An interfacial self-assembled carbonyl reductase and its application in the synthesis of ethyl (r)-3-hydroxy-3-phenylpropionate
A technology of ethyl phenylpropionate and carbonyl reductase, which is applied in the direction of oxidoreductase, microbial-based methods, enzymes, etc., can solve the problems of high price, low substrate processing amount, and containing precious metals, and achieve an increase in conversion rate , Improve catalytic efficiency, reduce the effect of enzyme activity loss
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Embodiment 1
[0030] Example 1 Screening and identification of Candida tropicalis CGMCC No.15016
[0031] 1. Strain screening process:
[0032] (1) culture medium
[0033] Enriched plate medium (g / L): Glucose 20, (NH 4 ) 2 SO 4 1, KH 2 PO 4 0.5,K 2 HPO 4 1.5, NaCl1, MgSO 4 0.1, agar 20, the solvent is deionized water, and the pH value is natural.
[0034] Slant Medium (g / L): Glucose 20, (NH 4 ) 2 SO 4 1, KH 2 PO 4 0.5,K 2 HPO 4 1.5, NaCl 1, MgSO 4 0.1, agar 20, the solvent is deionized water, and the pH value is natural.
[0035] Seed medium (g / L): glucose 20, yeast powder 3, (NH 4 ) 2 SO 4 1, KH 2 PO 4 0.5,K 2 HPO 4 1.5, NaCl1, MgSO 4 0.1, the solvent is deionized water, and the pH value is natural. The composition of the fermentation medium is the same as that of the seed medium.
[0036] (2) Culture method
[0037] Incline culture: culture at 30°C for 3-5 days.
[0038] Seed cultivation: transfer the cultivated slant into a 100mL Erlenmeyer flask f...
Embodiment 2
[0062] Embodiment 2: the preparation of carbonyl reductase
[0063] 1. Wet bacteria
[0064] (1) Inoculate Candida tropicalis CGMCC No.15016 into the slant medium and culture at 30°C for 3-5 days to obtain slant bacteria; slant medium composition: glucose 20g / L, ammonium sulfate 1g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1.5g / L, sodium chloride 1g / L, magnesium sulfate 0.1g / L, agar 20g / L, the solvent is deionized water, and the pH value is natural.
[0065] (2) Inoculate the bacterium on the slope into a 100 mL Erlenmeyer flask filled with 25 mL of seed medium, and culture at 30° C. and 120 r / min for 24 hours to obtain seed liquid. Seed medium composition: glucose 46g / L, yeast juice 25g / L, potassium dihydrogen phosphate 4g / L, dipotassium hydrogen phosphate 4g / L, sodium chloride 0.1g / L, solvent is deionized water, pH value is natural.
[0066] (3) Put the seed solution cultivated for 24 hours into a 1000 mL Erlenmeyer flask filled with 250 mL ...
Embodiment 3
[0075] Example 3 Preparation and Application of Interfacial Self-Assembly Carbonyl Reductase
[0076] (1) Preparation of interfacial self-assembled carbonyl reductase
[0077] Add 5mg of carbonyl reductase enzyme solution (180U / mg) prepared in Example 2 into 6.25ml, pH 7.9, 0.05M Tris-HCl buffer solution to prepare 0.8g / L enzyme solution, then add 3.75g / L poly Styrene (Mw 10000Da) toluene solution 10ml was reacted for 1 hour in the dark at a shaker speed of 80r / min at 30°C. After the reaction is completed, centrifuge at 1200r / min for 10min, and the interfacial self-assembling enzyme is distributed on the two-phase interface of water and organic phase (that is, the upper layer is toluene, the middle is the interfacial self-assembling enzyme, and the lower layer is Tris-HCl buffer solution). The buffer and organic solvent were removed, and the middle layer was washed 3 times with toluene and pH 7.9, 0.05M Tris-HCl buffer solution successively to remove free carbonyl reductase a...
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