A New Microplate Screening Method for the Simultaneous Activity Quantification of Feruloyl Esterases, Tannases, and Chlorogenate Esterases

Abstract

Feruloyl, chlorogenate esterases, and tannases are enzymes useful in phenolic modifications of pharmaceutical relevance as protectors against several degenerative human diseases. Therefore, there is a growing interest in discovering new sources of these enzymes. However, traditional methods for their activity measurements are time-consuming and poorly adapted for high-throughput screening. In this study, a successful new microplate high-throughput screening method for the simultaneous quantification of all mentioned activities is demonstrated. This method allows the detection of activities as low as 1.7 mU ml(-1). Furthermore, reaction rates increased proportionally with the amount of enzyme added, and no interferences with the other commercial hydrolases tested were found. The utility of the method was demonstrated after simultaneously screening feruloyl, chlorogenate esterase, and tannase activities in solid state fermentation extracts obtained during the kinetics of production of 20 fungal strains. Among these, seven strains were positive for at least one of the esterase activities tested. This result shows the potential for the rapid routine screening assays for multiple samples of moderate low to high enzymatic levels.

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

“…Of the 136 stably transformed lines based on PCR detection of the faeB gene and Southern blot (Additional file 2), 18 showed detectable levels of faeB activity (Figure 2A,B; data shown for representative lines 2 V, ER28 and 3A covering one line from each genotype) using a microplate rapid screening assay [17]. Of the 18 faeB -expressing lines, 11 were targeted to apoplast, five targeted the vacuole and two targeted the endoplasmic reticulum.…”

“…The concentrate was resuspended in 3 ml of 2.5 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.2) and reisolated by centrifugation into a final volume of less than 1 ml. For preliminary assessment of feruloyl esterase activity in transgenic lines, enzyme was qualitatively estimated using a microplate screening assay [17] by measuring the hydrolysis of ethyl ferulate (50 mM ethyl ferulate, 5 mM of p -nitrophenol in isopropanol, diluted in nine volumes of 2.5 mM MOPS, pH 7.2). To test the samples, 10 mg (in triplicate) of concentrated plant protein extracts in 20 μl of 2.5 mM MOPS (pH 7.2) were placed in each microplate well, and 100 μl of substrate was added immediately before readings.…”

A New Native Source of Tannase Producer, Penicillium sp. EZ-ZH190: Characterization of the Enzyme

“…It hydrolyzes the ester and depside linkages of tannic acid to produce glucose and gallic acid. The enzyme has wide applications in food, beverage, brewing, cosmetics, and chemical industries (7)(8)(9). It is mainly used for the preparation of gallic acid, coffee flavored soft drinks, instant tea, high grade leather tannin, clarification of fruit juice and bear, food detannification, and to clean up highly polluting tannin from the leather industry effluent (7,10,11).…”

A practical high-throughput screening system for feruloyl esterases: Substrate design and evaluation

“…1) [14] as well as substrates based on (2-chloro-4-nitrophenyl)-1,2,4-butanetriol that require additional non-enzymatic reactions of periodate oxidation and beta-elimination to trigger the liberation of the chromophore [15]. A microplate screening based on the pH indicator, 4-nitrophenol, has also been reported [16]. However, the establishing and evaluation of a reliable and practical high-throughput screening system is still required, which would otherwise facilitates the directed evolution of FAEs, as well as the novel enzyme mining from environmental microorganisms [17,18].…”