Crystal structure and function analysis of 6-phosphogluconate dehydrogenase in Mycobacterium tuberculosis.

6-phosphogluconate dehydrogenase (6PGDH) is an essential enzyme in energy metabolism and redox reactions, and represents a potential drug target for the development of therapies targeting trypanosomes, plasmodium, or other pathogens. Tuberculosis, caused by Mycobacterium tuberculosis, is a contagious disease that severely affects human health, with approximately one-third of the world's population infected. However, the protein structure, exact oligomeric state, and catalytic mechanism of 6PGDH in Mycobacterium tuberculosis (Mt6PGDH) have remained largely unknown. In this study, we successfully purified and determined the structure of Mt6PGDH, revealing its function as a tetramer in both solution and crystal states. Through structural comparisons, we clarified the tetramer formation mechanism and the oligomeric organization of short-chain 6PGDHs. Additionally, we identified key residues for coenzyme recognition and catalytic activity. This work not only deepens our understanding of the enzymatic function of Mt6PGDH but also lays a foundation for the development of drugs targeting this enzyme.

Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from Geobacillus kaustophilus.

Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans-urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus (GkHAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The GkHAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that GkHAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global GkHAL folding. These findings suggest that GkHAL could be a promising candidate for potential biotechnological applications.

Isolation of Chalcomoracin as a Potential α-Glycosidase Inhibitor from Mulberry Leaves and Its Binding Mechanism.

Diabetes is a chronic metabolic disease, whereas α-glucosidases are key enzymes involved in the metabolism of starch and glycogen. There is a long history of the use of mulberry leaf (the leaf of Morus alba) as an antidiabetic herb in China, and we found that chalcomoracin, one of the specific Diels-Alder adducts in mulberry leaf, had prominent α-glucosidase inhibitory activity and has the potential to be a substitute for current hypoglycemic drugs such as acarbose, which have severe gastrointestinal side effects. In this study, chalcomoracin was effectively isolated from mulberry leaves, and its α-glucosidase inhibition was studied via enzymatic kinetics, isothermal titration (ITC) and molecular docking. The results showed that chalcomoracin inhibited α-glucosidase through both competitive and non-competitive manners, and its inhibitory activity was stronger than that of 1-doxymycin (1-DNJ) but slightly weaker than that of acarbose. ITC analysis revealed that the combination of chalcomoracin and α-glucosidase was an entropy-driven spontaneous reaction, and the molecular docking results also verified this conclusion. During the binding process, chalcomoracin went into the "pocket" of α-glucosidase via hydrophobic interactions, and it is linked with residues Val544, Asp95, Ala93, Gly119, Arg275 and Pro287 by hydrogen bonds. This study provided a potential compound for the prevention and treatment of diabetes and a theoretical basis for the discovery of novel candidates for α-glycosidase inhibitors.

Conformational Plasticity of the Active Site Entrance in E. coli Aspartate Transcarbamoylase and Its Implication in Feedback Regulation.

Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a "textbook example" for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase.

Controlling the Kinetics of an Enzymatic Reaction through Enzyme or Substrate Confinement into Lipid Mesophases with Tunable Structural Parameters.

Lipid liquid crystalline mesophases, resulting from the self-assembly of polymorphic lipids in water, have been widely explored as biocompatible drug delivery systems. In this respect, non-lamellar structures are particularly attractive: they are characterized by complex 3D architectures, with the coexistence of hydrophobic and hydrophilic regions that can conveniently host drugs of different polarities. The fine tunability of the structural parameters is nontrivial, but of paramount relevance, in order to control the diffusive properties of encapsulated active principles and, ultimately, their pharmacokinetics and release. In this work, we investigate the reaction kinetics of p-nitrophenyl phosphate conversion into p-nitrophenol, catalysed by the enzyme Alkaline Phosphatase, upon alternative confinement of the substrate and of the enzyme into liquid crystalline mesophases of phytantriol/H2O containing variable amounts of an additive, sucrose stearate, able to swell the mesophase. A structural investigation through Small-Angle X-ray Scattering, revealed the possibility to finely control the structure/size of the mesophases with the amount of the included additive. A UV-vis spectroscopy study highlighted that the enzymatic reaction kinetics could be controlled by tuning the structural parameters of the mesophase, opening new perspectives for the exploitation of non-lamellar mesophases for confinement and controlled release of therapeutics.

Enzymatic kinetics regarding reversible metabolism of CS-0777, a sphingosine 1-phosphate receptor modulator, via phosphorylation and dephosphorylation in humans.

1. CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes. 2. The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498 µM to 1060 µM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate. 3. The Michaelis constants of four human ALPs and microsomes were in the range from 10.9 µM to 32.1 µM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes. 4. FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.

Species-Specific Inactivation of Triosephosphate Isomerase from Trypanosoma brucei: Kinetic and Molecular Dynamics Studies.

Human African Trypanosomiasis (HAT), a disease that provokes 2184 new cases a year in Sub-Saharan Africa, is caused by Trypanosoma brucei. Current treatments are limited, highly toxic, and parasite strains resistant to them are emerging. Therefore, there is an urgency to find new drugs against HAT. In this context, T. brucei depends on glycolysis as the unique source for ATP supply; therefore, the enzyme triosephosphate isomerase (TIM) is an attractive target for drug design. In the present work, three new benzimidazole derivatives were found as TbTIM inactivators (compounds 1, 2 and 3) with an I50 value of 84, 82 and 73 µM, respectively. Kinetic analyses indicated that the three molecules were selective when tested against human TIM (HsTIM) activity. Additionally, to study their binding mode in TbTIM, we performed a 100 ns molecular dynamics simulation of TbTIM-inactivator complexes. Simulations showed that the binding of compounds disturbs the structure of the protein, affecting the conformations of important domains such as loop 6 and loop 8. In addition, the physicochemical and drug-like parameters showed by the three compounds suggest a good oral absorption. In conclusion, these molecules will serve as a guide to design more potent inactivators that could be used to obtain new drugs against HAT.

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae).

Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000-5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis.

The enhancement mechanisms of mucin and lactoferrin on α-amylase activity in saliva: Exploring the interactions using QCM-D and molecular docking.

α-Amylase activity differs between individuals and is influenced by dietary behavior and salivary constituents, but limited information is available on the relationship between α-amylase activity and saliva components. This study investigated the impact of salivary proteins on α-amylase activity, their various correlations, the effect of mucin (MUC5B and MUC7) and lactoferrin on the enzymatic kinetics of α-amylase, and the mechanisms of these interactions using the quartz crystal microbalance with dissipation (QCM-D) technique and molecular docking. The results showed that α-amylase activity was significantly correlated with the concentrations of MUC5B (R2 = 0.42, p < 0.05), MUC7 (R2 = 0.35, p < 0.05), and lactoferrin (R2 = 0.35, p < 0.05). An in vitro study demonstrated that α-amylase activity could be significantly increased by mucins and lactoferrin by decreasing the Michaelis constant (Km) of α-amylase. Moreover, the results from the QCM-D and molecule docking suggested that mucin and lactoferrin could interact with α-amylase to form stable α-amylase-mucin and α-amylase-lactoferrin complexes through hydrophobic interactions, electrostatic interactions, Van der Waals forces, and hydrogen bonds. In conclusion, these findings indicated that the salivary α-amylase activity depended not only on the α-amylase content, but also could be enhanced by the interactions of mucin/lactoferrin with α-amylase.

New prenylated indole-benzodiazepine-2,5-diones with α-glucosidase inhibitory activities from the mangrove-derived Aspergillus spinosus.

Mangrove-derived fungi have been demonstrated to be promising source of structurally diverse and widely active secondary metabolites. During our search for new bioactive compounds, eight new indole-benzodiazepine-2,5-dione derivatives asperdinones A-H (1-8) and two known congeners (9 and 10) were isolated from the culture extracts of the mangrove-derived fungus Aspergillus spinosus WHUF0344 guided by one strain many compounds (OSMAC) and the heteronuclear 1H, 13C single-quantum coherence (HSQC) based small molecule accurate recognition technology (SMART) strategies. The structures and absolute configurations of the new compounds were elucidated by detailed spectroscopic analyze and electronic circular dichroism (ECD) calculations. The putative biosynthetic pathway of these compounds was proposed. Compounds 1-10 were evaluated for their antibacterial and α-glucosidase inhibitory activities. None of compounds showed antibacterial activity. Compounds 2-6 and 8 exhibited moderate inhibitory effects against α-glucosidase with IC50 values in the range of 24.65-312.25 µM. Besides, both 3 and 4 inhibited α-glucosidase variedly. Furthermore, the molecular docking study showed that compounds 2-4 were perfectly docking into the active sites of α-glucosidase. This study not only enriched the chemical diversity of secondary metabolites from the mangrove-derived fungi, but also provided potential hit compounds for further development of α-glucosidase inhibitors.

Collagen derived Gly-Pro-type DPP-IV inhibitory peptides: Structure-activity relationship, inhibition kinetics and inhibition mechanism.

Our previous study has demonstrated that both the amino acid at N3 position and peptide length affected the DPP-IV inhibitory activity of Gly-Pro-type peptides. To further elucidate their molecular mechanism, a combined approach of QSAR modeling, enzymatic kinetics and molecular docking was used. Results showed that the QSAR models of Gly-Pro-type tripeptides and Gly-Pro-type peptides containing 3-12 residues were successfully constructed by 5z-scale descriptor with R2 of 0.830 and 0.797, respectively. The lower values of electrophilicity, polarity, and side-chain bulk of amino acid at N3 position caused higher DPP-IV inhibitory activity of Gly-Pro-type peptides. Moreover, an appropriate increase in the length of Gly-Pro-type peptides did not change their competitive inhibition mode, but decreased their inhibition constants (Ki values) and increased interactions with DPP-IV. More importantly, the interactions between the residues at C-terminal of Gly-Pro-type peptides containing 5 âˆ¼ 6 residues with S2 extensive subsites (Ser209, Phe357, Arg358) of DPP-IV increased the interactions of Gly residue at N1 position with the S2 subsites (Glu205, Glu206, Asn710, Arg125, Tyr662) and decreased the acylation level of DPP-IV-peptide complex, and thereby increasing peptides' DPP-IV inhibitory activity.

Kinetic-conceptual model of the hydrolysis of bovine plasma proteins - ALCALASE® 2.9L: The role of inhibition by product.

In the present work, the inhibitory effect of the peptide fractions, obtained through enzymatic hydrolysis of bovine plasma was evaluated, on the enzyme used in the reaction (Alcalase 2.4 L). In this sense, Ultra-filtered peptide fractions of different molecular sizes (A: Fraction>10; B: Fraction 10-3 kDa; and C: Fraction <3 kDa), were used to verify the impact on the total hydrolysis rate. The Fractions between 3 and 10 kDa were refined to fit a conceptual kinetic model which considers inhibition by product and substrate. Additionally, the inactivation of the enzyme through the reaction time was evaluated and its effects incorporated into the model. It was shown that some peptides released in the successive stages of the reaction can in turn inhibit the activity of the hydrolyzing enzyme. The model evaluated suggests a time-varying expression of inhibition parameters as a function of the initial substrate concentration in the reaction. This is based on the kinetic changes of the product profiles for each reaction time in the evaluated operating conditions (S0 variable). A greater inhibitory effect due to the products is evidenced when the reaction occurs with a higher load of the initial substrate (S0 = 20 g/L).

Interaction between Changan Granule and its main components in the plasma and CYP450 enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Changan Granule (CAG) is a Chinese patent drug developed based on an empirical prescription in accordance with the formulation theory of Traditional Chinese Medicine. The prescription is composed of eight herbal drugs which have been traditionally used by Chinese people for a long history. It has effects of invigorating spleen and supplementing qi, as well as regulating liver and ceasing diarrhea, and is indicated for the treatment of irritable bowel syndrome (IBS). AIM OF THE STUDY: This study was aimed to investigate the interaction between CAG and its main components and cytochrome P450 (CYP450) enzymes so as to characterize the major metabolites and metabolic enzymes and evaluate the safety concerns to its clinical use. MATERIALS AND METHODS: Both in vivo and in vitro experiments using such as diarrhea-predominant IBS (IBS-D) rat model, HepG2 cells, and human liver microsomes (HLM) were carried out to investigate the interaction between CAG and its main components and CYP450 enzymes. Real-time quantitative PCR (qPCR), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and cocktail probes were employed to qualitatively or quantitatively measure the metabolites and metabolic enzymes. RESULTS: CAG inhibited the enzyme activities of CYP1A2, CYP2E1, CYP2D6, CYP2C9, and CYP3A4 and the mRNA expressions of CYP2E1, CYP2C9, CYP3A4, and CYP2D6 in vitro. CAG down-regulated the increased expression of CYP1A2 and up-regulated the decreased expression of CYP3A1 in vivo. Twenty-two metabolites were characterized from the main components of CAG after incubation with HLM in vitro. CYP2D6, CYP2E1, CYP3A4 and CYP2C9 were identified as the characteristic metabolic enzymes. CONCLUSIONS: This study provides a reference for clinical application of CAG in safety. CAG and CYP450 enzymes are interacted. CAG is mainly metabolized by CYP2E1 and CYP2D6. The expression of CYP2E1 and CYP2D6 are more susceptible to be influenced by CAG in comparison with that of CYP3A4, CYP2C9 and CYP1A2. It implies the potential risk of interaction when CAG is taken together with the drugs metabolized by CYP2E1 and CYP2D6.

New pyrrolopyridine-based thiazolotriazoles as diabetics inhibitors: enzymatic kinetics and in silico study.

Aim: To synthesize pyrrolopyridine-based thiazolotriazoles as a novel class of α-amylase and α-glucosidase inhibitors and to determine their enzymatic kinetics. Methodology: Pyrrolopyridine-based thiazolotriazole analogs (1-24) were synthesized and characterized through proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance and high-resolution electron ionization mass spectrometry. Results: All synthesized analogs displayed good inhibitory potential of α-amylase and α-glucosidase ranging 17.65-70.7 µM and 18.15-71.97 µM, respectively, compared with the reference drug, acarbose (11.98 µM and 12.79 µM). Analog 3 was the most potent among the synthesized analogs, having α-amylase and α-glucosidase inhibitory activity at 17.65 and 18.15 µM, respectively. The structure-activity relationship and binding modes of interactions between selected analogs were confirmed via docking and enzymatic kinetics studies. The compounds (1-24) were tested for cytotoxicity against the 3T3 mouse fibroblast cell line and were observed to be nontoxic.

Three recombinant peroxidases as a degradation agent of aflatoxin M1 applied in milk and beer.

The aim of this work was to estimate the effects of three recombinant peroxidases (rPODs) on the degradation of aflatoxin M1 (AFM1) in a model solution and were applied in milk and beer to study the AFM1 degradation. Besides, the contents of AFM1 in model solution, milk and beer were evaluated, and the kinetic parameters of rPODs were determined (Michaelis-Menten constant - Km and maximal velocity - Vmax). The optimized reaction conditions (The degradation was over 60 %) for these three rPODs in the model solution were, respectively as follows: pH were 9, 9, and 10; hydrogen peroxide concentrations were 60, 50, and 60 mmol/L; at an ionic strength of 75 mmol/L and reaction temperature of 30 °C with 1 mmol/L K+ or 1 mmol/L Na+. These three rPODs (1 U /mL) presented the maximum activity for degradation of AFM1 of 22.4 %, 25.6 %, and 24.3 % in milk, while 14.5 %, 16.9 %, and 18.2 % in beer, respectively. Meanwhile, the survival rate of Hep-G2 cells raised about 1.4 times after treated with peroxidase-generated AFM1 degradation products. Therefore, POD may be a promising alternative to reduce the pollution of AFM1 in model solution, milk, beer, and minimize their impact on the environment in humans.

Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes.

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.

Fabrication of lipase-loaded particles by coacervation with chitosan.

Coacervation of the lipase from Aspergillus oryzae (AOL) with chitosan was a feasible way to fabricate lipase-loaded particles and the optimum conditions were phase separation pH 5.5, chitosan to AOL mass ratio 1:5, and temperature 25 °C in the absence of NaCl, which conferred an AOL loading efficiency of up to 95.48% and activity recovery of 69.60%. The AOL-chitosan coacervates were highly porous and more susceptible to weight loss upon heating. Coacervation with chitosan increased the activity of AOL and shifted its optimum pH from 7.0 to 6.0, but exerted no effect on its optimum temperature (45 °C). Thermal deactivation kinetics analysis revealed that the coacervated AOL was more thermal stable, while the Michaelis-Menten kinetics analysis indicated that coacervation with chitosan increased the Vmax of AOL by 2.4 folds, but decreased its substrate affinity by 3.6 folds. Hence, the AOL-chitosan coacervates are potential in the construction of Pickering emulsion-based lipase catalysis systems.

Identification, kinetics and thermodynamic analysis of novel ß-galactosidase from Convolvulus arvensis seeds: An efficient agent for delactosed milk activity.

The ß-galactosidase was extracted and purified from 100 g of C. arvensis seeds using a variety of protein purification procedures such as ammonium sulphate fractionation, gel filtration, and finally chromatography on a cationic ion exchanger. The effects of metal ions, kinetics parameters, and glycoprotein nature were determined, as well as the optimal pH and temperature of the purified enzyme. With a high specific activity (72 units/mg), ß-galactosidase was isolated to a 24-fold apparent electrophoretic homogeneity. The molecular mass of ß-galactosidase was determined as monomeric, which was further confirmed by SDS-PAGE and MALDI-TOF/MS analysis, with a 45 kDa molecular weight. The enzyme has a Km of 0.33 mM and a Vmax of 42 µmol/min Lactose in milk was reduced by 38.5 and 70 % after 4 h of incubation with ß-galactosidase from C. arvensis. The ß-galactosidase thermal inactivation kinetic parameters ΔH°, ΔS°, and ΔG° were calculated, indicating that the enzyme undergoes significant unfolding events during denaturation. Using ß-galactosidase from C. arvensis seeds, lactose hydrolysis in milk up to approx. 50 % was observed. The findings indicate the potential use of C. arvensis seeds for the production of low/delactosed milk for lactose-intolerant population.

Magnolol and Luteolin Inhibition of α-Glucosidase Activity: Kinetics and Type of Interaction Detected by In Vitro and In Silico Studies.

Magnolol and luteolin are two natural compounds recognized in several medicinal plants widely used in traditional medicine, including type 2 diabetes mellitus. This research aimed to determine the inhibitory activity of magnolol and luteolin on α-glucosidase activity. Their biological profile was studied by multispectroscopic methods along with inhibitory kinetic analysis and computational experiments. Magnolol and luteolin decreased the enzymatic activity in a concentration-dependent manner. With 0.075 µM α-glucosidase, the IC50 values were similar for both compounds (~ 32 µM) and significantly lower than for acarbose (815 µM). Magnolol showed a mixed-type antagonism, while luteolin showed a non-competitive inhibition mechanism. Thermodynamic parameters suggested that the binding of magnolol was predominantly sustained by hydrophobic interactions, while luteolin mainly exploited van der Waals contacts and hydrogen bonds. Synchronous fluorescence revealed that magnolol interacted with the target, influencing the microenvironment around tyrosine residues, and circular dichroism explained a rearrangement of the secondary structure of α-glucosidase from the initial α-helix to the final conformation enriched with ß-sheet and random coil. Docking studies provided support for the experimental results. Altogether, the data propose magnolol, for the first time, as a potential α-glucosidase inhibitor and add further evidence to the inhibitory role of luteolin.