hymecromone and 4-methylumbelliferyl-butyrate

Acetylcholinesterase (AChE) and general-esterase (GE) activities are important to understand detoxification processes of xenobiotics. The assays to quantify them have employed different substrates, inhibitors, types of experiments (in vitro and in vivo) and model organisms. The aim of this work was to give a systematic overview of the effect of the above factors on the outcome of AChE and GE activity measurements. We showed that AChE activity could be measured with the substrate acetylthiocholine iodide (AChI) but not with acetylcholine bromide (AChB) and only in in vitro assays. For GE activity, Michaelis-Menten kinetics differed between the substrates 4-methylumbellifery butyrate (4-MUB) and 1-naphtyl acetate (1-NA) in the measurements of in vitro activity, but their inhibition curves and IC

Topics: Acetylcholinesterase; Acetylthiocholine; Animals; Benzenaminium, 4,4'-(3-oxo-1,5-pentanediyl)bis(N,N-dimethyl-N-2-propenyl-), Dibromide; Chironomidae; Cholinesterase Inhibitors; Daphnia; Enzyme Assays; Esterases; Hymecromone; Kinetics; Naphthols; Xenobiotics

Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase's thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2's thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions. Graphical Abstract.

Topics: 2-Propanol; Acetone; Bacterial Proteins; Catalytic Domain; Cyclohexanes; Enzyme Stability; Ethanol; Geobacillus; Hymecromone; Lipase; Methanol; Molecular Dynamics Simulation; Protein Conformation; Solvents; Temperature; Toluene

Multi-compartmentalized biphasic proteinosomes were self-assembled using a single-step double Pickering emulsion procedure, and exploited for enzyme-mediated interfacial catalysis, polysaccharide shell templating, and hydrogel functionalization.

Topics: Acrylic Resins; Alginates; Animals; Artificial Cells; Cattle; Dextrans; Glucose; Glucose Oxidase; Hexanols; Hydrogels; Hymecromone; Lipase; Nanoconjugates; Serum Albumin, Bovine; Water

Tuberculosis (TB) is a global health threat with nearly 500 000 new cases of multidrug-resistant TB estimated to occur every year, so new drugs are desperately needed. A number of current antimycobacterial drugs work by interfering with the biosynthesis of key components of the mycolylarabinogalactan (mAG). In light of this observation, other enzymes involved in the synthesis of the mAG should also serve as targets for antimycobacterial drug development. One potential target is the Antigen 85 (Ag85) complex, a family of mycolyltransferases that are responsible for the transfer of mycolic acids from trehalose monomycolate (TMM) to the arabinogalactan. Virtual thiophenyl-arabinoside conjugates were docked to antigen Ag85C (PDB code: 1va5 ) using Glide. Compounds with good docking scores were synthesized by a Gewald synthesis followed by linking to 5-thioarabinofuranosides. The resulting thiophenyl-thioarabinofuranosides were assayed for inhibition of mycoyltransferase activity using a 4-methylumbelliferyl butyrate fluorescence assay. The conjugates showed K(i) values ranging from 18.2 to 71.0 μM. The most potent inhibitor was soaked into crystals of Mycobacterium tuberculosis antigen 85C and the structure of the complex determined. The X-ray structure shows the compound bound within the active site of the enzyme with the thiophene moiety positioned in the putative α-chain binding site of TMM and the arabinofuranoside moiety within the known carbohydrate-binding site as exhibited for the Ag85B-trehalose crystal structure. Unexpectedly, no specific hydrogen bonding interactions are being formed between the arabinofuranoside and the carbohydrate-binding site of the active site suggesting that the binding of the arabinoside within this structure is driven by shape complementarily between the arabinosyl moiety and the carbohydrate binding site.

Topics: Acyltransferases; Antigens, Bacterial; Antitubercular Agents; Catalytic Domain; Crystallography, X-Ray; Drug Design; Galactans; Glycoconjugates; Hymecromone; Kinetics; Molecular Docking Simulation; Mycobacterium tuberculosis; Spectrometry, Fluorescence; Structure-Activity Relationship; Substrate Specificity

Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8±0.1 and 5.7±0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8±0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8-8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7±0.3 vs. 0.71±0.05 s(-1) for the hoki enzyme) and lower activation energy (2.0±0.4 vs. 7.6±0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.

Topics: Amino Acid Sequence; Animals; Calcium; Catalysis; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Digestive System; Fractional Precipitation; Gadiformes; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Lipase; Molecular Sequence Data; Paraoxon; Salmon; Sequence Analysis, Protein; Sodium Cholate; Temperature

Topics: Bacteria; Hymecromone; Lipase; Luminescent Measurements

Moraxella catarrhalis can easily be differentiated from other oxidase-positive, gram-negative cocci with tributyrine, 4-methylumbelliferyl butyrate, or indoxyl acetate. All M. catarrhalis give positive reactions, and all Neisseria spp. give negative reactions. The 4-methylumbelliferyl butyrate tube test and indoxyl acetate strip test provide same-day identification of M. catarrhalis isolates.

Topics: Acetylesterase; Acinetobacter; Bacterial Typing Techniques; Carboxylic Ester Hydrolases; Evaluation Studies as Topic; Humans; Hymecromone; Indoles; Moraxella; Moraxella catarrhalis; Neisseria; Species Specificity; Substrate Specificity; Triglycerides

Branhamella catarrhalis can be distinguished from Neisseria spp. by the presence of butyrate esterase. This enzyme can be rapidly detected when 4-methylumbelliferyl butyrate is used as the substrate. All B. catarrhalis strains tested gave a positive fluorescence reaction within 5 min, while Neisseria spp. remained negative, even after 18 h of incubation.

Topics: Carboxylic Ester Hydrolases; Hymecromone; Moraxella catarrhalis; Neisseria; Umbelliferones