A kind of method for preparing β-haloether and β-halohydrin catalyzed by peroxidase
A technology of peroxidase and peroxidase is applied in the biological field to achieve the effects of being beneficial to large-scale production, simple raw materials and mild reaction conditions
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[0082] The preparation method of UPO enzyme in the following examples is as follows:
[0083] Pichia pastoris cell cultures containing rAaeUPO were clarified by centrifugation at 8000 rpm for 2 hours at 4°C. The supernatant was filtered through a 20 μm filter and kept at -80°C. rAaeUPO activity was determined by using the ABTS assay in NaPi buffer pH 5.0;
[0084] Protein purification: The supernatant was concentrated and dialyzed against 100 mM sodium phosphate, pH 7. AaeUPO was purified in one step using an NGC chromatography system (Biorad). Separations were performed on Q Sepharose FF 30-mL cartridges at a flow rate of 5 mL / min. After 90 mL, the retained protein was eluted with a 0-50% NaCl gradient in 450 mL, followed by a 50-100% gradient in 50 mL and 100% NaCl in 75 mL peroxidase activity followed by peroxidase activity in H 2 O 2 In the presence of ABTS oxidation, the appropriate fractions were pooled, concentrated and dialyzed against 100 mM sodium phosphate buff...
Embodiment 1
[0085] Embodiment 1, the preparation of compound 1-bromo-2-ethoxycyclohexane:
[0086]
[0087] In a 100mL reaction flask, add 31.5mL of pH=6 phosphate buffer solution (100 mM sodium dihydrogen phosphate), add absolute ethanol (13.5mL) to the above buffer solution, and then add 0.93mL of phosphate buffer solution with a mass fraction of 30 % hydrogen peroxide solution (concentration is 5.38mol / L, the concentration in the reaction system is 100mM), 0.59g potassium bromide and 0.820g cyclohexene, finally add 150μL VCPO enzyme (the raw material enzyme concentration is 13.5mg / mL, the reaction The enzyme concentration in the system was 500 nM). After about 3 hours of reaction, add 0.59g potassium bromide, 0.93mL hydrogen peroxide (concentration is 5.38mol / L, the concentration in the reaction system is 100mM) and 0.820g cyclohexene, and add three times in total. Add in separate batches to ensure maximum catalytic activity of the enzyme.
[0088] The above reaction system was st...
Embodiment 2
[0089] Embodiment 2, the preparation of compound (2-bromo-1-propoxyethyl) benzene:
[0090]
[0091]In a 100mL reaction flask, add 31.5mL of phosphate buffer solution (same pH=6 as above), then add 13.5mL of absolute ethanol to dissolve in the buffer solution, and then add 0.93mL of 30% hydrogen peroxide solution in sequence (the concentration is 5.38mol / L, the concentration in the reaction system is 100mM), 0.59g potassium bromide and 0.16g styrene, and finally 150 μL VCPO enzyme (the same as the above enzyme concentration is 13.5mg / mL, the enzyme concentration in the reaction system is 500nM). After about 3 hours of reaction, 0.59 g of potassium bromide, 0.93 mL of hydrogen peroxide (concentration of 5.38 mol / L) and 0.16 g of styrene were added three times in total.
[0092] The above reaction system was stirred in a water bath at 30°C for 9 hours, then the reaction was terminated, 100 mL of ethyl acetate was added for extraction twice, the organic phases were combined, ...
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