Method for synthesizing aromatic aldehyde perfume compound by enzyme method
A technology for the synthesis of aromatic aldehydes by enzymatic methods, applied in the direction of oxidoreductase, fermentation, etc., can solve the problem of low conversion rate and achieve the effect of simple operation and mild reaction conditions
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Embodiment 1
[0064] Example 1: Preparation of PeAAOx
[0065] Recombinant expression of PeAAOx by E. coli system: The plasmid pFLAG1-PeAAOx was transformed into E. coli competent cells E.coli BL21 star (DE3), and after thermal activation, it was evenly coated on ampicillin-resistant LB cells with a coating rod On solid medium, then placed at 37°C for 12-18h incubation until a single colony was clearly observed. The monoclonal colonies on the transformed plates were picked in 5 mL of resistant LB liquid medium and cultured overnight at 37°C and 180 rpm. The bacterial liquid was scaled up to 100 mL of resistant TB liquid medium according to the proportion of 1%, and cultivated at 37° C. and 180 rpm for 2-3 hours until the OD600 was 0.7-0.8. Scale up to 500 mL of TB liquid medium at a ratio of 2.5%, and culture at 37°C and 180 rpm for 2-3 hours until OD600 is 0.8-1.0. 1 mM IPTG was added for induction and cultured at 37°C and 180 rpm for 4 h.
[0066] The fermentation broth was centrifuged...
Embodiment 2
[0068] Example 2: Comparison of PeAAOx-coupled AaeUPO cascade catalysis and single-enzyme catalysis
[0069] (1) PeAAOx-coupled AaeUPO cascade catalysis experiment: add phosphate buffer solution (50mM, pH 7.0), PeAAOx enzyme solution (final concentration: 30 μM), and AaeUPO enzyme solution (final concentration: 2 μM), and then added the substrate veratrol (final concentration of 50 mM, the substrate was pre-dissolved in acetonitrile), and the total volume of the reaction was 1 mL. The reaction flask was placed on a round-hole reaction rack, and then the whole was placed in a constant temperature oil bath for 6, 12, 24 and 36 hours at a reaction temperature of 40 °C and a stirring speed of 500 rpm.
[0070] PeAAOx single-enzyme catalysis experiment: add phosphate buffer solution (50mM, pH 7.0), PeAAOx enzyme solution (final concentration: 32μM), and substrate veratrol (final concentration: 50mM) into a 4mL transparent glass reaction flask. The substrate was predissolved in ace...
Embodiment 3
[0077] Example 3: The effect of PeAAOx enzyme addition on the oxidation of veratrol
[0078]
[0079] According to the method of Example 2, the reaction flask was placed in a constant temperature oil bath, and the reaction was carried out under the conditions of 30° C. and 500 rpm. After the reaction was completed, it was extracted with ethyl acetate containing 25mM n-dodecane internal standard, and dried with anhydrous sodium sulfate. After centrifugal 3min at 12000rpm, get the upper organic phase and carry out gas phase detection (detection method is the same as in Example 2, and the gas chromatogram is as follows: Figure 5 shown).
[0080] The result of the reaction is Image 6 Shown: It can be seen from the figure that the reaction effect is the best when the amount of PeAAOx enzyme is 30 μM. After 24 hours of reaction, the product concentration reaches 35 mM, and the substrate conversion rate is 70%.
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