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Siliceous mesocellular foam (MCF) is versatile support material for enzyme immobilization. This material is attractive for its robust, well-defined pore structure with interconnected, ultra large pores that facilitate diffusion. Excellent activity and extremely low level of leaching (metal or enzyme) were attained with these robust supports. MCF was synthesized by hydrothermal procedure using tetraethylorthosilicate (TEOS), amphiphilic block co-polymers Pluronic P123 and Trimethyl benzene as auxilary chemical under acidic conditions. The amino groups have been grafted to as-synthesized mesoporous silica by 3-aminopropyl trimethoxysilane (3-APTS) and glutaraldehyde was then coupled with amino functionalized silica. The physicochemical properties of the prepared materials were characterized by Nitrogen adsorption – desorption, FTIR, XPS, CPMAS NMR techniques. Meso cellular foams having ultra large pore diameter of 241 A. Results showed that glutaraldehyde bound silica showed highest stability for enzyme binding.

This work identified a summary of new chalcone derivatives (E)-2-(4-acetamidophenoxy)-N-(3-(3-(4-substitutedphenyl) acryloyl) phenyl) acetamide by the condensation of 2-(4-acetamidophenoxy)-N-(3-acetylphenyl) acetamide with various aromatic aldehydes. Synthesized compounds were characterized by IR and 1H NMR spectroscopy. Several bacterial species and also candida albicans were tested for the antimicrobial activity. Compared to the standard drug streptomycin and clotrimazole against bacterial and fungal species, 5e, 5c and 5d have shown strong antibacterial and moderate antifungal activity. The aim of the anti - microbial agent enzyme was to investigate and describe the interactions of the identified hits within the target enzyme binding pocket using the synthesized composite against glucosamine-6 phosphorus synthase. The docking results enhanced the behaviour of new derivatives as promising antimicrobials. The simulation of (E)-2-(4-acetamidophenoxy)-N-(3-(3-(2-chlorophenyl) acryloyl) phenyl acetamide (5a) in BRCA1 resulted in the creation of two hydrogen bond interactions with bond distance (2.13 A) and it was observed that the best binding energy value for −9.07Kcal / mol

During acidic pretreatment of renewable lignocellulosic biomass, lignin depolymerization/repolymerization reactions often occur, which intensifies lignin inhibition on cellulose hydrolysis generally through lignin-derived inhibitors formation, unproductive binding and physical blockage effect. In order to improve the enzymatic digestibility of dilute acid pretreated hardwood, in this work various additives were compared and their inhibition mitigating effects were examined. It was revealed that 2-naphthol addition reduced the formation of inhibitory derivatives from lignin, thus increasing the enzymatic hydrolysis yield of the whole slurry after dilute acid pretreatment. The addition of phenolic acids largely diminished enzyme binding effect of the insoluble lignin. Moreover, the 2-naphthol-7-sulfonate additive led to the most pronounced mitigation on physical blockage effect of lignin, as XPS and SEM results showed the significant removal of surface lignin. This work revealed the different mechanisms of various additives in mitigating lignin inhibition, and provided a better understanding on utilizing proper additives to improve biomass saccharification for biorefinery applications.

Background:. Circular RNA (circRNA) is a type of closed circular noncoding RNA (ncRNA), mostly formed by back-splicing or alternative splicing of pre-messenger RNA (mRNA). The aim of this study was to explore the expression profile of circRNA in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and discover potential molecular markers of AS. Methods:. The circRNA microarray technology was used to detect the expression of circRNAs in the peripheral blood of 6 patients with AS and 6 healthy controls (HC). To screen the differentially expressed circRNAs by fold change (FC) and P value, these differentially expressed circRNAs were analyzed by bioinformatics. In 60 cases of AS and 30 cases of HC, 4 circRNAs were subjected to real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and their correlation with various clinical indicators was analyzed. Finally, the receiver operating characteristic (ROC) curve was used to analyze their potential as AS diagnostic markers. Results:. The microarray results showed that there were 1369 significantly differently expressed (P 1.5) circRNAs between the AS and HC groups (675 upregulated and 694 downregulated). The results of bioinformatics analysis suggested that they were mainly involved in “enzyme binding,” “adenosine ribonucleotide binding,” “MAPK signaling pathway”, etc. The RT-qPCR results showed that the expressions of hsa_circRNA_001544 (U = 486.5, P

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A2 (PLA2 ) activity. Therefore, the regulation of PLA2 catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA2 due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA2 inhibition. A more detailed analysis of the plasma-treated PLA2 identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation.

Significance 3-benzylidene chroman-4-ones share close homology with naturally occurring bioactive compounds. Objectives This study evaluated the antifungal, antioxidant and anticancer activities of novel 3-benzylidene chromanone analogues with respect to their structure-activity relationships. Methods Compounds 45e-64e were synthesized inhouse. Aspergillus niger (MTCC 1344) Aspergillus flavus and Botrytis cinareae were the fungal strains tested. Computational docking analysis were carried out for Vanin-1, estrogen receptor and Akt proteins using Auto-dock vina. Free radical scavenging, total antioxidant capacity was analyzed using spectrophotometric methods. MCF-7(breast cancer) cell line was used for anticancer assays. Flow cytometry was used to detect cell cycle and apoptosis. Results Out of the twenty compounds screened, compounds 47e,50e,52e,57e and 61e that possessed either methoxy and ethoxy/methyl/isopropyl group exhibited very good activity against all fungi. Compounds possessing methoxy group alone showed moderate activity and compounds devoid of methoxy, and ethoxy groups did not show any activity. When computationally analyzed against target proteins for antioxidant properties, the compounds exhibited excellent binging efficacy to Vanin-1 and estrogen receptors. These predictions were translated in the in vitro free-radical scavenging and antioxidant assays. The compounds exhibited anti-proliferative efficacy in breast cancer cell line, increased the sub-G0/G1 cell cycle populations and total apoptosis in MCF-7 cells. Additionally, the compounds also depicted excelling binging energy when computationally analyzed for Akt enzyme binding. Conclusion In summary, our study identified potential analogues of 3-benzylidene chroman-4-one molecules with excellent anti-fungal, anti-oxidant and anticancer activities which demands further research for drug developments.

Background The molecular mechanism of Astragali Radix in the treatment of children with nephrotic syndrome (NS) is unclear. This study aimed to use network pharmacology to explore this potential mechanism. Methods The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to identify the main active ingredients of Astragali Radix. The PharmMapper, Online Mendelian Inheritance in Man (OMIM), and GeneCards databases were then used to identify the active ingredients of Astragali Radix. The String database and Cytoscape software were used to construct the protein-protein network. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID Database. Results In the TCMSP Database, a total of 20 chemical constituents of Astragali Radix were screened. After removing the duplicates and false positive genes, 394 targets of these active ingredients were obtained from PharmMapper. By comparing the NS-related genes in the GeneCards and OMIM Databases, a total of 39 potential NS-related targets were ultimately identified. The protein-protein-interaction network included 39 nodes and 366 edges. The top 5 proteins were albumin (ALB), serine/threonine kinase (AKT1), epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and matrix metallopeptidase 9 (MMP9). The GO analysis showed that the target genes were mainly involved in biological processes (e.g., signal transduction, the positive regulation of cell proliferation, and the positive regulation of migration). The cellular components included a plasma membrane, extracellular exosome, and extracellular space. The molecular functions included protein binding, zinc-ion binding, protein tyrosine kinase activity, and enzyme binding. The KEGG analysis showed that the treatment of NS by Astragali Radix mainly involved pathways in cancer, proteoglycans in cancer, the phosphatidylinositol 3-kinase and protein kinase B (PI3K-Akt) signaling pathway, the rennin-angiotensin-system (Ras) signaling pathways, and Forkhead box protein O1 (FoxO) signaling pathways. Conclusions In the present study, the network pharmacology method was used to explore the potential targets and pathways of Astragali Radix in the treatment of NS. We also provided future research directions for the treatment of NS with a complex pathogenesis.

Abstract The regulation of the activity and selectivity of phospholipase A2 (PLA2), which is capable of cleaving fatty acid in the second position (sn-2) of the phospholipid, is carried out through the membrane-binding and catalytic sites of the enzyme. For hydrolytic activity, PLA2 must first bind to the phospholipid membrane, and the binding efficiency depends on the composition of the membrane. The membrane-binding site of PLA2 is formed by several tens of amino acids and its composition differs from enzyme to enzyme; hydrophobic and positively charged amino acids play a key role in the interaction. In this work, we investigated the interaction of PLA2 from bee venom with phospholipid bilayers of palmitoyl oleoylphosphatidylcholine (POPC) containing different amounts of palmitoyloleoylphosphatidylglycerol (POPG). On the basis of the measurements of the protein intrinsic fluorescence and the anisotropy of the fluorescence of the lipid probe we propose the construction of lipid–protein interaction maps, which reflect both the efficiency of protein binding and changes in the structure of the membrane. These changes cause alterations in the fluorescence anisotropy of the label, which in turn is a measure of the mobility of the lipid environment of the fluorescent probe. Analysis of interaction maps showed that there is a relationship between lipid mobility and enzyme binding efficiency: the optimum interaction of PLA2 with membranes from a POPC/POPG mixture lies in the region of the highest lipid mobility, and not in the region of the highest negative charge. This dependence complements the existing understanding of the process of recognition of the membrane surface by the enzyme and the selection of lipids by the enzyme already bound to the membrane. The proposed mapping method can be extended to other membrane-active proteins.

Aims: Design and in silico studies of 2,5-disubstituted triazole and thiadiazole derivatives as Pteridine Reductase 1 inhibitors. With a view to develop effective agents against Leishmaniasis, 2-substituted-5-[(1H-benzimidazol-2yl) methyl] azole derivatives (A1-A12) were designed against the target enzyme Pteridine reductase 1. Methodology: The series was designed by targeting Pteridine reductase 1 which is an enzyme responsible for folate and pterin metabolism. Based on thorough study of the enzyme structure and structural features of ligands required for optimum interaction with the enzyme, a series of 12 compounds consisting of 2,5-disubstituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives was designed. In silico studies were carried out which included docking studies (using V Life software) to understand binding of the compounds with enzyme PTR1, ADMET studies, drug likeness studies for physicochemical properties and bioactivity studies to understand the possible mechanism of action of the compounds. These studies were undertaken using online softwares, molinspiration and admetSAR web servers. Results: Compounds A10 and A12 gave the best docking scores of -59.9765 and -60.4373 respectively that were close to dihydrobiopterin (original substrate). All the compounds complied with Lipinski’s rule of five. Most of the compounds displayed favorable ADMET properties. Conclusion: The 2,5-disubstituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives exhibited good binding affinity for PTR1 enzyme (PDB code: 1E92). The docking scores indicated that enzyme binding may be governed by the nature and size of the substituents on the azole ring. The compounds display well-defined drug-like and pharmacokinetic properties based on Lipinski’s rule of five with additional physicochemical and ADMET parameters. Bioactivity studies suggested the possible drug mechanism as enzyme inhibition. Hence, this study provides evidence for consideration of valuable ligands in 2,5-disubstituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives as potential pteridine reductase 1 inhibitor and further in vitro and in vivo investigations may prove its therapeutic potential.

Traditional Chinese medicine (TCM) has been utilized for the treatment of colon cancer. Qizhen decoction (QZD), a potential compound prescription of TCM, possesses multiple biological activities. It has been proven clinically effective in the treatment of colon cancer. However, the molecular mechanism of anticolon cancer activity is still not clear. This study aimed to identify the chemical composition of QZD. Furthermore, a collaborative analysis strategy of network pharmacology and cell biology was used to further explore the critical signaling pathway of QZD anticancer activity. First, ultraperformance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) was performed to identify the chemical composition of QZD. Then, the chemical composition database of QZD was constructed based on a systematic literature search and review of chemical constituents. Moreover, the common and indirect targets of chemical components of QZD and colon cancer were searched by multiple databases. A protein-protein interaction (PPI) network was constructed using the String database (https://www.string-db.org/). All of the targets were analyzed by Gene Oncology (GO) bioanalysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the visual network topology diagram of "Prescription-TCM-Chemical composition-Direct target-Indirect target-Pathway" was constructed by Cytoscape software (v3.7.1). The top molecular pathway ranked by statistical significance was further verified by molecular biology methods. The results of UPLC-Q-TOF/MS showed that QZD had 111 kinds of chemical components, of which 103 were unique components and 8 were common components. Ten pivotal targets of QZD in the treatment of colon cancer were screened by the PPI network. Targets of QZD involve many biological processes, such as the signaling pathway, immune system, gene expression, and so on. QZD may interfere with biological pathways such as cell replication, oxygen-containing compounds, or organic matter by protein binding, regulation of signal receptors or enzyme binding, and affect cytoplasm and membrane-bound organelles. The main antitumor core pathways were the apoptosis metabolic pathway, the PI3K-Akt signal pathway, and so on. Expression of the PI3K-Akt signal pathway was significantly downregulated after the intervention of QZD, which was closely related to the inhibition of proliferation and migration of colon cancer cells by cell biology methods. The present work may facilitate a better understanding of the effective components, therapeutic targets, biological processes, and signaling pathways of QZD in the treatment of colon cancer and provide useful information about the utilization of QZD.

In this research, we explored various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), and evaluated the increase in surface area and its effect on enzyme binding potential. We also manipulated incubation and reaction conditions and assessed the subsequent effects on activity and stability of β-galactosidase, with comparisons between various solid support materials and free (dissolved) enzyme. Nylon-6 fiber membranes were created by electrospinning and were compared with other materials as solid supports for enzyme binding. The other materials included polyvinylidene fluoride 5-kDa nanofiltration dairy membranes, nylon-6 pellets, and silica glass beads. Scanning electron microscopy revealed the large surface area of NFM, which correlated with greater enzyme activity compared with the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate o-nitrophenyl-β-d-galactopyranoside. Compared with the other solid supports, NFM had greater maximum enzyme binding potential. Across pH conditions ranging from 3.5 to 6.0 (including the optimal pH of 4.0-5.0), enzyme activity was maintained on the membrane-immobilized samples, whereas free enzyme did not maintain activity. Altering the storage temperature (4, 22, and 50°C) affected enzyme stability (i.e., the ability of the enzyme to maintain activity over time) of free and polyvinylidene fluoride membrane samples. However, NFM samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above the maximum enzyme binding capacity had no significant effect on enzyme stability for membrane-immobilized samples; however, both had lower mean stability than free enzyme by approximately 74%. With further development, β-galactosidase immobilized on NFM or other membranes could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis-creating products that are reduced in lactose and increased in sweetness, with no requirement for "added sugars" on the nutrition label and no enzyme listed as final product ingredient.

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active‐site flexibility and transglycosylation. Residues of the BCX “thumb” were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts., Shifting the balance: Glycoside hydrolases are attractive tools for the enzymatic synthesis of carbohydrates. We used the xylanase from B. circulans (BCX) to study the interplay between active‐site flexibility and the transglycosylation reaction. A point mutation that enhances the flexibility and dynamics of BCX active site shifts the balance of the enzymatic reaction toward transglycosylation.

The bacteriolysin lysostaphin (Lst) and endolysin PlyPH are potent modular lytic enzymes with activity against clinically-relevant Gram-positive Staphylococcus aureus and Bacillus cereus, respectively. Both enzymes possess an N-terminal catalytic domain and C-terminal binding domain, with the latter conferring significant enzyme specificity. Lst and PlyPH show reduced activity in the presence of bacterial growth-supporting conditions, such as complex media. Here, we hypothesize that Lst and PlyPH bind poorly to their targets in growth media, which may influence their use in antimicrobial applications in the food industry, as therapeutics, and for control of microbial communities. To this end, binding of isolated Lst and PlyPH binding domains to target bacteria was quantified in the presence of three increasingly complex media - phosphate buffered saline (PBS), defined growth medium (AAM) and undefined complex medium (TSB) by surface plasmon resonance (SPR) and flow cytometry. Evaluation of binding kinetics by SPR demonstrated that PlyPH binding was particularly sensitive to medium composition, with 8-fold lower association and 3.4-fold lower dissociation rate constants to B. cereus in TSB compared to PBS. Flow cytometry studies indicated a decrease in the binding-dependent fluorescent populations of S. aureus and B. cereus, for lysostaphin binding domain and PlyPH binding domain, respectively, in TSB compared to PBS. Enzyme binding behavior was consistent with the enzymes' catalytic activity in the three media, thereby suggesting that compromised enzyme binding could be responsible for poor activity in more complex growth media.

A non-covalent supramolecular enzyme system, which is formed by non-covalent interactions of an enzyme with substrate analogs, shows better enzyme catalytic activity than the enzyme itself. A non-covalent supramolecular laccase-dye substrate analog system (SL-DSA) was designed to confirm the enzyme catalyzing mechanism. A range of spectral and electrochemical methods showed that the non-covalent interaction is important in the catalytic degradation reaction of 13 dyes. The decolorization rate was 10–54% higher than with laccase (LAC) alone. Thus, the SL-DSA has better catalytic activity than LAC itself in the three-step degradation reaction of dyes. The enzymatic mechanism of SL-DSA identified may act to supplement the mechanism of the enzyme binding directly with the substrate.

The genus Foeniculum is known for its wide ethnobotanical use in the Mediterranean region. Herein, we explored the compositional differences of volatile oils and headspace aroma of Florence fennel (Foeniculum vulgare var. azoricum (Mill.) Thell.) based on its different organs and various geographical origins via gas chromatography coupled with mass spectrometry (GC-MS). Sixty-seven volatile components were detected with phenylpropenes and monoterpenes, including trans-anethole, limonene, α-pinene, trans-β-ocimene, fenchyl acetate, and fenchone, as major constituents. Phenylpropenes were dominant in fennel hydro-distilled oils, whereas monoterpenes were dominant in most of the headspace aroma. The infraspecific variability was assessed using the unsupervised multivariate data analysis tools PCA and HCA, resulting in segregate clustering of accessions from different organs and locations with trans-anethole, limonene, trans-β-ocimene, fenchone, myristicin, and apiole as major phytomarkers contributing to this segregation. The antiviral activities of samples against hepatitis A and C viruses were investigated using the plaque reduction assay, HAV 3C proteinase and HCV NS5B polymerase inhibitory assays with a percentage inhibition between 66% and 85% and IC50 values from 1.8 to 26.7 μg mL-1. In silico molecular docking scores in latter enzyme binding pockets revealed key allosteric interactions with trans-β-ocimene and β-fenchyl acetate showing the best Gibb's free energy. Florence fennel exhibited interesting new perspectives for medicinal and industrial applications.

The inhibition properties of 10 tea polyphenols against α-glucosidase were studied through inhibition assay, inhibition kinetics, fluorescence quenching and molecular docking. It was found that the inhibitory activity of polyphenols with a 3 and/or 3' galloyl moiety (GM) was much higher than that without a GM. The GM could enter into the active site of α-glucosidase and bind with the catalytic amino acid residues through hydrogen bonding and π-conjugation, thus playing an important role in the competitive inhibition of catechins and theaflavins. The positive linear correlations among the constants characterizing the inhibitory activity and binding affinity of tea polyphenols to α-glucosidase indicate that enzyme inhibition by polyphenols is caused by the binding interactions between them, and that the combination of the characterization methods for polyphenol-glucosidase binding is reasonable. In addition, the in vivo hypoglycemic effects of galloylated polyphenols suggest that the GM may be considered as a pharmaceutical fragment for the alleviation of type II diabetes symptoms through α-glucosidase inhibition.

Simultaneous exfoliation of crystalline α-zirconium phosphate (α-ZrP) nanosheets and enzyme binding, induced by shearing, without the addition of any toxic additives is reported here for the first time. These materials were thoroughly characterized and used for applications. The bulk α-ZrP material (20 mg mL-1) was exfoliated with low concentrations of a protein such as bovine serum albumin (BSA, 3 mg mL-1) in a shear reactor at 10k rpm for

The pathogenesis of slow-transit constipation (STC) remains largely unclear, with the roles of microRNAs (miRs/miRNAs) yet to be determined. Co-expression network analysis of miRNAs in STC is crucial to elucidating potential underlying mechanisms. Weighted gene correlation network analysis was performed in the miRNA expression profile of STC (GSE57969). The key miRNA target genes were further functionally enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). A Protein-Protein Interaction (PPI) network was constructed, with a total of 12 color-clustered modules determined. Seven key miRNAs were established, including five miRNAs from the turquoise module (hsa-miR-20b, hsa-miR-128, hsa-miR-129-3p, hsa-miR-30b and hsa-miR-340), one miRNA from the blue module (hsa-miR-619) and one from the black module (hsa-miR-486-3p). A total of 2,077 key miRNA target genes were predicted. GO analysis revealed that the ‘protein modification process’ and ‘cellular protein modification process’ were the most significantly enriched processes in the ‘Biological Processes’ category, whereas the ‘nucleoplasm’ in ‘Cellular Components’ and ‘enzyme binding’ in ‘Molecular Functions’ were the most significantly enriched processes. The ‘cAMP signalling pathway’ was the top KEGG pathway. The hub genes identified from the PPI network included calmodulin (CALM)2, CALM1, histone deacetylase (HDAC)3, glycogen synthase kinase 3 β, HDAC9, heat-shock protein family A member 8, G-protein subunit γ (GNG)13, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1, GNG10 and GNG7. This bioinformatics analysis demonstrated co-expressed miRNA networks with insightful genes and pathways associated with STC.

Aldose reductase (AR) is the first enzyme of the polyol pathway that has physiological importance under hyperglycaemic conditions. The article has been focussed on AR enzyme inhibition by selected compounds. For this purpose, thein vitroinhibitory effects of various compounds on commercially available recombinant human AR (rAR) enzyme activity were investigated. The IC(50)values of compounds on rAR inhibition effect were found for 6-hydroxy flavone, syringic acid, diosmetin, 6-fluoroflavone, 7-hydroxy-4 '-nitroisoflavone, myricetin as 2.05, 2.97, 15.75, 16.1, 49.5, and 63 mu M, respectively. 6-Hydroxy flavone and syringic acid competitively inhibited rAR with respect to the NADPH with K(i)values 0.509 +/- 0.036 and 0.842 +/- 0.012 mu M. In addition, docking studies were performed to evaluate the potential enzyme binding positions of the compounds. Ourin vitroandin silicoresults indicated that the 6-hydroxy flavone may be a good lead compound in the development of AR inhibitors to prevent diabetic complications., Agri Ibrahim Cecen University Scientific Research [SYO.18.003], This study was financially supported by Agri Ibrahim Cecen University Scientific Research [Project number: SYO.18.003].

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl−]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl−]i, respectively. Cell shrinkage and a decrease in [Cl−]iincrease the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl−]iactivate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl−]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl−]ibut can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl−]ibut is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK’s carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.

The selection of effective pretreatment method for exploiting lignocellulosic biomass plays an important role for sustainable biofuel production as it accounts the highest share of production cost. The present study aims to compare the efficiency of glycerol thermal pretreatment (GTP) over hydrothermal and alkali pretreatments of paddy (rice) straw as a feedstock. The efficacy of pretreatment was assessed by analyzing the reducing sugar (RS) yield, holocellulose digestibility and component degradation. The study was further elaborated for enzyme kinetics, FTIR spectra, crystallinity, accurate mass and energy balance. GTP showed 71.52% RS yield with 94.36% holocellulose digestibility, which was higher than that of alkali and hydrothermal pretreatments. GTP was more effective in lignin and hemicellulose removal with little damage to cellulose. The enzyme kinetic study further proved the effectiveness of GTP and showed the enhanced enzyme adsorption and hydrolysis on exposing enzyme binding sites. Crystallinity of cellulose revealed the effectiveness of GTP by XRD studies. FTIR study gives emphasis on alteration in lignocellulose structure due to pretreatment. The mass and energy balances further confirmed the reliability of GTP pretreatment.

Unlike the canonical base pairs AT and GC, the molecular properties of mismatches such as hydrogen bonding and stacking interactions are strongly dependent on the identity of the neighbouring base pairs. As a result, due to the sheer number of possible combinations of mismatches and flanking base pairs, only a fraction of these have been studied in varying experiments or theoretical models. Here, we report on the melting temperature measurement and mesoscopic analysis of contiguous DNA mismatches in nearest-neighbours and next-nearest neighbour contexts. A total of 4032 different mismatch combinations, including single, double and triple mismatches were covered. These were compared with 64 sequences containing all combinations of canonical base pairs in the same location under the same conditions. For a substantial number of single mismatch configurations, 15%, the measured melting temperatures were higher than the least stable AT base pair. The mesoscopic calculation, using the Peyrard–Bishop model, was performed on the set of 4096 sequences, and resulted in estimates of on-site and nearest-neighbour interactions that can be correlated to hydrogen bonding and base stacking. Our results confirm many of the known properties of mismatches, including the peculiar sheared stacking of tandem GA mismatches. More intriguingly, it also reveals that a number of mismatches present strong hydrogen bonding when flanked on both sites by other mismatches. To highlight the applicability of our results, we discuss a number of practical situations such as enzyme binding affinities, thymine DNA glycosylase repair activity, and trinucleotide repeat expansions., A comprehensive experimental and theoretical evaluation of all DNA mismatch contexts, providing an insight into the intra-molecular interactions.

DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale.

The use of selective covalent inhibitors with low binding affinity and high reactivity with the target enzyme is a promising way to solve a long-standing problem of the "undruggable" RAS-like proteins. Specifically, compounds of the ARS family that prevent the activation of the GDP-bound G12C mutant of Kirsten RAS (KRAS) are in the focus of recent experimental research. We report the first computational characterization of the entire reaction mechanism of the covalent binding of ARS-853 to the KRASG12C·GDP complex. The application of molecular dynamics, molecular docking and quantum mechanics/molecular mechanics approaches allowed us to model the inhibitor binding to the protein and the chemical reaction of ARS-853 with Cys12 in the enzyme binding site. We estimated a full set of kinetic constants and carried out numerical kinetic analysis of the process. Thus, we were able to compare directly the physicochemical parameters of the reaction obtained in silico and the macroscopic parameters observed in experimental studies. From our computational results, we explain the observed unusual dependence of the rate constant of covalent complex formation, kobs, on the ARS concentration. The latter depends both on the non-covalent binding step with the equilibrium constant, Ki, and on the rate constant of covalent adduct formation, kinact. The calculated ratio kinact/Ki = 213 M-1 s-1 reproduces the corresponding experimental value of 250 ± 30 M-1 s-1 for the interaction of ARS-853 with KRASG12C. Electron density analysis in the reactive region demonstrates that covalent bond formation occurs efficiently according to the Michael addition mechanism, which assumes the activation of the C[double bond, length as m-dash]C bond of ARS-853 by a water molecule and Lys16 in the binding site of KRASG12C. We also refine the kinact and Ki constants of the ARS-107 compound, which shares common features with ARS-853, and show that the decrease in the kinact/Ki ratio in the case of ARS-107 is explained by changes in both Ki and kinact constants.

The combination of experimental and computational strategies has been of great value in the identification and development of metabolism of drugs. Nowadays modern drug design, molecular docking methods are helpful in exploring the ligand conformations adopted within the binding sites of macro-molecular targets such as DNA, proteins, and enzymes, there by reducing cost, time and wayward efforts of chemist. Since the development of the algorithms in the 1980s, molecular docking became an important tool in drug discovery like investigation of crucial molecular events, including ligand binding modes and the corresponding intermolecular interactions that stabilize the ligand-receptor complex, can be conveniently performed. In present study we have tried to investigate the drug binding pocket of various cytochrome (CYP) enzymes found in humans. All structures of drugs are optimized at B3LYP/6-31** level of theory using Gaussian program suite. Docking of substrate-enzyme duo was done using AUTODOCK 4.0. Computational docking revealed that almost all drugs have same binding pocket with varied binding affinities due to change in interactions and interacting distance from heme prosthetic moiety with transition metal iron as chelating ion.

Human ceruloplasmin (hCP) is a multi-copper oxidase with ferroxidase and amine oxidase activities. Molecular dynamics simulation (MDS) and docking analysis of biogenic monoamines with ceruloplasmin explain the role of Asp1025, Glu935, Glu272, Glu232 and Glu230 together with the binding site water molecules (referred as conserved water molecules) in the stabilization of neurotransmitter (Serotonin, Norepinephrine and Epinephrine) molecules within the binding cavity of hCP. Conserved water molecules are found at specific positions interacting with the protein structures that have sequence similarity. The ethylamine side chain nitrogen atom (N1) of neurotransmitter molecules interacts with water molecules in the binding cavity formed by Asp1025, Glu935 and Glu232 residues. These residues form an acidic triad mimicking a substrate binding cavity. The hydroxyl groups attached to the catechol ring of epinephrine and norepinephrine have been stabilized by Asp230 and Asp232 residues. Data suggests that the recognition of biogenic amines mediates through the N+(amine) ...Asp1025-His1026-CuCis-His path. The potential recognition path of biogenic monoamines to trinuclear copper cluster supported by active site water molecules (referred as conserved water molecules) is described in this report.

Objective. Arsenic trioxide (Pishuang, Pishi, arsenolite, As2O3, and CAS 1327-53-3), a naturally occurring and toxic mineral as a drug for more than 2000 years in China, has been found to have a valuable function in hepatocellular carcinoma (HCC) in recent years. However, its exact mechanism remains to be elucidated. Therefore, this study was intended to explore the potential anti-HCC mechanism of arsenic trioxide through network pharmacology. Methods. The potential targets of arsenic trioxide were collected from PubChem and TargetNet. HCC targets were obtained from the GeneCards database. Then, a protein-protein interaction (PPI) network of arsenic trioxide and HCC common targets was established using STRING. GO and KEGG pathway enrichment analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Finally, an arsenic trioxide-target-pathway-HCC network was built by Cytoscape 3.2.1, and network topological analysis was carried out to screen the key candidate targets. Results. A total of 346 corresponding targets of arsenic trioxide and 521 HCC-related targets were collected. After target mapping, a total of 52 common targets were obtained. GO analysis showed that the biological process was mainly involved in the negative regulation of cellular senescence, response to tumor necrosis factor, and cellular response to hypoxia. Molecular functions included NF-kappa B binding, enzyme binding, p53 binding, and transcription factor binding. Cellular components mainly were replication fork, ESC/E(Z) complex, RNA polymerase II transcription factor complex, and organelle membrane. KEGG pathways were mainly enriched in the PI3K-Akt signaling pathway, VEGF signaling pathway, p53 signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, AMPK signaling pathway, NF-kappa B signaling pathway, FoxO signaling pathway, ErbB signaling pathway, and MAPK signaling pathway. In the arsenic trioxide-target-pathway-HCC network, targets such as AKT1, RAF1, RELA, TP53, and PTEN had a higher degree. Conclusions. Our study showed that key targets of arsenic trioxide were mainly involved in multiple biological processes and pathways. It provided a theoretical basis for the screening of drug targets.

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ∼330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increased hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

Practical laboratory proxies that correlate to vaccine efficacy may facilitate trials, identify nonresponders, and inform about boosting strategies. Among clinical and laboratory markers, assays that evaluate antibodies that inhibit receptor‐binding domain (RBD) ligation to angiotensin‐converting enzyme‐2 receptor (receptor‐binding inhibition [RBI]) may provide a surrogate for viral neutralization assays. We evaluated RBI before and after a median of 34 days (interquartile range [IQR]: 33–40) of the second dose of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Sinovac's CoronaVac (CN) or AstraZeneca/Oxford's AZD1222 (AZ) vaccines in 166 individuals. Both vaccines elicited high inhibitory titers in most subjects, 95% (158/166), with signal inhibition above 30% and 89% (127/143) with more than fourfold increase from prevaccination titers, but titers tend to decrease over time. Both postvaccination inhibitory titers (95%, IQR 85%–97% for AZ vs. 79%, IQR 60%–96% for CN, p = 0.004) and pre/post‐titer increase (AZ 76%, IQR 51%–86% for AZ vs. 47%, IQR 24%–67% for CN, p

Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach, termed native MOWChIP-seq, produces high-quality binding profiles using 1000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.

This work aimed to construct 3D-QSAR CoMFA and CoMSIA models for a series of 31 FAAH inhibitors, containing the 1,3,4-oxadiazol-2-one moiety. The obtained models were characterized by good statistical parameters: CoMFA Q2 = 0.61, R2 = 0.98, CoMSIA Q2 = 0.64, R2 = 0.93. The CoMFA model field contributions were 54.1% and 45.9% for steric and electrostatic fields, respectively. In the CoMSIA model, electrostatic, steric, hydrogen bond donor, and hydrogen acceptor properties were equal to 34.6%, 23.9%, 23.4%, and 18.0%, respectively. These models were validated by applying the leave-one-out technique, the seven-element test set (CoMFA r2test-set = 0.91, CoMSIA r2test-set = 0.91), a progressive scrambling test, and external validation criteria developed by Golbraikh and Tropsha (CoMFA r20 = 0.98, k = 0.95, CoMSIA r20 = 0.98, k = 0.89). As the statistical significance of the obtained model was confirmed, the results of the CoMFA and CoMSIA field calculation were mapped onto the enzyme binding site. It gave us the opportunity to discuss the structure–activity relationship based on the ligand–enzyme interactions. In particular, examination of the electrostatic properties of the established CoMFA model revealed fields that correspond to the regions where electropositive substituents are not desired, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one moiety. This highlights the importance of heterocycle, a highly electronegative moiety in this area of each ligand. Examination of hydrogen bond donor and acceptor properties contour maps revealed several spots where the implementation of another hydrogen-bond-donating moiety will positively impact molecules’ binding affinity, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one ring. On the other hand, there is a large isopleth that refers to the favorable H-bond properties close to the terminal phenoxy group of a ligand, which means that, generally speaking, H-bond acceptors are desired in this area.

Diabetes mellitus is a main global health apprehension. Macrovascular illnesses, neuropathy, retinopathy, and nephropathy are considered some of its severe hitches. Gliptins are a group of hypoglycemic agents that inhibit dipeptidyl peptidase-IV (DPP-IV) enzyme and support blood glucose-lowering effect of incretins. In the current research, synthesis, characterization, docking, and biological evaluation of fourteen Schiff’s bases 5a–f and 9a–h were carried out. Compound 9f revealed the best in vitro anti-DPP-IV activity of 35.7% inhibition at a concentration of 100 μM. Compounds 9c and 9f with the highest in vitro DPP-IV inhibition were subjected to the in vivo glucose-lowering test using vildagliptin as a positive inhibitor. Vildagliptin, 9c, and 9f showed significant reduction in the blood glucose levels of the treated mice after 30 min of glucose administration. Moreover, induced fit docking showed that these derivatives accommodated the enzyme binding site with comparable docking scores. Schiff’s bases can serve as promising lead for the development of new DPP-IV inhibitors.

The photoactivatable modified oligonucleotides were used to investigate direct contacts formed by the type IIE EcoRII restriction endonuclease and the T/A bases of its recognition site (5'-CCT/AGG). EcoRII dimer consists of a central catalytic core, made of two C-terminal endonuclease-like domains (EcoRII-C) from different subunits, and two N-terminal effector DNA binding domains (EcoRII-N). According to co-crystal structure of isolated EcoRII-C with DNA catalytic dimer EcoRII-C flips nucleotides of the central T/A pair into the enzyme binding pockets. Нere, photocross-linking technique was used to investigate the direct contacts formed by extrahelical T/A bases in the protein pockets of full-length EcoRII within the pre-reactive EcoRII–DNA complex obtained in the presence of Ca2+ in solution. Photoreactive zero-length agent 5-iodo-2'-deoxyuridine (IdU) was introduced as single substituent into the central T/A position of EcoRII recognition site or into the flanking nucleotide sequences of 14-mer DNA substrate. The substitution of only dT or dA residues in EcoRII recognition site resulted in formation of photocross-links upon irradiation only in the presence of Ca2+. Proteolytic digestion of the enzyme-oligonucleotide conjugates followed by MALDI-MS analysis have allowed to identify the 224VEYD227 EcoRII region involved in the formation of the cross-links. This region belongs to the central part of H-10 α-helix. Y226 residue was suggested to form cross-link with T or A bases of EcoRII site replaced by IdU within the pre-reactive complex. The flipped base pair protein pockets of EcoRII seem to accommodate equally well both A and T bases of the DNA substrate. Altogether, IdU-containing photoactivatable DNA substrates have allowed to trap the flipped bases in complex with full-length EcoRII before DNA cleavage in the solution and to identify direct enzyme–DNA contacts important for high specificity of EcoRII for the Т/A nucleotides providing a highly specific cleavage reaction., This work was supported by the Russian Foundation for Fundamental Investigations under Grant no. 19-04-00533.

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein (“CasKAS”). We demonstrate this method in both in vitro and in vivo contexts.

Valonea tannin is a natural product readily extracted from acorn shells that has been suggested to have potential skin whitening properties. This study investigated the tyrosinase inhibition activity of extracted valonea tannin and the associated structure–function activity. Nuclear magnetic resonance spectroscopy and molecular weight analysis with gel permeation chromatography revealed that valonea tannin could be characterized as a hydrolysable tannin with galloyl, hexahydroxydiphenoyl and open formed-glucose moieties and an average molecular weight of 3042 ± 15 Da. Tyrosinase inhibition assays demonstrated that valonea tannin was 334 times more effective than gallic acid and 3.4 times more effective than tannic acid, which may relate to the larger molecular size. Kinetic studies of the inhibition reactions indicated that valonea tannin provided tyrosinase inhibition through mixed competitive–uncompetitive way. Stern–Volmer fitted fluorescence quenching analysis, isothermal titration calorimetry analysis and in silico molecule docking showed valonea tannin non-selectively bound to the surface of tyrosinase via hydrogen bonds and hydrophobic interactions. Inductively coupled plasma-optical emission spectroscopy and free radical scavenging assays indicated the valonea tannin had copper ion chelating and antioxidant ability, which may also contribute to inhibition activity. These results demonstrated the structure–function activity of valonea tannin as a highly effective natural tyrosinase inhibitor that may have commercial application in dermatological medicines or cosmetic products.

The biological activities of Eleutherine plicata Herb. have been linked to isoeleutherin and eleutherin naphthoquinones. However, there are few reports in the literature regarding the cytotoxic and genotoxic potential of these compounds. There are reports in the literature that the inhibition of topoisomerase II (TOPO II) is involved in the toxicity of these compounds, as it causes damage to cellular DNA. In this study, we evaluated the genotoxicity and mutagenicity of these compounds using a bioassay on Allium cepa and micronuclei. We also performed an in silico evaluation of the toxic potential of these molecules using the PreADMET server. Finally, to assess whether binding to TOPO II influences toxicity, we used molecular docking and molecular dynamics (MD) simulations. In silico studies of prediction have demonstrated the identical toxicity profiles and mutagenicity for Algae, Daphnia, and fish. However, eleutherin proved to be more genotoxic, increasing the mitosis index, aberration index, and micronucleus, bud, and bridge were observed during metaphase. The results of docking and MD simulations demonstrated that the compounds were able to interact with the residues present in the enzyme binding pocket. Throughout the MD trajectories, the compounds showed molecular stability and the free energy results prove that the compounds formed a stable complex with TOPO II. These results provide new insights into the genotoxic and mutagenic potential of isoeleutherin and eleutherin.

Carbonic anhydrases are enzymes capable of transforming carbon dioxide into bicarbonate to maintain functionality of biological systems. Synthetic isolation and implementation of carbonic anhydrases into membrane have recently raised hopes for emerging and efficient strategies that could reduce greenhouse emission and the footprint of anthropogenic activities. However, implementation of such enzymes is currently challenged by the resulting membrane’s wetting capability, overall membrane performance for gas sensing, adsorption and transformation, and by the low solubility of carbon dioxide in water, the required medium for enzyme functionality. We developed the next generation of enzyme-based interfaces capable to efficiently adsorb and reduce carbon dioxide at room temperature. For this, we integrated carbonic anhydrase with a hydrophilic, user-synthesized metal–organic framework, we showed how the framework’s porosity and controlled morphology contribute to viable enzyme binding to create functional surfaces for the adsorption and reduction of carbon dioxide. Our analysis based on electron and atomic microscopy, infrared spectroscopy, and colorimetric assays demonstrated the functionality of such interfaces, while Brunauer–Emmett–Teller analysis and gas chromatography analysis allowed additional evaluation of the efficiency of carbon dioxide adsorption and reduction. Our study is expected to impact the design and development of active interfaces based on enzymes to be used as green approaches for carbon dioxide transformation and mitigation of global anthropogenic activities.

In this study, a method for direct screening and identification of α-glucosidase inhibitors (AGIs) from extracts of natural products was established based on polyamidoamine (PAMAM) coated magnetic microspheres. A facile route to synthesize the magnetic PAMAM was employed and α-glucosidase was successfully covalently attached to its surface through cross linking of glutaraldehyde. Using the enzyme-loaded magnetic microspheres, potential inhibitors were fished out from crude extracts directly, followed by structure confirmation. The inhibitory activities of the screened components were further investigated by the enzyme-loaded magnetic microspheres. The Fe3O4 @PAMAM@α-Glu microspheres displayed favorable dispersibility, fast magnetic separation, large enzyme binding amount (42.9 μg•mg−1) and high enzyme activity. Moreover, the α-glucosidase on the surface of PAMAM coating maintained high storage stability and remarkable reusability. Taking advantage of specific interaction of the α-glucosidase with AGIs, the materials could selectively capture a known AGI (+)-catechin under the interference of an inactive compound salicylic acid, with a binding capacity as high as 15.4%. Additionally, using the Fe3O4 @PAMAM@α-Glu microspheres in the inhibition assay, the enzymatic reaction could be stopped by magnetic separation instead of the traditional addition of Na2CO3 solution, which not only eliminated the disturbance of termination reagent to the results, but also reused the immobilized α-glucosidase. The screening and inhibitory activity verification of potential ligands in Radix Paeoniae Rubra (“Chi-shao” in Chinese) extracts were achieved by using Fe3O4 @PAMAM@α-Glu microspheres, demonstrating practical applicability of our method. Therefore, the magnetic PAMAM-based screening approach could be a feasible and alternative strategy for discovering enzyme inhibitors from natural product extracts.