Michaelis-Menten Research Articles

Optimization and characterization studies of poultry waste valorization for peptone production using a newly Egyptian Bacillus subtilis strain

Valorization of poultry waste is a significant challenge addressed in this study, which aimed to produce cost-effective and sustainable peptones from poultry waste. The isolation process yielded the highly potent proteolytic B.subtilis isolate P6, identified through 16S rRNA gene sequencing to share 94% similarity with the B.subtilis strain KEMET024 (GenBank accession number PP694485.1) and deposited in MIRCEN culture collection, Cairo, Egypt as EMCC 998871. It reached optimal production levels during 24 h of incubation, with biomass at 2.5 g/L, protease activity at 455 U/mL, and total amino acid (TAA) concentration at 208 mg/mL. For screening the most significant factors for peptone production, the Plackett–Burman design identified meat and bone meal concentration as the main significant factor influencing total amino acid reaching 420 mg/mL. BOX-Behnken design optimized peptone production increasing its production level by twofold to reach 2850 U/mL of protease activity and 580 mg/mL of total amino acids. The produced peptone demonstrated a superior amino acid profile compared to commercial peptones, with a remarkably higher total amino acid content of 621.556 mg/g and elevated levels of essential amino acids like aspartic acid (37.745%), glutamic acid (90.876%), glycine (117.272%), and alanine (50.373%). Characterization revealed optimal pH and temperature conditions of around pH 8 and 50–60°C, respectively, for the proteolytic activity. The Michaelis–Menten and Lineweaver–Burk plots determined a Km of 0.5 mg/mL and Vmax of 174.08 U/mL suggesting cooperative substrate binding and providing insights into the enzyme’s maximum rate and affinity. The produced peptone exhibited minimal cytotoxicity at lower concentrations (≤ 1 mg/mL), with cell viability exceeding 94% against normal human skin fibroblast (HSF) cells. However, higher concentrations (≥ 3 mg/mL) displayed increased cytotoxic effects. Moreover, the results strongly indicate that the produced peptone, particularly at 0.5% concentration, is an effective nitrogen source for B. subtilis cultivation, demonstrating its potential for biotechnological applications. This study successfully valorized poultry waste by developing a sustainable and cost-effective alternative to commercial peptones, contributing to waste valorization and sustainable biotechnological processes.Graphical abstract

Kinetically-derived maximal dose (KMD) confirms lack of humanrelevance for high-dose effects of octamethylcyclotetrasiloxane (D4).

The kinetically-derived maximal dose (KMD) is defined as the maximum external dose at which kinetics are unchanged relative to lower doses, e.g., doses at which kinetic processes are not saturated. Toxicity produced at doses above the KMD can be qualitatively different from toxicity produced at lower doses. Here, we test the hypothesis that high-dose-dependent toxicological effects of octamethylcyclotetrasiloxane (D4) occur secondary to kinetic overload. Octamethylcyclotetrasiloxane (D4) is a volatile, highly lipophilic monomer used to produce silicone polymers, which are ingredients in many consumer products and used widely in industrial applications and processes. Chronic inhalation at D4 concentrations 104 times greater than human exposures produces mild effects in rat respiratory tract, liver weight increase and pigment accumulation, nephropathy, uterine endometrial epithelial hyperplasia, non-significant increased uterine endometrial adenomas, and reduced fertility secondary to inhibition of rat-specific luteinizing hormone (LH) surge. Mechanistic studies indicate a lack of human relevance for most of these effects. Respiratory tract effects arise in rats due to direct epithelial contact with mixed vapor/aerosols and increased liver weight is a rodent-specific adaptative induction of drug-metabolizing hepatic enzymes. D4 is not mutagenic or genotoxic, does not interact with dopamine receptors, and interacts at ERα with potency insufficient to cause uterine effects or to alter the LH surge in rats. These mechanistic findings suggest high-dose-dependence of the toxicological effects secondary to kinetic overload, a hypothesis that can be tested when appropriate kinetic data are available that can be probed for the existence of a KMD. We applied Bayesian analysis with differential equations to information from kinetic studies on D4 to build statistical distributions of plausible values of the Km and Vmax for D4 elimination. From those distributions of likely Km and Vmax values, a set of Michaelis-Menten equations were generated that are likely to represent the slope function for the relationship between D4 exposure and blood concentration. The resulting Michaelis-Menten functions were then investigated using a change-point methodology known as the "kneedle" algorithm to identify the probable KMD range. We validated our Km and Vmax using out of sample data. Analysis of the Michaelis-Menten elimination curve generated from those Vmax and Km values indicates a KMD with an interquartile range of 230.0-488.0ppm [2790-5920mg/m3; 9.41-19.96µM]. The KMD determined here for D4 is consistent with prior work indicating saturation of D4 metabolism at approximately 300ppm [3640mg/m3; 12.27µM] and supports the hypothesis that many adverse effects of D4 arise secondary to high-dose-dependent events, likely due to mechanisms of action that cannot occur at concentrations below the KMD. Regulatory methods to evaluate D4 for human health protection should avoid endpoint data from rodents exposed to D4 above the KMD range and future toxicological testing should focus on doses below the KMD range.

Functional Characterization of the SHIP1-Domains Regarding Their Contribution to Inositol 5-Phosphatase Activity

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a multidomain protein consisting of two protein–protein interaction domains, the Src homology 2 (SH2) domain, and the proline-rich region (PRR), as well as three phosphoinositide-binding domains, the pleckstrin homology-like (PHL) domain, the 5-phosphatase (5PPase) domain, and the C2 domain. SHIP1 is commonly known for its involvement in the regulation of the PI3K/AKT signaling pathway by dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) at the D5 position of the inositol ring. However, the functional role of each domain of SHIP1 for the regulation of its enzymatic activity is not well understood. To determine the contribution of the individual domains to catalytic activity, the full-length protein was compared with truncated constructs lacking one or more domain(s), regarding the substrate turnover (kcat) and catalytic efficiency (kcat/Km) towards ci8-PtdIns(3,4,5)P3. With this approach, it was possible to verify the allosteric activation of SHIP1 mediated by the C2 domain as described previously, while the PHL domain seemed instead to have a negative effect regarding catalytic efficiency. The full-length SHIP1 clearly displayed the highest turnover and the second-highest catalytic efficiency, showing the role of the SH2 domain and PRR not only in protein–protein interactions but also in catalysis. The SH2 domain increased substrate turnover but negatively affected catalytic efficiency. The linker between the SH2 and the PHL domains decreased the turnover number but positively influenced the catalytic efficiency. The PRR increased both the substrate turnover and the protein’s catalytic efficiency. The regression analysis of the Michaelis–Menten graph revealed SHIP1 to be an allosteric enzyme, with the PRR and the linker being the most involved domains in that regard. In summary, our data indicate a complex regulation of the enzymatic activity of SHIP1 by its individual domains. While the C2 domain and PRR at the carboxy-terminus have a positive effect on enzymatic activity, the SH2 and PHL domain at the amino-terminus inhibit catalytic efficiency.

Sensitive and selective colorimetric detection of thiophanate-methyl based on a novel Ru-Fe3O4 nanozyme with enhanced peroxidase-like activity.

Anovel Ru-Fe3O4 nanozyme with enhanced peroxidase-like (POD-like) activity was synthesized through a hydrothermal method. Ru-Fe3O4 nanozyme was effectively utilized for the detection of thiophanate-methyl (TM) using a colorimetric technique. The POD-like activity of Ru-Fe3O4 was found to be superior compared to Fe3O4, Rh-Fe3O4, and Pd-Fe3O4. Ru-Fe3O4 providedexcellent POD-like activity with Michaelis constants of 0.00645mM and 0.66714mM for substrates of 3,3',5,5'-tetramethylbenzidine and H2O2, respectively. The density functional theory calculation showed that Ru-Fe3O4 had better H2O2 decomposition reactivity compared toFe3O4. Compared withFe3O4, the adsorbed H2O2 molecules underwent decomposition more readily on the Ru-Fe3O4 catalytic surface facilitating the occurrence of the H2O2 catalytic reaction, further suggesting the excellent POD-like activity of Ru-Fe3O4. The coordination and electrostatic interactions of Ru-Fe3O4 and TM promoted their binding, contributing to TM covering Ru-Fe3O4 catalytic site and inhibiting the POD-like activity of Ru-Fe3O4. As a result, the sensitive and selective detection of TM using Ru-Fe3O4 by a colorimetric method was achieved. The Ru-Fe3O4 nanozyme displayed a good linear correlation, with a linear detection range and a detection limit of 0.1-100 and 0.03μg/mL, respectively. The Ru-Fe3O4 nanozyme was used for the determination of TM in soil and water samples with success.

Exploiting unique NP1 interface: Oriented immobilization via electrostatic and affinity interactions in a tailored PDA/PEI microenvironment enhanced by concanavalin A.

Enzyme immobilization techniques are crucial for enhancing enzyme stability and catalytic efficiency. Traditional methods such as physical adsorption and simple covalent binding often fail to maintain enzyme activity and stability. In this study, an innovative multi-level immobilization strategy was proposed to achieve efficient targeted immobilization of nuclease P1 (NP1) by fine-tuning the surface microenvironment. Molecular simulation results revealed that the distinctive electrostatic distribution and the specific placement of basic amino acids, such as lysine, on the NP1 surface caused dopamine to preferentially adsorb on areas away from NP1's active site. This selective adsorption facilitated the directed immobilization of NP1, while the positively charged environment generated by the co-deposited surface further enhanced NP1's adsorption capacity. This multilevel modification was found to significantly optimize the physicochemical environment of the immobilized surface through surface characterization and enzymatic testing. This strategy greatly improves enzyme activity (3590.0 U/mg), stability, and reusability (70% after 10 cycles). In particular, NP1 on this surface exhibited an optimal Michaelis constant (Km) of 34.0mM and a maximum reaction rate of 5.5mMmin-1, demonstrating the remarkable effect of the modification strategy in enhancing the enzyme catalytic performance. The present study provides an efficient and stable immobilization platform for enzyme catalytic applications by precisely modulating the surface microenvironment and the oriented immobilization strategy, which has an important potential for practical applications. This stable and reusable NP1 platform allows for efficient DNA/RNA cleavage, facilitating its application in industrial biocatalysis, biomedical enzyme-based processes, and biosensors.

Porphyrin modified ZnCo2O4 nanospheres as the excellent peroxidase/oxidase dual nanozymes for colorimetric sensing of cholesterol

5,10,15,20-Tetrakis(4-carboxyl phenyl)porphyrin (H2TCPP) modified ZnCo2O4 nanospheres (H2TCPP-ZnCo2O4) were prepared by a two-step hydrothermal method. Compared with pure ZnCo2O4, H2TCPP-ZnCo2O4 possessed higher peroxidase-like activity and fast catalyze the chromogenic substrate 3,3,5,5-tetramethylbenzidine (TMB) into oxTMB by H2O2 with a blue change. The catalytic behaviors of H2TCPP-ZnCo2O4 is consistent with the Michaelis–Menten equation. Several techniques including electrochemical, fluorescent probes and capture agents were used to investigate the catalytic mechanism, which was identified as electron transfer and superoxide radical (•O2−), due to the synergetic effect between porphyrin molecules and ZnCo2O4. Based on the nanoperoxidase activity of H2TCPP-ZnCo2O4, a facile colorimetric sensing platforms was designed for the determination of H2O2 and cholesterol. The linear range for H2O2 determination is 100 to 200μM, with a detection limit of 76.297μM as well as cholesterol is 100 to 900μM, with a detection limit of 40.027μM

Single-atom nanozyme-based catalytic ROS clearance for efficiently alleviating eczema.

Single-atom nanozymes (SAzymes) with excellent biological catalytic activity have emerged as promising candidates for advancing biomedical applications. Herein, we synthesized a RuN4-SAzyme by thermal decomposition. In in vitro experiments, the RuN4-SAzyme demonstrated exceptional catalytic efficiency in mimicking the activity of peroxidase, with a Michaelis-Menten constant (Km) for 3,3',5,5'-tetramethylbenzidine reaching 0.077 mM, reflecting a high substrate affinity. Moreover, the RuN4-SAzyme also exhibited catalase- and superoxide dismutase-like activities, as well as nicotinamide adenine dinucleotide oxidase-like activity, effectively neutralizing reactive oxygen species. Significantly, in vivo experiments demonstrated that RuN4-SAzymes can effectively alleviate eczema-like symptoms by clearing inflammatory factors, reducing oxidative stress, and promoting macrophage polarization from M1 to M2, showing comparable efficacy to the clinical drug tacrolimus.

Construction and enzymatic characterization of a monomeric variant of dimeric amylosucrase from Deinococcus geothermalis

Amylosucrase (ASase; E.C. 2.4.1.4), a member of glycoside hydrolase family 13 (GH13), produces α-1,4-glucans and sucrose isomers using sucrose as its sole substrate. This study identifies and characterizes the dimeric structure of ASase from Deinococcus geothermalis (DgAS), highlighting essential amino acid residues for maintaining the dimeric state. The monomeric form, DgAS R30A, exhibited a higher affinity for sucrose compared to the wild-type (WT), especially during the formation of the ASase–glucose intermediate complex and subsequent hydrolysis. Notably, DgAS R30A produced a higher proportion of α-glucans with a degree of polymerization (DP) of ≤20 and fewer α-glucans with a DP of ≥31. This suggested that the reduced surface area of the oligosaccharide binding site in the monomeric form led to decreased binding of longer-chain maltooligosaccharides, favoring the formation of shorter DP α-glucans. Kinetic analysis revealed significantly lower Michaelis constants (Km) for DgAS R30A's total and hydrolysis activities, with the overall performance (Kcat/Km) values for DgAS R30A exceeded those of the WT at all sucrose concentrations. Here, we report the first high-resolution homodimeric DgAS structure, revealing conserved active site residues and a unique dimerization interface. This study enhances our understanding of the molecular factors influencing the oligomeric state and enzyme activities.

Nanomineralzyme as a novel sustainable class of nanozyme: Chalcopyrite-based nanozyme for the visual detection of total antioxidant capacity in citrus fruit.

Chemically-synthesized Nanozymes that are widely used as alternatives to enzymes face challenges such as high precursor costs, complex preparation processes, and limited catalytic efficiency. To overcome these drawbacks, we introduce naturally derived nanozymes, nanomineralzymes, as a promising alternative, offering benefits like affordability, cost-effectiveness, and scalability. Chalcopyrite (CP, CuFeS2) was sourced from a mineral deposit, and CP nanoparticles were produced by milling. These nanoparticles exhibited strong peroxidase-like activity, achieving a low Michaelis-Menten constant using 3,3',5,5'-tetramethylbenzidine as a substrate. Characterizations revealed the presence of cuprous, cupric, ferrous, and ferric ions in the CP mineral. The proposed mechanism involves an enhanced Fenton and Fenton-like process due to the metal ions' multi-valence states. CP nanozyme activity was inhibited to produce radicals due to hydrogen atom transfer and single electron transfer with ascorbic acid, glutathione and cysteine. The CP mineralzyme-based total antioxidant capacity probe was successfully used for detection of TAC in citrus fruits.

Enzyme-modified Pt nanoelectrodes for glutamate detection.

We present here a glutamate oxidase (GluOx)-modified platinum (Pt) nanoelectrode with a planar geometry for glutamate detection. The Pt nanoelectrode was characterized using electrochemistry and scanning electron microscopy (SEM). The radius of the Pt nanoelectrode measured using SEM is ∼210 nm. GluOx-modified Pt nanoelectrodes were generated by dip coating GluOx on the Pt nanoelectrode in a solution of 0.9% (wt%) bovine serum albumin (BSA), 0.126% (wt%) glutaraldehyde, and 100 U mL-1 GluOx. An increase in current was observed at +0.7 V vs. Ag/AgCl/1 M KCl with adding increasing concentrations of glutamate. Two-sample t-test results showed that there is a significant difference for current at +0.7 V between the blank and the added lowest glutamate concentration, as well as between adjacent glutamate concentrations, confirming that the increase in current is related to the increased glutamate concentration. The experimental current-concentration curve of glutamate detection fitted well to the theoretical Michaelis-Menten curve. At the low concentration range (50 μM to 200 μM), a linear relationship between the current and glutamate concentration was observed. The Michaelis-Menten constants of Imax and Km were calculated to be 1.093 pA and 0.227 mM, respectively. Biosensor efficiency (the ratio of glutamate sensitivity to H2O2 sensitivity) is calculated to be 57.9%. Enzact (Imax/H2O2 sensitivity, an indicator of the amount of enzyme loaded on the electrode) of the GluOx-modified Pt nanoelectrode is 0.243 mM. We further compared the sensitivity of a GluOx-modified Pt nanoelectrode with a GluOx-modified carbon fiber microelectrode (7 μm diameter and a sensing length of ∼350 μm). Glutamate detection on the GluOx-modified carbon fiber microelectrode fitted well to a Michaelis-Menten like response. Based on the fitting, the GluOx-modified carbon fiber microelectrode exhibited an Imax of 0.689 nA and a Km of 301.2 μM towards glutamate detection. The best linear range of glutamate detection on the GluOx-modified carbon fiber microelectrode is from 50 μM to 150 μM glutamate. The GluOx-modified carbon fiber microelectrode exhibited a higher potential requirement for glutamate detection compared to the GluOx-modified Pt nanoelectrode.

Structural basis for membrane association and catalysis by phosphatidylserine synthase in Escherichia coli.

Phosphatidylserine synthase (PssA) is essential in the biosynthesis of phosphatidylethanolamine, a major phospholipid of bacterial membranes. A peripheral membrane protein PssA can associate with the cellular membrane in its active state or exist in the cytosol in an inactive form. The membrane-bound enzyme acts on cytidine diphosphate diacylglycerol (CDP-DG) to form cytidine monophosphate and a covalent intermediate, which is subsequently targeted by serine to produce phosphatidylserine. Here, we present two crystal structures of Escherichia coli PssA, one complexed with CDP-DG and the other without. The lipid-bound structure mimics the Michaelis complex before the formation of a covalent intermediate, revealing key determinants for substrate recognition and catalysis. Notably, membrane-free PssA is in a monomer-dimer equilibrium, with only the monomer capable of associating with the membrane, suggesting a regulatory mechanism for phospholipid biosynthesis dependent on the oligomerization state of the enzyme.

Intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism.

The Parkinson's disease (PD)-linked protein Leucine-Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lies within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM and the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity, suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together, our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

Metabolic rearrangement enables adaptation of microbial growth rate to temperature shifts.

Temperature is a key determinant of microbial behaviour and survival in the environment and within hosts. At intermediate temperatures, growth rate varies according to the Arrhenius law of thermodynamics, which describes the effect of temperature on the rate of a chemical reaction. However, the mechanistic basis for this behaviour remains unclear. Here we use single-cell microscopy to show that Escherichia coli exhibits a gradual response to temperature upshifts with a timescale of ~1.5 doublings at the higher temperature. The response was largely independent of initial or final temperature and nutrient source. Proteomic and genomic approaches demonstrated that adaptation to temperature is independent of transcriptional, translational or membrane fluidity changes. Instead, an autocatalytic enzyme network model incorporating temperature-sensitive Michaelis-Menten kinetics recapitulates all temperature-shift dynamics through metabolome rearrangement, resulting in a transient temperature memory. The model successfully predicts alterations in the temperature response across nutrient conditions, diverse E. coli strains from hosts with different body temperatures, soil-dwelling Bacillus subtilis and fission yeast. In sum, our model provides a mechanistic framework for Arrhenius-dependent growth.

PURIFICATION AND CHARACTERIZATION OF MUCOR JANSENII LIPASE ISOLATED FROM COCOA PROCESSING PLANT EFFLUENTS IN ILE-OLUJI, ONDO STATE, NIGERIA

This study purified and characterized lipases secreted by a fungus Mucor jansenii isolated from the effluent of a cocoa processing plant. This was done in an effort to investigate the fungus' ability to produce lipase for industrial and biotechnological uses. The fungus isolated was identified macroscopically and microscopically using Lactophenol cotton blue stain. The enzyme produced was purified partly by ion-exchange chromatography on QAE-Sephadex A-25. Specific activity of the partially purified Mucor jansenii B6 lipase was 680.33 units/mg protein, 350.11 units/mg protein, and 342.19 units/mg protein, respectively, for isoforms A, B, and C. Partly purified M. jansenii B6 lipase had a molecular weight of 127 kDa, as determined by gel fitment on the Sephacryl S-200 column. The optimum pH and temperature for enzyme activity were 11.0 and 50 oC, respectively. The enzyme activity was enhanced by Ba2+, Na+, Ca2+, and Mg2+ at 5 mM and 10 mM, while Al3+ reduced the catalytic activity. Activity of the enzyme increased in acetone but decreased in ethyl acetate. The Michaelis constant (Km) and maximum velocity (Vmax) of the enzyme were found to be 0.5 mM and 16.6 units/mg protein, respectively. The study concluded that Mucor jansenii B6 lipase is a potential alkaline and thermostable lipase suitable for industrial and biotechnological applications.

Modulating Biomimetic Peroxidase Activity of CuMOFs Through Ligand Engineering: Sensitive Colorimetric Detection of H2O2 and Glutathione

AbstractNatural peroxidase enzymes are widely utilized for various diagnostic applications, yet their inherent instability and susceptibility to external factors pose challenges. This study introduces a ligand engineering approach to fine‐tune the peroxidase enzyme‐like activity of copper‐based metal‐organic frameworks (MOFs). We have rationally modulated the enzyme‐mimicking activity of a series of Cu‐BDC‐X based MOFs via employment of various ligands (BDC = 1,4‐benzene dicarboxylic acid; CuMOF‐1, X═H2O; CuMOF‐2, X═4,4′‐bipyridine and CuMOF‐3, X═N‐methylimidazole). As compared to its analogues, CuMOF‐3 exhibited an enhanced catalytic activity, most likely due to N‐methylimidazole‐induced structural distortion that fine‐tunes the metal's electronic environment and improves substrate interactions. Additionally, this modification allowed CuMOF‐3 to be miniaturized to the nanoscale, enhancing its catalytic performance. Mechanistic investigations revealed the generation of •OH as reactive oxygen species (ROS) to accelerate tetramethylbenzidine oxidation, promoting peroxidase‐like activity. The observed catalytic behavior of CuMOF‐3 followed Michaelis–Menten kinetics, exhibiting lower Km value compared to those of natural peroxidase enzymes. Under optimized conditions, CuMOF‐3 facilitated the development of a colorimetric assay for sensitive detection of H2O2 and glutathione (GSH) in various spiked food samples. This study uniquely demonstrates the impact of tailored ligand combinations and MOF geometry on optimizing peroxidase‐mimicking activity, providing a new direction for the design of high‐performance colorimetric diagnostic tools in the food processing industry.

High catalytic nickel−platinum nanozyme enhancing colorimetric detection of Salmonella Typhimurium in milk

Colorimetric qualitative and sensitive quantitative detection of Salmonella Typhimurium (S. Typhimurium) holds significant importance for ensuring food safety and preventing foodborne illnesses. In the study, an ultra-high catalytic activity and biocompatible nickel-platinum nanoparticle (NiPt NP) nanozyme is successful synthesized to prepare a NLISA strategy for the detection of S. Typhimurium. The synthesized NiPt NPs exhibit high oxidase-like catalytic efficiency, with a Michaelis constant (Km) of 0.493 mM, similar to that of natural horseradish peroxidase (HRP). The maximal reaction velocity (Vmax) was determined to be 1.97 × 10-7 M·s-1 exhibiting a 1.97-fold higher than that of the HRP (1.0 × 10-7 M·s-1). Meanwhile, the antibody employed in this NiPt NPs-based NLISA exhibits exceptional capture efficacy, generating a stable immune complex with S. Typhimurium. The NiPt NPs-based NLISA demonstrates sensitivity, specificity, convenience, and cost-efficiency for the detection of S. Typhimurium. Under optimal conditions, this NiPt NPs-based NLISA demonstrates a quantitative range of 103∼106 cfu/mL with a detection limit as low as 103 cfu/mL. A single-blind experimental testing detects different concentrations of S. Typhimurium spiked skim milk, indicating the application potential of the proposed NLISA in real samples. In all, this research provides novel insights into the synthesis of nanozymes with excellent catalytic activity and their applications in S. Typhimurium biosensing.

Nitrogen form differently modulates nitrogen uptake and utilization and related gene expression between two tea cultivars

Nitrogen (N) is essential for the growth and development of tea plants, and ammonium (NH4+) and nitrate (NO3-) are crucial N sources for tea yield and amino acid contents. However, the uptake and utilization of different N forms in tea plants are different. Reasonable N form is an important means to enhance the growth and development of tea plants. Therefore, supplying suitable N forms may be effective way to optimize N use efficiency. A hydroponic trial was conducted with 'Chuancha No.2′ (CC) and 'Emeiwenchun' (EW) tea cultivars with N form treatments: NH4+ and NO3-. The results showed that there were significant difference in the NH4+/NO3- uptake kinetics, dynamic changes in 15N abundance, enzyme activities, and related gene expression in N metabolism between CC and EW after NH4+/NO3- treatment. In CC and EW, NH4+ and NO3- uptake followed Michaelis-Menten kinetics at low N concentrations (< 1 mmol l-1), but CC exhibited a slightly higher uptake rate than EW when supplied with 2 mmol l-1 NH4+. In a 0–24 h 15N tracing experiment, CC roots accumulated 15N faster than EW under NH4+. NH4+-fed CC exhibited higher GS activities and higher expression levels of CsAMTs and genes involved in N utilization, such as CsNR, CsGS2, CsGDH1, and CsTS1, compared to EW. When NO3- was provided, EW roots accumulated more 15N than CC roots from 8 to 24 h, which could be attributed to the higher NR activities and higher expression levels of CsNRT1.5, CsNR, CsGS1.1 and CsGS1.2 than those in CC. In summary, the two tea varieties exhibited distinct characteristics of N uptake and utilization, as well as gene expression patterns under NH4+/NO3- treatments. CC demonstrated an advantage in NH4+ uptake and assimilation, while EW exhibited an advantage in NO3-.

The NADH-dependent flavin reductase ThdF follows an ordered sequential mechanism though crystal structures reveal two FAD molecules in the active site.

Two-component flavin-dependent monooxygenases are of great interest as biocatalysts for the production of pharmaceuticals and other relevant molecules, as they catalyze chemically important reactions such as hydroxylation, epoxidation and halogenation. The monooxygenase components require a separate flavin reductase, which provides the necessary reduced flavin cofactor. The tryptophan halogenase Thal from Streptomyces albogriseolus is a well-characterized two-component flavin-dependent halogenase. Thal exhibits some limitations in terms of halogenation efficiency, also caused by unproductive enzyme-substrate complexes with reduced flavin adenine dinucleotide (FAD). Since the reductase components have an important regulatory function for the activity and efficiency of the monooxygenase by controlling the supply of reduced flavin, here we studied the so far uncharacterized flavin reductase ThdF from the same gene cluster in S. albogriseolus, which potentially cooperates with Thal. A crystal structure of ThdF in complex with both substrates, FAD and NADH, revealed their orientation for hydride transfer. We obtained two further ThdF structures with two FAD molecules bound to the active site, suggesting a ping-pong bi-bi mechanism. In contrast, steady-state enzyme kinetics clearly showed that ThdF catalyzes flavin reduction via an ordered sequential mechanism, with FAD being bound first and FADH2 released last. Compared to related flavin reductases, ThdF has a low kcat and low KM value. The inhibition of ThdF by NAD+ might limit Thal's halogenation activity when the cellular NADH level is low. These results provide first insights into how the efficiency of Thal could be controlled by flavin reduction at the reductase ThdF.

Process Understanding of Transamination Reaction in Chiral Pharmaceutical Intermediate Production Catalyzed by an Engineered Amine Transaminase

AbstractChiral amines are key building blocks for the synthesis of many active pharmaceutical ingredients (APIs). Biocatalytic routes offer significant advantages to provide sustainable access to such motifs on commercial scale, with sacubitril valsartan sodium hydrate as a recent example. In this study a deeper mechanistic and kinetic understanding of the central biocatalytic step in the synthesis of sacubitril valsartan sodium hydrate, applying the evolved transaminase CDX‐043, was gained. The equilibrium of the transamination reaction was investigated in detail, and two kinetic models (ping‐pong two‐substrate kinetics and Michaelis–Menten double substrate kinetics) were established, considering substrate and product inhibition. The determined equilibrium constant indicates that the equilibrium lies strongly on the product side. The results of the kinetic studies demonstrate that the transaminase reaction is in conformity with the Michaelis–Menten double substrate kinetic model. Product inhibition was found to be more severe than substrate inhibition. The application of a plug flow reactor (PFR) was shown to be the preferred reactor setup to reduce the occurring inhibition.

Enhancing the photo-induced oxidase-like activity of fluorescein with methyl viologen for colorimetric detection of organophosphorus pesticide

Overcoming the intrinsic low activity of most photoinduced oxidase mimics has been extremely challenging. In this work, we developed a methyl viologen (MV2+) mediated strategy to enhance the oxidase-like activity of fluorescein. The presence of MV2+ gives it a high affinity for TMB with a low Michaelis-Menten constant (Km) of 0.053 mM, which is about 2.8 times lower than that of fluorescein and with a remarkable catalytic constant (Kcat) as 0.2490 s−1, which is 3 times as high as that of fluorescein. Fluorescein diacetate (FDA) without oxidase-like activity can be hydrolyzed in situ to produce fluorescein in the presence of carboxylesterases (CaE). Based on the inhibition of CaE activity by organophosphorus pesticides (OP), a novel colorimetric signal biosensor was established with a wide linear range from 1.0 to 200 ng/mL. This work not only provides a convenient and feasible strategy for enhancing the activity of photoinduced oxidase mimics but also blazes a new pathway for the sensitive detection of OP.