Acyltransferases are enzymes that are capable of catalyzing the transesterification of non-activated esters in an aqueous environment and therefore represent interesting catalysts for applications in various fields. However, only a few acyltransferases have been identified so far, which can be explained by the lack of a simple, broadly applicable high-throughput assay for the identification of these enzymes from large libraries. Here, we present the development of such an assay that is based on the enzymatic formation of oligocarbonates from dimethyl carbonate and 1,6-hexanediol. In contrast to the monomers used as substrates, the oligomers are not soluble in the aqueous environment and form a precipitate which is used to detect enzyme activity by the naked eye, by absorbance or by fluorescence measurements. With activity detected and thus confirmed for the enzymes Est8 and MsAcT, the assay enabled the first identification of acyltransferases that act on carbonates. It will thus allow for the discovery of further efficient acyltransferases or of more efficient variants via enzyme engineering.
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 coli co-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−1 was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.
Summary
The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo‐, regio‐ and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram‐negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole‐cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity VMAX(app) for the oxidation of cyclohexanol with the ADH whole‐cell biocatalysts was determined as 59.9 mU ml−1. For the oxidation of cyclohexanone with the CHMO whole‐cell biocatalysts a VMAX(app) of 491 mU ml−1 was obtained. A direct conversion of cyclohexanol to ε‐caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole‐cell biocatalysts. Gas chromatography was applied to quantify the yield of ε‐caprolactone. 1.12 mM ε‐caprolactone was produced using ADH and CHMO displaying whole‐cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD578nm 10. Furthermore, the reaction cascade as applied provided a self‐sufficient regeneration of NADPH for CHMO by the ADH whole‐cell biocatalyst.
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