Anticholine esterase activity of phosphonium bis-zwitterions based on 2-cyanoacrylates

Phosphonium bis-zwitterions based on bis-2-cyanoacrylates are weak reversible inhibitors of choline esterases with a diverse anticholine esterase effect. This can find use in additional classification of choline esterases. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2192–2194, November, 1999.     

Micro two-dimensional gel electrophoresis of serum and testis esterases from different strains of mice

Two-dimensional electrophoresis with time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension was applied to the separation of native molecular forms of esterases from serum and testis of four strains of mice (C57BL/6J, Swiss OF1, F1 hybrid derived from these two populations and Tfm). In Phast System, a modified pH 3-9 gradient, a linear 8-25% gel gradient and a migration time corresponding to 300 Vh, were found to provide the best conditions for esterase analysis. About 70 esterase-active fractions could be separated with good reproducibility. The variants were characterized by their pI (3.9-7.35), their relative mobility and the visual estimation of their susceptibility towards neuraminidase and different esterase inhibitors. In the two tissues, the distribution of the esterase variants corresponded to a 50-500 kDa molecular mass range of calibration proteins, but most of the serum and testis-specific isoforms were confined to the 59-72 kDa range. All serum variants contained a terminal N-acetylneuraminic acid residue, whereas only the testicular esterases in common with those in serum appeared sensitive to neuraminidase. Cholinesterases with a low relative mobility and carboxylesterases with a high relative mobility were detected in serum, while carboxylesterases accounted for the greatest part in the testis which also contained cholinesterases and acetylesterases. Minor interspecies differences were found between C57BL/6J and Swiss OF1 esterases. The expression of two variants which differed between these two species seemed intermediate for the hybrid originating from these two populations. Two new spots were detected in the two-dimensional map of esterases from the strain bearing the Tfm mutation.     

Isoelectric focusing in immobilized pH gradients of phosphoglucomutase and esterases from the spiny lobster

A method is described for detecting polymorphisms of cephalothorax and tail homogenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) and esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0-8.0 interval for phosphoglucomutase and in the pH 3.5-5.0 and 4.2-4.9 ranges for esterases, both enzymes appeared as polymorphic band patterns. These could be explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2, 3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilized pH gradients than in polyacrylamide gel electrophoresis.     

用于测量农药残留的小麦酯酶的选择

为研制探测农药残留的生物传感器，研究了农药乐果[O,O-二甲基S－（N－甲基胺基甲酰甲戎）二硫代磷酸酯]对各种小麦植物酯酶的抑制，从小麦中提取植物酶，以农药乐果为抑制剂，采用分光光度法研究了乐果对各种小麦酯酶活性的影响；研究显示，不同品种小麦酯酶对农药乐果的敏感度不同，在所研究的品种中，豫麦39和小麦周9对乐果较敏感，研究结果说明了选择小麦酶源的必要性。     

Insight into Substrate Preference of Two Chimeric Esterases by Combining Experiment and Molecular Simulation

Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed via domain swapping between two ancient thermophilic esterases, were investigated on their molecular simulation(including homology modeling, substrates docking and substrate binding affinity validation) and enzymatic assay(specific activities and activation energies calculating). Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated, the substrate binding domains or binding pockets are important but not the only factor for substrate preference.     

4.3 Degradation and modification of cellulose acetates by biological systems

A survey is given on recent findings in the enzymology of cellulose acetate degradation. Acetyl esterases have been identified as the principal enzymes, initiating cellulose acetate degradation as a prerequisite for endoglucanase-catalyzed cellulose acetate depolymerisation. Acetyl esterases are provided by nature to deacetylate naturally occurring partly acetylated polysaccharides, i.e. xylan and chitin. Accordingly they are not designed to attack high DS cellulose acetate. Under these circumstances acetyl esterases require a pretreatment of cellulose acetate, leading to some reduction in DS, in case highly substituted material should be degraded. One of these treatments is composting under the conditions of which a partial deacetylation may occur under the action of heat and high pH, facilitating the accessibility for acetyl esterases. However from the present knowledge it cannot be excluded that certain microbial specialists exist, being capable to degrade high DS cellulose acetate.     

An enzymatic method for the detection of esters

An enzymatic method for the detection of esters has been developed. The use of various esterases as an analytical tool has been examined. It has been found that lipase can be used for the detection of esters under specified conditions. The relative rates of hydrolysis observed in the case of various esters have been explained on the basis of the generally accepted mechanism. The enzymatic method has been compared with the hydroxamic acid test.     

Novel ferrocenyl phosphonate derivatives. Inhibition of serine hydrolases by ferrocene azaphosphonates

Owing to their unique properties, ferrocene compounds are gaining increasing interest for biological applications, particularly as enzyme inhibitors. Phosphonate derivatives including a ferrocenyl moiety were synthesized by reaction of dimethyl‐ and diphenylphosphite with ferrocenyl methyl maleimide. The ferrocenyl diphenyl phosphonate complex was characterized by X‐ray diffraction. The ability of these organometallic compounds to inhibit the enzymatic activity of the serine esterases acetyl‐ and butyrylcholinesterase was investigated. It appeared that the new ferrocenyl phosphonates inhibited both enzymes by competitive, mixed or non competitve mechanisms with inhibition constants in the range 35–1000 µM. Both compounds also behave as slow time‐dependent inactivators of butyrylcholinesterase. The presence of the ferrocenyl entity seems then to have a dramatic effect on the biochemical behavior of the systems. Copyright © 2010 John Wiley & Sons, Ltd.     

Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates

The adrenaline test for enzymes is a general back-titration procedure to detect 1,2-diols, 1,2-aminoalcohols and α-hydroxyketones reaction products of enzyme catalysis by colorimetry. The method was used to profile a series of esterases and lipases for their esterolytic activity on a series of carbohydrate and polyol acetates. Substrates were prepared by peracetylation and used for parallel microtiter-plate analysis of enzyme activities. This method can be used to achieve a rapid and automated characterization of a set of enzymes during HTS screening.     

Inhibition and labeling of enzymes and abzymes by phosphonate diesters

Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymaticactivity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of try psin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permitanalysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDSPAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretretment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.     

Identification of a metagenome-derived esterase with high enantioselectivity in the kinetic resolution of arylaliphatic tertiary alcohols

35 metagenome-derived esterases bearing a GGG(A)X motif were screened for activity and enantioselectivity in the hydrolysis of a range of tertiary alcohol acetates. Most of the active esterases showed little or no enantioselectivity in the hydrolysis of the terpinyl acetate, linalyl acetate and 3-methylpent-1-yn-3-yl acetate. However, one esterase showed excellent enantioselectivity (E > 100) in the kinetic resolution of 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate as confirmed by a preparative scale reaction.     

Microbial enzymes mined from the Urania deep-sea hypersaline anoxic basin

We created a metagenome expression library from the brine:seawater interface of the Urania hypersaline basin, screened it for esterases, and characterized five of these. Two had no significant sequence homology to known esterases, hydrolyzed both carboxylesters and thioesters, and exhibited unusual, habitat-specific characteristics (preference for high hydrostatic pressure and salinity). One has an unusual structural signature incorporating three catalytic active centers mediating distinct hydrolytic activities and an adaptive tertiary-quaternary structure that alters between three molecular states, according to the prevailing physicochemical conditions. Some of the esterases have high activities, specificities, enantioselectivities, and exceptional stability in polar solvents, and they are therefore potentially useful for industrial biotransformations. One possesses the highest enantioselectivity toward an ester of the important chiral synthon solketal (E: 126[S]; 98%ee).     

Enantiomeric resolution of 2-aryl propionic esters with hyperthermophilic and mesophilic esterases: contrasting thermodynamic mechanisms

The enantiomeric resolution of 2-aryl propionic esters by hyperthermophilic and mesophilic esterases was found to be governed by contrasting thermodynamic mechanisms. Entropic contributions predominated for mesophilic esterases from Candida rugosa and Rhizomucor miehei, while enthalpic forces controlled this resolution by the esterase from the extremely thermoacidophilic archaeon, Sulfolobus solfataricus P1. This disparity in thermodynamic mechanism can be attributed to the differences in conformational flexibility of mesophilic and thermophilic enzymes as they relate to the temperature range (4-70 degrees C) examined.     

Asymmetric hydrolysis of enol esters with two esterases from Marchantia polymorpha

Two esterases participating in the asymmetric hydrolysis of α-alkylated enol acetates to α-chiral ketones were isolated from the cultured cells of Marchantia polymorpha. These two esterases had opposite enantioselectivities and both of them reversed the stereoselectivity of protonation into the enol intermediate in the hydrolysis when the chain length and the bulkiness of α-substituents were increased.     

应用激光解吸质谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析蛇毒精氨酸酯酶的纯度和分子量

应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和SDS-聚丙烯酰胺凝胶电泳对吉林省两地市的同种白眉蝮蛇蛇毒中具有抗栓塞药效的精氨酸酯酶进行了分析和比较。MALDI-TOF-MS法具有快速、准确度高、灵敏度高的优点,两种方法结合,互为补充,取得了令人满意的结果,MALDI-TOF-MS完全可以直接用作蛇毒成分分离过程中重要的研究手段。     

Molecular polymorphism of human enzymes as the basis of individual sensitivity to drugs. Supercomputer-assisted modeling as a tool for analysis of structural changes and enzymatic activity of proteins

The nature of individual sensitivity to drugs associated with molecular polymorphism of human enzymes is discussed. The influence of molecular polymorphism on the activity of key human esterases, in particular, cholinesterases and carboxylesterase, responsible for hydrolytic metabolism of ester-containing drugs, is analyzed. A method was developed, which involves supercomputer-assisted modeling as a tool for assessment of molecular mechanism of the impact of point mutations on the catalytic activity of enzymes. This work is a part of a study aimed at elaboration of the concept and methods of personalized medicine.     

Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.     

N-Methyl Indoxyl Esters As Fluorometric Substrates For Lipase

Abstract

The results of a study of 6 N-methyl indoxyl esters as substrates for lipase indicated N-methyl indoxyl myri-state to be the best substrate for the analysis of this enzyme. Using this ester from 0.0002 to 4.0 units per ml of porcine pancreas lipase can be determined in the presence of several other esterases with an accuracy and precision of about 1.5%. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Determination of rat plasma esterase-1 (ES-1) activity by scanning densitometry of gradient polyacrylamide gels with zymogram detection.

There is no specific assay for rat plasma esterase-1 (ES-1) activity. Plasma contains many esterases, while known substrates do not discriminate between esterases. With gel electrophoresis, plasma esterase isozymes can be separated. Thus, a method consisting of gradient polyacrylamide gel electrophoresis, visualization of the enzyme with a staining technique based on substrate conversion, and densitometric scanning of the stained gel has been developed for quantitative measurement of rat plasma ES-1 activity. ES-1 activities were expressed as total peak areas. Reproducibility of the method was found to be about 10% (expressed as apparent between-gel coefficient of variation). When the ES-1 zone areas was expressed relative to that of a plasma ES-1 standard, reproducibility was about 3%. The kinetics of catalysis of alpha-naphthyl acetate hydrolysis by ES-1 could be determined with the gel scanning assay; the Km was 0.76 mM. At the alpha-naphthyl acetate concentration of 2.69 mM, total peak areas of the ES-1 zone were linearly associated with the staining time (up to at least 40 min) and amount of plasma (up to 26.25 microL). The pH of the staining buffer influences the ES-1 zone area, the largest areas being obtained when the pH ranged between 7.0 and 7.8. With propionate as acyl moiety of the alpha-naphthyl ester substrate, ES-1 zone areas were higher than with either acetate, butyrate or hexanoate.