Conformation Of Enzyme Research Articles

Effects of Octacosanol Isolated from Moringa oleifera Leaves on Inhibiting the Activity of Pancreatic Lipase.

Moringa oleifera Lam., a perennial species of the Moringaceae family, is esteemed for its multifaceted nutritional, medicinal, and economic properties. M. oleifera leaves are abundant in bioactive compounds with potential health benefits, yet existing structural and functional studies of these bioactives remain insufficiently comprehensive. This study aimed to isolate the active compound from M. oleifera leaves and investigate its mechanism of inhibiting lipid absorption and its potential as a pancreatic lipase inhibitor. In the present study, octacosanol (OCT) was isolated and purified from M. oleifera leaves, demonstrating notable pancreatic lipase inhibitory activity with an IC50 of 7.87 ± 0.72 μg/mL, and effectively suppressing lipid uptake in HepG2 cells. Simulated digestion assays indicated that OCT retained 73.5% of its pancreatic lipase inhibitory activity, with a recorded inhibition rate of 63.62%. Molecular docking analyses revealed that OCT binds to pancreatic lipase with an affinity comparable to orlistat and stronger than that of 4-nitrophenyl palmitate. The binding of OCT to key active sites (Ser152, His263, and Asp176) likely disrupts the enzyme's conformation, decreasing its substrate affinity. Additionally, OCT significantly attenuated lipid absorption and the synthesis of total cholesterol and triglycerides. This study elucidates the lipid-lowering mechanism of OCT and provides a theoretical foundation for its potential application in food production and biomedicine.

Enhancing the Stability and Activity of Enzymes through Layer-by-Layer Immobilization with Nanocomposite Hydrogel

Enhancing the Stability and Activity of Enzymes through Layer-by-Layer Immobilization with Nanocomposite Hydrogel

Formation Mechanisms of Protein Coronas on Food-Related Nanoparticles: Their Impact on Digestive System and Bioactive Compound Delivery.

The rapid development of nanotechnology provides new approaches to manufacturing food-related nanoparticles in various food industries, including food formulation, functional foods, food packaging, and food quality control. Once ingested, nanoparticles will immediately adsorb proteins in the biological fluids, forming a corona around them. Protein coronas alter the properties of nanoparticles, including their toxicity, cellular uptake, and targeting characteristics, by altering the aggregation state. In addition, the conformation and function of proteins and enzymes are also influenced by the formation of protein coronas, affecting the digestion of food products. Since the inevitable application of nanoparticles in food industries and their subsequent digestion, a comprehensive understanding of protein coronas is essential. This systematic review introduces nanoparticles in food and explains the formation of protein coronas, with interactions between proteins and nanoparticles. Furthermore, the potential origin of nanoparticles in food that migrate from packaging materials and their fates in the gastrointestinal tract has been reviewed. Finally, this review explores the possible effects of protein coronas on bioactive compounds, including probiotics and prebiotics. Understanding the formation mechanisms of protein coronas is crucial, as it enables the design of tailored delivery systems to optimize the bioavailability of bioactive compounds.

Immobilization of snailase and β-glucosidase on L-aspartic acid-modified magnetic amorphous ZIF for efficiently and sustainably producing ginsenoside compound K.

Immobilization of snailase and β-glucosidase on L-aspartic acid-modified magnetic amorphous ZIF for efficiently and sustainably producing ginsenoside compound K.

Integrating Enzymes with Reticular Frameworks To Govern Biocatalysis.

Integrating enzymes with reticular frameworks offers promising avenues for access to functionally tailorable biocatalysis. This Minireview explores recent advances in enzyme-reticular framework hybrid biocomposites, focusing on the utilization of porous reticular frameworks, including metal-organic frameworks, covalent-organic frameworks, and hydrogen-bonded organic frameworks, to regulate the reactivity of an enzyme encapsulated inside mainly by pore infiltration and in situ encapsulation strategies. We highlight how pore engineering and host-guest interfacial interactions within reticular frameworks create tailored microenvironments that substantially impact the mass transfer and enzyme conformation, leading to biocatalytic rate enhancement, or imparting enzymes with non-native biocatalytic functions, including substrate selectivity and new activity. Additionally, the feasibility of leveraging the photothermal effect of a framework to optimize the local reaction temperature and photoelectric effect to elicit diverse photoenzyme-coupled reactions is also summarized in detail, which can expand the functional repertoire of biocatalytic transformations under light irradiation. This Minireview underscores the potential of reticular frameworks as tunable and reliable platforms to govern biocatalysis, offering pathways for engineering sustainable, efficient, and selective biocatalytic reactors in pharmaceutical, environmental, and energy-related applications.

Nanoscale size impact of nanoparticle interaction and activity studies with urease.

Herein, we provide insights into the size-dependent interactions of silver nanoparticles (AgNPs) with urease and their implications for enzyme inhibition. AgNPs with a size of 5 nm exhibited the strongest binding affinity of 66 nM, resulting in significant enzyme attachment, interfering enzyme conformation, and a consequent loss of activity. Mid-sized AgNPs, i.e., 20 and 50 nm, exhibited binding affinities of 712 and 616 nM, causing only slight structural alterations. In contrast, 100 nm AgNPs demonstrated a high binding affinity of 171 nM accompanied by a favorable enthalpic contribution and a pronounced inhibitory effect.

Elucidating on the quaternary structure of viper venom phospholipase A2 enzymes in aqueous solution.

Elucidating on the quaternary structure of viper venom phospholipase A2 enzymes in aqueous solution.

Preservative effects and mechanism of condensed tannins from Cercis chinensis Bunge leaves on fresh-cut lotus root.

This study aimed to investigate the potential of condensed tannins isolated from Cercis chinensis Bunge leaves as natural preservatives for fruits and vegetables. The research demonstrated that C. chinensis leaves condensed tannins (CLCT) significantly delay the browning process and reduce nutritional loss in fresh-cut lotus roots. Prodelphinidins were identified as the primary component of CLCT through electrospray ionization mass spectrometry and high-performance liquid chromatography analyses, with minor proportions of procyanidins and high levels of galloylation. The structural units predominantly consisted of (epi)gallocatechin. CLCT exhibited substantial inhibitory activity against tyrosinase, which may involve interactions with copper ions at the enzyme's active site and alterations in enzyme conformation, resulting in reversible inhibition. Additionally, CLCT displayed remarkable antioxidant and antibacterial properties, effectively scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'- azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and significantly inhibiting the growth of Pseudomonas aeruginosa and Staphylococcus aureus. Overall, the potential of CLCT as a natural preservative and tyrosinase inhibitor underscores its promising application in preserving fruits and vegetables.

Understanding Acute Hemolytic Anemia Severity Through Computational Analysis of G6PDChatham Variant: Designing a New Activator as a Potential Drug

Glucose-6-phosphate dehydrogenase deficiency (G6PDD) is a major enzymatic disease affecting human red blood cells (RBCs), causing hemolytic anemia due to the diminish of the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) synthesis and altered redox balance within erythrocytes. This study sought to correct the defect in G6PDChatham (Ala355Thr) caused by the loss of interactions (hydrogen bonds and salt bridges) by docking the AG1 molecule at the dimer interface, thus restoring these lost interactions. The enzyme conformation was then analyzed before and after AG1 binding using molecular dynamics simulation (MDS). The reasons behind the severity of acute hemolytic anemia (AHA) were explained using several parameters, such as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), hydrogen bonds, salt bridges, radius of gyration (Rg), solvent accessible surface area (SASA), and covariance matrix analysis. Structural alterations in G6PDChatham, including the absence of interactions in a key region of the variant structure, can significantly impact protein stability and function, subsequently contributing to disease severity. Upon AG1 binding, these missing interactions were resorted to correct the structural defect of the variant. This restoration improves dimer stability and restores G6PD function. To develop new G6PD activators, several new analogues (SY7, SY8, SY9, and SY10) were rationally developed by substituting the linker region of the AG1 structure with other functional groups using the Avogadro software. These compounds were successfully synthesized and docked with G6PDChatham where the best binding affinity ranged between -8.0 and -9.1 kcal/mol. SY8, a promising G6PD activator, is predicted to be easily metabolized and excreted, making it less likely to cause toxicity. Its high drug score, drug-likeness, and favorable safety profile make it a strong candidate for synthesis and cellular testing. The toxicity risk assessment supported the overall drug score, increasing confidence in finding additional small-molecule activators for G6PDD disorder. Amidst the absence of effective treatments, such discovery hopes to improve the lives of those with AHA by assisting the development of appropriate pharmaceuticals for G6PDD.

Exploring CRISPR-Cas9 HNH-Domain-Catalyzed DNA Cleavage Using Accelerated Quantum Mechanical Molecular Mechanical Free Energy Simulation.

The target DNA (tDNA) cleavage catalyzed by the CRISPR Cas9 enzyme is a critical step in the Cas9-based genome editing technologies. Previously, the tDNA cleavage from an active SpyCas9 enzyme conformation was modeled by Palermo and co-workers (Nierzwicki et al., Nat. Catal. 2022 5, 912) using ab initio quantum mechanical molecular mechanical (ai-QM/MM) free energy simulations, where the free energy barrier was found to be more favorable than that from a pseudoactive enzyme conformation. In this work, we performed ai-QM/MM simulations based on another catalytically active conformation (PDB 7Z4J) of the Cas9 HNH domain from cryo-electron microscopy experiments. For the wildtype enzyme, we acquired a free energy profile for the tDNA cleavage that is largely consistent with the previous report. Furthermore, we explored the role of the active-site K866 residue on the catalytic efficiency by modeling the K866A mutant and found that the K866A mutation increased the reaction free energy barrier, which is consistent with the experimentally observed reduction in the enzyme activity.

Engineering enzyme conformation within liquid-solid hybrid microreactors for enhanced continuous-flow biocatalysis

The artificial engineering of an enzyme’s structural conformation and dynamic properties to promote its catalytic activity and stability outside cellular environments is highly pursued in industrial biotechnology. Here, we describe an elegant strategy of combining the rationally designed liquid-solid hybrid microreactor with a tailor-made polyethylene glycol functional ionic liquid (PEG-IL) microenvironment to exercise a high level of control over the configuration of enzymes for practical continuous-flow biocatalysis. As exemplified by a lipase driven kinetic resolution reaction, the obtained system exhibits a 2.70 to 30.35-fold activity enhancement compared to their batch or traditional IL-based counterparts. Also, our results demonstrate that the thermal stability of encapsulated lipase can be significantly strengthened in the presence of PEG groups, showcasing a long-term continuous-flow stability even up to 1000 h at evaluated temperature of 60 oC. Through systematic experiment and molecular dynamics simulation studies, the conformational changes of the active site cavity in the modified lipases are correlated with enzymatic properties alteration, and the pronounced effects of PEG-groups in stabilizing enzyme’s secondary structures by delaying unfolding at elevated temperatures are identified. We believe that this study will guide the design of high-performance enzymatic systems, promoting their utilization in real-world biocatalysis applications.

A Haloalkane Dehalogenase DhaA Nanoparticle Based on Pullulan Conjugation and Polyethyleneimine Adsorption.

Haloalkane dehalogenase DhaA is a member of the α/β-hydrolase superfamily and can degrade the halogenated compounds. However, the enzyme could not tolerate harsh and extreme environmental conditions, such as high temperature, extreme pH, and hypersaline, which limits its practical applications. Pullulan is a hydrophilic polysaccharide and acts as an additive to improve the enzyme stability. Polyethyleneimine (PEI) is a protein stabilizer and a polymer with a high density of ionizable amino groups. In the present study, DhaA was covalently conjugated with acetylated pullulan and adsorbed with PEI by electrostatic interactions to form nanoparticles (PEI-pullulan-DhaA). As compared with DhaA, PEI-pullulan-DhaA essentially maintained the enzymatic activity of DhaA, along with slight change in the kinetic parameters and enzyme conformation. The conjugated pullulan tends to form a large hydrated layer around DhaA. PEI, a cationic polymer, generated an amphiphilic microenvironment around DhaA. Pullulan conjugation and PEI adsorption could significantly improve the stability of DhaA against high temperature and low pH by structural stabilization of DhaA. PEI-pullulan-DhaA could also tolerate the hypersaline, organic solvents, and long-term storage. Thus, PEI-pullulan-DhaA has a strong environmental stability and is promising for industrial and environmental applications.

Fabrication of Thermo-Responsive Polymer-MOF@cellulase Composites with Improved Catalytic Performance for Hydrolysis of Cellulose.

Metal-organic frameworks (MOFs) are considered as an ideal enzyme support because of their porous structural superiority. However, MOFs@enzyme composites have usually compromised their hydrolysis efficiency due to the narrow space inducing unfavourable enzyme conformations. Herein, a thermo-responsive poly(N,N-dimethylacrylamide) (PD) was fixed onto the surface of UiO-66-NH2 (UiO) through a post-synthetic modification protocol. Using poly(2-vinyl-4,4 dimethylazlactone) (V) as a linker, PVD-UiO@cellulase composites were fabricated after cellulase was immobilized onto the UiO surface through covalent bonding. The composites conferred favorable cellulase conformations, boosting hydrolysis efficiency and stability, which relied on the soft PVD shell and confinement effect yielded by the curled PVD chains at high temperatures. Compared with free cellulase, the proposed composites exhibited a 33.1-fold enhancement of the Kcat values at 50 °C. The PVD-UiO@cellulase composites were applied to the hydrolysis of cellulose in the stalks and leaves of Epipremnum aureum. The results highlight the potential of smart PVD-UiO@cellulase composites in the hydrolysis of cellulose, affording a valuable platform for the preparation of unique MOFs@enzyme composites and their industrial applications.

Amino-Alkyl Group Dual-Functional Modification Synergistically Regulated Lipase-Carrier Interactions and Enhanced Phytosterol Ester Synthesis Efficiency.

Precisely controlling enzyme conformation to enhance catalytic performance is a highly sought-after yet challenging goal in the immobilization of biocatalysts. Excessively strong enzyme-carrier interactions can restrict enzyme dynamics and reduce catalytic efficiency, while excessively weak interactions may lead to enzyme leakage, thereby reducing reusability. In this study, we developed a novel strategy to finely regulate the interaction between the carrier and the enzyme through the adjustment of the ratio of amino and octadecyl functional groups. The expressed activity of the novel immobilized lipase, CRL@AOMR, was 1.32- and 2.34-fold higher than that of the monofunctional macroporous resin. Moreover, the synthesis of various phytosterol esters in solvent-free systems was conducted as a model reaction to investigate the utilization of CRL@AOMR in different reactions. Under optimized conditions, an impressive yield of 96.1% for phytosterol oleate was achieved and a yield of 76.2% was maintained even after six cycles of utilization (288 h). This study demonstrates the potential feasibility of developing immobilization strategies via dual modification of amino and alkyl groups, which is a potential general strategy for other enzymes with surface lysine.

Janus Separation-Sensing Membrane Hosted with Enzyme@MOF Nanoreactor for Real-Time Blood Sensing.

Plasma separation, rich in biomarkers crucial for diagnosis, is conventionally achieved via high-speed centrifugation, a method hindered by its blood usage, lengthy processes, and complex operations, which delays detection. We introduced a novel real-time blood sensing method based on a Janus membrane and enzymes @MOFs. Asymmetric driving of the janus membrane can realize spontaneous separation of plasma and prevent hemolysis during direct separation. Glucose oxidase (GOx), uric acid oxidase (UOx) and horseradish peroxidase (HRP) were encapsulated in a hydrophilic organometallic framework (MOFs) to construct an enzyme cascade nanoreactor. Embedding enzyme in hydrophilic MOFs not only retains the natural conformation of free enzyme but also improves the brittleness of enzyme, endows MOFs with new biological functions, and expands its sensing application. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen and a custom app for color interpretation, we achieved real-time visualization of glucose (Glu) and uric acid (UA) at a 50 μM limit. The system accurately analyzed serum samples, matching commercial kits and showing promise for portable, personalized diagnostics.

Rational Engineering of a β-Glucosidase (H0HC94) from Glycosyl Hydrolase Family I (GH1) to Improve Catalytic Performance on Cellobiose.

The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate p-nitrophenyl β-d-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. We report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 μmol/min/mg), a 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in catalytic efficiency (236.65 mM-1 s-1) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase β-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.

Effect of surfactants on enzymatic hydrolysis of PET

Effect of surfactants on enzymatic hydrolysis of PET

Inhibitive Mechanism of Loquat Flower Isolate on Tyrosinase Activity and Melanin Synthesis in Mouse Melanoma B16 Cells.

Melanin naturally exists in organisms and is synthetized by tyrosinase (TYR); however, its over-production may lead to aberrant pigmentation and skin conditions. Loquat (Eriobotrya japonica (Thunb.) Lindl.) flowers contain a variety of bioactive compounds, while studies on their suppressive capabilities against melanin synthesis are limited. Loquat flower isolate product (LFP) was obtained by ethanol extraction and resin purification, and its inhibitory efficiency against TYR activity was investigated by enzyme kinetics and multiple spectroscopy analyses. In addition, the impact of LFP on melanin synthesis-related proteins' expression in mouse melanoma B16 cells was analyzed using Western blotting. HPLC-MS/MS analysis indicated that LFP was composed of 137 compounds, of which 12 compounds, including flavonoids (quercetin, isorhamnoin, p-coumaric acid, etc.) and cinnamic acid and its derivatives, as well as benzene and its derivatives, might have TYR inhibitory activities. LFP inhibited TYR activity in a concentration-dependent manner with its IC50 value being 2.8 mg/mL. The inhibition was an anti-competitive one through altering the enzyme's conformation rather than chelating copper ions at the active center. LFP reduced the expression of TYR, tyrosinase-related protein (TRP) 1, and TRP2 in melanoma B16 cells, hence inhibiting the synthesis of melanin. The research suggested that LFP had the potential to reduce the risks of hyperpigmentation caused by tyrosinase and provided a foundation for the utilization of loquat flower as a natural resource in the development of beauty and aging-related functional products.

Analysis of the Behavior of Deep Eutectic Solvents upon Addition of Water: Its Effects over a Catalytic Reaction.

This study presents the potential role of deep eutectic solvents (DESs) in a lipase-catalyzed hydrolysis reaction as a co-solvent in an aqueous solution given by a phosphate buffer. Ammonium salts, such as choline chloride, were paired with hydrogen bond donors, such as urea, 1,2,3-propanetriol, and 1,2 propanediol. The hydrolysis of p-nitrophenyl laureate was carried out with the lipase Candida antarctica Lipase B (CALB) as a reaction model to evaluate the solvent effect and tested in different DES/buffer phosphate mixtures at different % w/w. The results showed that two mixtures of different DES at 25 % w/w were the most promising solvents, as this percentage enhanced the activities of CALB, as evidenced by its higher catalytic efficiency (kcatKM). The solvent analysis shows that the enzymatic reaction requires a reaction media rich in water molecules to enable hydrogen-bond formation from the reaction media toward the enzymatic reaction, suggesting a better interaction between the substrate and the enzyme-active site. This interaction could be attributed to high degrees of freedom influencing the enzyme conformation given by the reaction media, suggesting that CALB acquires a more restrictive structure in the presence of DES or the stabilized network given by the hydrogen bond from water molecules in the mixture improves the enzymatic activity, conferring conformational stability by solvent effects. This study offers a promising approach for applications and further perspectives on genuinely green industrial solvents.

Inhibitory effect and molecular mechanism on tyrosinase and browning of fresh-cut apple by longan shell tannins

Inhibitory effect and molecular mechanism on tyrosinase and browning of fresh-cut apple by longan shell tannins