Michaelis-Menten Research Articles

High-activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane.

Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6±20mgL-1 with a specific activity of 96±0.32 Umg-1, resulting in a total enzyme activity of 19137±1131UL-1, demonstrating significant potential for industrial applications.

Convergence in simulating global soil organic carbon by structurally different models after data assimilation.

Current biogeochemical models produce carbon-climate feedback projections with large uncertainties, often attributed to their structural differences when simulating soil organic carbon (SOC) dynamics worldwide. However, choices of model parameter values that quantify the strength and represent properties of different soil carbon cycle processes could also contribute to model simulation uncertainties. Here, we demonstrate the critical role of using common observational data in reducing model uncertainty in estimates of global SOC storage. Two structurally different models featuring distinctive carbon pools, decomposition kinetics, and carbon transfer pathways simulate opposite global SOC distributions with their customary parameter values yet converge to similar results after being informed by the same global SOC database using a data assimilation approach. The converged spatial SOC simulations result from similar simulations in key model components such as carbon transfer efficiency, baseline decomposition rate, and environmental effects on carbon fluxes by these two models after data assimilation. Moreover, data assimilation results suggest equally effective simulations of SOC using models following either first-order or Michaelis-Menten kinetics at the global scale. Nevertheless, a wider range of data with high-quality control and assurance are needed to further constrain SOC dynamics simulations and reduce unconstrained parameters. New sets of data, such as microbial genomics-function relationships, may also suggest novel structures to account for in future model development. Overall, our results highlight the importance of observational data in informing model development and constraining model predictions.

A study of nonlinear fractional-order biochemical reaction model and numerical simulations

This article depicts an approximate solution of systems of nonlinear fractional biochemical reactions for the Michaelis–Menten enzyme kinetic model arising from the enzymatic reaction process. This present work is concerned with fundamental enzyme kinetics, utilised to assess the efficacy of powerful mathematical approaches such as the homotopy perturbation method (HPM), homotopy analysis method (HAM), and homotopy analysis transform method (HATM) to get the approximate solutions of the biochemical reaction model with time-fractional derivatives. The Caputo-type fractional derivatives are explored. The proposed method is implemented to formulate a fractional differential biochemical reaction model to obtain approximate results subject to various settings of the fractional parameters with statistical validation at different stages. The comparison results reveal the complexity of the enzyme process and obtain approximate solutions to the nonlinear fractional differential biochemical reaction model.

Unveiling the anticancer potential of L-Asparaginase (ASNase) from novel Streptomyces isolate HB2AG: Purification and characterization study

L-asparaginase (ASNase) obtained from microbial origins is a significant enzyme in the commercial sphere, crucial for the treatment of acute lymphoblastic leukemia. In the current investigation, we isolated ASNase through techniques involving ammonium sulfate precipitation and DEAE sepharose anion exchange column chromatography, revealing a molecular weight of 35 kDa. The enzyme exhibited its peak activity at pH 8 and 40°C, maintaining approximately 99% of its enzymatic effectiveness. The Michaelis constant (Km) and maximum velocity (Vmax) were determined to be 0.0021 M and 714.3 µM/min, respectively. ASNase was found to be non-toxic on HEK293 cells and selectively effective against breast cancer, hepatocellular carcinoma cells, colorectal adenocarcinoma cells, Human lung cancer cell line, Neck squamous cells, and Human head carcinoma cells. Hepatocellular carcinoma cells exhibited remarkable responsiveness to ASNase, displaying an IC50 of 23 µg/mL. This led to observable changes in nuclear morphology indicative of apoptosis, as evidenced by DAPI and Annexin V-FITC staining. ASNase also showed cell migration potential on HEK293 cells. Streptomyces HB2AG ASNase’s dynamic functioning across pH and temperature ranges, as well as its stability at 40°C and cytotoxic potential, make it a promising pharmaceutical candidate for various applications.

Transport of glycinin, the major soybean allergen, across intestinal epithelial IPEC-J2 cell monolayers.

Soybean allergen entering the body is the initial step to trigger intestinal allergic response. However, it remains unclear how glycinin, the major soybean allergen, is transported through the intestinal mucosal barrier. The objective of this study was to elucidate the pathway and mechanism of glycinin hydrolysatetransport through the intestinal epithelial barrier using IPEC-J2 cell model. Purified glycinin was digested by in vitro static digestion model. The pathway and mechanism of glycinin hydrolysates transport through intestinal epithelial cells were investigated by cellular transcytosis assay, cellular uptake assay, immunoelectron microscopy and endocytosis inhibition assay. The glycinin hydrolysates were transported across IPEC-J2 cell monolayers in a time/dose-dependent manner following the Michaelis equation. Immunoelectron microscopy showed a number of glycinin hydrolysates appeared in the cytoplasm, but no glycinin hydrolysates were observed in the intercellular space of IPEC-J2 cells. The inhibitors, colchicine, chlorpromazine and methyl-β-cyclodextrin,significantly inhibited the cellular uptake of glycinin hydrolysates. The glycinin hydrolysates crossed IPEC-J2 cell monolayers through the transcellular pathway. Both clathrin- and caveolae-dependent endocytosis were involved in the epithelial uptake of the hydrolysates. These findings provided potential targets for the prevention and treatment of soybean allergy.

Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan.

Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl-glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 23-(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the -1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi-functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary β-domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering.

Fibrinolytic enzyme from Arthrospira platensis: Kinetic and thermodynamic investigation

Fibrinolytic enzymes from microorganisms have been investigated by their potential application as thrombolytic agents. Previous studies show that the fibrinolytic enzyme from Arthrospira platensis (FEAP) is stable at human physiological temperature (<50 °C) and pH (6.0–10.0). Understanding of the kinetic/thermodynamic characteristics is important to make the advances for industrial applications feasible. Therefore, a kinetic/thermodynamic analysis of FEAP was performed on the fibrin hydrolysis and enzyme thermoinactivation. Results showed two Michaelis-Menten-type profiles with two plateaus, revealing high affinity for the substrate with low Michaelis constant values (0.02 and 6.37 µg∙mL−1). The activation energy of the hydrolysis catalyzed by FEAP and the standard enthalpy variations of reversible enzyme unfolding were 49.65 and 107.01 kJ∙mol−1, respectively. When the temperature increased from 40 to 70 °C, the deactivation rate constant increased from 0.0050 to 0.0134 min−1, while the half-life decreased from 138.62 to 51.72 min. These results allowed to estimate the activation energy (E*d = 27.50 kJ∙mol−1), enthalpy (24.64 ≤ ΔH*d ≤ 24.89 kJ∙mol−1), entropy (−209.86 ≤ ΔS*d ≤ −210.10 J∙mol−1∙K−1), and Gibbs free energy (90.61 ≤ ΔG*d ≤ 96.74 kJ∙mol−1) of thermal denaturation. The data suggest that FEAP is a promising and thermostable biocatalyst that could be exploited for industrial applications.

Convex Representation of Metabolic Networks with Michaelis–Menten Kinetics

Polyhedral models of metabolic networks are computationally tractable and can predict some cellular functions. A longstanding challenge is incorporating metabolites without losing tractability. In this paper, we do so using a new second-order cone representation of the Michaelis–Menten kinetics. The resulting model consists of linear stoichiometric constraints alongside second-order cone constraints that couple the reaction fluxes to metabolite concentrations. We formulate several new problems around this model: conic flux balance analysis, which augments flux balance analysis with metabolite concentrations; dynamic conic flux balance analysis; and finding minimal cut sets of networks with both reactions and metabolites. Solving these problems yields information about both fluxes and metabolite concentrations. They are second-order cone or mixed-integer second-order cone programs, which, while not as tractable as their linear counterparts, can nonetheless be solved at practical scales using existing software.

Mathematical model and electronic system for real-time O2 control in storage boxes: Development and validation under fluctuating temperatures

Modified atmosphere storage containers with gas-permeable membranes are effective in reducing post-harvest losses, mainly at low temperatures but become ineffective in controlling O2 and CO2 at temperatures above 3 °C. This study aimed to develop a method to control O2 in a storage box. This method uses a diffusion tube, that blocks air from entering the box but allows for air exchange when an air blower is activated. The blower ON frequency (BOF) was modelled as a function of temperature, considering the type and amount of produce, blower properties, tube dimension, and the O2 setpoint. The effects of temperature and gas composition on respiration rate kinetics were analysed with Michaelis-Menten and Arrhenius equations. The model was used to program the microcontroller to control the blower. Validation was performed using a 190 L box containing 25 kg of broccoli. The developed model maintained the desired O2 concentration under fluctuating temperatures. The BOF values ranged from 25.6 to 549.2 s h−1, for temperature fluctuations between 4 and 23 °C. The time required to reach the 3% O2 in the box differed depending on the blower's state. With the blower turned ON from the beginning, it took 24.9 h. However, when the blower was initially turned OFF, it took 11.1 h. Despite temperature changes, the system maintained O2 at 3.8 ± 0.29% and CO2 at 14.4 ± 0.66%. The system is promising for commercial use and best suited for CO2 tolerant produce because it requires a separate mathematical model and control mechanism.

Conformational and Electronic Variations in 1,2- and 1,5a-Cyclophellitols and their Impact on Retaining α-Glucosidase Inhibition.

Glycoside hydrolases (glycosidases) take part in myriad biological processes and are important therapeutic targets. Competitive and mechanism-based inhibitors are useful tools to dissect their biological role and comprise a good starting point for drug discovery. The natural product, cyclophellitol, a mechanism-based, covalent and irreversible retaining β-glucosidase inhibitor has inspired the design of diverse α- and β-glycosidase inhibitor and activity-based probe scaffolds. Here, we sought to deepen our understanding of the structural and functional requirements of cyclophellitol-type compounds for effective human α-glucosidase inhibition. We synthesized a comprehensive set of α-configured 1,2- and 1,5a-cyclophellitol analogues bearing a variety of electrophilic traps. The inhibitory potency of these compounds was assessed towards both lysosomal and ER retaining α-glucosidases. These studies revealed the 1,5a-cyclophellitols to be the most potent retaining α-glucosidase inhibitors, with the nature of the electrophile determining inhibitory mode of action (covalent or non-covalent). DFT calculations support the ability of the 1,5a-cyclophellitols, but not the 1,2-congeners, to adopt conformations that mimic either the Michaelis complex or transition state of α-glucosidases.

Effect of Food Composition on the PK of Isoniazid Quantitatively Explained Using Physiologically Based Biopharmaceutics Modeling.

This work shows the utilization of a physiologically based biopharmaceutics model (PBBM) to mechanistically explain the impact of diverse food types on the pharmacokinetics (PK) of isoniazid (INH) and acetyl-isoniazid (Ac-INH). The model was established and validated using published PK profiles for INH along with a combination of measured and predicted values for the physico-chemical and biopharmaceutical propertied of INH and Ac-INH. A dedicated ontogeny model was developed for N-acetyltransferase 2 (NAT2) in human integrating Michaelis Menten parameters for this enzyme in the physiologically based pharmacokinetic (PBPK) model tissues and in the gut, to explain the pre-systemic and systemic metabolism of INH across different acetylator types. Additionally, a novel equation was proposed to calculate the luminal drug degradation related to the presence of reducing sugars, using individual sugar molar concentrations in the meal. By incorporating luminal degradation into the model, adjusting bile salt concentrations and gastric emptying according to food type and quantity, the PBBM was able to accurately predict the negative effect of carbohydrate-rich diets on the PK of INH.

Targeted screening of multiple anti-inflammatory components from Chrysanthemi indici Flos by ligand fishing with affinity UF-LC/MS.

Chrysanthemi indic Flos (CIF) has been commonly consumed for the treatment of inflammation and related skin diseases. However, the potential bioactive components responsible for its anti-inflammatory and sensitive skin (SS) improvement activities, and the correlated mechanisms of action still remain unknown. In this work, it was firstly found that the CIF extract (CIFE) displayed arrestive free radical scavenging activity on DPPH and ABTS radicals, with no significant difference with positive control Trolox (p > 0.05). Then, compared to the negative group, CIFE markedly decreased the productions of the pro-inflammatory cytokines (IL-1β, IL-6, PEG2, TNF-α, IFN-γ, NO) in LPS induced RAW264.7 cells in a dose-dependent manner (p < 0.01). Besides, CIFE strongly inhibited the COX-2 and hyaluronidase (HAase) with the IC50 values of 1.06 ± 0.01μg/mL and 12.22 ± 0.39μg/mL, indicating higher inhibitory effect than positive control of aspirin of 6.33 ± 0.05μg/mL (p < 0.01), and comparable inhibitory effect with indometacin of 0.60 ± 0.03μg/mL, and ascorbic acid of 11.03 ± 0.41μg/mL (p > 0.05), respectively. Furthermore, kinetic assays with Lineweaver-Burk plot (Michaelis Menten equation) suggested that CIFE reversibly inhibited the COX-2 and HAase, with a mixed characteristics of competitive and non-competitive inhibition. Thereafter, multi-target affinity ultrafiltration liquid chromatography-mass spectrometry (UF-LC/MS) method was employed to fast fish out the potential COX-2 and HAase in CIFE. Herein, 13 components showed various affinity binding degrees to the COX-2 and HAase, while those components with relative binding affinity (RBA) value higher than 3.0, such as linarin and chlorogenic acid isomers, were deemed to be the most bioactive components for the anti-inflammatory and SS improvement activities of CIFE. Finally, the interaction mechanism, including binding energy, inhibition constant, docking sites, and the key amino acids involved in hydrogen bonds between the potential ligands and COX-2/HAase were simulated and confirmed with the molecule docking analysis. In summary, this study showcased the prominent anti-inflammatory and SS improvement activities of CIF, which would provide further insights on this functional medicinal plant to be a natural anti-SS remedy.

Bioinspired bimetallic metal–organic framework nanozyme with laccase-mimicking activity for detection and removal of phenolic contaminants

Laccase, despite its eco-friendly nature, faces constraints due to cost and inherent vulnerability, restricting its widespread application. Therefore, pursuing the development of a robust laccase mimetic displaying exceptional activity remains an ongoing endeavor. This research synthesized a bimetallic metal–organic framework nanozyme (Cu-Mn MOF) with enhanced laccase-mimicking activity via a one-step solvothermal method. The diverse Cu redox valence state of the nanozyme mimics the multi-copper active center of laccase, which endows Cu-Mn MOF with laccase-mimicking activity. Incorporating Mn introduces a novel metal center into the MOF, creating a catalytic microenvironment. Bimetallic Cu-Mn synergistic interplay accelerates the electron transfer rate, resulting in enhanced laccase-mimicking activity. The Michaelis constant of Cu-Mn MOF for 2,4-dichlorophenol (2,4-DP) registers at 0.080 mM, displaying remarkable stability and recyclability even under stringent conditions, thereby achieving a comprehensive surpass of laccase. Accordingly, Cu-Mn MOF was utilized to degrade phenolic compounds and design a smartphone sensing platform to facilitate portable and visual detection of phenol. Utilizing the occurrence of Cu-S coordination, which accelerates the electron transfer rate, a thiourea-enhanced Cu-Mn MOF was designed to improve the degradation rate. The limit of detection (LOD) for smartphone platform is 0.159 μM, with a linear detection range of 1–40 μM. The detection method was applied to determine the phenol in spiked samples with the spiked recoveries rate of 96.05–104.30 % and the relative standard deviations less than ± 4 %. The outcomes of these applications reveal the vast application potential of Cu-Mn MOF in environmental engineering.

Characterization of Human Organic Anion Transporter 4 (hOAT4) and Mouse Oat5 (mOat5) As Functional Orthologs for Renal Anion Uptake and Efflux Transport.

Organic anions (OAs) are compounds including drugs or toxicants that are negatively charged at physiologic pH and are typically transported by organic anion transporters (OATs). Human OAT4 (SLC22A11) is expressed in the apical membrane of renal proximal tubules. Although there is no rodent ortholog of hOAT4, rodents express Oat5 (Slc22a19), an anion exchanger that is also localized to the apical membrane of renal proximal tubule cells. The purpose of this study was to determine the functional similarity between mouse Oat5 and human OAT4. Chinese hamster ovary (CHO) cells expressing SLC22A11 or Slc22a19 were used to assess the transport characteristics of radiolabeled ochratoxin (OTA). We determined the kinetics of OTA transport; the resulting Michaelis constant (Kt) and maximal rate of mediated substrate transport (Jmax) values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 μM, respectively, and Jmax 4.4 and 3.9 pmol/cm2, respectively. For the profile of OTA inhibition by OAs, IC50 values were determined for several clinically important drugs and toxicants. The resulting IC50 values ranged from 9 μM for indomethacin to ∼600 μM for the diuretic hydrochlorothiazide. We measured the efflux of OTA from preloaded cells; both hOAT4 and mOat5 supported the efflux of OTA. These data support the hypothesis that OAT4 and Oat5 are functional orthologs and share selectivity for OTA both for reabsorption and secretion. SIGNIFICANCE STATEMENT: This study compares the selectivity profile between human organic anion transporter (OAT4) and mouse Oat5. Our data revealed a similar selectivity profile for ochratoxin A (OTA) reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, although not genetic orthologs, behave as functional orthologs for both uptake and efflux. These data will be instrumental in selecting an appropriate animal model when studying the renal disposition of anionic drugs and toxicants.

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5′-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3′-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3′-monophosphate (3′-AMP) to adenosine-3′,5′-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3′-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 μM, and the turnover number, kcat, was 8 s−1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3′-AMP were determined to be −25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

Structure/function relationship of Atlantic bonito (Sarda sarda Bloch) myoglobin and comparison with Atlantic and Tinker mackerel myoglobins

Atlantic bonito meat has economic potential as an alternative to mackerel consumption. Thus, considering the presence of myoglobin (Mb) in red fish muscles, we report the characterisation of Atlantic bonito Mb compared to Atlantic and Tinker mackerel Mbs since this haemoprotein is implicated in lipid oxidation and fish meat preservation. A plethora of biochemical approaches were employed to purified Mb from Atlantic bonito and determine the autoxidation rate constant (0.189 ± 0.009 h−1), melting temperature (Tm = 72.84 ± 1.02 °C) and pseudoperoxidase activity in different conditions (pH and several cations). Atlantic and Tinker mackerel Mbs showed a lower Tm (∼66.85 °C), while oxyMb autoxidation rate constant was higher for Atlantic mackerel (∼1.08-fold) and lower for Tinker mackerel (∼1.35-fold) compared to Atlantic bonito. This Mb had a Michaelis-Menten constant (Km) of 38.63 ± 1.89 μM, ∼2.49-fold and 2.27-fold lower than Atlantic and Tinker mackerel Mbs, respectively. Atlantic bonito Mb primary structure has 146 amino acid residues with the N-terminal acetylated and 25 amino acid substitutions with respect to Atlantic and Tinker mackerel Mbs. In silico analysis revealed that 7 out of 25 substitutions are close to the haem-pocket, while 18 out of 25 are far from this region. All substitutions, except H20, L70 and L81 are exposed on the protein globular surface. Overall, the results of this research provide new information for future studies that will be useful to the fish industry for preservation of frozen or canned Atlantic bonito meat considering the presence of Mb as a reactive haemoprotein.

Tailoring the interfacial microenvironment of magnetic metal-organic frameworks using amino–acid-based ionic liquids for lipase immobilization

Modifying the carrier interface is a promising method to improve the microenvironment of immobilized enzymes and enhance their activity and stability. In this work, using proline as amino acid, magnetic metal-organic frameworks (MOFs) were modified with an amino-acid-based ionic liquid (AAIL) with two hydroxyl groups followed by adsorption of porcine pancreatic lipase (PPL). The activity recovery of the prepared immobilized lipase (MMOF-AAIL/PPL) was up to 162 % higher than that of MMOF-PPL (70.8 %). The Michaelis constant of MMOF-AAIL/PPL was 0.0742 mM lower than that of MMOF-PPL, but the catalytic efficiency was 0.0223 min−1 which was higher than MMOF-PPL. Furthermore, MMOF-AAIL/PPL maintained 85.6 % residual activity after stored for 40 days and its residual activity was 71.9 % while that for MMOF-PPL was 58.8 % after incubated in 6 M urea for 2 h. Particularly, after ten consecutive cycles, the residual activity of MMOF-AAIL/PPL still reached 84.4 %. In addition, the magnetic properties of the support facilitate the separation process which improves the utilization efficiency of immobilized enzymes.

Determination of low concentrations of glucose through colorimetric analysis using CoFe2O4 magnetic catalyst and SAT-3.

Natural enzyme mimics have attracted attention as alternatives to natural peroxidases. Among these, magnetic nanoparticles, especially ferrites, have attracted attention because of their unique electronic and physical structures, which are expected to be applied in various fields, including high-frequency magnetic materials, biomaterials, gas sensors, and semiconductor photocatalysts. The structural properties of the synthesized catalysts were investigated using X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The prepared CoFe2O4 exhibited a spinel ferrite structure and formed a wood-flake-like bulk structure. In this study, magnetic CoFe2O4 was prepared using a precipitation method as a natural enzyme mimetic. CoFe2O4 showed excellent peroxidase-like activity, as demonstrated by the Michaelis-Menten constant (Km) and the maximum velocity (Vmax). The linear ranges of the calibration curves for H2O2 and glucose were in the range of 0-500µM, and the detection limits were 1.83 and 5.91µM, respectively. This analytical method was applied for the determination of glucose in human serum, and the results were satisfactory and consistent with certified values. The performance of this sensor was comparable to or superior to those of several other sensors commonly used for glucose analysis, indicating that its practical application is feasible.

Enhancing the recovery of complex amino acids from excess sludge via low-intensity ultrasound-assisted enzymatic hydrolysis

The recovery of high-value products such as complex amino acids from activated sludge is a crucial alternative for the recycling of excess sludge, although several challenges still need to be addressed, especially in terms of extraction efficiency and product quality. Herein, a promising approach involving low-intensity ultrasound-assisted enzymatic hydrolysis (UEH) was utilized to obtain complex amino acids from sludge protein. Compared with the values obtained for single enzymatic hydrolysis (EH), after a 20-minute pre-treatment at ambient temperature using low-intensity ultrasound (0.15 W/mL), the enzyme activity increased by 28.7 %, and the degree of protein hydrolysis (DH) correspondingly rose by 93.3 %. The application of ultrasound during subsequent EH resulted in a decrease in DH, with the extent of the decrease increasing over time. This suggested that the primary cause was conformational changes of enzymes rather than mass transfer. Moreover, both the Michaelis–Menten model (R2 > 0.99) and protein hydrolysis model (R2 > 0.96) accurately simulated EH and UEH, indicating that UEH exhibits superior enzyme–substrate affinity and a higher rate of protein hydrolysis. Furthermore, after separation and purification, the total content of seven essential amino acids in the dry material exceeded that in traditional feed sources such as soybean meal. Additionally, the safety test and energy consumption analysis demonstrated that UEH has good potential for the extraction of complex amino acids from excess sludge.

Peroxidase like Zn doped Prussian blue facilitates salinity tolerance in winter wheat through seed dressing

Salt stress severely limits the growth and yield of wheat in saline-alkali soil. While nanozymes have shown promise in mitigating abiotic stress by scavenging reactive oxygen species (ROS) in plants, their application in alleviating salt stress for wheat is still limited. This study synthesized a highly active nanozyme catalyst known as ZnPB (Zn-modified Prussian blue) to improve the yield and quality of wheat in saline soil. According to the Michaelis-Menten equation, ZnPB demonstrates exceptional peroxidase-like enzymatic activity, thereby mitigating oxidative damage caused by salt stress. Additionally, studies have shown that the ZnPB nanozyme is capable of regulating intracellular Na+ efflux and K+ retention in wheat, resulting in a decrease in proline and soluble protein levels while maintaining the integrity of macromolecules within the cell. Consequently, field experiments demonstrated that the ZnPB nanozyme increased winter wheat yield by 12.15 %, while also significantly enhancing its nutritional quality. This research offers a promising approach to improving the salinity tolerance of wheat, while also providing insights into its practical application.