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

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.

Engineering of a thermophilic dihydroxy-acid dehydratase to enhance its dehydration ability on glycerate to pyruvate and its application in in vitro synthetic enzymatic biosystems

“…Finally, the R3 mutant with 10-times higher specific activity than the wild type SsDHAD was obtained. The activity improvement level was much higher than that of the previous study of Begander et al by a random mutation which gave an activity enhancement on glycerate by 1.5times (Begander et al, 2020). The semi-rational design enabled the enhancement of dehydration activity of SsDHAD toward glycerate to pyruvate based on computer algorithms.…”

“…The extreme low activity of DHAD on glycerate hinders the production of bio-based products and biofuels by many in vitro synthetic enzymatic biosystems based on non-phosphorylative Entner Doudoroff (np-ED) pathway (Ahmed et al, 2005;Xie et al, 2018). Even though the dehydratation activity of SsDHAD was improved by 1.5-times through non-rational design protein engineering (Begander, Huber, Döring, Sperl, & Sieber, 2020), still cannot meet the requirement of further application.…”

High-throughput screening, next generation sequencing and machine learning: advanced methods in enzyme engineering

“…More recently, Begander and colleagues 11 developed a similar two-step reaction scheme to assess the enzymatic activity of a d -glycerate dehydratase. In this work, the second reaction uses the same set of enzymes to convert pyruvate into Bindschedler's green dye.…”

Nanofibrous Membrane with Encapsulated Glucose Oxidase for Self-Sustained Antimicrobial Applications

“…Moisture from agar would wet both paper towel and membrane so that Glu can be released to react with GOx to produce H 2 O 2 . H 2 O 2 generated would then react with DA-64 with the help of HRP to produce Bindschedler's green color [42] as shown in Figure 4. The color intensities developed on agar plates with and without additional supplement of 0.8% w/v glucose were compared.…”