Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones
© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:23 |
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| Enthalten in: |
Chembiochem : a European journal of chemical biology - 23(2022), 7 vom: 05. Apr., Seite e202100468 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Peng, Martin [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 07.04.2022 Date Revised 21.07.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/cbic.202100468 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a C−C coupling | |
| 650 | 4 | |a enzyme catalysis | |
| 650 | 4 | |a immobilization | |
| 650 | 4 | |a lyases | |
| 650 | 4 | |a protein engineering | |
| 650 | 7 | |a Benzaldehydes |2 NLM | |
| 650 | 7 | |a Ketones |2 NLM | |
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| 700 | 1 | |a Engqvist, Martin K M |e verfasserin |4 aut | |
| 700 | 1 | |a Niemeyer, Christof M |e verfasserin |4 aut | |
| 700 | 1 | |a Rabe, Kersten S |e verfasserin |4 aut | |
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