was studied. The use of various oxo components in the Passerini multicomponent reaction (P‐MCR) has been evaluated. The first example of a semicyclic O,O‐acetal employed as an aldehyde equivalent in the P‐MCR has been reported. Finally, the established protocol was successfully applied for the enantioselective synthesis of 3‐hydroxy‐lactams, which play a crucial role in the synthesis of the number of bioactive compounds. [ABSTRACT FROM AUTHOR]

In this paper, a new analytical method is reported for the steady state concentrations of glucose, oxygen and gluconic acid in a sensitive composite membrane that contains nanoparticles of an anionic polymer glucose oxidase and catalyse embedded in a hydrophobic polymer. The theoretical model involves a system of nonlinear coupled reaction-diffusion equations. This polynomial approximation method is based on the Genocchi polynomials together with their operational matrices of derivative. Also the convergence analysis of the proposed method is discussed. The obtained GPM solutions are compared with the Adomian decomposition method (ADM) solutions. Satisfactory agreement with ADM and numerical results is observed. Moreover, the use of GPM is found to be simple, efficient, flexible and less computation costs. [ABSTRACT FROM AUTHOR]

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla. [ABSTRACT FROM AUTHOR]

The aim of this study is to produce new medium-long-medium structured triacylglycerols (MLM-STAGs) containing caprylic acid (CA) at the sn -1,3 positions, and long chain saturated fatty acids (LCSFAs) and bioactive fatty acids at the sn -2 position via two-step enzymatic process by Lipozyme TL-IM, using autotrophic microalgae ( Isochrysis galbana ) TAGs. In the first step, the ethanolysis of microalgae oil TAGs for 2-monoacylglycerols (2-MAGs) production are performed in the presence of water content of 5 wt% at ethanol/oil mole ratio of 24:1 with Lipozyme TL-IM at 25 °C for 12 h. In the larger-scale production of 2-MAGs, the 2-MAGs yield and the purity of the purified MAGs attained are 31.44-36.08 wt% and 91.57 mol%, respectively. In the second reaction, these 2-MAGs are esterified with caprylic acid (CA) in n -hexane system to synthesize almost pure MLM-STAGs with the recycled Lipozyme TL-IM under the optimized conditions. The final MLM-STAGs contain 64.28% CA and 13.35% ω-3 polyunsaturated fatty acids. The CA-LCSFAs-CA (43.01%) and novel CA-stearidonic acid (SDA, C 18:4ω-3 )-CA (23.88%) are the major MLM-STAGs present. In all, Isochrysis galbana -derived oil containing SDA is a promising source for producing high-value microalgal bioproducts by two-step enzymatic process in a cost-effective manner. [ABSTRACT FROM AUTHOR]

Microparticles have attracted much attention for medical, analytical and biological applications. Calcium carbonate (CaCO3) templating method with the advantages of having narrow size distribution, controlled morphology and good biocompatibility that has been widely used for the synthesis of various protein-based microparticles. Despite CaCO3 template is biocompatible, most of the conventional methods to create stable protein microparticles are mainly driven by chemical crosslink reagents which may induce potential harmful effect and remains undesirable especially for biomedical or clinical applications. In this article, we demonstrate the fabrication of protein microparticles and microcapsules with an innovative method using biomolecular tools such as enzymes and affinity molecules to trigger the assembling of protein molecules within a porous CaCO3 template followed by a template removal step. We demonstrated the enzyme-assisted fabrication of collagen microparticles triggered by transglutaminase, as well as the affinity-assisted fabrication of BSA-biotin avidin microcapsules triggered by biotin-avidin affinity interaction, respectively. Based on the different protein assemble mechanisms, the collagen microparticles appeared as a solid-structured particles, while the BSA-biotin avidin microcapsules appeared as hollow-structured morphology. The fabrication procedures are simple and robust that allows producing protein microparticles or microcapsules under mild conditions at physiological pH and temperature. In addition, the microparticle morphologies, protein compositions and the assemble mechanisms were studied. Our technology provides a facile approach to design and fabricate protein microparticles and microcapsules that are useful in the area of biomaterials, pharmaceuticals and analytical chemistry. [ABSTRACT FROM AUTHOR]

Although there are several reports showing the employment of aqueous two-phase systems (ATPS) for biotransformation in large-scale, the industrial application of ATPS is still hampered by their drawbacks such as slow diffusive mass transfer, long settling time for phase separation, and batch processes. Here, we propose for the first time a continuous-flow process based on a suitably designed ATPS parallel-laminar flow in a double Y-branched microfluidic device using for enzymatic catalysis. In a model urease reaction, polyethylene glycol (PEG) 8 kDa and dextran (Dex) 500 kDa are selected as polymer solutions, in which the enzymes settle mainly in the Dex phase while the products partition mainly to the PEG phase during short residence time. On account of rapid micro-transport, as well as integrate one single step of biocatalysis and separation process, obvious intensification of enzymatic catalysis in such ATPS microfluidic platform is demonstrated. Compared with the conventional ATPS in a beaker under gentle stirring, the enzymatic reaction rate in ATPS microfluidic is 500 times higher. Furthermore, the effects of flow rates, substrate concentration, residence time, and recycling number on the reaction rate and conversion rate are evaluated, respectively. Our study provides an all-aqueous, micro-scale and coupled reaction-separation platform for the process intensification of enzymatic catalysis, which has not only theoretical significance, but also practical value for biotransformation-based fields, such as biomedical, pharmaceuticals and food applications. [ABSTRACT FROM AUTHOR]

Azelaic acid (AzA) and its derivatives have been known to be effective in the treatment of acne and various cutaneous hyperpigmentary disorders. The esterification of azelaic acid with lauryl alcohol (LA) to produce dilaurylazelate using immobilized lipase B from Candida antarctica (Novozym 435) is reported. Response surface methodology was selected to optimize the reaction conditions. A well-fitting quadratic polynomial regression model for the acid conversion was established with regards to several parameters, including reaction time and temperature, enzyme amount, and substrate molar ratios. The regression equation obtained by the central composite design of RSM predicted that the optimal reaction conditions included a reaction time of 360 min, 0.14 g of enzyme, a reaction temperature of 46 °C, and a molar ratio of substrates of 1:4.1. The results from the model were in good agreement with the experimental data and were within the experimental range (R2 of 0.9732).The inhibition zone can be seen at dilaurylazelate ester with diameter 9.0±0.1 mm activities against Staphylococcus epidermidis S273. The normal fibroblasts cell line (3T3) was used to assess the cytotoxicity activity of AzA and AzA derivative, which is dilaurylazelate ester. The comparison of the IC50 (50% inhibition of cell viability) value for AzA and AzA derivative was demonstrated. The IC50 value for AzA was 85.28 μg/mL, whereas the IC50 value for AzA derivative was more than 100 μg/mL. The 3T3 cell was still able to survive without any sign of toxicity from the AzA derivative; thus, it was proven to be non-toxic in this MTT assay when compared with AzA [ABSTRACT FROM AUTHOR]

General quality characteristics including formaldehyde content of iced and un-iced sea foods were assessed in this study. Formation of formaldehyde in sea food is by enzymatic reaction and oxidation of lipids as a result of the activity of microbes. Great attention has been paid to volatile aldehydes like formaldehyde in aquatic products. Results showed that formaldehyde levels were significantly higher in un-iced fishes than that of the immediately iced fishes and it was in the range of 0.001 - 0.32 mg/kg in iced fishes and it was in the range of 10.64 - 18.75 mg/kg in un-iced fishes. Study showed that seafood contained high amount of formaldehyde because of natural production by postmortem enzymatic reaction. This study clarified that improperly stored seafood is a source of formaldehyde to human being. Sensory and microbiological qualities were good in fresh fishes than that of formaldehyde contained fishes. Normally sea foods were in good quality until the formaldehyde content and microbiological counts were below the permissible levels. [ABSTRACT FROM AUTHOR]

Here, we present a miniaturized lab-on-a-chip detecting system for an all-electric and label-free analysis of the emulsion droplets incorporating the nanoscopic silicon nanowires-based field-effect transistors (FETs). We specifically focus on the analysis of β-galactosidase e.g., activity, which is an important enzyme of the glycolysis metabolic pathway. Furthermore, the efficiency of the synthesis and action of β-galactosidase can be one of the markers for several diseases, e.g., cancer, hyper/hypoglycemia, cell senescence, or other disruptions in cell functioning. We measure the reaction and reaction kinetics-associated shift of the source-to-drain current Isd in the system, which is caused by the change of the ionic strength of the microenvironment. With these results, we demonstrate that the ion-sensitive FETs are able to sense the interior of the aqueous reactors; thus, the conjunction of miniature nanosensors and droplet-based microfluidic systems conceptually opens a new route toward a sensitive, optics-less analysis of biochemical processes.

The micro sequential injection analysis / lab-on-valve (µSIA-LOV) system is a miniaturized SIA system resulting from the implementation of a lab-on-valve (LOV) atop of the selection valve. It integrates the detection cell and the sample processing channels into the same device, promoting the reduction of reagent consumption and waste generation, the improvement of the versatility, and the reduction of the time of analysis. All of these characteristics are really relevant to the implementation of enzymatic reactions. Additionally, the evaluation of cholesterol in serum samples is widely relevant in clinical diagnosis, since higher values of cholesterol in human blood are actually an important risk factor for cardiovascular problems. An automatic methodology was developed based on the µSIA-LOV system in order to evaluate its advantages in the implementation of enzymatic reactions performed by cholesterol esterase, cholesterol oxidase and peroxidase. Considering these reactions, the developed methodology was also used for the evaluation of cholesterol in human serum samples, showing reliable and accurate results. The developed methodology presented detection and quantification limits of 1.36 and 4.53 mg dL−1 and a linear range up to 40 mg dL−1. This work confirmed that this µSIA-LOV system is a simple, rapid, versatile, and robust analytical tool for the automatic implementation of enzymatic reactions performed by cholesterol esterase, cholesterol oxidase, and peroxidase. It is also a useful alternative methodology for the routine determinations of cholesterol in real samples, even when compared with other automatic methodologies.

This paper concerns the synthesis of structured triglycerides containing different proportions and positions from medium- (M) and long-chain (L) fatty acids on a glycerol backbone. Structured triglycerides of MLM type were synthesized by utilizing canarium oil and incorporating caprylic acid (C8:0) as a source for the medium chain (M) fatty acids. Synthesis was performed through a two-step enzymatic reaction, with ethanolysis as the first step and esterification as the second step. Both reactions use the sn-1,3 specific lipase as a catalyst, which has specific activity at positions sn-1 and sn-3 of the triglyceride structure. The results from high-performance liquid chromatography showed that the stereospecific distribution of fatty acids in the structured triglyceride was 29.52±0.59 and 44.28±0.88 mol% of caprylic acid in the positions of sn-1,2,3 and sn-1,3, respectively. Furthermore, analysis of the physicochemical properties of both the native canarium oil and the structured triglycerides using an independent-sample t-test at p < 0.05 indicated that the two samples were significantly different for saponification number, iodium number, and average molecular weight. The results of this study showed that canarium oil can be exploited as a starting material for functional food application. [ABSTRACT FROM AUTHOR]