Esterase

About: Esterase is a research topic. Over the lifetime, 7622 publications have been published within this topic receiving 168270 citations.

Optimization of FDA–PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa

Abstract: A rapid toxicity test based on inhibition of esterase activity in the harmful freshwater microalgae – Microcystis aeruginosa was developed using flow cytometry. The hydrolysis rate of fluorescein diacetate (FDA) by intracellular esterase to fluorescein was used to indicate the metabolic activity of algae. Uptake of FDA was optimized at different concentrations and incubation times. Propidium iodide (PI) was utilized to assess cell membrane integrity. The optimized FDA/PI staining dosages were 10 mg/L and 10 μM, respectively, lower than the reported concentrations. Correspondingly, the proper incubation time was 14–21 min at the optimal FDA dosage determined in this study. A new procedure based on optimized FDA/PI condition, called “whole algal culture flow cytometry with fluorescence triggering”, was developed for short-term bioassays. This new procedure, taking account of working conditions such as pH and impure cultures, is able to avoid algal cell damages in sample preparation and separate algal cells from non-algal particles by fluorescence triggering. This newly-developed procedure was then used to assess the toxicity of copper on M. aeruginosa in a short-term exposure (36 h). As copper concentrations increased, it was found that the esterase activity decreased in a concentration-dependent manner with increased membrane fragments. Moreover, esterase activity was a good indicator of copper toxicity in M. aeruginosa . The EC 50 value based on mean fluorescence intensity (MFI) was 123.3 μg/L (95% confidence limits 101.5–146.2 μg/L). Therefore, the new-developed procedure could be used for sublethal endpoints detection, and has the potential to be a rapid and cost-effective bioassay for selecting M. aeruginosa control methods or exploring the M. aeruginosa activity inhibition mechanism.

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 1. A theory of the ionic control of a complex multi-enzyme system.

Abstract: A theory is proposed to explain the physical bases of the ionic control of the activity of an enzyme system, located on a plant cell surface, and probably involved in cell-wall synthesis and extension. The model, which is based on various previously published experimental results, involves several assumptions: a cell-wall pectin methyl esterase de-esterifies pectins and thus creates the fixed negative charges of the cell wall; various enzymes incorporate uncharged carbohydrates in cell-wall material; cell-wall extension implies the sliding of cellulose microfibrils; the enzymes responsible for carbohydrate incorporation are activated by protons in the pH range 4-8 and have very similar pH dependencies: the cell-wall pectin methyl esterase is inhibited by protons in the same pH range. The mathematical derivation of this model, written in the form of a hypercycle, indicates that it is equivalent to a set of two antagonistic enzyme reactions: an enzyme reaction conditioned by pectin methyl esterase which results in the increase of fixed charge density of the cell wall; a number of 'growth enzymes', which produce extension and building up of the cell wall and therefore a decrease of charge density. The mathematical study of this model shows it may display a very high co-operativity of its response to slight changes of pH. This cooperativity means that the cell wall charge density may dramatically increase or decrease, within a very narrow pH range. The steep response of this system appears to be the direct consequence of different pH sensitivities of pectin methyl esterase and of the other cell-wall enzymes involved in cell growth. Calcium, which tightly binds to the cell wall, may diminish or even suppress this abrupt charge transition. This model suggests a novel theory of the ionic control of cell-wall expansion. The very basis of this theory is the existence of an electrostatic potential difference, delta psi, between the inside and the outside of the cell wall. When this delta psi value is large, the local proton concentration is high. Therefore the enzymes involved in cell wall extension and building up are active, but pectin methyl esterase is not. Therefore, the cell wall extends and the charge density decreases. The delta psi value then declines, as well as the local proton concentration. Under these conditions, the pectin methyl esterase becomes activated, whereas the 'growth enzymes' are not. This activation of pectin methyl esterase restores the initial, or an even higher, electrostatic potential difference, which in turn results in a decrease of local pH.(ABSTRACT TRUNCATED AT 400 WORDS)

Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences.

Abstract: Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

Abstract: EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Here we report for the first time the 2.1-A resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.