The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/ mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

No abstract

Im Zuge der Erarbeitung der Richtlinie VDI 4254 Blatt 2, die die Emissionsmessung von Endotoxinen an Betriebsanlagen beschreibt, wurden Vergleichsmessungen mit Limulus-Amöbozyten-Lysat(LAL)- und rekombinanter-Faktor-C(rFC)-Tests durchgeführt und Verfahrenskenngrößen für das vollständige Messverfahren nach der Richtlinie VDI 4219 bestimmt. Hierfür fanden an einer zwangsbelüfteten Hähnchenmastanlage zum Zeitpunkt hoher bakterieller Emissionen 25 parallele Probenahmen (Doppelbestimmungen) mit dem Impingement-Verfahren statt. Die Endo- toxinaktivitäten wurden von fünf Laboren mithilfe von LAL-und rFC-Tests verschiedener Hersteller gemessen. Beide Methoden erwiesen sich als grundsätzlich für die Analytik von Emissionsproben geeignet, lieferten jedoch Konzentrationsangaben in unterschiedlicher Höhe. Da der rFC-Test keine Pfeilschwanzkrebse benötigt und die Messunsicherheit niedriger ist als beim LAL-Test, wurde für die Aktivitätsbestimmung von Endotoxinen in Emissionsproben nach der Richtlinie VDI 4254 Blatt 2 ausschließlich die Analytik mit dem rFC-Test beschrieben und standardisiert.

No abstract