Trichoderma asperellum and application thereof in synthesizing (R)-[3,5-dual (trifluoromethyl) phenyl] ethanol
A technology of Trichoderma aculeatus and trifluoromethyl, which is applied in the field of biocatalysis and can solve the problems that are not suitable for industrial applications
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Embodiment 1
[0057] Example 1: Obtaining wet mycelium
[0058] The seed medium formula is as follows: glucose 25g / L, peptone 27.5g / L, (NH 4 ) 2 SO 4 3g / L, KH 2 PO 4 1g / L, MgSO 4 1.25g / L, ZnSO 4 ·7H 2 O 0.008g / L, prepared with distilled water, pH 6.0.
[0059] The formula of the fermentation medium is the same as that of the seed medium, which is prepared with tap water;
[0060] Insert the mature slant strain into a 250mL shake flask containing 70mL seed culture medium, cultivate at 30°C and 200r / min for 19 hours, and then transfer the seed solution to a 70mL fermentation at an inoculum volume ratio of 5% Culture medium in a 250mL shake flask at 30°C and 200r / min for 21 hours. After the cultivation, the fermentation broth was centrifuged and washed with phosphate buffer, and the wet mycelium was collected for use.
Embodiment 2
[0061] Example 2: Detection method of product concentration
[0062] After the completion of the biotransformation reaction, the reaction solution was extracted with an equal volume of ethyl acetate (10 mL), and the concentration of the product and unreacted substrate in the extract was analyzed by gas chromatography and quantified by the internal standard method. The internal standard is dodecane. Take 1mL extract and add 2μL dodecane for analysis. The specific operating conditions of gas chromatography are: injector temperature 250℃, detector temperature 250℃, column temperature 80℃, hold for 2min, raise temperature at 4℃ / min to 180℃ for 10min, carrier gas is nitrogen, flow rate is 1.5mL / min , The split ratio is 1:20, and the injection volume is 1μL. According to the spectrum obtained by gas chromatography, the relative correction factor method is used to calculate the concentration of the product in the reaction solution, and then the yield (Yield) and the ee value of the pr...
Embodiment 3
[0064] The wet mycelium obtained in Example 1 was suspended in 10 mL of phosphate buffer (pH 5.7). The concentration of the wet mycelium by dry weight was 35 g / L; 10 mmol / L of [3,5-bis( Trifluoromethyl)phenyl]ethanone was used as a substrate, and 50g / L glucose was added as an auxiliary substrate, and the reaction was placed in a shaker at 30°C and 200r / min for 10 hours. Using the detection method of Example 2, the concentration of the product (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol is 4.2 mmol / L, the optical purity ee value is 94.7%, and the yield is 42.2% (thorn The gas chromatogram of the bioreduction reaction extract of Trichoderma ZJPH0810 strain is shown figure 2 ).
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