Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones
Artikel i vetenskaplig tidskrift, 2022

Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T50) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3 mmol ⋅ L-1 ⋅ d−1. Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.


C−C coupling

enzyme catalysis


protein engineering


Martin Peng

Karlsruher Institut für Technologie (KIT)

Dominik L. Siebert

Karlsruher Institut für Technologie (KIT)

Martin Engqvist

Chalmers, Biologi och bioteknik, Systembiologi

Christof M. Niemeyer

Karlsruher Institut für Technologie (KIT)

Kersten S. Rabe

Karlsruher Institut für Technologie (KIT)


1439-4227 (ISSN) 1439-7633 (eISSN)

Vol. 23 7 e202100468


Biokemi och molekylärbiologi

Biokatalys och enzymteknik

Organisk kemi





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