Strains and reagents
The recombinant E. coli cell was prepared and determined its initial reaction rate, yield, enantiomeric purity and relative activity according to our previous methods (Wei et al. 2017). MAA, (R/S)-HBME and tert-butyldimethyl chlorosilane (TBDMCS) were purchased from Sigma-Aldrich (USA). All other reagents and solvents were of analytical grade and used without further purification.
Cultivation of E. coli BL21(DE3)-pETduet1-gaccr-gdh cells
E. coli BL21(DE3)-pETduet1-gaccr-gdh strain culture: E. coli BL21(DE3)-pETduet1-gaccr-gdh was inoculated into 50 mL LB broth with 100 μg/mL ampicillin and cultured at 37 ℃ and 160 rpm for 12 h. Secondly, 1 mL of the strain liquid was added to 100 mL of LB broth with 100 μg/mL ampicillin and incubated at 37 ℃ and 160 rpm for approximately 4 h. When the optical density at 600 nm (OD 600) of the culture reached 1.2, the temperature was changed to 20 ℃, and then 40 μL IPTG (500 mM) was injected. Subsequently, the cells were continued to incubate at 20 ℃ and 160 rpm. After18 h of incubation, the cells were harvested by centrifugation (8000 rpm, 4 ℃, 5 min) and washed twice with normal saline.
Immobilization of E. coli BL21(DE3)-pETduet1-gaccr-gdh cells
120 mg the engineering cells (wet weight) and 4 mL sodium alginate solution (2.5%, w/v) were mixed. Then, the mixture was dropwise added to 2% (w/v) CaCl2 solution using a 5 mL syringe. After hardened for 30 min, the beads were collected and washed with Tris–HCl buffer (200 mM, pH 7.0) to obtain calcium alginate-immobilized cells, named as CA-immobilized cells. CA-immobilized cells were added to chitosan solution (0.9%, w/v, pH 5.0) for 20 min, and then were collected by filtration and washed with Tris–HCl buffer (200 mM, pH 7.0) to obtain the immobilized cells with chitosan and calcium alginate, named CS/CA-immobilized cells.
To study the total number of immobilized microbial cells within the beads, three beads were selected at random from a sample (Greenberg et al. 1995). The selected beads were ruptured to make homogenous solution and the solution was diluted 10 times. The cells within the diluted solutions were allowed to grow in petri dishes containing nutrient agar and incubated at 37 ℃ for 24 h. The cell density was obtained by colony on petri dishes.
The catalytic activity of immobilized cells was assessed by the asymmetric reduction of MAA to (R)—HBME. The reaction conditions were as follows: 4 mL Tris–HCl buffer (200 mM, pH 7.0), 100 mM MAA, 150 mM glucose, 40 °C, 200 rpm. After the reaction period, 50 μL of sample was taken, and then 200 μL of ethyl acetate was added to extract the substrate and product from the sample. The mixture was shaken with a vortex mixer for 3 min, centrifuged (12,000 r/min, 5 min). Finally, the supernatant was taken and stored at 4 ℃ for GC analysis.
Preparation of (R)-HBME from MAA reduction catalyzed by the immobilized cells
In the typical experiments, the catalysts including the free cells, CA-immobilized cells and CS/CA-immobilized cells were added to 10 mL reactor, respectively. Then, 4 mL Tris–HCl solution (200 mM, pH 7.0) consisting of 100 mM MAA and 200 mM glucose were added above reactor. The reaction was carried out at 40 ℃ and 200 rpm. Aliquots were regularly withdrawn to determine the level of the product during the reaction. The initial reaction rate was calculated according to the level of HBME at 30 min. The yield of product was equal to the determined concentration of (R)-HBME divided by the theoretical concentration of (R)-HBME.
Effect of immobilization method on the catalytic performances of the cells.
Based on the above approach preparing (R)-HBME, the catalytic performances of the free cells, CA-immobilized cells, and CS/CA-immobilized cells were compared. During the reaction, samples were analyzed to determine the yield of product by gas chromatography.
Thermal stability of the immobilized cells
The two catalysts including the free cells and CA-immobilized cells were added to 4 mL Tris–HCl solution (200 mM, pH 7.0), respectively, and incubated at the different temperature (30, 40, 50, and 60 ℃) and different time (0, 1, 2, 3, 4, 5, 6 h). The catalytic activity of the catalysts was determined on basis of the above approach in “Immobilization of E. coli BL21(DE3)-pETduet1-gaccr-gdh cells”. The relative activity was calculated as the ratio of the activity of treated CA-immobilized cells exposing to different temperature and pH after required incubation time and activity of the untreated CA-immobilized cells. The activity of the untreated CA-immobilized cells was set as 100%. The relative activity of free cells was calculated in the same way.
pH stability of the immobilized cells
The pH stability was investigated by pre-incubating the CA-immobilized cells in different pH buffers range of 4.0–8.0 using various buffer systems at 50 mM. The incubated temperature is 4 ℃. The buffers were as follows: citrate–phosphate (pH 4.0–7.0) and Tris–HCl (pH 7.0–8.0). After incubation, immobilized cells were added to reaction mixture solution and reacted for a period of time at 40 ℃. During the incubation process, the catalyst was taken out to determine the catalytic activity according to the above approach.
The free cells and CA-immobilized cells were stored in Tris–HCl buffer (200 mM, pH 7.0) at 4 ℃. The catalytic activity of the stored catalysts at the different stored time was determined on basis of the above approach.
The reusability of the free cells and CA-immobilized cells were compared by determining the initial rate during repeated usages. The asymmetric reduction reaction catalyzed by free cells and immobilized cells was 2 h and 4 h per batch, respectively. CA-immobilized cells were obtained by 0.22 μm water microporous membrane filtration, and the free cells were collected by centrifugation (8000 rpm, 5 min, 4 ℃). These deposits were washed twice with Tris–HCl (200 mM, pH 7.0). The recovered catalysts were continued to use for the next batch. The activity of the catalysts in the first cycle was defined as 100%.
Effect of MAA levels on the biocatalytic synthesis of (R)-HBME
4 mL Tris–HCl buffer (200 mM, pH 7.0) reaction systems consisted of CA-immobilized cells containing 240 mg wet cells, MAA, and glucose. The level of MAA ranged from 300 to 700 mM, and the molar ratio of glucose to MAA was at 1:2. The pH of the reaction solution was adjusted with Na2CO3 and His powder of 2:1 (mass ratio), and the pH was adjusted to 7.0 every 0.5 h. The reaction was conducted at 40 ℃ and 200 rpm. When reaction for 30 min, samples were withdrawn to determine the initial rate, and after the reaction completed, samples were taken out to measure the yield of the product.
The product was analyzed by an Agilent 6890 N gas chromatograph equipped with a flame ionization detector (FID). The optimal experimental conditions of gas chromatography were as follows: CP-Chiralsil-Dex-CB (USA) column, the inlet temperature at 190 °C, column temperature at 90 °C and detector temperature at 210 °C; flow rate of carrier gas at 1.6 mL/min; split ratio of 30:1. The retention time of MAA and (R)-HBME was 4.85 and 6.11 min, respectively.