Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida

Abstract

Background: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction.

Results: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL^{- 1}. 2-HPP titer, yield and productivity using the free cells were 1.2 g L^{- 1}, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g^{- 1} DCW h^{- 1}, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product.

Conclusions: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.

Keywords: 2-Hydroxypropiophenone; Benzoylformate decarboxylase (BFD); Biotransformation; Cell immobilization; Pseudomonas putida; αHydroxyketones.

Fig. 1

Effect of acetaldehyde concentration on the biotransformation efficiency. The experiments were carried out in a 100 mL Erlenmeyer flask containing fixed benzaldehyde concentration (50 mM) and varying concentrations of acetaldehyde at 30 °C and 200 rpm. Bioconversion was carried out aerobically at 30 °C and pH 7 with shaking at 200 rpm. Data are results of triplicate experiments ± SD

Fig. 2

Effect of reaction time on 2-HPP formation. Reactions were performed in a 100 mL Erlenmeyer flask containing 50 mM benzaldehyde and 600 mM acetaldehyde at 30 °C and 200 rpm. Data are results of triplicate experiments ± SD

Fig. 3

2-HPP production after 3 h with different cell concentrations. Reactions were performed in a 100 mL Erlenmeyer flask containing 50 m M benzaldehyde and 600 mM acetaldehyde at 30 °C and 200 rpm. Data are results of triplicate experiments ± SD

Fig. 4

Effect of benzaldehyde concentration on 2-HPP formation, unconsumed benzaldehyde and benzyl alcohol production. Cell concentration, acetaldehyde concentration, and reaction time were set to 0.033 g WCW/mL, 600 mM and 3 h, respectively. Data are results of triplicate experiments ± SD

Fig. 5

2-HPP profile at varying pH values. The reaction was performed at 20 mM benzaldehyde, 600 mM acetaldehyde, 0.033 g WCW/mL, 30 °C, and 200 rpm for 3 h. Data are results of triplicate experiments ± SD

Fig. 6

The profile of 2-HPP production by the encapsulated P. putida. A: 2-HPP production by a benzaldehyde concentration of 20 mM and with 0.033 g WCW/mL of immobilized cells load. B: 2-HPP production by a benzaldehyde concentration of 40 mM and with 0.033 g WCW/mL of immobilized cells load. C: 2-HPP production by a benzaldehyde concentration of 20 mM and with 0.055 g WCW/mL of immobilized cells load. Biotransformation was carried out aerobically with a constant acetaldehyde concentration of 600mM, 200 beads in 6 mL buffer at 30 ^°C and pH 7 with shaking at 200 rpm. Data are results of triplicate experiments ± SD