Michaelis-Menten Research Articles

Trypsin from digestive tract of harpiosquillid mantis shrimp: Molecular characteristics and the inhibition by chitooligosaccharide and its catechin conjugate

AbstractTrypsin from the digestive tract of harpiosquillid mantis shrimp (HMS) was purified using ammonium sulfate precipitation and a soybean trypsin inhibitor‐CNBr‐activated Sepharose 4B affinity column. The purified trypsin (PTRP‐HMS) had a purity of 30.4‐fold, and a yield of 14.5% was obtained. PTRP‐HMS had the molecular weight of 23.0 kDa as examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and only one isoform was detected by native‐PAGE. Its optimal temperature and pH were 55°C and 8.5, respectively. TLCK almost completely inhibited the activity of trypsin. The PTRP‐HMS had a Michaelis–Menten constant (Km) and catalytic constant (Kcat) of 0.87 mM and 13.04 s−1, respectively, toward Nα‐benzoyl‐l‐arginine 4‐nitroanilide hydrochloride. When chitooligosaccharide (COS) and COS‐catechin (COS‐CAT) conjugates were examined for inhibition toward the PTRP, the latter exhibited higher efficacy in inhibiting the trypsin. Both COS and COS‐CAT conjugate showed mixed‐type inhibition kinetics. As a consequence, COS and COS‐CAT conjugate could be used as natural additives for inhibiting trypsin in whole HMS, thus retarding the softening and lengthening the shelf‐life of HMS during the iced storage.Practical ApplicationHarpiosquillid mantis shrimp (HMS) is of high demand due to its delicacy. However, its meat undergoes rapid softening within 2–3 days when stored in ice. Understanding causative proteolytic enzymes, especially trypsin from digestive tract, paves a way for preventing their negative impact on HMS eating quality. Employment of safe inhibitors, for example, chitooligosaccharide (COS) or COS conjugated with catechin, could inhibit HMS trypsin. Overall, softening of whole HMS containing trypsin in its digestive tract can be impeded, especially when treated with COS‐CAT. This finding is beneficial for the HMS local vendor or exporter, in which HMS quality can be maintained.

Structure and Function of Sabinene Synthase, a Monoterpene Cyclase That Generates a Highly Strained [3.1.0] Bicyclic Product.

Sabinene is a plant natural product with a distinctive strained [3.1.0] bicyclic ring system that is used commercially as a spicy and pine-like fragrance with citrus undertones. This unusual monoterpene has also been studied as an antifungal and anti-inflammatory agent as well as a next-generation biofuel. In order to understand the molecular determinants of [3.1.0] bicyclic ring formation in sabinene biosynthesis, we now report three X-ray crystal structures of sabinene synthase from Western red cedar, Thuja plicata (TpSS), with open and partially closed active site conformations at 2.21-2.72 Å resolution. We additionally report the complete biochemical characterization of sabinene synthase, including steady-state kinetics, active site mutagenesis, and product array profiling. The catalytic metal ion requirement is unexpectedly broad for a class I terpene cyclase: optimal catalytic activity was measured using Mn2+ or Co2+, with more modest activity observed using Mg2+ or Ni2+. Kinetic parameters were determined for both full-length TpSS and a deletion variant lacking the putative N-terminal plastidial targeting sequence, designated ΔTpSS. Monoterpene product profiles for both indicated similar product arrays independent of the catalytic metal ion used, with sabinene comprising nearly 90% of the total products generated. Site-directed mutagenesis was utilized to probe the function of active site residues, and several mutants yielded altered product arrays. Most notably, the G458A substitution converted ΔTpSS into a high-activity α-pinene synthase. α-Pinene contains a bicyclic [3.1.1] ring system; structural and mechanistic analyses suggest a molecular rationale for the reprogrammed transannulation reaction, leading to the alternative bicyclic product.

A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes.

The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS 'urzymes'. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes. AlphaFold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to our designed HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment and the spliced open reading frame GlyCA1-2. Both exhibit robust single-turnover burst sizes and ATP consumption rates higher than those previously published for HisCA urzymes and comparable to those for LeuAC and TrpAC. GlyCA is more than twice as active in glycine activation by adenosine triphosphate as the full-length GlyRS-B α2 dimer. Michaelis-Menten rate constants for all three substrates reveal significant coupling between Exon2 and both substrates. GlyCA activation favors Class II amino acids that complement those favored by HisCA and LeuAC. Structural features help explain these results. These minimalist GlyRS catalysts are thus homologous to previously described urzymes. Their properties reinforce the notion that urzymes may have the requisite catalytic activities to implement a reduced, ancestral genetic coding alphabet.

Gas Chromatography-Mass Spectrometry and Fourier Transform Infrared Spectroscopy Analysis of Potential α-Glucosidase Inhibitors from Terminalia catappa Leaf Extracts

Aims: Postprandial hyperglyceamia is often caused by insulin insufficiency or cellular glucose uptake complications, and conventional treatments often yield undesirable side effects. The aim of the current work was to assess the α-glucosidase inhibitory activities of T. catappa leaf extracts and use spectroscopic techniques to partially characterize possible inhibitors. Study Design: Phytochemical screening, enzyme inhibition assay and spectrometric and spectroscopic characterization of bioactive compounds from leaf extract and fractions of T. catappa. Place and Duration of Study: The entire work was carried out at the Department of Applied Chemistry and Biochemistry, University for Development Studies, Ghana within nine months. Methodology: T. catappa leaf extract and fractions were qualitatively screened for phytochemicals and their biological activity assessed in α-glucosidase inhibition assay. The potential enzyme inhibitors were characterized by Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared (FT-IR) Spectroscopy. Results: Phytochemical screening of the extract and the various solvent fractions revealed the presence of alkaloids, flavonoids, saponins, flavanol glycosides, phenolics, and terpenoids. The α-glucosidase inhibition potential of the crude leaf extract was concentration-dependent with a half-maximal inhibitory concentration (IC50) of 337.5 µg/ml. Solvent fractions from the crude extract demonstrated α-glucosidase inhibitory activity with IC50 values ranging from 170.40 µg/ml for ethyl acetate (EtOAc) fraction to 71.90 µg/ml for n-hexane (n-Hex) fraction. Acarbose, the control drug, demonstrated significant α-glucosidase inhibition activity in a similar pattern with IC50 of 6.16 µg/ml. The enzyme inhibition kinetics parameters, specifically the Michaelis constant (KM) and Maximum reaction velocity (Vmax) values, suggested that the inhibition of α-glucosidase by T. catappa extract followed a competitive mode. GC-MS and FT-IR analysis of the n-Hexane fraction identified the compounds Eugenol, Urs-12-en-24-oic acid, 3-oxo-, methyl ester (+)-, phytol, trans-isoeugenol, α-amyrin, and squalene as the potential antidiabetic agents that might be responsible for reducing postprandial blood glucose levels. Conclusion: These findings show that T. catappa leaf extract contains α-glucosidase inhibitors and therefore serves as a valuable resource in the discovery of natural anti-diabetic agents.

Discovery of a new lead molecule to develop a novel class of human factor XIIa inhibitors.

Factor XIIa (FXIIa) is a plasma serine protease within the contact activation pathway. Inhibiting FXIIa could offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks that accompany the use of existing anticoagulants. Therefore, we investigated the anticoagulant properties of an amidine-containing molecule (inhibitor 1) to identify a potential lead molecule for subsequent development of FXIIa inhibitors. Results indicated that inhibitor 1 primarily inhibits human FXIIa with an IC50 value of ~30 µM. The inhibitor demonstrated variable selectivity against thrombin, factor IXa, factor Xa, factor XIa, and activated protein C. Michaelis-Menten kinetics indicated that the molecule is an active site inhibitor of FXIIa. Molecular modeling studies revealed that the molecule recognizes residues His57, Asp189, and Ala190 in FXIIa's active site. The inhibitor selectively and concentration-dependently prolonged the clotting time of human plasma under activated partial thromboplastin time assay conditions. The inhibitor did not exhibit significant cytotoxicity in human HEK293 cells and the in silico pharmacokinetics and toxicology data were comparable to known anticoagulants. This study introduces inhibitor 1 as a lead platform for further development as an anticoagulant to provide a more effective and safer approach to preventing and treating thromboembolic diseases.

Reconstruction of exposure to volatile organic compounds from venous blood concentration and an uncertain physiologically-based pharmacokinetic model

Physiologically-based pharmacokinetic modeling was applied to determine exposures to volatile organic compounds, specifically focusing on m-xylene. Passive diffusion was used to describe permeation through the skin. The proposed model agreed with the experimental data and allowed researchers to monitor the concentration profiles in different compartments. The study also focused on the impact of parameter uncertainty on the model predictions. Local and global sensitivity analyses evaluated the influence of partition parameters, diffusion coefficients in the skin, and metabolic parameters on the blood concentration. Both methods show that the Michaelis-Menten kinetics and the lean tissue:blood partition coefficients contributed the most to the total variability. A reverse dosimetry approach used the measured biomarker level to estimate the exposure dose in four hours. The results aligned with experimental data when simulations were conducted using random parameters selected within twenty-five percent of the mean.

Soft plant root structure-media flow interactions: Exploring the adverse effect of lead contamination in North-Eastern Indian rice

We experimentally investigate the effect of lead (Pb2+) contamination on the roots of an Assamese rice line variety Lachit using a heavy metal analyzing fluidic tool. To demonstrate the adverse effects of lead contamination on rice seedlings in a controlled environment, we have performed a number of multidisciplinary experiments. Also, we develop a numerical model in this endeavor to predict the Michaelis–Menten kinetics parameters, which are used to depict the lead transport phenomenon following soft root structure-media flow interactions. We show that increased inlet lead concentration of the media solution leads to a reduction in root growth exponentially in the developed fluidic device. As supported by the Raman spectra analysis, the drastic metabolic changes are visible under lead contamination. Our results revel that, in comparison to the control condition, lead accumulation results in a decrease in the uptake of nitrogen and also, the metallic nutritional components (K+, Na+, and Ca2+). Under lead contamination, the average osmotic pressure difference at the root surface is seen to be less than in the control situation. The inferences drawn from the current research shed light on the detrimental effects of lead contamination on rice roots, which have the potential to significantly lower agricultural yields and threaten food security in areas where rice is the primary food source.

Enlightening the blind spot of the Michaelis–Menten rate law: The role of relaxation dynamics in molecular complex formation

Enlightening the blind spot of the Michaelis–Menten rate law: The role of relaxation dynamics in molecular complex formation

Enhanced activity of split trehalase biosensors by interspecies domain combineering.

The split trehalase biosensor has potential as a versatile diagnostic technology. Split enzymes are engineered proteins, divided into inactive fragments, which can reassemble and regain activity when brought together by an analyte. The split TreA biosensor requires no sample processing and produces stable signals (in the form of glucose). Split trehalase reagents can function in blood, but periplasmic trehalase of E.coli requires blood acidification for maximal activity. The objective of this study was to obtain split trehalase with near physiological pH optimum. For this purpose, periplasmic trehalases of Cellvibrio spp. with higher activity at neutral pH, were split in analogy with the E. coli TreA into hood and catalytic domains. However, these split trehalases displayed self-complementation due to spontaneous reassembly. In contrast, when catalytic domains of Cellvibrio trehalases were combined with E.coli hood domains, these hybrids displayed conditional complementation capacity when split trehalase fragments fused to immunoglobulin-binding protein G (STIGA) were used to quantify immunoglobulin concentrations. Other hybrid combinations of Cellvibrio spp. had increased activity compared to the cognate pairs, albeit with strong self-complementation. A mutagenesis analysis of residues responsible for self-complementation led to uncoupling of self-complementation from allostery. The Michaelis-Menten kinetics of Cellvibrio enzymes and fragment pairs confirmed improved activity of a mutated hybrid pair of Cellvibrio hood and catalytic domains at physiological pH. In conclusion, the improvements in pH optimum and activity, demonstrated with STIGA, can now be leveraged to enhance other variations of the split trehalase biosensor platform, broadening its utility for testing clinical samples.

Properties and kinetic behavior of free and immobilized laccase from Oudemansiella canarii: Emphasis on the effects of NaCl and Na2SO4 on catalytic activities

Studies have highlighted the great potential of Oudemansiella canarii laccase in degrading synthetic dyes for reducing their toxicity. Immobilization of enzymes improves usability in degradation processes and the present work succeeded in immobilizing this laccase onto MANAE-agarose. Immobilization improved pH, thermal, and storage stabilities. Both, free and immobilized enzymes presented Michaelis-Menten kinetics with the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with Km values of 0.056 ± 0.003 and 0.195 ± 0.022 mM, respectively. Immobilization increased Vmax 1.27-fold. NaCl caused incomplete (hyperbolic) inhibition, which was satisfactorily described by the one-substrate one-modifier mechanism. Immobilization reduced the maximal inhibition by NaCl from 80.2 to 55.7 %. The effect of Na2SO4 was predominantly stimulation, but inhibition of the free enzyme occurred at high substrate concentrations. Stimulation of the immobilized enzyme by Na2SO4 was much more pronounced. It strongly depended on the substrate concentration and was much stronger (up to 300 %) at low substrate concentrations. The combined effects of substrate and sulfate on the immobilized laccase could be satisfactorily described by the one-substrate one-modifier mechanism. The modified response of the immobilized O. canarii laccase to NaCl and Na2SO4 considerably favors its use as a tool in bioremediation processes because environmental contamination by salts frequently represents a strong operational challenge.

Alterations in nitrogen metabolism caused by heavy metals in the acid-tolerant microalga Coccomyxa onubensis

The microalga Coccomyxa onubensis is an extremophile microorganism with a unique ecosystem (Río Tinto, Huelva, Spain) that contains high amounts of contaminants, including heavy metals, sulphates, and nitrates, in acidic environments (pH 2.5). The current work presents an evaluation of the capacity of Coccomyxa onubensis to assimilate different nitrogen sources under Cu2+, Cd2+, AsO33−, AsO43− and Hg2+ stress, and the metabolic implications of these stressors. The results showed that ammonium consumption was less affected than nitrate consumption when microalgae were cultivated with heavy metals (except cadmium). The activities of enzymes involved in nitrogen metabolism, such as nitrite reductase (NiR; EC:1.7.7.1), glutamine synthetase (GS; EC:6.3.2.1) and glutamate dehydrogenase (GDH; EC:1.4.1.2) were characterised to determine the Michaelis-Menten constant (Km) and optimal temperature and pH values, being 45, 40 and 60 °C and pH values of 7.5, 6.0 and 9.0 for NiR, GS, and GDH, respectively. The effects of different heavy metals on these enzymes were assessed at multiple levels, and the results showed that the enzymatic activity of NiR was downregulated, specially under copper stress, maintaining 23 % of control NiR activity at 2 mM Cu2+. The enzymatic activity of GS was upregulated at low concentrations under cadmium and mercury stress (115–120 % of control cultures GS activity at 25 μM Cd2+ and 50 nM Hg2+, respectively) and downregulated at high concentrations of these elements. GDH activity was upregulated in the presence of Cu2+, Cd2+, and Hg2+, with increases up to 192, 155 and 154 % at 1 mM Cu2+, 300 μM Cd2+, and 250 nM Hg2+, respectively. These results provide a better explanation of the effects of heavy metal stress on N metabolism in Coccomyxa onubensis, which may be used as a model eukaryotic organism of the Tinto River acidophilic ecosystem.

Multifrequency-STD NMR unveils the first Michaelis complex of an intramolecular trans-sialidase from Ruminococcus gnavus

RgNanH is an intramolecular trans-sialidase expressed by the human gut symbiont Ruminococcus gnavus, to utilise intestinal sialylated mucin glycan epitopes. Its catalytic domain, belonging to glycoside hydrolase GH33 family, cleaves off terminal sialic acid residues from mucins, releasing 2,7-anhydro-Neu5Ac which is then used as metabolic substrate by R. gnavus to proliferate in the mucosal environment. RgNanH is one of the three intramolecular trans-sialidases (IT-sialidases) characterised to date, and the first from a gut commensal organism. Here, saturation transfer difference NMR (STD NMR) in combination with computational techniques (molecular docking and CORCEMA-ST) were used to elucidate the specificity, kinetics and relative affinity of RgNanH for sialoglycans and 2,7-anhydro-Neu5Ac. We propose the first 3D model for the Michaelis complex of an IT-sialidase. This confirms the sialic acid to be the main recognition element for the interaction in the enzymatic cleft and highlights the crucial role of Trp698 to make CH-π stacking with the galactose residue of the substrate 3′-sialyllactose. The same contact is shown not to be possible for 6′-sialyllactose, due to geometrical constrains of the α-2,6 linkage. Indeed 6′-sialyllactose is not a substrate, even though it is shown to bind to RgNanH by STD NMR. These findings corroborate the role of Trp698 for the α-2,3 specificity of trans-sialidases. In this structural study, the use of Differential Epitope Mapping STD NMR (DEEP-STD NMR) approach allowed the validation of the proposed 3D models in solution. These structural approaches are shown to be instrumental in shedding light on the molecular mechanisms underpinning enzymatic reactions in the absence of enzyme-substrate X-ray structures.

Multiple NADPH-cytochrome P450 reductases from Lycoris radiata involved in Amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids (AAs), such as galanthamine and lycorine, are natural products of Lycoris radiata possessing various pharmacological activities including anti-acetylcholinesterase, anti-inflammatory, and antitumour activities. Elucidating the biosynthesis of these special AAs is crucial for understanding their production and potential modification for improved clinical application, of which cytochrome P450 enzymes catalyse the formation of key alkaloid skeletons and subsequent modification processes, with the NAPDH cytochrome P450 reductases (CPRs) serving as essential redox partners. This study identified three CPRs, LrCPR1, LrCPR2, and LrCPR3, encoding 700, 697 and 695 amino acids, respectively, which belong to Class II CPRs. The LrCPRs reduced cytochrome c and ferricyanide in an NADPH-dependent manner, and their activities all followed the typical Michaelis-Menten curve. In yeast, the co-expression of LrCPRs and CYP96T6 produced the galantamine-like alkaloid namely N-demethylnarwedine, suggesting that they support the catalytic activity of CYP96T6. Quantitative analysis of the transcriptional expression profiles showed that LrCPRs were expressed in all the examined tissues of L. radiata, and their gene expression patterns are consistent with other genes that may be involved in the biosynthetic pathway of AAs, including cinnamate 4-hydroxylase and phenylalanine ammonia-lyase. Our study firstly provides the functional characterization of LrCPRs in L. radiata, which will contribute to the discovery of biosynthetic pathways and heterologous production of AAs.

Nickel-doped cuprous oxide nanocauliflowers with specific peroxidase-like activity for sensitive detection of hydrogen peroxide and uric acid

Copper-based nanomaterials have the properties of mimetic enzymes and can be used as excellent candidates for colorimetric sensing due to their environmental friendliness, low cost, and high abundance. In this paper, Ni-doped Cu2O nano cauliflower (Ni-Cu2O) was synthesized for the first time and applied to the detection of H2O2 and uric acid (UA) in human serum and urine. It was found that the proportion of Ni incorporation controls the morphology and the catalytic effect of Ni-Cu2O. The catalytic mechanism was studied by X-ray photoelectron spectroscopy, free radical capture experiments, photoluminescence spectroscopy, and steady-state kinetic analysis, which verified the redox reactions involving electron transfer and active substances. The results showed that Ni-Cu2O could catalyze the formation of reactive oxygen species (•OH, O2•−, 1O2, h+) from H2O2, which could oxidize 3,3′, 5,5′-tetramethylbenzidine (TMB) to oxTMB, and the color changed from colorless to blue. The Michaelis-Menten constant (Km) and the maximum initial velocity (Vmax) of Ni-Cu2O were 1.8 mM and 15.2×10−8 M/s, respectively. Based on the excellent peroxidase-like (POD) activity of Ni-Cu2O, a colorimetric sensing platform combined with TMB was proposed to sensitively detect H2O2 and UA in a wide range, and the detection limits were as low as 0.17 μM and 0.22 μM, respectively. This study creates a platform for using the Cu-based cauliflowers as a biosensor to detect UA in the medical and biomedicine fields.

Aggregation-induced emissive copper nanoclusters with peroxidase-like activity for colorimetric detection of cholesterol

Accurate and point-of-care cholesterol detection is of paramount significance for the prevention of cardiovascular diseases. The colorimetric assay based on peroxidase is a commonly used approach for cholesterol detection, without requiring any complicated biomolecular labeling or sophisticated instrumentation. Copper nanoclusters (CuNCs), exhibiting luminescent properties and peroxidase activity, have garnered significant attention in biomedical application recently. Herein, the glutathione-stabilized copper nanoclusters (GSH-CuNCs) were prepared with an easy one-pot method, employing glutathione as both a reducing agent and stabilizer. An optimization of the GSH-CuNCs preparation was carried out to obtain the highest peroxidase-like activity. UV-Vis absorption was measured to explore the steady-state kinetics of the GSH-CuNCs-catalyzed oxidation of 3,3',5,5' - tetramethylbenzidine (TMB) by H2O2. A colorimetric method for cholesterol detection was developed by combining the catalytic reaction of CuNCs and the enzymatic oxidation of cholesterol with cholesterol oxidase (ChOx). Under the optimized conditions, the UV-Vis absorbance of oxidized TMB (oxTMB) is proportional to the concentration of cholesterol within the range of 6.2-187.5 μM, and the limit of detection (LOD) is determined to be 3.0 μM. More importantly, cholesterol levels can be directly distinguished with the naked eye. Furthermore, the practicality of the method for detecting cholesterol in human serum has been verified with promising results. As expected, this simple, cost-effective, and easy-to-operate colorimetric method for cholesterol detection has potential applications in clinical diagnosis and provides valuable insights into the colorimetric sensing based on CuNCs.

Vanadium doped ZIF-L derived V-NC peroxidase-like nanozymes for tannic acid sensing

Transition metal-based nitrogen doping carbon nanozyme has attracted great attention owning to their high potential to replace natural enzymes. In this study, we prepared novel vanadium doped ZIF-L derived N-doped carbon nanozyme (V-NC) via facilely pyrolysis strategy. The cross-shaped morphology endows V-NC with unique V-Nx active sites, hierarchical pore character and large specific surface area, leading to its good peroxidase-mimetic activity. The steady-state kinetics trials revealed the low Km values of V-NC for TMB and H2O2, demonstrating its good affinity to TMB and H2O2. Mechanism research revealed that the excellent peroxidase-like activity of V-NC resulted from co-participation of •OH and O2•−, which were confirmed by reactive oxygen species (ROS) scavenging experiments and EPR trials. Under optimized operational conditions, and by virtue of the antioxidant properties of tannic acid (TA), a colorimetric sensing assay based on the peroxidase-like activity of V-NC was developed for sensitive detection of TA in the 0.1–2.5 µM linear range with low detection limit of 19.8 nM and the relative standard deviation (RSD, n = 3) of below 5%. This work renders a new idea for designing and developing high activity N-doped carbon nanozyme, and provides an accurate and feasible assay for the analysis of TA in real samples.

Purification and characterization of α-galactosidases from Penicillium griseoroseum for efficient soymilk hydrolysis

Soybean utilization is limited by the presence of raffinose oligosaccharides (RFO), which are not digested by humans and cause gastrointestinal discomfort. This study explores the potential of α-galactosidases from Penicillium griseoroseum for RFO hydrolysis in soymilk. Two distinct α-galactosidase enzymes, designated α-Gal1 and α-Gal2, were purified using a combination of ion-exchange chromatography and native polyacrylamide gel electrophoresis. Both enzymes exhibited characteristics of multimeric proteins and displayed similar biochemical properties. Optimal activity was observed at a pH range of 4.5–5.0 and a temperature range of 40–45 °C. Notably, α-Gal1 demonstrated high thermostability with a half-life of 16 h at 40 °C. The α-galactosidases displayed different substrate affinitiesfor the substrates ρ-NP-αGal, o-NP-αGal, rD-raffinose, d-stachyose, and mD-melibiose. The Michaelis-Menten constant (Km) values for α-Gal1 were 1.06, 1.31, 28.74, 19.88, and 4.77 mmol/L, respectively, while those for α-Gal2 were 0.8, 1.26, 30.46, 21.74 and 5.01 mmol/L, respectively. Both α-Gal1 and α-Gal2 were strongly inhibited by metal ions (Ag⁺, Cu2⁺, Fe2⁺, and Hg2⁺) and moderately inhibited by d-melibiose. Importantly, both enzymes efficiently hydrolyzed RFOs, achieving complete d-stachyose elimination from soymilk after a 6-h incubation. These findings propose the promising application of these α-galactosidases in industrial soymilk production, potentially enhancing its nutritional value and alleviating gastrointestinal issues in consumers.

Identification of positions in human aldolase a that are neutral for apparent KM

According to evolutionary theory, many naturally-occurring amino acid substitutions are expected to be neutral or near-neutral, with little effect on protein structure or function. Accordingly, most changes observed in human exomes are also expected to be neutral. As such, accurate algorithms for identifying medically-relevant changes must discriminate rare, non-neutral substitutions against a background of neutral substitutions. However, due to historical biases in biochemical experiments, the data available to train and validate prediction algorithms mostly contains non-neutral substitutions, with few examples of neutral substitutions. Thus, available training sets have the opposite composition of the desired test sets. Towards improving a dataset of these critical negative controls, we have concentrated on identifying neutral positions – those positions for which most of the possible 19 amino acid substitutions have little effect on protein structure or function. Here, we used a strategy based on multiple sequence alignments to identify putative neutral positions in human aldolase A, followed by biochemical assays for 147 aldolase substitutions. Results showed that most variants had little effect on either the apparent Michaelis constant for substrate fructose-1,6-bisphosphate or its apparent cooperativity. Thus, these data are useful for training and validating prediction algorithms. In addition, we created a database of these and other biochemically characterized aldolase variants along with aldolase sequences and characteristics derived from sequence and structure analyses. This database is publicly available at https://github.com/liskinsk/Aldolase-variant-and-sequence-database.

Soft-sensor model development for CHO growth/production, intracellular metabolite, and glycan predictions.

Efficaciously assessing product quality remains time- and resource-intensive. Online Process Analytical Technologies (PATs), encompassing real-time monitoring tools and soft-sensor models, are indispensable for understanding process effects and real-time product quality. This research study evaluated three modeling approaches for predicting CHO cell growth and production, metabolites (extracellular, nucleotide sugar donors (NSD) and glycan profiles): Mechanistic based on first principle Michaelis-Menten kinetics (MMK), data-driven orthogonal partial least square (OPLS) and neural network machine learning (NN). Our experimental design involved galactose-fed batch cultures. MMK excelled in predicting growth and production, demonstrating its reliability in these aspects and reducing the data burden by requiring fewer inputs. However, it was less precise in simulating glycan profiles and intracellular metabolite trends. In contrast, NN and OPLS performed better for predicting precise glycan compositions but displayed shortcomings in accurately predicting growth and production. We utilized time in the training set to address NN and OPLS extrapolation challenges. OPLS and NN models demanded more extensive inputs with similar intracellular metabolite trend prediction. However, there was a significant reduction in time required to develop these two models. The guidance presented here can provide valuable insight into rapid development and application of soft-sensor models with PATs for ipurposes. Therefore, we examined three model typesmproving real-time product CHO therapeutic product quality. Coupled with emerging -omics technologies, NN and OPLS will benefit from massive data availability, and we foresee more robust prediction models that can be advantageous to kinetic or partial-kinetic (hybrid) models.

Species Differences in Ezetimibe Glucuronidation

Background: Peclinical and clinical studies have revealed that ezetimibe, an approved cholesterol-absorption inhibitor, is rapidly and extensively metabolized to a more potent metabolite, ezetimibe glucuronide. Since different species are commonly used in the pharmacokinetic and pharmacodynamic studies of ezetimibe, it is essential to determine the species difference in glucuronidation of ezetimibe in order to accurately evaluate ezetimibe’s pharmacokinetics and pharmacodynamics. The purpose of the study was to compare species differences in ezetimibe glucuronidation rates using intestinal microsomes from humans, rats, mice, monkeys, and dogs. Method: Intestinal microsomes from different species were used to assess the ezetimibe glucuronidation rates. Multiple substrate concentrations at 0.5, 2, 5, 10, 20, 30, 40, and 50 µM were tested and fitted into the Michaelis–Menten model to determine the enzyme kinetic parameters. Results: The results showed that the glucuronidation rates with these tested species were significantly different. Kinetic studies revealed that the maximum metabolic rate (Vmax) was higher in monkeys (3.87 ± 0.22 nmol/mg/min) than that in rat (2.40 ± 0.148 nmol/mg/min) and mouse (2.23 ± 0.10 nmol/mg/min), and then human (1.90 ± 0.08 nmol/mg/min) and dog (1.19 ± 0.06 nmol/mg/min). The CLint was an 8.17-fold difference among these species, following the order of mouse > dog > human > rat = monkey. Conclusions: The study revealed that the rate of ezetimibe glucuronidation in the intestine was different in different species and has an impact on ezetimibe glucuronidation in the intestine. When analyzing the pharmacodynamics, pharmacokinetics, or toxicology of ezetimibe using different models, these species differences must be taken into consideration.