Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters

Abstract

β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening.

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).