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

Buß, O.; Jager, S.; Dold, S.M.; Zimmermann, S.; Hamacher, K.; Schmitz, K.; Rudat, J.

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.


Zugehörige Institution(en) am KIT Institut für Bio- und Lebensmitteltechnik (BLT)
Publikationstyp Zeitschriftenaufsatz
Jahr 2016
Sprache Englisch
Identifikator DOI: 10.1371/journal.pone.0146104
ISSN: 1932-6203
URN: urn:nbn:de:swb:90-517000
KITopen ID: 1000051700
Erschienen in PLoS ONE
Band 11
Heft 1
Seiten e0146104
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