Method for preparing beta-halogenated ether and beta-halogenated alcohol under catalysis of peroxidase
A peroxidase and peroxidase technology, which is applied in the biological field to achieve the effects of simple raw materials, mild reaction conditions and good stability
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[0082] The preparation method of UPO enzyme in the following examples is as follows:
[0083] Pichia pastoris cell culture broth containing rAaeUPO was 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 using the ABTS assay in NaPi buffer at 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 NGC chromatography system (Biorad). Separation was performed on a Q Sepharose FF 30-mL cartridge at a flow rate of 5 mL / min. After 90 mL, the retained protein was eluted with a gradient of 0-50% NaCl in 450 mL, followed by a gradient of 50-100% in 50 mL and 100% NaCl in 75 mL of peroxidase activity followed by an H 2 o 2 Oxidation in the presence of ABTS, appropriate fractions were pooled, concentrated and dialyzed against 100 mM sodium phosphate buffer (pH 7). Pur...
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 (sodium dihydrogen phosphate 100 mM), add absolute ethanol (13.5mL) to the above buffer solution, and then sequentially add 0.93mL with a mass fraction of 30 % hydrogen peroxide solution (concentration is 5.38mol / L, concentration in the reaction system is 100mM), 0.59g potassium bromide and 0.820g cyclohexene, finally add 150μL VCPO enzyme (concentration of raw material enzyme is 13.5mg / mL, reaction The enzyme concentration in the system is 500nM). After reacting for about 3 hours, 0.59g potassium bromide, 0.93mL hydrogen peroxide (concentration: 5.38mol / L, concentration in the reaction system: 100mM) and 0.820g cyclohexene were added for a total of three times. Add in separate batches to ensure maximum catalytic activity of the enzyme.
[0088] The above reaction system was stirred and reacted at 30°C for ...
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 (Concentration is 5.38mol / L, the concentration in the reaction system is 100mM), 0.59g potassium bromide and 0.16g styrene, finally add 150μL VCPO enzyme (the same as the above enzyme concentration is 13.5mg / mL, the enzyme concentration in the reaction system is 500nM). After reacting for about 3 hours, 0.59 g of potassium bromide, 0.93 mL of hydrogen peroxide (concentration: 5.38 mol / L) and 0.16 g of styrene were added three times in total.
[0092] The above reaction system was stirred and reacted in a water bath at 30°C for 9 hours, then the reaction was terminated, and 100 mL of ethyl acetate was added for extraction twice in total. The organic pha...
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