Table 1.
Overview of statistical measures for evaluation of HTS assays.
σ = standard deviation; μ = mean; m = median; pos = data set of positive controls; neg = data set of negative controls; n = sample size; Fk = cumulative density function for data set k; maxj = maximum distance between two distributions; SSMD = Strictly standardized mean difference; xi = ith value of the (ordered) data set x.
Table 2.
For comparison of enzyme activity equal protein concentrations (mg/mL) were used. The concentrations of all hydrolase solutions were determined by Bradford assay. Stock solutions of hydrolases were 10 mg/mL. All enzymes but pNB-Est13 were were purchased comerially. pNB-Est13 was hetrologous expressed in E. coli.
Table 3.
Criteria for performance evaluation of HTS-assays [43, 47, 51, 71, 86, 87].
Fig 1.
Overview of assays for hydrolysis of β-keto esters.
(A) Hydrolysis reaction of β-keto ethylester catalyzed by hydrolase. (B) pH-assay: photometric detection of pH change due to acid formation and deprotonation with bromothymol blue. Ethanol based assays: (C) Oxidative luminescence assay using alcohol oxidase, horseradish peroxidase and ethanol (D) Photometric detection of ethanol by oxidation by dehydrogenases under conversion of NAD+to NADH.
Fig 2.
Histogram of positive and negative controls of different HTS assays.
For each control 44–48 values were measured a) pH-indicator assay at 440 nm b) pH-indicator assay at 620 nm c) luminescence ethanol assay (mean luminescence intensity) d) luminescence ethanol assay (integrated luminescence intensity) e) photometric ethanol assay at 340 nm.
Fig 3.
Matrix of different statistical parameters for evaluation of HTS assays.
For each parameter the assays were ranked from the best (green) to the worst assay (red). The assays were grouped by the kind of detection. Ethanol quantification: Lum(int), Lum(mean), UV/Vis (ethanol dehydrogenase assay). pH-indicator assay at 440 and at 620 nm.
Fig 4.
Screening of different β-keto esters against different hydrolases with pH-indicator assay.
(for abbreviations see Table 3) The concentration of substrates was 2.0 mM in 2.5 mM sodium phosphate buffer (pH 7.0). The reactions were carried out at 30°C for 30 min. The activities (μmol/min) were normalized to μmol actives sites per s. (grey: no measurement possible) Para-nitrobenzyl-esterase 13 (pNB-Est13) was purified by us (described in methods). a) Structure of ethyl benzoylacetate with different substituents used as substrates in the screening. b) Activity matrix for 8 different enzymes (ABCL, ALM, ALFP, CRL, pNB-Est13, PPL, RML and TLL) against the respective substrates. The turnover number is in [1/s], for illustration turnover values were color coded from blue (low) to red (high).