Ping-pong Mechanism Research Articles

Asymmetric Sulfoxidation of Thioether Catalyzed by Soybean Pod Shell Peroxidase to Form Enantiopure Sulfoxide in Water-in-Oil Microemulsions: A Kinetic Model.

Esomeprazole with chiral sulfoxides structure is used to treat gastric ulcer disease. Soybean pod shell peroxidase (SPSP) is a peroxidase extracted from soybean pods shells which are one of the most abundant natural resources in the world. In the production of chiral sulfoxides catalyzed by SPSP, it is very important to establish the reaction kinetic model and explore the reaction mechanism for the development of the process, however, there is no report on the establishment of the model. Asymmetric sulfoxidation reactions catalyzed by SPSP in water-in-oil microemulsions were carried out, and the King-Altman approach was used to establish a kinetic model. A yield of 91% and e.e. value of 96% for esomeprazole were obtained at the activity of SPSP of 3200 U ml-1 and 50 °C for 5 h. The mechanism with a two-electron reduction of SPSP-I is accompanied with a single-electron transfer to SPSP-I and nonenzymatic reactions, indicating that three concomitant sub-mechanisms contribute to the asymmetric oxidation involving five enzymatic and two nonenzymatic reactions, which can represent the asymmetric sulfoxidation of organic sulfides to form enantiopure sulfoxides. With 5.44% of the average relative deviation, a kinetic model fitting experimental data was developed. The enzymatic reactions may follow ping-pong mechanism with substrate inhibition of H2 O2 and product inhibition of esomeprazole, while nonenzymatic reactions follow a power law. Those results indicate that SPSP with a lower cost and higher thermal stability may be used as an effective substitute for horseradish peroxidase.

A green and facile approach to a graphene-based peroxidase-like nanozyme and its application in sensitive colorimetric detection of L-cysteine.

A facile and green approach to the preparation of peroxidase-like nanozymes by reducing and functionalizing graphene oxide (rGO) with Ganoderma polysaccharide (GP) has been achieved in this work. Our results showed that the as-fabricated nanozyme, namely rGO-GP, possessed the excellent property of simulating peroxidase with higher catalytic activity compared with GO or rGO obtained by using chitosan, which may be due to the better dispersion of rGO-GP in the solution. Steady-state kinetics studies further showed that the catalytic process conformed to Michaelis-Menten equation and ping-pong mechanism. Benefiting from the excellent peroxidase property of rGO-GP, we have also successfully established a highly sensitive and selective colorimetric detection approach to trace detection of L-cysteine (L-Cys). The limit of detection (LOD) of L-cysteine is 0.1μM and the linear detection range is 2-30μM. Furthermore, the nanozyme was successfully applied for detecting L-cysteine in serum. This work therefore demonstrates the advantages of rGO-GP as an effective nanozyme in both its green synthesis and detecting application.

Peroxidase-Like Platinum Clusters Synthesized by Ganoderma lucidum Polysaccharide for Sensitively Colorimetric Detection of Dopamine

The sensitive and selective detection of dopamine (DA) is very important for the early diagnosis of DA-related diseases. In this study, we reported the colorimetric detection of DA using Ganoderma lucidum polysaccharide (GLP) stabilized platinum nanoclusters (Ptn-GLP NCs). When Pt600-GLP NCs was added, 3,3’,5,5’-tetramethylbenzidine (TMB) was rapidly catalyzed and oxidized to blue oxTMB, indicating the peroxidase-like activity of Pt600-GLP NCs. The catalytic reaction on the substrate TMB followed the Michaelis-Menton kinetics with the ping-pong mechanism. The mechanism of the colorimetric reaction was mainly due to the formation of hydroxyl radical (•OH). Furthermore, the catalytic reaction of Pt600-GLP NCs was used in the colorimetric detection of DA. The linear range for DA was 1–100 μM and the detection limit was 0.66 μM. The sensitive detection of DA using Pt-GLP NCs with peroxidase-like activity offers a simple and practical method that may have great potential applications in the biotechnology field.

New Paradigm, Equations, Algorithm, and Computer software of Mass‐action Law Based Biodynamics, Pharmacodynamics and Bioinformatics (MAL‐BD/PD/BI) for Econo‐Green Biomedical R&D and Regulatory Guidance

The prevailing divergent, empirical biomedical R&D heavily rely on statistics for variation, probability, correlation, and significance that may lead to inefficiency or high cost-effectiveness. An alternative precision/quantitative approach with Nature's biochemical, biophysical and bio-mathematical principle, leads to the unified fundamental Doctrine of the Median (DoM), that serves as the as the largest common denominator to simplify biomedical complexity and diversity. Instead of using many data points to fit the empirical curves or models, we can reversely, using only few dose data-points to fit DoM, with MAL equations, algorithms and auto-computer simulations of diagnostic plots using CompuSyn software, to achieve quantitative, automated, digitalized or indexed conclusions, in vitro, in animals and in clinical trials. Thus, experimental observations-first priority has become MAL theory-first compliance endeavor. From MAL to DoM to MAL-BD/PD/BI, took over 17 years to complete that involve the derivation of over 300 bio-reaction rate equations via the un-precedent pattern analysis, combinatory analysis and mathematical induction-and-deduction. MAL-BD/PD/BI reveals three main paradigms: (1) The Median-effect equation (MEE) as the unified theory that indicates i. Dose and Effect are interchangeable, ii. Median dose (Dm) is the universal reference point and dynamic-order common link, iii. Ki can never greater than IC50, iv. Dose-effect curves (DEC) can be linearized into straight lines, with slope (m) and x-intercept (log Dm) of the Median-effect plot (MEP), and v. The DEC can be generated via MEP with a minimum of only two data points (the MTDP theory) by computer simulation, where the 3rd point is dose-zero, and the 4th point is the universal Dm point. Thus, for data-points availability, 2 becomes 4, 3 becomes 5, and 4 becomes 6. This is a breakthrough for in vivo study/trials. (2) The Combination Index equation (CIE) where CI<1, =1, and >1 indicates, synergism, additive effect, and antagonism, respectively. (3) The Dose reduction index equation (DRIE), where DRI>1, =1, and <1, indicate favorable, no dose-reduction, and unfavorable dose-reduction, respectively, in drug combinations. The diagnostic plots provide needed BI. The terms of DoM, MEE and CIE are all dimensionless ratios, these indicating that the theory is independent to drug's physical entities (chemicals, biologicals, natural products, radiation, temperature, oxygen-tension, and pH, etc), units (ug, nM, mg/ml, mg/kg, IU, Rad, multiple of infections, etc) or mechanisms of actions (competitive, noncompetitive, uncompetitive, sequential, ordered, ping-pong, and random mechanisms). As of Jan 1, 2021, the article introduced MAL-BD/PD/BI algorithms (Chou TC, Pharmacol. Rev. 58:621-681, 2006) received 3,813 citations in 1,030 biomedical journals. In addition, the article introducing the CI algorithm and computer software (Chou TC and Talalay P. Adv. Enz. Regul. 22:27-55, 1984) received 6,937 citations in 1,338 biomedical journals globally. These confirmed the board applications to nearly all disciplines of biomedical R&D.

Palladium Nanoparticles/Graphdiyne Oxide Nanocomposite with Excellent Peroxidase-like Activity and Its Application for Glutathione Detection

In this study, palladium nanoparticles loaded graphdiyne oxide (Pd/GDYO) nanocomposite were fabricated by in-situ reduction of palladium chloride in the dispersion of GDYO, and characterized by Raman spectra, transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The synthesized Pd/GDYO was first found to have catalytic activities similar to those of the peroxidase enzyme, which can catalyze the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine(TMB) in the presence of hydrogen peroxide(H2O2). Steady-state kinetic studies showed that the catalytic reaction of Pd/GDYO follows a ping-pong mechanism, and Pd/GDYO has a stronger activity to TMB with a Michaelis constant(Km) value of 5.32×10−4 mmol/L. Based on the shielding effect of glutathione(GSH) on the Pd/GDYO-H2O2-TMB reaction system, a colorimetric detection method for GSH was developed with a wide linear range from 0.1 µmol/L to 40 µmol/L and a limit of detection of 0.1 µmol/L. The method was successfully applied for fast and accurate detection of GSH in injection powder drugs. It was expected that this peroxidase-like Pd/GDYO nanocomposite would have wide applications in the fields of biomedicine and environmental chemistry.

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too.

Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.

A Superfamily-wide Activity Atlas of Serine Hydrolases in Drosophila melanogaster.

The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of chemical proteomics techniques, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlases for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, we initially report a bioinformatics analysis toward the identification and categorization of nonredundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as a proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult D. melanogaster, and we propose new research directions, resources, and tools that this study can provide to the fly community.

A New Approach to Synthesis of Benzyl Cinnamate: Kinetic and Thermodynamic Investigation

Background: Benzyl cinnamate is widely used in many fragrance compounds. The traditional methods to obtain benzyl cinnamate, including chemical synthesis or extraction from leaves, have many drawbacks. Recently, lipase-catalyzed synthesis of benzyl cinnamate has provided us with a promising alternative due to its high catalytic efficiency, mild reaction conditions, and green and environmentally friendly process. In this work, the purpose is to investigate the kinetics and thermodynamics of enzymatic synthesis of benzyl cinnamate. Objective: The objective of the current study was to investigate the kinetics and thermodynamics of the enzymatic synthesis of benzyl cinnamate. Results: The results showed that the reaction followed the Ping-Pong mechanism leading to substrate inhibition. Furthermore, the kinetic and thermodynamic parameters were estimated. The inhibition constant KiB decreased with temperature enhancement, implying that the inhibition of benzyl alcohol on lipase could be depressed at high temperatures. Moreover, the activation energy of the first-step reaction (52.46 kJ/mol) was much higher than that of the second-step reaction (12.97 kJ/mol), demonstrating that the first-step reaction was the rate-limiting reaction. The esterification process was found to be endothermic, with an enthalpy value (ΔH) of +55.7 kJ/mol and entropy value (ΔS) of +170.2 J/mol K, respectively. Based on the change of Gibbs free energy (ΔG), enzymatic esterification of cinnamic acid changed from non-spontaneous to spontaneous reaction when temperature was raised to above 53ºC. Conclusion: This obtained information could be utilized to optimize the biosynthesis of benzyl cinnamate.

Induction and catalytic properties of grasshopper (Zonocerus variegatus) glutathione transferase fed on different food plants

In order to establish the role of diet on the induction and catalytic properties of glutathione transferase (GST) in insects, variegated grasshopper (Zonocerus variegatus) was exposed to different food plants separately for 30 days and the properties of the induced enzyme were then investigated.Insects fed on cassava (M. esculenta) leaves had the highest GST induction followed by insects fed on bitter leaf (V. amygdalina). Z. variegatus that fed in the wild on different food plants had the least suggesting that allelochemicals in the food plants have a compensatory toxicity-alleviating actions on one another. 1-Chloro-2,4-dinitrobenzene (CDNB) was the best substrate for all the induced GST however, the mode of binding of the substrate to the induced enzyme was not the same. GST from M. esculenta-fed insect showed ping-pong kinetic mechanism whereas GSTs from V. amygdalina and T. procumbens-fed insects showed random sequential mode of substrate binding. Catalytic efficiency (kcat/Km) of GST from M. esculenta-fed insects was 3‐8-fold higher than other induced enzymes. Commercial insecticides- cypermethrin and lindane had an inhibition constant, Ki, of 0.13±0.004 mM and 0.68±0.09 mM, respectively, suggesting that the concentration as used in the field (0.03 mM for cypermethrin and 0.3 mM for lindane) would have little effect on the insect's GST.The study concluded that higher GST activity are induced in insects that fed on monotonous diets than those that fed on various food plants. Hindgut appears to be the primary organ of detoxication. The catalytic properties of the induced enzymes are different from one another.

Choline Chloride-urea Deep Eutectic Mixture Water for the Synthesis of an Amphiphilic Compound of Glyceryl Monocaffeate.

The Lipase-catalyzed synthesis of glyceryl monocaffeate (GMC) in choline chloride-urea of natural deep eutectic solvent (NADES) media is reported to provide amphiphilic character to caffeic acid (CA). The modification of CA into GMC could potentially increase its solubility and widen the application of CA's biological activities in water and oil-based systems. The high conversion was achieved when the reaction was carried out with the addition of more than 20 %v/v water, at a high molar ratio of glycerol and 40°C. It was found that the lipase-catalyzed transesterification of ethyl caffeate (EC) and glycerol in choline chloride-urea of DES media obeyed ping-pong bi-bi mechanism with Vmax = 10.9 mmol.min-1, KmEC = 126.5 mmol and KmGly = 1842.7 mmol.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi–bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi–bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity.

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs (AA-tRNAs) to catalyse cyclodipeptide formation in a ping-pong mechanism. Despite intense studies of these enzymes in past years, the tRNA regions of the two substrates required for CDPS activity are poorly documented, mainly because of two limitations. First, previously studied CDPSs use two identical AA-tRNAs to produce homocyclodipeptides, thus preventing the discriminative study of the binding of the two substrates. Second, the range of tRNA analogues that can be aminoacylated by aminoacyl-tRNA synthetases is limited. To overcome the limitations, we studied a new model CDPS that uses two different AA-tRNAs to produce an heterocyclodipeptide. We also developed a production pipeline for the production of purified shortened AA-tRNA analogues (AA-minitRNAs). This method combines the use of flexizymes to aminoacylate a diversity of minitRNAs and their subsequent purifications by anion-exchange chromatography. Finally, we were able to show that aminoacylated molecules mimicking the entire acceptor arms of tRNAs were as effective a substrate as entire AA-tRNAs, thereby demonstrating that the acceptor arms of the two substrates are the only parts of the tRNAs required for CDPS activity. The method developed in this study should greatly facilitate future investigations of the specificity of CDPSs and of other AA-tRNAs-utilizing enzymes.

A new electrochemical strategy for the synthesis of a new type of sulfonamide derivatives

This study is the first report of electrochemical generation of hydroxyimino-cyclohexa-dien-ylidene haloniums and their application in the synthesis of new halo-N-hydroxysulfonamide derivatives. These compounds were obtained in a one-pot process based on the reaction of halonium acceptors with arylsulfinic acids. The method is easy to carry out, as it is performed using the carbon electrodes in a simple undivided cell. The protocol has a broad substrate scope with a tolerance for a variety of functional groups. The proposed mechanism is a ping-pong type reaction mechanism, which in its first stage the halonitroarene is reduced at the cathode to related hydroxylamine and in the second stage the cathodically generated hydroxylamine by oxidation at the anode and participating in disproportionation reaction is converted to the halonium acceptor.

2D CTAB-MoSe2 Nanosheets and 0D MoSe2 Quantum Dots: Facile Top-Down Preparations and Their Peroxidase-Like Catalytic Activity for Colorimetric Detection of Hydrogen Peroxide.

We report the facile and economic preparation of two-dimensional (2D) and 0D MoSe2 nanostructures based on systematic and non-toxic top-down strategies. We demonstrate the intrinsic peroxidase-like activity of these MoSe2 nanostructures. The catalytic processes begin with facilitated decomposition of H2O2 by using MoSe2 nanostructures as peroxidase mimetics. In turn, a large amount of generated radicals oxidizes 3,3,5,5-tetramethylbenzidine (TMB) to produce a visible color reaction. The enzymatic kinetics of our MoSe2 nanostructures complies with typical Michaelis–Menten theory. Catalytic kinetics study reveals a ping–pong mechanism. Moreover, the primary radical responsible for the oxidation of TMB was identified to be Ȯ2− by active species-trapping experiments. Based on the peroxidase mimicking property, we developed a new colorimetric method for H2O2 detection by using 2D and 0D MoSe2 nanostructures. It is shown that the colorimetric sensing capability of our MoSe2 catalysts is comparable to other 2D materials-based colorimetric platforms. For instance, the linear range of H2O2 detection is between 10 and 250 μM by using 2D functionalized MoSe2 nanosheets as an artificial enzyme. Our work develops a systematic approach to use 2D materials to construct novel enzyme-free mimetic for a visual assay of H2O2, which has promising prospects in medical diagnosis and food security monitoring.

Generalized enzymatic mechanism of catalysis by tetrameric l-asparaginases from mesophilic bacteria

The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

Biodiesel production catalysed by low-cost liquid enzyme Eversa® Transform 2.0: Effect of free fatty acid content on lipase methanol tolerance and kinetic model

A low-cost liquid lipase from genetically modified Aspergillus oryzae (Eversa® Transform 2.0) was used in this study for biodiesel production. The catalytic performance of this enzyme was evaluated using refined palm oil adjusted with different free fatty acid (FFA) content. The methanol-to-oil molar ratio was varied from 1:1 to 8:1 to determine the reaction rate, ester conversion and methanol tolerance of this enzyme at different FFA content. The reaction was carried out at low temperature (40 °C) for 24 h using very low enzyme concentration (0.2 wt%). The results showed that the enzyme could tolerate a higher dosage of methanol and could increase reaction rate and conversion when higher FFA content was present in the feedstocks. A biodiesel conversion of 97% could be obtained when the feedstocks contained ≥80 wt% FFA. Furthermore, a new semi-empirical model based on the Ping-Pong Bi-Bi mechanism has been developed to estimate the reaction kinetics of biodiesel production from feedstocks containing a mixture of triacylglycerol and FFA. In conclusion, Eversa® Transform 2.0 can be used for the production of biodiesel from low-quality feedstocks containing high FFA content in a sustainable and economical manner.

One-pot three-step enzymatic ROP in situ to form polycaprolactone from cyclohexanone: Optimizing and kinetic modeling

A one-pot ring-opening polymerization (ROP) to form poly ε-caprolactone (PCL) from cyclohexanone (CHN) mediated by immobilized T. laibacchii lipase was proposed. The optimal conditions by using response surface methodology (RSM) were established as follows: the supplement of lipase, 23.55 mg, the reaction temperature, 59.4 °C and the molar ratio of urea hydrogen peroxide (UHP) to CHN, 1.31:1, under which the yield of PCL and number-average molar mass (Mn) at a high CHN concentration of 1.25 M were 96.5% and 2912, respectively. The one-pot ROP consists three steps: first, ethyl acetate (EtOAc) reacts with UHP to generate peracetic acid catalyzed by lipase; then, CHN is lactonized to ε-caprolactone (CL) with peracetic acid in situ generated in nonenzymatic, and acetic acid formed reacts with UHP to form peracetic acid; finally, CL suffers from enzymatic ROP to in situ generate PCL. There are three factors contributing to the high yield: cross-linked immobilized lipase employed, the supplement of lipase added in step 3 of ROP and optimal conditions adopted. The initial concentration of UHP was reduced from 2.5 M to 1.625 M owing to acetic acid reacting with UHP to yield peracetic acid which can help obtain a high yield of PCL. A kinetic model for this one-pot three-step enzymatic ROP reaction was developed, which fitted the observed data well. The enzymatic reaction mediated by lipase may obey the ping-pong mechanism with substrate inhibition of UHP and acetic acid, while the nonenzymatic reaction may obey a power law. Using the kinetic model, the contribution of the reaction of UHP with EtOAc and acetic acid to the yield of peracetic acid can be distinguished, contributing to better comprehend the reaction process.

Kinetics of Enzymatic Synthesis of Cyanidin-3-Glucoside Lauryl Ester and Its Physicochemical Property and Proliferative Effect on Intestinal Probiotics.

The interest in anthocyanins used in food, cosmetic, and pharmaceutical industries has increased the research in order to improve their stability while maintaining bioactivity. In this work, cyanidin-3-glucoside lauryl ester (Cy3glc-C12) was enzymatically synthesized, using Novozym 435 as a catalyst, as well as to obtain a kinetic model for the bioprocess. Its liposolubility, UV–VIS absorbance property, thermostability, and potential proliferative effect on intestinal probiotics were also studied. The maximum conversion yield (68.7 ± 2.1%) was obtained with a molar ratio (substrate:donor) of 1:56, 435 16.5 g/L Novozym, temperature of 56 °C, and a time of 28 h via the acylation occurred at 6′′-OH position of the glucoside. The kinetics of the reaction is consistent with a ping-pong bi-bi mechanism and the parameters of the respective kinetic equations are reported. Compared with native Cy3glc, the liposolubility, pH resistivity and thermostability of Cy3glc-C12 were significantly improved. The growth kinetics of Bifidobacteria and Lactobacillus was established based on the Logistic equation, and Cy3glc-C12 could promote their proliferation especially during the logarithmic growth, in which lower pH and more bacteria population were found compared with those of media without anthocyanins. This research provided a reference for the industrial production of Cy3glc-C12 and extended its application to natural products in lipophilic systems.

Deglycosylation of the Isoflavone C-Glucoside Puerarin by a Combination of Two Recombinant Bacterial Enzymes and 3-Oxo-Glucose.

A human intestinal bacterium strain related to Dorea species, PUE, can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose. We reported previously that 3″-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation, and we characterized a bacterial enzyme, the DgpB-DgpC complex, that cleaved the C-glycosidic bond in 3″-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of the C-3″ hydroxyl in puerarin has not been identified. In this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride acceptor. In the redox reaction, NAD(H) functioned as the cofactor, which bound tightly but noncovalently to DgpA. Kinetics analysis of DgpA revealed that the reaction proceeded via a ping-pong mechanism. Enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, the DgpB-DgpC complex, and 3-oxo-glucose. In addition, the metabolite derived from the sugar moiety in the 3″-oxo-puerarin-cleaving reaction catalyzed by the DgpB-DgpC complex was characterized as 1,5-anhydro-d-erythro-hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a β-elimination-like mechanism.IMPORTANCE One important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more-absorbable aglycones and further degradation products that show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O-deglycosylation have been reported, enzymes responsible for C-deglycosylation are still limited. In this study, we characterized enzymes involved in the C-deglycosylation of puerarin from a human intestinal bacterium, PUE. Here, we report the purification and characterization of a recombinant oxidoreductase involved in C-glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C-deglycosylation.

Biochemical characterization of NADH:FMN oxidoreductase HcbA3 from Nocardioides sp. PD653 in catalyzing aerobic HCB dechlorination.

Nocardioides sp. PD653 genes hcbA1, hcbA2, and hcbA3 encode enzymes that catalyze the oxidative dehalogenation of hexachlorobenzene (HCB), which is one of the most recalcitrant persistent organic pollutants (POPs). In this study, HcbA1, HcbA2, and HcbA3 were heterologously expressed and characterized. Among the flavin species tested, HcbA3 showed the highest affinity for FMN with a K d value of 0.75±0.17 µM. Kinetic assays revealed that HcbA3 followed a ping-pong bi-bi mechanism for the reduction of flavins. The K m for NADH and FMN was 51.66±11.58 µM and 4.43±0.69 µM, respectively. For both NADH and FMN, the V max and k cat were 2.21±0.86 µM and 66.74±5.91 sec-1, respectively. We also successfully reconstituted the oxidative dehalogenase reaction in vitro, which consisted of HcbA1, HcbA3, FMN, and NADH, suggesting that HcbA3 may be the partner reductase component for HcbA1 in Nocardioides sp. PD653.