Enzyme Substrate Research Articles

Photo-enzyme-polymerized hydrogel platform exhibits photo-switchable redox reversibility for diabetic wound healing

Redox homeostasis catalyzed by series oxidoreductases is important to maintain normal physiological functions, including mutually contradictory reactive oxygen species generation or reduction by separated enzyme regulation during cellular metabolism. The unidirectional enzyme regulation of redox homeostasis by biocatalytic reaction is an emerging catalytic medicine approach to disease biomimetic treatments. Bioinspired by photosynthesis, an efficient photo-enzyme-coupled hydrogel platform (Gel) with a spatio-temporally controlled hydrogel network was constructed through photoenzymatic-initiated radical polymerization in the absence of the corresponding enzyme substrate. Then, the coupling mechanism of light-induced electron transfer-enzyme activity center allosteric was explored through free radical analysis and theoretical calculations. The photo-switchable redox performance has been demonstrated based on the special anaerobic dehydrogenases, the flavin adenine dinucleotide (FAD)-centered dihydrolipoamide dehydrogenase (DLD) platform. At last, both in vitro and in vivo biological effects have been verified to evaluate the bidirectional oxidation/antioxidant modulation for the complicated wound healing in diabetic mice and wound-infected animals. The photoenzymatic coupling dehydrogenase-laden hydrogel platform proposed in this work provides ideas for the engineering design of enzymes, and the physical-biochemical regulatory mechanisms also offers a theoretical basis for controllable activation of enzyme activity, allowing for potential biomedical applications in metabolic processes.

A raman spectroscopic investigation of conformation of flavin adenine dinucleotide, a coenzyme of d-amino acid oxidase

Based on silver nanoparticles application, surface-enhanced Raman scattering spectra of D-amino acid oxidase from pig kidney were recorded and analyzed. Spectral parameters characteristic of conformational changes in cofactor flavin adenine dinucleotide during activation of the enzyme by D-amino acids were revealed. It was found that enzyme substrate specificity determines the amount of time from the start of of the conformational changes of flavin adenine dinucleotide until they no longer occur: in the presence of D-alanine, registration of the said conformational changes takes up just a few seconds, while it takes 10 min in the presence of D-serine .

Design and Mechanism Insight of Monodispersed AuCuPt Alloy Nanozyme with Antitumor Activity.

The abrogation of the self-adaptive redox evolution of tumors is promising for improving therapeutic outcomes. In this study, we designed a trimetallic alloy nanozyme AuCuPt-PpIX (ACPP), which mimics up to five naturally occurring enzymes: glucose oxidase (GOD), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione peroxidase (GPx). Facilitated by these enzyme-mimicking traits, the constructed ACPP nanozymes can not only disrupt the established redox homeostasis in tumors through a series of enzymatic cascade reactions but also achieve cyclic regeneration of the relevant enzyme substrates. Density functional theory (DFT) calculations have theoretically explained the synergistic effect of multimetallic doping and the possible mechanism of enzymatic catalysis. The doped Cu and Pt sites are conducive to the adsorption, activation, and dissociation of reactant molecules, whereas the Au sites are conducive to desorption, which significantly improves catalytic efficiency via a synergistic effect. Additionally, ACPP nanozymes can improve the effect of protoporphyrin (PpIX)-enabled sonodynamic therapy (SDT) by alleviating hypoxia and initiating ferroptosis by inducing lipid peroxidation (LPO) and inhibiting GPX4 activity, thus achieving multimodal synergistic therapy. This study presents a typical paradigm to enable the use of multimetallic alloy nanozymes for the treatment of tumor cells with self-adaptive properties.

Characterization and Antibacterial Activity of Soil Actinomycetes from Diverse Land Use Systems in Meru South, Eastern Kenya

Aim: The increasing emergence and global spread of antibiotic-resistance by microorganisms pose a severe public health threat. Continuous exploration of different environments is required to find new sources of antibacterial compounds. Therefore, the study isolated, characterized and identified potential Actinomycetes candidates with antibacterial activity against bacterial pathogens from diverse land use systems.
 Study Design: Soil samples from land use systems were collected using a cross-sectional survey technique using line transect sampling in order to isolate Actinomycetes. A 30 × 7× 3 factorial experiment with a completely randomized design was used for evaluating Actinomycetes isolates with antibacterial activity.
 Methodology: The in-vitro cultivation of Actinomycetes was evaluated using four selective media. The Actinomycetes isolates were characterized using morphological, biochemical, and molecular markers. The antibacterial activity screening of crude extracts was conducted against six bacterial pathogens using the agar well diffusion method. The antibacterial activity of Actinomycetes isolates were analyzed using Analysis of Variance. The molecular identification was performed using 16S rRNA sequence homology.
 Results: The morphological analysis showed variations in colony morphology, including differences in color, size, and texture. Biochemical tests provided insights into the metabolic capabilities of the isolates, indicating variations in enzymatic activities and substrate utilization. Antagonistic activity of Actinomycetes extracts exhibited significant differences (P =.05) against test bacterial pathogens. Notably, isolate C52 from degraded forest zone showed the highest antibacterial activity against Staphylococcus aureus (12.21 mm), isolate L6 (16.23 mm) against Listeria monocytogenes and isolate C50 (15.5 mm) against Raoutella planticola. Streptomyces celluloflavus, S. griseobrunneus, S. pratensis, S. crystallinus, and S. eurocidicus were identified in the study.
 Conclusion: The crude extracts obtained from Actinomycetes showed significant antibacterial activity against the selected test organisms. This suggests the presence of bioactive compounds with potential antibacterial properties within these extracts.

Rationally Engineered hCES2A Near-Infrared Fluorogenic Substrate for Functional Imaging and High-Throughput Inhibitor Screening.

Human carboxylesterase 2A (hCES2A) is an important endoplasmic reticulum (ER)-resident enzyme that is responsible for the hydrolytic metabolism or activation of numerous ester-bearing drugs and environmental toxins. The previously reported hCES2A fluorogenic substrates suffer from limited emission wavelength, low specificity, and poor localization accuracy, thereby greatly limiting the in situ functional imaging of hCES2A and drug discovery. Herein, a rational ligand design strategy was adopted to construct a highly specific near-infrared (NIR) substrate for hCES2A. Following scaffold screening and recognition group optimization, HTCF was identified as a desirable NIR fluorophore with excellent photophysical properties and high ER accumulation ability, while several HTCF esters held a high potential to be good hCES2A substrates. Further investigations revealed that TP-HTCF (the tert-pentyl ester of HTCF) was an ideal substrate with ultrahigh sensitivity, excellent specificity, and a substantial signal-to-noise ratio. Upon the addition of hCES2A, TP-HTCF could be rapidly hydrolyzed to release HTCF, a chemically stable product that emitted bright fluorescent signals at around 670 nm. A TP-HTCF-based biochemical assay was then established for the high-throughput screening of potent and cell-active hCES2A inhibitors from an in-house compound library. Furthermore, TP-HTCF displayed high imaging resolution for imaging hCES2A in living cells as well as mouse liver slices and tumor-xenograft mice. Collectively, this study demonstrates a rational strategy for developing highly specific fluorogenic substrates for an ER-resident target enzyme, while TP-HTCF can act as a practical tool for sensing hCES2A in living systems.

Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex Double-Stranded DNA Detection.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12 (Cas12) system is attracting interest for its potential as a next-generation nucleic acid detection tool. The system can recognize double-stranded DNA (dsDNA) based on Cas12-CRISPR RNA (crRNA) and induce signal transduction by collateral cleavage. This property is expected to simplify comprehensive genotyping. Here, we report a solid-phase collateral cleavage (SPCC) reaction by CRISPR/Cas12 and its application toward one-pot multiplex dsDNA detection with minimal operational steps. In the sensor, Cas12-crRNA and single-stranded DNA (ssDNA) are immobilized on the sensing surface and act as enzyme and reporter substrates, respectively. We also report a dual-target dsDNA sensor prepared by immobilizing Cas12-crRNA and a fluorophore-labeled ssDNA reporter on separate spots. When a spot captures a target dsDNA sequence, it cleaves the ssDNA reporter on the same spot and reduces its fluorescence by 42.1-57.3%. Crucially, spots targeting different sequences do not show a reduction in fluorescence, thus confirming the one-pot multiplex dsDNA detection by SPCC. Furthermore, the sequence specificity has a two-base resolution, and the detectable concentration for the target dsDNA is at least 10-9 M. In the future, the SPCC-based sensor array could achieve one-pot comprehensive genotyping by using an array spotter as a reagent-immobilizing method.

Engineered β-glycosidase from Hyperthermophilic Sulfolobus solfataricus with Improved Rd-hydrolyzing Activity for Ginsenoside Compound K Production.

Hyperthermophilic Sulfolobus solfataricus β-glycosidase (SS-βGly), with higher stability and activity than mesophilic enzymes, has potential for industrial ginsenosides biotransformation. However, its relatively low ginsenoside Rd-hydrolyzing activity limits the production of pharmaceutically active minor ginsenoside compound K (CK). In this study, first, we used molecular docking to predict the key enzyme residues that may hypothetically interact with ginsenoside Rd. Then, based on sequence alignment and alanine scanning mutagenesis approach, key variant sites were identified that might improve the enzyme catalytic efficiency. The enzyme catalytic efficiency (kcat/Km) and substrate affinity (Km) of the N264D variant enzyme for ginsenoside Rd increased by 60% and decreased by 17.9% compared with WT enzyme, respectively, which may be due to a decrease in the binding free energy (∆G) between the variant enzyme and substrate Rd. In addition, Markov state models (MSM) analysis during the whole 1000-ns MD simulations indicated that altering N264 to D made the variant enzyme achieve a more stable SS-βGly conformational state than the wild-type (WT) enzyme and corresponding Rd complex. Under identical conditions, the relative activities and the CK conversion rates of the N264D enzyme were 1.7 and 1.9 folds higher than those of the WT enzyme. This study identified an excellent hyperthermophilic β-glycosidase candidate for industrial biotransformation of ginsenosides.

Integrated Production of Xylitol, Ethanol, and Enzymes from Oil Palm Empty Fruit Bunch through Bioprocessing as an Application of the Biorefinery Concept

Oil palm empty fruit bunch (OPEFB), an abundant source of lignocellulosic biomass waste, is rich in hemicellulose and is converted into xylose for xylitol production. The remaining cellulose-rich residue can be efficiently hydrolyzed into glucose, which serves as a substrate for bioethanol and enzymes. This process aligns with an integrated biorefinery model aimed at optimizing the utilization of OPEFB. This study optimizes a two-stage enzymatic hydrolysis fermentation for OPEFB conversion into value-added products. Using a 4% NaOH pretreatment, lignin was degraded while preserving hemicellulose and cellulose. This hydrolysis yielded 12.27 g/L of xylose and 36.86 g/L of glucose. Ethanol production, using varied fermentation media, achieved maximum concentrations of 0.043 g/L for xylitol and 21.35 g/L for ethanol, with substrate-to-product yields of 0.005 g/g and 0.374 g/g, respectively. Furthermore, enzyme production by Aspergillus niger was assessed on multiple parameters, recording a peak cellulase activity of 55.16 ± 20.24 U/mL and enzyme weight of 42.748 kDa. The OPEFB substrate yielded the highest protein content of 0.00942 ± 0.00010 mg/mL. These findings demonstrate the feasibility and efficiency of the two-stage enzymatic hydrolysis strategy in facilitating integrated biorefinery processes for efficient and sustainable OPEFB utilization.

Orthogonal Enzyme-Substrate Design Strategy for Discovery of Human Protein Palmitoyltransferase Substrates.

Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.

A BAHD acyltransferase contributes to the biosynthesis of both ethyl benzoate and methyl benzoate in the flowers of Lilium oriental hybrid 'Siberia'.

Lily is a popular flower worldwide due to its elegant appearance and pleasant fragrance. Floral volatiles of lily are predominated by monoterpenes and benzenoids. While a number of genes for monoterpene biosynthesis have been characterized, the molecular mechanism underlying floral benzenoid formation in lily remains unclear. Here, we report on the identification and characterization of a novel BAHD acyltransferase gene that contributes to the biosynthesis of two related floral scent benzoate esters, ethyl benzoate and methyl benzoate, in the scented Lilium oriental hybrid 'Siberia'. The emission of both methyl benzoate and ethyl benzoate in L. 'Siberia' was found to be tepal-specific, floral development-regulated and rhythmic. Through transcriptome profiling and bioinformatic analysis, a BAHD acyltransferase gene designated LoAAT1 was identified as the top candidate gene for the production of ethyl benzoate. In vitro enzyme assays and substrate feeding assays provide substantial evidence that LoAAT1 is responsible for the biosynthesis of ethyl benzoate. It was interesting to note that in in vitro enzyme assay, LoAAT1 can also catalyze the formation of methyl benzoate, which is typically formed by the action of benzoic acid methyltransferase (BAMT). The lack of an expressed putative BAMT gene in the flower transcriptome of L. 'Siberia', together with biochemical and expression evidence, led us to conclude that LoAAT1 is also responsible for, or at least contributes to, the biosynthesis of the floral scent compound methyl benzoate. This is the first report that a member of the plant BAHD acyltransferase family contributes to the production of both ethyl benzoate and methyl benzoate, presenting a new mechanism for the biosynthesis of benzoate esters.

A fluorometric method for measuring homocysteine thiolactonase activity

The enzyme homocysteine thiolactonase (HTase) is involved in the metabolism of homocysteine, an amino acid. As a byproduct of the metabolism of methionine, a secondary amino acid, homocysteine (Hcy), a sulfur-containing amino acid, is produced. Hcy levels in the blood are linked to higher risks of heart disease, stroke, and other disorders. To create Hcy, HTase catalyzes the hydrolysis of homocysteine thiolactone (HTL). This process helps control the body's homocysteine levels and prevents the accumulation of homocysteine in the blood.This essay describes a precise, easy-to-follow procedure for measuring homocysteine thiolactonase (HTase) activity. This protocol (NAM-HTase) determines HTase activity via incubation with γ-thiobutyrolactone, an enzyme substrate, in a pH-appropriate solution at 37 °C for five minutes. It then uses thiol fluorometry to monitor the produced thiol groups. The newly formed thiol groups are reacted with the fluorescent probe N-(9-acridinyl)maleimide (NAM) and quantified using fluorometric intensity at an Ex/Em of 360/432 nm to evaluate HTase activity. The method is linear for 4-mercaptobutyric acid and homocysteine, from 0.005 ± 0.001 to 6 ± 0.04 µM. By comparing the HTase activity in matched samples to that of a standard method, this new protocol was found suitable for clinical applications. Its accuracy compared to other methodologies was shown by a correlation of 0.9995 between its values and those of a standard method. Using the H+ ion releasing method in matched samples, analysis of variance was used to verify the novel method against HTase activity. In conclusion, the proposed NAM-HTase method successfully measured HTase activity with high reliability.

Synthesis and Enzymatic Conversion of Amino Acids Equipped with the Silanetriol Functionality: A Prelude to Silicon Biodiversification

AbstractSynthetic routes are reported for the three analogues of the simplest L‐2‐amino‐dicarboxylic acids, aspartate, glutamate, and aminoadipate, in which the silanetriol group (Si(OH)3) replaces the distal carboxyl group (CO2H). Direct access to the silanetriol amino acids relied either on catalytic hydrosilylation of a terminal alkene using triethoxysilane, or on alkylation of a glycine equivalent anion by triallyl(iodomethyl)silane. In both cases, acid hydrolysis afforded the silanetriol amino acids. These were shown to self‐assemble into siloxane Si‐O clusters as their concentration in water increased in the pH range of 1–12. Such reversible cross‐linking did not prevent silanetriol amino acids from serving as substrates of an aminotransferase enzyme, boding well for their utilization as microbial nutrients to encompass silicon in future stages of metabolism and polypeptide edifices.

Product Profiles of Promiscuous Enzymes Can be Altered by Controlling In Vivo Spatial Organization.

Enzyme spatial organization is an evolved mechanism for facilitating multi-step biocatalysis and can play an important role in the regulation of promiscuous enzymes. The latter function suggests that artificial spatial organization can be an untapped avenue for controlling the specificity of bioengineered metabolic pathways. A promiscuous terpene synthase (nerolidol synthase) is co-localized and spatially organized with the preceding enzyme (farnesyl diphosphate synthase) in a heterologous production pathway, via translational protein fusion and/or co-encapsulation in a self-assembling protein cage. Spatial organization enhances nerolidol production by ≈11- to ≈62-fold relative to unorganized enzymes. More interestingly, striking differences in the ratio of end products (nerolidol and linalool) are observed with each spatial organization approach. This demonstrates that artificial spatial organization approaches can be harnessed to modulate the product profiles of promiscuous enzymes in engineered pathways in vivo. This extends the application of spatial organization beyond situations where multiple enzymes compete for a single substrate to cases where there is competition among multiple substrates for a single enzyme.

Purification and Characterization of Glucosamine Oxidase from Aspergillus Terreus NUK-1204 for Production of Glucosaminic Acid

D-Glucosaminic acid (2-amino-2-deoxy-D-gluconic acid) is a valuable glucosamine derivative from the oxidation of the glucosamine (2-amino-2-deoxy-D-glucose). It can be produced via the fermentation of certain microorganisms, which involves complicated purification task, though. In an attempt to find an enzyme suited to glucosaminic acid production, the study presented a purified homogeneous glucosamine oxidase deriving from the wheat bran culture of a soil-isolated fungal strain, Aspergillus terreus NUK-1204. Maximum activity was observed in the wheat bran solid culture after five days of NUK-1204 growth, following its release from cells at 0.011 unitmL -1culture filtrate. The new kind of oxidase comprises a single polypeptide chain with a molecular mass of 54 kDa, containing FAD and exhibiting absorption maxima at 274, 372 and 439 nm. The enzyme was stable in 4.5-11 pH range, with an optimal reaction pH at 10.5 and optimal reaction temperature at 30°C. It remained stable at 20-50°C for 1 hr. The relative activity of glucosamine oxidase towards glucosamine and N-acetyl-glucosamine was 100:7, demonstrating high substrate specificity. It doesn’t inhibit glucosamine. To the best of our knowledge, this is the first report ever on the discovery of a glucosamine oxidase based on enzyme substrate specificity.

Long-Term Evaluation of Biomarkers in the Czech Cohort of Gaucher Patients.

A personalized treatment decision for Gaucher disease (GD) patients should be based on relevant markers that are specific to GD, play a direct role in GD pathophysiology, exhibit low genetic variation, reflect the therapy, and can be used for all patients. Thirty-four GD patients treated with enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) were analyzed for platelet count, chitotriosidase, and tartrate-resistant acid phosphatase activity in plasma samples, and quantitative measurement of Lyso-Gb1 was performed in dried blood spots. In our ERT and SRT study cohorts, plasma lyso-GL1 correlated significantly with chito-triosidase (ERT: r = 0.55, p < 0.001; SRT: r = 0.83, p < 0.001) and TRAP (ERT: r = 0.34, p < 0.001; SRT: r = 0.88, p < 0.001), irrespective of treatment method. A platelet count increase was associated with a Lyso-Gb1 decrease in both treatment groups (ERT: p = 0.021; SRT: p = 0.028). The association of Lyso-Gb1 with evaluated markers was stronger in the SRT cohort. Our results indicate that ERT and SRT in combination or in a switch manner could offer the potential of individual drug effectiveness for particular GD symptoms. Combination of the key biomarker of GD, Lyso-Gb1, with other biomarkers can offer improved response assessment to long-term therapy.

Chemoproteomic Approaches to Studying RNA Modification-Associated Proteins.

The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for N6-methyladenosine (m6A) and N1-methyladenosine (m1A) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both m6A and m1A to mediate transcript destabilization. Furthermore, m6A also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.

Conserved residues at the family and subfamily levels determine enzyme activity and substrate binding in glycoside hydrolase family 13

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering.

A green approach to obtaining concentrated lipophilic bioactives from oil-rich extracts via biocatalytic alcoholysis in supercritical carbon dioxide

A novel green method to concentrate β-carotene was developed by (i) ethanolysis of triolein using a commercial immobilized lipase (Novozym 435) in a supercritical carbon dioxide (SC-CO2) bioreactor, and (ii) isolation of the produced fatty acid ethyl esters (FAEEs) from the reaction mixtures by taking the advantage of the tunable solvent properties of SC-CO2. The effects of enzymatic reaction conditions (temperature (45–65 °C), pressure (8–15 MPa), enzyme load (10–20%), and substrate mole ratio of triolein to ethanol (1:3–1:9)) on the conversion were investigated and optimized based on the FAEE content of the reaction products. The reaction carried out at 55 °C, 15 MPa, 20% enzyme load, and 1:6 substrate mole ratio in 8 h resulted in the highest FAEE content (93.3%). The isolation of FAEEs from the reaction mixtures was studied using both static and continuous SC-CO2 extractions at various temperatures (40–60 °C) and pressures (10–20 MPa). The highest FAEEs yield (89.9%) was achieved with a continuous extraction at 40 °C and 20 MPa for 4 h, where the FAEE extract (96.3% purity) has a great potential to be used as biodiesel, a sustainable alternative to petroleum diesel. Owing to the high extraction yield of FAEEs from the reaction mixture, the concentration of β-carotene was significantly increased by 9.3-fold. The proposed approach is a green, food-grade approach to produce β-carotene concentrate that can be utilized in smaller amounts to develop functional foods with health-promoting effects.

Antifungal activity and identification of bioactive peptide from Etawa crossbreed goat (Capra hircus) milk protein hydrolyzed using trypsin enzyme

There had been a growing interest in biopreservation in recent years due to the increasing demand for food products that were free of synthetic preservatives and therefore safer to consume. This made it necessary to explore natural bioactive with antifungal properties that can inhibit microbial spoilage and extend shelf life. Etawa crossbreed goat milk (Capra hircus) peptide fraction has antifungal activity. Therefore, this study aimed to determine the peptide with antifungal activity resulting from the casein and whey proteins of goat milk hydrolysis by trypsin. The hydrolyzate was fractionated using a Strong Cationic Exchange (SCX) catridge and the antifungal activity test was performed against Aspergillus sp. using the disc diffusion method. The most active peptide fraction with the highest antifungal activity was identified using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Based on the results, the ratio of the enzyme substrates used to hydrolyze casein and whey proteins is 1:10; 1:20; 1:30 and 1:40. The greatest degree of hydrolysis of casein and whey proteins was obtained at a 1:40 enzyme-substrate ratio of 66.8% and 75.4%, respectively. The analysis of Sodium Dodecylsulfate - Polyacrylamide Gel Electrophoresis (SDS PAGE) showed that goat milk casein contained αS1-casein and β-casein, while the whey proteins contained beta-lactoglobulin and α-lactalbumins. The casein and whey protein hydrolysates were fractionated using an SCX with pH variations of 3–9. The Goat Milk Casein (GMC) 5, 6, 7 dan 8 peptide fractions inhibited Aspergillus sp, and the most active was a fraction from GMC6 with an inhibition zone of 26.95 ± 0.63 mm and Minimum Inhibitory Concentration (MIC) value of 31.25 μg/mL. Furthermore, the morphology of Aspergillus sp. mycelia after treatment with peptide fraction showed that the mycelia were wrinkled and withered. The fractions with the highest antifungal activity contained 4 peptide sequences, namely YNVPQLEIVPK, KENNINELSK, GLSPEVPNENLLR, and YLGYLEQLLK. These results showed that peptide from goat milk had potential applications as a natural antifungal compound.

Synthesis of naproxen thiadiazole urea hybrids and determination of their anti-melanoma, anti-migration, tyrosinase inhibitory activity, and molecular docking studies

Novel sixteen naproxen urea compounds were synthesized bearing the thiadiazole ring. Their inhibitory activities against tyrosinase were investigated. 3o was discovered to be the most potent inhibitor of tyrosinase, with an IC50 value of 35.0 µM. The kinetic parameters were used to determine the type of enzyme inhibition. The results showed that 3o was an uncompetitive inhibitor with the Ki value of 62.2 µM. Additionally, the cytotoxic effects of the synthesized compounds on melanoma (B16F10), mouse embryonic (3T3) and the healthy 3T3 cell lines were also investigated. According to the cytotoxicity results, 3e (IC50= 2.17 µM) showed the highest cytotoxicity on the B16F10 cells. Furthermore, the effects of selected compounds on the migration rate of melanoma cells were investigated. In addition, molecular modeling studies were also performed and the results showed the possible interactions between the uncompetitive inhibitor 3o with the Tyrosinase-L-Tyrosine enzyme substrate complex.