A Novel High-Throughput Assay Enables the Direct Identification of Acyltransferases

Abstract

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.

Sequence‐Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases

“…In order to gain more insight into the acyltransferase efficiency of the newly identified acyltransferases, we needed a more sophisticated assay that provides information about the relative rates of acyl transfer to acyl‐donor hydrolysis. However, our oligocarbonate assay provides only qualitative information. Mestrom et al.…”

“…We recently reported a high‐throughput assay for identifying more enzymes with acyltransferase activity in water . By screening several hydrolases using this assay, we identified a member of the family of bacterial hormone‐sensitive lipases (bHSLs), Est8, as a promiscuous acyltransferase .…”

Discovery and Design of Family VIII Carboxylesterases as Highly Efficient Acyltransferases

“…We previously showed that the acyltransferase-catalyzed formation of oligocarbonates from dimethyl carbonate and 1,6-hexanediol opacifies an initially transparent, aqueous solution. [9] However, not all promiscuous acyltransferases are expected to produce oligocarbonates from these substrates. In this study, we used a general emulsion-based assay, employing 2-phenylethanol and vinyl acetate as substrates, to screen the hydrolases available in our laboratory.…”

Enzymatic Ring‐Opening Polymerization of Lactones: Traditional Approaches and Alternative Strategies

“…We believe that more studies will be dedicated in the future to explore the synthesis of polymers in reaction media other than traditional approaches. Herein, the discovery of new hydrolases, which can perform polyesterifications in aqueous media, will play an important role as has been recently documented by Bornscheuer and co‐workers . In addition, reaction engineering strategies as shown for the eROPs running in continuous flow systems with “packed” immobilized enzymes will be of high interest in the future for high‐throughput optimization of reaction conditions for high productivities in a resource and time efficient way.…”