Abstract
BackgroundPublished biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM. Enzyme deactivation was identified as the major limitation and was attributed to adduct formation of the aldehyde substrate with amino acid residues of the reductase.ResultsA single point mutant of Candida tenuis xylose reductase (CtXR D51A) with very high catalytic efficiency (43·103 s−1 M−1) for (S)-2-phenylpropanal was found. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst expressing the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 gsubstrate/gcell-dry-weight was selected as a suitable compromise between product ee and the conversion ratio. A catalyst loading of 40 gcell-dry-weight was used to convert 1 M racemic 2-phenylpropanal into 843 mM (115 g/L) (S)-phenylpropanol with 93.1% ee.ConclusionThe current industrial production of profenols mainly relies on hydrolases. The bioreduction route established here represents an alternative method for the production of profenols that is competitive with hydrolase-catalyzed kinetic resolutions.
Highlights
Published biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM
Candida tenuis Xylose reductase (CtXR), which converts xylose to xylitol in the central sugar metabolism of Candida tenuis, is a member of the aldo–keto reductase (AKR) superfamily
Like many other AKRs, CtXR has broad substrate specificity, and several substrate-binding site mutants with improved specificity for aromatic ketones were used in the synthesis of (R)ethyl mandelates and (S)-phenylethanols [18, 19]
Summary
Enzymes and strains Racemic 2-phenylpropanal (98%), racemic 2-phenylpropanol (97%), acetophenone (99%) and racemic 1-phenylethanol (≥ 98%) were purchased from Sigma-Aldrich/ Fluka (Vienna, Austria); (S)-2-phenylpropanal (95%) and (R)-2-phenylpropanal (95%) were from Accela (Prien – Chiemsee, Germany); 2-hydroxypropyl-β-cyclodextrin (HBC, batch number OH053931501) was from Carbosynth (Berkshire, UK); NAD+ (98%), acetonitrile (≥ 99) and ethyl acetate (≥ 99,9%) were from Roth (Karlsruhe, Germany). The protein expression of CtXR (wild-type and mutants D51A, W24F, W24Y, N310A, N310D) was described previously [24]. The construction of the co-expression strain was previously described by Rapp et al [19]. Cultivation of the co-expression strain was described previously [19], and is summarized in the Additional file 1. GC analyses, ethyl acetate (1 mL) was added to 1 mL of a reaction mixture in a 2 mL Eppendorf tube. The tubes were filled up to 10 mL with ethyl acetate, vortexed and centrifuged for 15 min, 25 °C and 3220 g for extraction. For NMR analyses, substrate/product present in ethyl acetate after extraction was transferred into round-flasks and evaporated under reduced pressure. The isolated substrate/product was directly dissolved in deuterated methanol (20 μL substrate/product + 680 μL solvent)
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