Figure 1.
The biomass of Acetobacter sp. CCTCC M209061 and the reduction activity of AcCR during its growing process.
Biomass (▪); Reduction activity (○).
Figure 2.
SDS-PAGE analysis of the purified AcCR.
Gel was stained with 0.05% Coomassie Blue R-250: lane 1, molecular weight markers; lane 2, the purified enzyme with a molecular mass of 27 kDa.
Table 1.
Purification of AcCR from Acetobacter sp. CCTCC M209061.
Figure 3.
Relative activity of AcCR at different temperatures.
(A) Reduction of 4′-chloroacetophenone using NADPH as cofactor (▪); Oxidation of isopropanol using NADP+ as cofactor (□); (B) Reduction of 4′-chloroacetophenone using NADH as cofactor (▾); Oxidation of isopropanol using NAD+ as cofactor (□); The activity was measured in the temperature range of 10–45°C. The pH value was 5.0 (NADH as cofactor) or 7.5 (NADPH as cofactor) for reduction of 4′-chloroacetophenone or 8.0 for oxidation of isopropanol, respectively. The relative activity of AcCR at each optimal temperature for reduction and oxidation reactions was defined as 100%. (C) Thermal stability (•). The residual activity of AcCR after 5 h incubation (buffer pH 6.5) at varying temperatures were measured at 25°C.
Figure 4.
Relative activity of AcCR at different pH values.
(A) Reduction of 4′-chloroacetophenone using NADPH as cofactor (▪); (B) Oxidation of isopropanol using NADP+ as cofactor (□); (C) Reduction of 4′-chloroacetophenone using NADH as cofactor (▾); (D) Oxidation of isopropanol using NAD+ as cofactor (□). The buffers used were 50 mM citrate-phosphate (pH 4.0–8.0), Tris-HCl (pH 7.5–8.5) and glycine–NaOH (pH 8.6–9.5) buffers. The enzyme's activity was measured in the pH range of 4.0–9.5 at 25°C for reduction of 4′-chloroacetophenone and at 35°C for oxidation of isopropanol. The relative activity of AcCR at each optimal pH for reduction and oxidation reactions was defined as 100%.
Figure 5.
(A) pH stability (▪); The residual activities after incubation at various pH values (pH 4.5–8.0) for 5 days at 4°C were measured at buffer pH 5.0 and 25°C (NADH as cofactor). (B) Storage stability (▾). The residual activities after incubation at pH 6.5 for 30 days at 4°C were measured at buffer pH 5.0 and 25°C (NADH as cofactor). The relative activity of AcCR without incubation was defined as 100%.
Table 2.
Effects of various additives on the activity of AcCR.
Figure 6.
Effects of different substrates on AcCR-catalyzed asymmetric reduction of prochiral ketones.
Table 3.
Kinetic parameters of AcCR for reduction of 4′-chloroacetophenone and oxidation of isopropanol.
Table 4.
Effects of coenzyme supply methods on AcCR-mediated asymmetric reduction of prochiral ketones.
Figure 7.
The gene sequences of AcCR from Acetobacter sp. CCTCC M209061.