Filters

20212

More filters

20222
Reset filters

Settings

Author: Markel, Ulrich × Subject: directed evolution ×

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Less Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance
    (Weinheim : Wiley-VCH, 2021) Cui, Haiyang; Eltoukhy, Lobna; Zhang, Lingling; Markel, Ulrich; Jaeger, Karl-Erich; Davari, Mehdi D.; Schwaneberg, Ulrich
    Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other α/β-hydrolases. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
  • Thumbnail Image
    Item
    A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT
    (Weinheim : Wiley-VCH, 2021) Markel, Ulrich; Lanvers, Pia; Sauer, Daniel F.; Wittwer, Malte; Dhoke, Gaurao V.; Davari, Mehdi D.; Schiffels, Johannes; Schwaneberg, Ulrich
    Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases. © 2020 The Authors. Published by Wiley-VCH GmbH