Organic-inorganic nanocrystal reductase to promote green asymmetric synthesis

Abstract

An acetophenone reductase from Geotrichum candidum (GcAPRD) was immobilized by the organic-inorganic nanocrystal method. The GcAPRD nanocrystal presented improved stability and recyclability compared with those of the free GcAPRD. Moreover, the GcAPRD nanocrystal reduced broad kinds of ketones with excellent enantioselectivities to produce beneficial chiral alcohols such as (S)-1-(3',4'-dichlorophenyl)ethanol with >99% yield and >99% ee. The robust and versatile properties of the GcAPRD nanocrystal demonstrated an approach to promote green asymmetric synthesis and sustainable chemistry.

Fig. 1. Optimization of GcAPRD nanocrystal preparation (a) metal ions, (b) metal ion (Co²⁺) concentration, (c) PBS concentration, (d) PBS pH, and (e) protein concentration. Left and right column depict the immobilization yields and relative reaction yields, respectively. The reaction yield of free GcAPRD was set to be 100%. *Very small precipitation was found. The preparation and reaction conditions are described in the section 2.4.

Fig. 2. SEM images of GcAPRD nanocrystal formed under various protein concentrations, (a and b) control (no enzyme), (c and d) 0.02 mg mL⁻¹, (e and f) 0.10 mg mL⁻¹, (g and h) 0.50 mg mL⁻¹, and (i and j) 2.00 mg mL⁻¹.

Fig. 3. The proposed formation mechanism of GcAPRD nanocrystal, comprised of 3 steps: (1) nucleation, (2) aggregation, and (3) anisotropic growth.

Fig. 4. Temperature and pH profile, and stability of free GcAPRD (orange-circle line) and GcAPRD nanocrystal (blue-square line). (a) temperature profile, (b) pH profile, and (c) storage stability. Enzyme activities of free GcAPRD and GcAPRD nanocrystal at 37 °C for (a), HEPES–NaOH buffer (pH 7.0) for (b), and day 0 for (c) were set to be 100%. For the pH profile, the enzyme activity was measured in MES–NaOH buffer (0.10 M) (dotted line), HEPES–NaOH buffer (0.10 M) (line), and glycine–NaOH buffer (0.10 M) (broken line). The activity assay is described in the section 2.4.

Fig. 5. Recyclability of GcAPRD nanocrystal. The reaction condition is described in the section 2.4.