L-glutamate Dehydrogenase Research Articles

Formate-Mediated Electroenzymatic Synthesis via Biological Cofactor NADH.

Synthetic biohybrid systems by coupling artificial system with nature's machinery may offer a disruptive solution to address the global energy crisis. We developed a versatile electroenzymatic pathway for the continuous synthesis of valuable chemicals, facilitated by formate-driven NADH regeneration. Utilizing a bismuth electrocatalyst, we achieved stable CO2 reduction to formate with approximately 90 % Faraday efficiency at a current density of 150 mA cm-2. The generated formate acts as a mediator to regenerate NADH, which is then coupled with immobilized redox enzymes-alcohol dehydrogenase (ADH), L-lactate dehydrogenase (LDH), and L-glutamate dehydrogenase (GDH)-to produce targeted chemicals at significant rates and exceptionally high turnover numbers (1.8×106 to 3.1×106). These achievements not only underscore the efficiency of the system but also its practical applicability in industrial settings. By leveraging in situ generated formate, this innovative approach demonstrates the potential of integrating electrocatalysis with enzymatic reactions for sustainable and efficient chemical production on a practical scale.

Bio-Inspired Photosynthesis Platform for Enhanced NADH Conversion and L-Glutamate Synthesis.

Inspired by the layered structure, light absorption, and charge carrier pathway of chloroplast thylakoids in natural photosynthesis, we propose a novel artificial photosynthesis platform, which is composed of layered structured vaterite as the scaffold with gold nanoparticles (AuNPs), photosensitizer eosin Y (EY), and redox enzyme L-glutamate dehydrogenase (GDH) as the functional components. The EY exhibited significantly enhanced light absorption and charge carrier generation due to the localized surface plasmon resonance (LSPR) around the AuNPs and light refraction within the layers. This artificial photosynthesis platform can regenerate reduced nicotinamide adenine dinucleotide (NADH) under visible light and promote the rapid conversion of α-ketoglutarate to L-glutamate (0.453 Mm/h). The excellent biocompatibility of layered vaterite significantly enhances the resistance of GDH to harsh conditions, including high pH (pH = 10) and elevated temperatures (37-57 °C).

Regulatory mechanism of the six-method massage antipyretic process on lipopolysaccharide-induced fever in juvenile rabbits: A targeted metabolomics approach

ObjectiveTo investigate the mechanism of the six-method massage antipyretic process (SMAP) and its influence on the body's metabolic state. MethodsThe random number table method was used to divide 24 New Zealand 2-month-old rabbits with qualified basal body temperature into a control group, model group and massage group (n = 8 per group). The model group and massage groups were injected with 0.5 μg/ml lipopolysaccharide (1 ml/kg) into the auricular vein, and the control group was injected with the same amount of normal saline at the same temperature. One hour after modelling, the massage group was given SMAP (opening Tianmen, pushing Kangong, rubbing Taiyang, rubbing Erhougaogu, clearing the Tianheshui and pushing the spine). The change of anal temperature 5 h after moulding was recorded to clarify the antipyretic effect. ResultsAfter modelling, the rectal temperature of the juvenile rabbits in the three groups increased. The rectal temperature of the model group was higher than that of the control group 5 h after modelling, and the rectal temperature of the massage group was lower than that of the model group (P < 0.05). The antipyretic mechanism is related to the regulation of the synthesis of phenylalanine, tyrosine and tryptophan, as well as the pentose phosphate pathway. Compared with the model group, the plasma interleukin (IL)-1, IL-6, interferon-gamma, toll-like receptor 4, nuclear factor κB, the mechanistic target of rapamycin complex 1, indoleamine 2,3-dioxygenase 1, aryl hydrocarbon receptor, liver aspartate transaminase (AST), alanine transaminase (ALT) and l-glutamate dehydrogenase (L-GLDH) expression in the massage group were significantly decreased (P < 0.05). Compared with the model group, the massage group had significantly reduced AST, ALT and L-GLDH expression in plasma (P < 0.05). ConclusionThe mechanism of SMAP therapy is related to regulating the expression of peripheral inflammatory factors and metabolic pathways.

High Selectivity Cofactor NADH Regeneration Organic Iridium Complexes Used for High-Efficiency Chem-Enzyme Cascade Catalytic Hydrogen Transfer.

Our research demonstrated that novel pentamethylcyclopentadienyl (Cp*) iridium pyridine sulfonamide complex PySO2NPh-Ir (7) could highly specifically catalyze nicotinamide adenine dinucleotide (NAD+) into the corresponding reducing cofactor NADH in cell growth media containing various biomolecules. The structures and catalytic mechanism of 7 were studied by single-crystal X-ray, NMR, electrochemical, and kinetic methods, and the formation of iridium hydride species Ir-H was confirmed to be the plausible hydride-transfer intermediate of 7. Moreover, benefiting from its high hydrogen-transfer activity and selectivity for NADH regeneration, 7 was used as an optimal metal catalyst to establish a chem-enzyme cascade catalytic hydrogen-transfer system, which realized the high-efficiency preparation of l-glutamic acid by combining with l-glutamate dehydrogenase (GLDH).

Azure A embedded in carbon dots as NADH electrocatalyst: Development of a glutamate electrochemical biosensor

Carbon nanodots modified with azure A (AA-CDs) have been synthesized and applied as redox mediator of bioelectrocatalytic reactions. A deep characterization of AA-CDs nanomaterial has been carried out, proving the covalent attachment of azure A molecules into the carbon dots nanostructure. Disposable screen-printed carbon electrodes (SPCE) have been modified with AA-CDs, through the action of chitosan polymer (Chit-AA-CDs/SPCE). The Chit-AA-CDs/SPCE electrocatalytic activity towards the oxidation of NADH has been proved, obtaining excellent results regarding the low oxidation potential achieved (−0.15 V vs. Ag) and low detection and quantification limits (LOD and LOQ) for NADH, 16 and 53 µM, respectively. The developed electrochemical platform has been applied for the construction of a glutamate biosensor by immobilizing L-glutamic dehydrogenase (GLDH/Chit-AA-CDs/SPCE). The morphology of GLDH/Chit-AA-CDs/SPCE platform was analysed by AFM at each different step of the electrode modification process. The resulting biosensing platform is capable of detect NADH enzymatically generated by GLDH in the presence of glutamate and NAD+. Good analytical parameters were obtained for glutamate analysis using GLDH/Chit-AA-CDs/SPCE, as LOD and LOQ of 3.3 and 11 µM, respectively. The biosensor has been successfully applied to the analysis of food and biological samples.

Photoelectrocatalytic biosynthesis fuelled by microplastics

Biocatalytic artificial photosynthesis integrates photocatalysis and redox biocatalysis to synthesize value-added chemicals using solar energy. However, this nature-inspired approach suffers from sluggish rates of reaction because of challenging water oxidation kinetics. Here we report photoelectrochemical biosynthetic reactions that use non-recyclable real-world poly(ethylene terephthalate) (PET) microplastics as an electron feedstock. A Zr-doped haematite photoanode extracts electrons from hydrolysed PET solutions obtained from post-consumer PET waste, such as drinks bottles, and transfers the electrons to the bioelectrocatalytic site. Carbon-based cathodes receive the electrons to activate redox enzymes (for example, unspecific peroxygenase, L-glutamate dehydrogenase and ene-reductase from the old yellow enzyme family) that drive various organic synthetic reactions. These reactions include oxyfunctionalization of C–H bonds, amination of C=O bonds and asymmetric hydrogenation of C=C bonds. These photoelectrocatalytic–biocatalytic hybrid reactions achieve total turnover numbers of 362,000 (unspecific peroxygenase), 144,000 (L-glutamate dehydrogenase) and 1,300 (old yellow enzyme). This work presents a photoelectrocatalytic approach for integrating environmental remediation and biocatalytic photosynthesis towards sustainable solar-to-chemical synthesis.

A Four-Step Enzymatic Cascade for Efficient Production of L-Phenylglycine from Biobased L-Phenylalanine.

Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we report a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, (S)-mandelate dehydrogenase (SMDH) from Pseudomonas putida, the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli. However, 10 mM L-Phe only afforded the synthesis of 7.21±0.15 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97±3.84 mM (6.04±0.58 g/L) L-Phg, reaching 99.9 % conversion. In summary, this work demonstrates a concise four-step enzymatic cascade for L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications.

Lignin-Induced CaCO3 Vaterite Structure for Biocatalytic Artificial Photosynthesis.

The vaterite phase of CaCO3 exhibits unique characteristics, such as high porosity, surface area, dispersivity, and low specific gravity, but it is the most unstable polymorph. Here, we report lignin-induced stable vaterite as a support matrix for integrated artificial photosynthesis through the encapsulation of key active components such as the photosensitizer (eosin y, EY) and redox enzyme (l-glutamate dehydrogenase, GDH). The lignin-vaterite/EY/GDH photobiocatalytic platform enabled the regeneration of the reduced nicotinamide cofactor under visible light and facilitated the rapid conversion of α-ketoglutarate into l-glutamate (initial conversion rate, 0.41 mM h-1; turnover frequency, 1060 h-1; and turnover number, 39,750). The lignin-induced vaterite structure allowed for long-term protection and recycling of the active components while facilitating the photosynthesis reaction due to the redox-active lignin. Succession of stability tests demonstrated a significant improvement of GDH's robustness in the lignin-vaterite structure against harsh environments. This work provides a simple approach for solar-to-chemical conversion using a sustainable, integrated light-harvesting system.

Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum.

Production of plastics from petroleum-based raw materials extensively contributes to global pollution and CO2 emissions. Biotechnological production of functionalized monomers can reduce the environmental impact, in particular when using industrial sidestreams as feedstocks. Corynebacterium glutamicum, which is used in the million-ton-scale amino acid production, has been engineered for sustainable production of polyamide monomers. In this study, wheat sidestream concentrate (WSC) from industrial starch production was utilized for production of l-lysine–derived bifunctional monomers using metabolically engineered C. glutamicum strains. Growth of C. glutamicum on WSC was observed and could be improved by hydrolysis of WSC. By heterologous expression of the genes xylA Xc B Cg (xylA from Xanthomonas campestris) and araBAD Ec from E. coli, xylose, and arabinose in WSC hydrolysate (WSCH), in addition to glucose, could be consumed, and production of l-lysine could be increased. WSCH-based production of cadaverine and 5-aminovalerate (5AVA) was enabled. To this end, the lysine decarboxylase gene ldcC Ec from E. coli was expressed alone or for conversion to 5AVA cascaded either with putrescine transaminase and dehydrogenase genes patDA Ec from E. coli or with putrescine oxidase gene puo Rq from Rhodococcus qingshengii and patD Ec . Deletion of the l-glutamate dehydrogenase–encoding gene gdh reduced formation of l-glutamate as a side product for strains with either of the cascades. Since the former cascade (ldcC Ec -patDA Ec ) yields l-glutamate, 5AVA production is coupled to growth by flux enforcement resulting in the highest 5AVA titer obtained with WSCH-based media.

Polydopamine-Assisted Rapid One-Step Immobilization of L-Arginine in Capillary as Immobilized Chiral Ligands for Enantioseparation of Dansyl Amino Acids by Chiral Ligand Exchange Capillary Electrochromatography.

Herein, a novel L-arginine (L-Arg)-modified polydopamine (PDA)-coated capillary (PDA/L-Arg@capillary) was firstly fabricated via the basic amino-acid-induced PDA co-deposition strategy and employed to constitute a new chiral ligand exchange capillary electrochromatography (CLE-CEC) method for the high-performance enantioseparation of D,L-amino acids (D,L-AAs) with L-Arg as the immobilized chiral ligand coordinating with the central metal ion Zn(II) as running buffer. Assisted by hydrothermal treatment, the robust immobilization of L-Arg on the capillary inner wall could be facilely achieved within 1 h, prominently improving the synthesis efficiency and simplifying the preparation procedure. The successful preparation of PDA/L-Arg coatings in the capillary was systematically characterized and confirmed using several methods. In comparison with bare and PDA-functionalized capillaries, the enantioseparation capability of the presented CLE-CEC system was significantly enhanced. Eight D,L-AAs were completely separated and three pairs were partially separated under the optimal conditions. The prepared PDA/L-Arg@capillary showed good repeatability and stability. The potential mechanism of the greatly enhanced enantioseparation performance obtained by PDA/L-Arg@capillary was also explored. Moreover, the proposed method was further utilized for studying the enzyme kinetics of L-glutamic dehydrogenase, exhibiting its promising prospects in enzyme assays and other related applications.

Constructive Optimization of a Multienzymatic Film Based on a Cascade Reaction for Electrochemical Biosensors.

The application of a multienzyme cascade reaction in electrochemical biosensors has the advantage of expanding the target substrates in addition to selectivity combining multiple enzymes on an electrode. However, the multienzyme system has the drawback of inefficient substance conversion because of the time-consuming passing of intermediates between the enzymes and/or diffusional loss of the intermediates. In this study, the optimal construction of a multienzymatic film in an ammonia detection sensor was investigated using a cascade reaction of l-glutamate oxidase and l-glutamate dehydrogenase as a model sensor. Three enzymatic films were prepared: (1) a mixed film designed to have a short diffusional distance between closely located enzymes, (2) a normal-sequential layered film arranged for the correct reaction pathway, and (3) a reverse-sequential layered film as a negative control. This was followed by comparison of the conversion efficiency of ammonia to hydrogen peroxide using time-dependent potentiometric measurements of a Prussian blue electrode determining the hydrogen peroxide amount. The results indicate that the conversion efficiency of the normal-sequential layered film was the highest among the three enzymatic films. The quantitative evaluation of the intermediate conversion efficiency of the cascade reaction showed that compared to the mixed film (34%), a higher conversion efficiency of 92% was obtained in the first enzymatic reaction step. These findings will promote the use of multienzymatic cascade reaction systems not only in biosensors and bioreactors but also in various industrial fields.

Efficient Synthesis of Hydroxytyrosol from l-3,4-Dihydroxyphenylalanine Using Engineered Escherichia coli Whole Cells.

Hydroxytyrosol is a high-value-added compound with a variety of biological and pharmacological activities. In this study, a whole-cell catalytic method for the synthesis of hydroxytyrosol was developed: aromatic amino acid aminotransferase (TyrB), l-glutamate dehydrogenase (GDH), α-keto acid decarboxylase (PmKDC), and aldehyde reductase (YahK) were co-expressed in Escherichia coli to catalyze the synthesis of hydroxytyrosol from l-3,4-dihydroxyphenylalanine (l-DOPA). The plasmids with different copy numbers were used to balance the expression of the four enzymes, and the most appropriate strain (pRSF- yahK- tyrB and pCDF- gdh- Pmkdc) was identified. After determination of the optimum temperature (35 °C) and pH (7.5) for whole-cell catalysis, the yield of hydroxytyrosol reached 36.33 mM (5.59 g/L) and the space-time yield reached 0.70 g L-1 h-1.

Construction of a novel bioanode for amino acid powered fuel cells through an artificial enzyme cascade pathway.

The construction of a novel bioanode based on L-proline oxidation using a cascade reaction pathway comprised of thermostable dehydrogenases. A novel multi-enzymatic cascade pathway, containing four kinds of dehydrogenases from thermophiles (dye-linked L-proline dehydrogenase, nicotinamide adenine dinucleotide (NAD)-dependent Δ1-pyrroline-5-carboxylate dehydrogenase, NAD-dependent L-glutamate dehydrogenase and dye-linked NADH dehydrogenase), was designed for the generation of six-electrons from one molecule of L-proline. The current density of the four-dehydrogenase-immobilized electrode, with a voltage of +450mV (relative to that of Ag/AgCl), was 226.8μA/cm2 in the presence of 10mM L-proline and 0.5mM ferrocene carboxylate at 50°C. This value was 4.2-fold higher than that of a similar electrode containing a single dehydrogenase. In addition, about 54% of the initial current in the multi-enzyme cascade bioanode was maintained even after 15days. Efficient deep oxidation of L-proline by multiple-enzyme cascade reactions was achieved in our designed electrode. The multi-enzyme cascade bioanode, which was built using thermophilic dehydrogenases, showed high durability at room temperature. The long-term stability of the bioanode indicates that it shows great potential for applications as a long-lived enzymatic fuel cell.

Amorphous Carbon Nitride as a Robust Photocatalyst for Biocatalytic Solar-to-Chemical Conversion

In situ regeneration of nicotinamide cofactor (NADH) is imperative because it is required as a reducing power for driving many redox enzymatic cycles useful in industry. Here, we report that amorphous carbon nitride (ACN) is a promising and robust photocatalyst for solar-driven biotransformation via NADH regeneration. Under visible light (λ > 420 nm), NADH regeneration yields by ACN reached 62.3% within an hour, whereas partially crystalline polymeric carbon nitride (CCN) hardly reduced NAD+ to NADH. Subsequently, the regenerated cofactor was consumed by l-glutamate dehydrogenase, a NADH-dependent enzyme, achieving the conversion of α-ketoglutarate with a turnover frequency of 2640 h–1. ACN showed excellent catalytic activity and long-term stability for light-driven biocatalysis; NADH regeneration efficiency after eight cycles remained above 92% of the first cycle’s efficiency, and the enzymatic reaction proceeded for more than 12 h without significant loss of ACN’s photoactivity. The remarkable photocata...

Charge-accumulative Potentiometric Measurements for Ammonia Detection Using an Enzymatic Cascade Reaction on a Prussian Blue Electrode

To detect ammonia using an electrochemical method, we developed a sensor chip consisting of l-glutamate dehydrogenase, l-glutamate oxidase, and a Prussian blue–carbon electrode. When the enzyme electrode was applied to potentiometric and amperometric ammonia measurements, the limits of detection were 0.2 µM and 2,100 µM, respectively. Thus, potentiometric measurements, in which the charge produced by the enzymatic reaction accumulates on the electrode, allow more sensitive detection of ammonia than amperometric measurements, in which the charge passes through the electrode.

Unbiased biocatalytic solar-to-chemical conversion by FeOOH/BiVO4/perovskite tandem structure

Redox enzymes catalyze fascinating chemical reactions with excellent regio- and stereo-specificity. Nicotinamide adenine dinucleotide cofactor is essential in numerous redox biocatalytic reactions and needs to be regenerated because it is consumed as an equivalent during the enzymatic turnover. Here we report on unbiased photoelectrochemical tandem assembly of a photoanode (FeOOH/BiVO4) and a perovskite photovoltaic to provide sufficient potential for cofactor-dependent biocatalytic reactions. We obtain a high faradaic efficiency of 96.2% and an initial conversion rate of 2.4 mM h−1 without an external applied bias for the photoelectrochemical enzymatic conversion of α-ketoglutarate to l-glutamate via l-glutamate dehydrogenase. In addition, we achieve a total turnover number and a turnover frequency of the enzyme of 108,800 and 6200 h−1, respectively, demonstrating that the tandem configuration facilitates redox biocatalysis using light as the only energy source.

Correlation of the kinetics of aggregation and inactivation of L-glutamate dehyrogenase during drying and particle formation of a levitated microdroplet

ABSTRACTMeasurement of single droplet drying in the acoustic levitator has been used to detect a correlation between the kinetics of aggregation and inactivation of the model protein l-glutamate dehydrogenase (GDH) during drying and particle formation. The droplet drying was performed at one set of conditions: drying gas temperature = 60°C; drying gas relative humidity = 1%, and drying gas velocity = 0.5 L/min. A centrifugation technique was used to remove insoluble aggregates from the rehydrated microparticles removed from the levitator chamber. Size exclusion chromatography could then be used to quantify the contents of nonaggregated native protein. During the constant-rate drying period up to the critical point of drying, there was retention of nonaggregated native structure. Only after the critical point of drying has been reached, a rapid decline in nonaggregated native content was observed owing to formation of insoluble aggregates. No sign of a soluble trimer of GDH was found. The kinetics of this behavior correlates closely with the kinetics of inactivation of the same enzyme in levitated microdroplets from the literature, i.e., retention of activity up to the critical point and rapid loss of activity after the critical point. The novelty of this work is that such a correlation between aggregation and inactivation can be detected during drying and particle formation on both sides of the critical point of drying.

A Simple α-Ketoglutarate Electrochemical Biosensor Based on Reduced MoS₂ Nanoparticle-Gold Nanoparticle Nanocomposite.

Convenient and sensitive methods for the detection of α-ketoglutarate (α-KG) will be beneficial for biomedical diagnosis and microbial fermentation industry. In this work, reduced MoS2 nanoparticles (rMNs) were prepared by ultrasonication and electrochemical reduction. The rMNs were then decorated with gold nanoparticles (Aunano) to fabricate rMNs-Aunano nanocomposite. The glass carbon electrode modified with rMNs-Aunano nanocomposite (rMNs-Aunano-GCE) showed excellent electro-catalytic oxidation toward NADH with a satisfied reproducibility and exhibited linear response in the range of 38.46-214.29 μM. Moreover, the L-Glutamic dehydrogenase modified rMNs-Aunano-GCE exhibited a sensitive and fast response to α-KG, with a linear response range of 11.12-52.94 μM and a detection limit of 6.25 μM. These findings will pave ways for the α-KG detection and the application of MoS2 or other two-dimensional materials in electrochemical sensing.

Dehydrogenase-Catalyzed Oxidation of Furanics: Exploitation of Hemoglobin Catalytic Promiscuity.

The catalytic promiscuity of hemoglobin (Hb) was explored for regenerating oxidized nicotinamide cofactors [NAD(P)+ ]. With H2 O2 as oxidant, Hb efficiently oxidized NAD(P)H into NAD(P)+ within 30 min. The new NAD(P)+ regeneration system was coupled with horse liver alcohol dehydrogenase (HLADH) for the oxidation of bio-based furanics such as furfural and 5-hydroxymethylfurfural (HMF). The target acids (e.g., 2,5-furandicarboxylic acid, FDCA) were prepared with moderate-to-good yields. The enzymatic regeneration method was applied in l-glutamic dehydrogenase (DH)-mediated oxidative deamination of lglutamate and for l-lactic-DH-mediated oxidation of l-lactate, which furnished α-ketoglutarate and pyruvate in yields of 97 % and 81 %, respectively. A total turnover number (TTON) of up to approximately 5000 for cofactor and an E factor of less than 110 were obtained in the bi-enzymatic cascade synthesis of α-ketoglutarate. Overall, a proof-of-concept based on catalytic promiscuity of Hb was provided for in situ regeneration of NAD(P)+ in DH-catalyzed oxidation reactions.

Correlation of uric acid, urea, ammonia and creatinine of seminal plasma with semen parameters and fertilization rate of infertile couples

Background: Ammonia, urea, uric acid, and creatinine are the major non-protein nitrogenous compounds (NPNs). It is reported that the concentration of NPNs in the seminal plasma of normal and infertile individuals is different and sperm is affected by NPNs. Objectives: The aim of this study was to determine the quantities of ammonia, urea, uric acid, and creatinine in seminal plasma and the correlation of these compounds with the fertilization rate after an intracytoplasmic sperm injection (ICSI). Methods: The levels of ammonia, urea, uric acid, and creatinine were determined in seminal plasma collected from 50 men. The ammonia and urea were determined by L-glutamate dehydrogenase and diacetyl monoxime method, respectively. Uric acid and creatinine were detected by enzymatic method and Jaffe reaction, respectively. The fertilization was evaluated around 16–18 hours post-ICSI on the appearance of 2 pronuclei and 2 polar bodies. The fertilization rate was calculated by the number of fertilized oocytes per the number of oocytes injected. Results: There was a significant negative correlation between seminal ammonia and sperm motility (P&lt;0.05). Urea and creatinine had a negative correlation with respect to the sperm count (P&lt;0.05). In addition, there was an inverse correlation between urea and uric acid of seminal plasma and sperm morphology (P&lt;0.05). A significant negative correlation was found between seminal uric acid and urea with respect to the percentage of the fertilization rate (P&lt;0.05). Conclusions: The findings of the present study showed that urea and uric acid in seminal plasma have a negative impact on the fertilization rate.