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Multi-enzyme cascade reactions capture the essence of nature’s efficiency by increasing the productivity of a process. Here we describe one such three-enzyme cascade for the synthesis of 6-hydroxyhexanoic acid. Whole cells of Escherichia colico-expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase (CHMO) for internal cofactor regeneration were used without the supply of external NADPH or NADP+. The product inhibition caused by the ε-caprolactone formed by the CHMO was overcome by the use of lipase CAL-B for in situ conversion into 6-hydroxyhexanoic acid. A stirred tank reactor under fed-batch mode was chosen for efficient catalysis. By using this setup, a product titre of >20 g L−1was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.

The “carrot and stick”principle could be combined with cell sorting to enable the selection of enantioselective esterase variants from a mutant library. Hence, the enormous diversity generated in directed evolution experiments is now easily accessible by this high‐throughput system. In line with the principle, the hydrolysis of 1glycerin supports cell growth, whereas the hydrolysis of 2leads to cell death.

Das Zuckerbrot‐und‐Peitsche‐Prinzipwurde auf die Selektion enantioselektiver Esterasevarianten aus einer Mutantenbibliothek übertragen und mit Zellsortierung kombiniert. Die durch gerichtete Evolution generierte Diversität lässt sich nun leicht mit diesem Hochdurchsatzsystem erschließen. Gemäß dem Selektionskonzept ergibt die Hydrolyse von 1Glycerin, das das Bakterienwachstum unterstützt, während das Produkt der Hydrolyse von 2zum Zelltod führt.

In this work, the immobilization of two hydrolases on plasma‐modified polypropylene carriers was investigated. Treating Accurel MP1001 with an oxygen plasma was found most suitable to increase the hydrophilicity and to allow for efficient immobilization. Thus, for lipase B fromCandida antarctica and for an esterase fromPyrobaculum calidifontis esterase (PestE) a 5‐fold and 14‐fold increase, respectively, in immobilization yield resulted compared to untreated carrier. In contrast to the oxygen‐modified support, modification of the polypropylene carrier with ammonia plasma showed no positive effect. Furthermore, it could be shown that immobilized PestE catalyzed enantioselective transesterification of α‐phenylethanol in vinyl acetate, whereas the free enzyme showed no activity. Both hydrolases could be recycled five times without significant loss of activity.

A procedure for the high-throughput screening of esterases is described. This includes enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E>100 toward an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains. This protocol can be completed in 3–4 days.

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