Method for catalyzing, reducing and producing (R)-4-substituent phenethyl alcohol utilizing Acetobacter
A kind of technology of acetic acid bacteria and acetophenone, which is applied in the field of asymmetric synthesis of biocatalyzed chiral compounds, can solve the problems of inhibition, low catalyst activity, decreased cell activity, etc., and achieve the effect of increasing stability and benefiting recycling
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Embodiment 1
[0060] Put 2g of Chinese kefir grains into a 250mL Erlenmeyer flask filled with 100mL of tomato medium, culture at 28°C for 24h, and transfer and culture continuously in tomato medium for 3 times under the same conditions. Bacterial solution was properly diluted and spread on solid separation medium by ten-fold dilution method, and cultured at 28°C for 24-36 hours to obtain a single colony. After purification, the slant was preserved for identification. Tomato medium: tomato juice 200mL / L, yeast extract 10g / L, glucose 10g / L, peptone 10g / L, pH 6.0.
[0061] The strain XZY003 identified as Acetobacter was transferred from the slant to the tomato medium, cultured at 30°C, 160r / min for 24h, centrifuged (3500r / min, 15min, 4°C), washed twice to obtain wet bacteria. Then disperse the wet cells in distilled water of equal weight, then add 4 times the weight concentration of the wet cells to 20g / L sodium alginate, stir evenly, and use 5 # Inject the above suspension into 200 mL of CaC...
Embodiment 2
[0063] The strain XZY003 identified as Acetobacter was transferred from the slant to the tomato medium, cultivated at 30°C, 160r / min for 24h, centrifuged (8000r / min, 10min, 4°C), washed twice, and wet cells were obtained. Then disperse the wet cells in distilled water of equal weight, and then add 6 times the weight concentration of the wet cells to 10g / L sodium alginate, stir evenly, and use 5 # Inject the above suspension into 200 mL of CaCl with a concentration of 20 g / L 2 solution, harden at 4°C for 4 hours, wash the hardened calcium alginate gel balls (diameter 2-3mm) with distilled water three times, and then transfer into 200mL containing CaCl 2 In the glucose solution, the final concentration is 0.5g / L CaCl 2 , 200g / L glucose, placed in 4 ℃ of refrigerators and preserved for later use, the immobilized Acetobacter cell granule of the wet thalline that obtains by embodiment 2, its diameter size is 2-3mm, spherical shape, general mechanical strength, its cell content 10...
Embodiment 3
[0065]The triethanolamine hydrochloride buffer solution (triethanolamine concentration in hydrochloric acid is 0.1mol / L, pH 7.0) of 4mL is packed in the Erlenmeyer flask of tool stopper, then add the immobilized Acetobacter cell of the wet thalline prepared in embodiment 2 respectively Particles and isopropanol and 4-bromoacetophenone form a mixture, and the concentrations of isopropanol and 4-bromoacetophenone in the mixture are 130.6mmol / L and 6mmol / L respectively, at 30°C, 180r / min, immobilized Acetobacter The weight-to-volume ratio of the cell particles to the mixture was 1.0 g / mL, and the reaction was carried out for 2 hours to obtain (R)-4-bromophenethyl alcohol. The yield of (R)-4-bromophenethyl alcohol was 85.4% by gas phase detection, and the enantiomeric purity of (R)-4-bromophenethyl alcohol was More than 99% e.e.
[0066] Gas phase detection method: The initial temperature of the chromatographic column is 145°C, and after maintaining for 10 minutes, it rises to 150...
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