Flow cytometry-based screening systems have successfully been used in directed evolution experiments. Herein, we report the first whole-cell, high-throughput screening platform for P450 monooxygenases based on flow cytometry. O-dealkylation of 7-benzoxy-3-carboxycoumarin ethyl ester (BCCE) by P450 BM3 generates a fluorescence coumarin derivative. After one round of directed evolution, P450 BM3 variants with up to 7-fold increased activity (P450 M3 DM-1: R255H) could be identified at a sampling rate of 500 events s −1 . The reported screening platform can likely be applied to directed evolution campaigns of any P450 monooxygenase that catalyzes the O-dealkylation of BCCE.

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.

The number of sulfhydryl groups of firefly luciferase was determined by spectrophotometric titration with p-mercuribenzoate in the presence and absence of a competitive inhibitor. Between six and seven sulfhydryls are titrated with p-mercuribenzoate in the native enzyme. In the presence of the inhibitor only four to five sulfhydryls will react with the p-mercuribenzoate. Four or five moles of p-mercuribenzoate can be reacted with the enzyme-inhibitor complex, and subsequent removal of the inhibitor results in recovery of 90% of the original enzymatic activity. Addition of 4 moles of p-mercuribenzoate to the enzyme in the absence of inhibitor results in complete loss of activity. The enzyme is also inhibited by dithiol reagents such as arsenite-2,3-dimercaptopropanol, CdC12, and y-(p-arsenosopheny1)-n-butyric acid. The data show that four or five of the enzyme sulfhydryls have no effect on the catalytic activity, but the two sulfhydryl groups which are "covered') by the inhibitor are essential in some way for the enzymatic reactions leading to light emission. The amino acid composition of the protein was determined and spectrographic analysis showed the absence of any metal in stoichiometric amounts, thereby excluding the possibility of a metal cofactor.Purified firefly luciferase (Green and McElroy, 1956), catalyzes the following reactions: LH2 + ATP + E E-LHZ-AMP + P-P (1) E-LH:!-AMP + O2 + products + light (2)

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