Application of Bacillus amyloliquefaciens esterase in resolution of N-BOC-DL-alpha-aminobutyric acid methyl ester
A technology of N-BOC-DL- and N-BOC-L-, which is applied in the field of biological enzyme catalysis, can solve the problems of low product purity, complicated conditions, and unfriendly chemical toxic substances.
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Embodiment 1
[0038] Example 1 Fermentation of Bacillus amyloliquefaciens WZZ002, whole genome extraction, esterase gene cloning and construction of engineering bacteria
[0039] 1. Fermentation conditions of Bacillus amyloliquefaciens WZZ002:
[0040] Slant medium composition: maltose 5g / L, beef extract 5g / L, peptone 5g / L, yeast extract 1g / L, anhydrous MgSO 40.5 g / L, agar powder 23g / L, solvent is deionized water, pH7.0.
[0041] Seed medium composition: maltose 5g / L, beef extract 5g / L, peptone 5g / L, yeast extract 1g / L, NaCl 0.5g / L, solvent is deionized water, pH7.0.
[0042] Bacillus amyloliquefaciens (B. amyloliquefaciens) CCTCC NO.M 2013366 was inoculated into the slant medium, and cultured at 37° C. for 12 hours to obtain slant strains.
[0043] Under aseptic conditions, the slant strains were inoculated into 50 mL of seed medium with an inoculation loop, and cultured on a constant temperature shaker at 30° C. and 200 rpm for 24 hours to obtain Bacillus amyloliquefaciens bacteria liqu...
Embodiment 2
[0054] The fermentation culture of the escherichia coli engineering bacterium B3 that embodiment 2 contains esterase
[0055] The Escherichia coli engineering bacterium B3 that embodiment 1 obtains is inoculated in LB medium, 37 ℃ cultures OD 600 to 0.5 (about 2 hours of culture), add IPTG to a final concentration of 0.02mM, and culture at 30°C for 10-12 hours. 300mL of bacterial solution was centrifuged at 8000rpm for 10min at 4°C to collect the bacterial cells, then washed twice with PBS buffer solution, centrifuged at 8000rpm for 10min to collect the bacterial cells. The collected bacteria were freeze-dried using a vacuum freeze dryer (cold trap -80°C, vacuum degree 20Pa) to obtain crude esterase enzyme powder, which was recorded as esterase B3 and stored in a refrigerator at 4°C. LB medium composition: tryptone 10g / L, yeast powder 5g / L, NaCl 5g / L, solvent is deionized water, pH is natural.
Embodiment 3
[0056] Example 3 Enzyme-catalyzed resolution of N-BOC-DL-α-aminobutyric acid methyl ester reaction
[0057] Weigh 0.05g of the esterase B3 prepared by the method of embodiment 2 in the 2mL EP tube, add 1mLNa 2 HPO 4 / NaH 2 PO 4 Buffer (pH7.5, 0.2mM) was used as the reaction solvent, and then 100uL of the substrate N-BOC-DL-α-aminobutyric acid methyl ester was added, with no esterase as the blank control, placed at 45°C, 1000rpm constant temperature mixing React in the homogenizer for 50min. After the reaction, the reaction solution was acidified with 2mM HCl, then 1mL of ethyl acetate was added, fully extracted by vortex shaker, and centrifuged (10000rpm, 5min) to obtain an organic phase containing N-BOC-L-α-aminobutyric acid methyl ester . 700 μL ethyl acetate layer was taken to detect the stereoselectivity and enzyme-catalyzed hydrolysis activity of the bacteria by gas chromatography.
[0058] Specific gas phase analysis conditions: using Agilent7890A gas chromatograph...
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