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An Enzymatic 2‐Step Cofactor and Co‐Product Recycling Cascade towards a Chiral 1,2‐Diol. Part II: Catalytically Active Inclusion Bodies
Author(s) -
Jäger Vera D.,
Piqueray Maja,
Seide Selina,
Pohl Martina,
Wiechert Wolfgang,
Jaeger KarlErich,
Krauss Ulrich
Publication year - 2019
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201900189
Subject(s) - chemistry , benzaldehyde , cofactor , alcohol dehydrogenase , enzyme , biocatalysis , benzyl alcohol , substrate (aquarium) , diol , combinatorial chemistry , alcohol , organic chemistry , catalysis , reaction mechanism , oceanography , geology
Optimal performance of multi‐step enzymatic one‐pot cascades requires a facile balance between enzymatic activity and stability of multiple enzymes under the employed reaction conditions. We here describe the optimization of an exemplary two‐step one‐pot recycling cascade utilizing the thiamine diphosphate (ThDP)‐dependent benzaldehyde lyase from Pseudomonas fluorescens ( Pf BAL) and the alcohol dehydrogenase from Ralstonia sp . ( R ADH) for the production of the vicinal 1,2‐diol (1 R ,2 R )‐1‐phenylpropane‐1,2‐diol (PPD) using both enzymes as catalytically active inclusion bodies (CatIBs). Pf BAL is hereby used to convert benzaldehyde and acetalydehyde to ( R )‐2‐hydroxy‐1‐phenylpropanone (HPP), which is subsequently converted to PPD. For recycling of the nicotinamide cofactor of the R ADH, benzyl alcohol is employed as co‐substrate, which is oxidized by R ADH to benzaldehyde, establishing a recycling cascade. In particular the application of the R ADH, required for both the reduction of HPP and the oxidation of benzyl alcohol in the recycling cascade is challenging, since the enzyme shows deviating pH optima for reduction (pH 6–10) and oxidation (pH 10.5), while both enzymes show only low stability at pH>8. This inherent stability problem hampers the application of soluble enzymes and was here successfully addressed by employing CatIBs of Pf BAL and R ADH, either as single, independently mixed CatIBs, or as co‐immobilizates (Co‐CatIBs). Single CatIBs, as well as the Co‐CatIBs showed improved stability compared to the soluble, purified enzymes. After optimization of the reaction pH, the R ADH/ Pf BAL ratio and the co‐solvent content, we could demonstrate that almost full conversion (>90%) was possible with CatIBs, while under the same conditions the soluble enzymes yielded at most >50% conversion. Our study thus provides convincing evidence that (Co‐)CatIB‐immobilizates can be used efficiently for the realization of cascade reactions, i. e. under conditions where enzyme stability is a limiting issue.