A kind of monoamine oxidase and its application in the synthesis of chiral azabicyclic compounds
A monoamine oxidase and compound technology, applied in the field of bioengineering, can solve the problems of low selectivity, low yield, incomplete reaction, etc.
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Embodiment 1
[0076] The enzyme activity assay of embodiment 1 monoamine oxidase
[0077] Dissolve 5mmol / L benzylamine in 50mmol / L phosphate buffer to obtain a substrate solution, take 1mL and add 10μL enzyme solution, react at 30°C for 20min, add 400μL dinitrophenylhydrazine (1mmol / L, dissolved in 3M HCl), and then Add 2.4mL of 1.25M NaOH, react for 5min, and detect the absorbance at a wavelength of 450nm.
[0078]Optical purity: Agilent1260 series HPLC, chiral column is ChiralpakAD-H (4.6×250mm, Diacel Chemical Ind.Ltd.), analyzed at 25°C, mobile phase is 90% n-heptane, 10% ethanol, 0.1% trifluoroacetic acid, The flow rate is 1 mL / min, and the detection wavelength is 220 nm. The retention time of the S-configuration product was 7.4min, and the retention time of the R-configuration product was 8.9min.
Embodiment 2
[0079] Embodiment 2 screening monoamine oxidase
[0080] Soil sample DNA (Chroma Spin TE-1000, Clontech Laboratories, Inc.USA) was collected from the campus of Wuhan University, partially digested with Sau3AI, and 1-4kb fragments were collected by electrophoresis, recovered and ligated to the BamHI site of pUC19 to obtain a plasmid library ; Transform the library into E.coli DH10b, smear it on an LB plate containing 100 μg / mL ampicillin, select positive clones and transfer it to a 96-deep well plate with 500 μL LB (100 μg / mL ampicillin), and incubate at 37°C for 4 hours Add 1mmol / L IPTG for induction, continue culturing overnight at 30°C, then take 10μL of each deep-well culture into a new 96-well plate containing 50mmol / L sodium phosphate buffer (pH7.5), freeze and thaw repeatedly at -80°C to freeze and thaw the bacteria For cleavage, add 5mmol / L benzylamine, incubate at 30°C for 30 minutes, then add 400μL dinitrophenylhydrazine (1mmol / L, dissolved in 3M HCl), then add 2.4mL1...
Embodiment 3
[0081] Construction and expression of embodiment 3 monoamine oxidase recombinant bacteria
[0082] Adopt primer PF (nucleotide sequence is SEQ ID NO: 3) and PR (nucleotide sequence is SEQ ID NO: 4) PCR amplification monoamine oxidase full-length gene (results see figure 1 ), the PCR product was digested with NdeI / XhoI and connected to pET28, and transformed into E.coli BL21(DE3), cultured on LB plates containing kanamycin (50μg / mL), and selected positive colonies to 100mL liquid LB culture Medium for culture, the overnight culture was transferred to fresh 1L LB culture medium and cultivated to OD 600 To 0.6 ~ 0.8, add IPTG200μM to induce protein expression, lower the temperature to 30°C and continue to culture for 24 hours. The bacteria were collected by centrifugation at 5000rpm, washed once with 0.2M pH7.0 sodium phosphate buffer, 1g of bacteria was resuspended in the above 5mL phosphate buffer, after ultrasonic disruption, the expression level was tested by SDS-PAGE.
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