Synthetic method of chiral 1,3-dihydroxyl-1-aryl acetone compound
A technology of arylacetone and hydroxypyruvate, applied in the field of biocatalysis, can solve the problems of narrow transketolase and low activity
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Embodiment 1
[0022] Embodiment 1: Construction and cultivation of genetically engineered bacteria
[0023]The specific construction and cultivation method of the genetically engineered bacteria producing pyruvate decarboxylase is as follows: the pyruvate decarboxylase nucleic acid sequence SEQ ID NO: 3 derived from Macrococcushajekii is gene-synthesized, constructed into a pET series vector, and placed in the host bacteria Escherichia coli for heterologous expression. Thaw the strains stored at -80°C, streak on the plate, and culture overnight in a constant temperature incubator at 37°C. Select a single colony on the plate and inoculate it into 20mL LB medium containing the corresponding antibiotic, cultivate it for about 12 hours as a seed solution, inoculate it into 700mL LB medium containing the corresponding antibiotic according to the inoculation amount of 1%, and inoculate it on a shaker at 37°C and 200rpm Grow to OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mmol / L t...
Embodiment 2
[0025] Example 2: Pyruvate decarboxylase catalyzes benzaldehyde and hydroxypyruvate to prepare 1,3-dihydroxy-1-phenylacetone.
[0026] (1) 0.26g hydroxypyruvate (25mM), 0.01g ThDP (0.1mM), 3.00g pyruvate decarboxylase genetically engineered bacteria, 20mM benzaldehyde (dissolved in 20% DMSO) were added in 80mL water, and the pH of the reaction system was adjusted to 8.0, react at 25°C for 24h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the extraction was confirmed by thin-layer chromatography. The organic solvent was removed by rotary evaporation to obtain the crude product, the substrate conversion rate and product yield were detected by gas phase, and the ratio of product and by-product isomers was detected by liquid phase HPLC. The test results are: the pyruvate decarboxylase catalyzes the R-configuration product, the ee value is >99%, the benzaldehyde substrate conversion rate is 61%, and the ratio ...
Embodiment 3
[0032] Example 3: Preparation of 1,3-dihydroxy-1-arylacetone by genetically engineered bacteria with pyruvate decarboxylase and D-amino acid oxidase.
[0033] With 2.00g D-amino acid oxidase genetically engineered bacteria, 3.00g pyruvate decarboxylase genetically engineered bacteria, 1.05g D-Serine (100mM), 0.02g MgCl 2 ·6H 2 Add O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) into 80mL water, adjust the pH of the reaction system to 7.0, incubate at 25°C, 200rpm for 1h, then add 20mM benzaldehyde (dissolved in 20% DMSO) to react for 24h . After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the extraction was confirmed by thin-layer chromatography. The organic solvent was removed by rotary evaporation to obtain the crude product, the substrate conversion rate and product yield were detected by gas phase, and the ratio of product and by-product isomers was detected by liquid phase HPLC. The test results are: ...
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