A kind of preparation method of cholesterol esterase
A technology of cholesterol esterase and cholesterol, applied in the field of enzyme engineering, can solve the problems of insufficient temperature tolerance to meet industrial needs, low concentration of organic solvents, etc., and achieve good acid-base stability and temperature stability
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Embodiment 1
[0046] Example 1: Burkholderia Cepacia ZWS15 fermentation enzyme
[0047] (1) Sliding culture: Burkholderia Cepacia ZWS15 is inoculated with the Burkholderia Cepacia Zws15 in the Solidia Cepacia, which is included in the LB solid medium, 37 ° C for 20-28 hours.
[0048] LB solid medium: 5 g of yeast 5g, protein 胨 10g, NaCl 10g, agar powder 20g per 1L distilled water.
[0049] (2) Seed culture: The strain cultured in step (1) was picked into a 50 mL LB liquid medium under sterile conditions, and the rotation was oscillated in a shaker of 37 ° C and a rotational speed of 200 rpm.
[0050] LB liquid medium: yeast powder 5g, protein 胨 10g, NaCl 10g were added to each 1L distilled water.
[0051] (3) Fermentation culture: Take step (2) The seed liquid after activation, inoculation into the fermentation medium, add cholesterol oleetic acid, induce cholester esterase, and induces cholesterol esterase, induction of cholesterol esterase Add Cape Tong X-100 to promote extracellular secretio...
Embodiment 2
[0056] Example 2: Purification and mass spectrometry of onion Berk Holder ZWS15 cholesterol esterase
[0057] (1) After the fermentation crude enzyme solution obtained in Example 1 was precipitated with ammonium sulfate, 10000 rpm was centrifuged at 4 ° C, and the precipitate was dissolved using pH 7.5, 50 mmol / L of Tris-HCl buffer, and used the same Solution for dialysis.
[0058] (2) After the Tris-HCl buffer with pH 7.5, 50 mmol / L, the DEAE ion exchange column was balanced, and the dialyzed sample was injected, and the chromatography column was rinsed with pH 7.5, 50 mmol / L of Tris-HCl buffer. Removing the miscellaneous protein of the unhalded column, ultimately with Tris-HCl buffer containing 1 mol / l NaCl, pH 7.5, 50 mmol / L, will be used in a linear elution in a linear elution in a linear elution of 1 ml / min. The column was eluted on the column, and the peak detected by ultraviolet was collected.
[0059] (3) The collected eluent was detected using SDS polyacrylami...
Embodiment 3
[0061] Example 3: Cholesterol esterase optimum pH and pH stability
[0062] (1) Determination of the optimum pH of cholesterol esterase
[0063] 1.5 ml of 0.2 mol / L of sodium acetate buffer (pH 3.0 ~ 5.5), 0.2 mol / L of potassium phosphate buffer (pH 5.5 ~ 7.5), 0.2 mol / L of Tris-HCl buffer (pH 7.5 ~ 9.0), 0.2mol / L NA 2 CO 3 -Nahco 3 Buffers (pH 9.0 ~ 11.0), 0.2 mol / L NaOH-NaCl buffer (pH 11.0 to 12.0), added 0.1 ml of the pure enzyme solution obtained in Example 2. 1 ml of 0.6 mmol / L cholesterol oleates were added to the substrate, and the activity of cholesterol esterase degradation of the substrate was determined at 37 ° C, and final results showed a percentage of the highest enzyme activity in the measurement value. Determination of relative enzyme activity Figure 4 Indicated. The optimum reaction of the enzyme is between pH 7.5 to 9.0.
[0064] (2) pH stability of cholesterol esterase
[0065] The buffer in 1.5 ml of step (1) was added to 0.1 mL of the pure enzyme ...
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