Determining the esterase activity of peptides and peptide assemblies.

Catalytic peptides are gaining attention as alternatives to enzymes, especially in industrial applications. Recent advances in peptide design have improved their catalytic efficiency with approaches such as self-assembly and metal ion complexation. However, the fundamental principles governing peptide catalysis at the sequence level are still being explored. Ester hydrolysis, a well-studied reaction, serves as a widely employed method to evaluate the catalytic potential of peptides. The standard colorimetric reaction involving para-nitrophenyl acetate hydrolysis acts as a benchmark assay, providing a straightforward and efficient screening method for rapidly identifying potential catalysts. However, maintaining standardized conditions is crucial for reproducible results, given that factors such as pH, temperature, and substrate concentration can introduce unwanted variability. This necessity becomes particularly pronounced when working with peptides, which often exhibit slower reaction rates compared to enzymes, making even minor variations significantly influential on the final outcome. In this context, we present a refined protocol for assessing the catalytic activity of peptides and peptide assemblies, addressing critical considerations for reproducibility and accuracy.

Purification of high molecular weight thermotolerant esterase from Serratia sp. and its characterization.

In the present study, an extracellular esterase from Serratia sp. was purified 24.46 fold using an initial ammonium sulphate precipitation step (optimized concentration of 30-40%), followed by Diethylaminoethyl cellulose (DEAE-cellulose) chromatography and size exclusion Sephadex G-200 column chromatography steps. The molecular weight of the esterase using native polyacrylamide gel electrophoresis (PAGE) was determined to be 236 kDa and by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 60 kDa suggesting that the enzyme was a tetramer of 4 subunits. The purified esterase was able to catalyze the hydrolysis of p-nitrophenyl esters, especially p-nitrophenyl acetate. Maximum esterase activity was achieved in 0.15 M Tris-HCl buffer of pH 8.5 at 50 °C after 10 min. The enzyme was stable for at least 8 h at 4 and 35 °C but the half-life was determined to be 4.5 h at 50 °C and 3 h at 60 °C. The esterase activity was inhibited by detergents (1 mM) (Triton X-100, Tween 60, Tween 80, ethylenediamine tetraacetic acid and SDS) except Tween 20. The esterase activity was inhibited by organic solvents (1 mM) such as ethanol, methanol, acetone, acetonitrile and was stable in the presence of glycerol, isopropanol but the organic solvent dimethyl sulfoxide (DMSO) significantly (p < 0.05) enhanced esterase activity. The matrix-assisted laser desorption ionization-time of flight mass spectrometry showed that the enzyme exhibited similarity with the pimeloyl-[acyl carrier protein] methyl ester esterase of Serratia marcescens.

Influence of Culture Conditions on the Production of Extracellular Esterase from Bacillus licheniformis and Its Characterization.

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. In the present study, thirty-seven bacterial isolates were isolated from soil contaminated with waste cooking oil, dairy waste etc. from Shimla and Solan district of H.P. Out of 37 isolates, the isolate RL-1, which gave maximum activity, was identified as Bacillus licheniformis MH061919. The optimization of various production parameters resulted in maximum activity at inoculum age of 24 h and inoculum size of 1.5% (v/v). Esterase gave considerable activity in production medium containing sodium chloride (0.5 % w/v), galactose (1%, w/v), coconut oil (2.0%, v/v) and beef extract (0.3%, w/v) at a temperature of 45℃ and pH 8.5.The enzyme production was enhanced by 3-fold after optimization of production parameters. Further, on optimizing reaction conditions, enzyme gave maximum activity at a temperature of 45℃ and pH 8.5. The para-nitrophenyl acetate (p-NPA) was found to be optimum substrate and metal ions and detergents have inhibitory effect on esterase activity.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.