Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity.

Benjamin Begander,Volker Sieber,Anna Huber,Josef Sperl,Manuel Döring

doi:10.3390/ijms21010335

Abstract

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.

Highlights

The increasing scarcity of fossil resources as well as an urgent demand for a reduction of global warming has enhanced the interest in sustainable products
The drivers of biocatalysis, work under energy-efficient and sustainable reaction conditions and are increasingly applied in industrial processes as they allow a combination of high turnover numbers with high selectivity [2]
The technique allows the generation of large mutant libraries, posing the need for a reliable and sturdy screening method that allows discriminating between the wildtype signal and potential hits, even at low starting activities

Summary

Introduction

The increasing scarcity of fossil resources as well as an urgent demand for a reduction of global warming has enhanced the interest in sustainable products. Novel production processes are needed, that allow the conversion of biomass to bulk and fine chemicals by simultaneously working under the conditions of green chemistry [1] To reach such processes, catalysis, and especially biocatalysis, is a central key. The drivers of biocatalysis, work under energy-efficient and sustainable reaction conditions and are increasingly applied in industrial processes as they allow a combination of high turnover numbers with high selectivity [2]. Despite these advantages, many enzymes isolated from natural origins show limited technical applicability due to low activity, low stability, or other parameters that do not allow economically feasible industrial processes [3]. While rational design intrinsically depends on the presence of a crystal structure and an understanding of the catalytic mechanism of an enzyme, directed evolution provides a powerful approach to optimize enzymes without a clearer

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