Fusarium oxysporum strain and its application in the preparation of 3β, 7α, 15α-trihydroxyandrost-5-en-17-one
A technology of Fusarium oxysporum and hydroxyandroster, which is applied in the field of biocatalysis to achieve the effects of high reaction yield, low cost and easy industrial production
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Embodiment 1
[0028] Slope culture: glucose 30g / L, peptone 12g / L, yeast extract 1g / L, potassium dihydrogen phosphate 2g / L, agar 30g / L, pH 4.5, sterilized at 121°C for 20 minutes, cooled after sterilization to make a slope , inoculated with Fusarium oxysporum strains, cultured at 30°C for 5 days, and used as slant activated seeds.
[0029] Initial fermentation culture: glucose 30g / L, peptone 12g / L, yeast extract 1g / L, potassium dihydrogen phosphate 0.9g / L, pH 5. Sterilize at 121°C for 20 minutes, cool after sterilization. Use an inoculation needle to inoculate the mycelia of the Fusarium oxysporum strain into a 500mL Erlenmeyer flask containing 110mL of fermentation medium. After 2 days, the fermentation broth was obtained.
[0030] Substrate addition: Dissolve the substrate DHEA in acetone (100 mg / mL), add it to the 500 mL Erlenmeyer flask containing the above fermentation broth at a concentration of 100 mg / bottle. min Fermentation reaction 96 hours.
[0031] Post-treatment of the transf...
Embodiment 2
[0036] The basic operation is as in Example 1, and the assay methods are all the assay methods of the conversion rate in Example 1.
[0037] (1) Optimization of carbon source
[0038] Other components of fixed initial fermentation medium (peptone 1.2%, yeast extract 0.1%, KH 2 PO 4 0.09%, pH5.5), only changing the type of carbon source (3%) using glucose, xylose, lactose, maltose, dextrin, starch, olive oil, oleic acid respectively. Inoculate the selected Fusarium oxysporum into these eight kinds of sterilized media, culture at 30°C for 48h, add 0.1% dehydroepiandrosterone, transform in a shaker at 30°C, 210r / min for 96h Determination. The result is as figure 1 As shown, the conversion yield of compound 1 is the best when using maltose as carbon source.
[0039] After determining the carbon source as maltose, other components of the fermentation medium (peptone 1.2%, yeast extract 0.1%, KH 2 PO 4 0.09%, pH 4.5), only change the concentration of maltose (1.25 ~ 7.25%). ...
Embodiment 3
[0061] Get three inoculation loop myceliums from the inclined surface with the inoculation needle of the Fusarium oxysporum strain described in the present invention and insert respectively in 7 bottles of 250ml Erlenmeyer flasks containing 50ml liquid medium, at 30°C, shaker speed 210r / min cultivated under conditions.
[0062] The substrate dehydroepiandrosterone was dissolved in acetone (100 mg / mL), added to the fermentation broth at a concentration of 50 mg / bottle, and reacted at 30° C. and 210 r / min. Take out 3 bottles every 24h, carry out HPLC measurement as the method described in embodiment 1, the impact of different reaction times on compound 1 conversion yield sees Figure 4 .
[0063] From Figure 4 It can be seen that the conversion yield of compound 1 showed an upward trend in the first 96 hours of conversion, reached the highest around 96 hours, and then began to decline slowly, so we chose 96 hours as the reaction time after adding the substrate.
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