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A high throughput whole cell flow cytometer screening toolbox was developed and validated by identifying improved variants (1.3-7-fold) for three hydrolases (esterase, lipase, cellulase). The screening principle is based on coupled enzymatic reaction using glucose derivatives which yield upon hydrolysis a fluorescent-hydrogel-layer on the surface of E. coli cells.

Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the µM range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4′-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5–9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased kcat for perhydrolytic activity compared with wild type. [ABSTRACT FROM PUBLISHER]

Correction for ‘A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels’ by Nina Lülsdorf et al., Chem. Commun., 2015, 51, 8679–8682. [ABSTRACT FROM AUTHOR]

Summary Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase ( Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton’s reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10 7 phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials. [ABSTRACT FROM AUTHOR]

Highlights: [•] Engineering of substrate binding pocket provided insights in protease's promiscuity. [•] The specificity of subtilisin Carlsberg is shifted towards peroxycarboxylic acid production. [•] Novel subtilisin Carlsberg variants have up to 5.5-fold increased perhydrolytic activity. [Copyright &y& Elsevier]