Method for purifying nitrilase
A technology of nitrilase and hydrolase, which is applied in the field of purification to obtain high-purity nitrilase, can solve the problems of not meeting the requirements of high value-added products, large loss of enzyme activity, and low catalytic efficiency, so as to improve the comprehensive utilization rate, The effect of simple production equipment and high purification efficiency
Examples
Embodiment 1
[0022] A method for purifying nitrilase, the specific method steps are as follows:
[0023] (1) Preparation of pretreatment solution
[0024] With commercially available crude nitrilase as raw material, according to the mass (g) of crude nitrilase: the volume (mL) ratio of buffer solution is the ratio of 1: 80, first crude nitrilase is added in the buffer solution, stirred After mixing well, stir for another 20 minutes to dissolve. Then the solution is pumped into a vacuum filter for vacuum filtration, and the filtrate and filter residue are collected respectively. The collected suction filtrate (that is to prepare the pretreatment liquid) is used for the next step; the collected suction residue can be used as a feed additive after being dried and pulverized. Wherein, the buffer solution is a borax-calcium chloride buffer solution with a pH of 8.0.
[0025] (2) Preparation of ultrafiltrate
[0026] After the (1) step was completed, the suction filtrate collected by the (1)...
Embodiment 2
[0032] A method for purifying nitrilase, with embodiment 1, wherein:
[0033] In the 1st step, the mass of crude nitrilase: the volume ratio of the buffer solution is 1g: 100mL, stirring and dissolving for 25 minutes, wherein the buffer solution is a borax-potassium chloride buffer solution with a pH of 9.0.
[0034] In the (2) step, the molecular weight cut-off of the ultrafilter is 55KDa, and the ultrafiltration pressure is 0.1MPa, and the first ultrafiltration is reduced to 10% of the original volume until the ultrafiltrate volume, and the ultrafiltration is until the ultrafiltration for the second time. The liquid hold-up volume is reduced to 10% of the original volume.
[0035] In the (3) step, the molecular weight cut-off of nanofilter is 500Da, and nanofiltration pressure is 0.3MPa, until the first nanofiltration retentate volume is reduced to 35% of original volume, and the second nanofiltration retentate volume is reduced to 20% of the original volume.
[0036]In th...
Embodiment 3
[0038] A method for purifying nitrilase, with embodiment 1, wherein:
[0039] In the 1st step, the mass of crude nitrilase: the volume ratio of the buffer solution is 1g: 120mL, stirring and dissolving for 30 minutes, wherein the buffer solution is a borax-water buffer solution with a pH of 9.18.
[0040] In the (2) step, the molecular weight cut-off of ultrafilter is 60KDa, and ultrafiltration pressure is 0.2MPa, and ultrafiltration is reduced to 12% of original volume till ultrafiltrate volume for the first time, and ultrafiltration is till ultrafiltration for the second time. The liquid hold-up volume is reduced to 12% of the original volume.
[0041] In the (3) step, the molecular weight cut-off of nanofilter is 600Da, and nanofiltration pressure is 0.4MPa, until the first nanofiltration retentate volume is reduced to 40% of original volume, and the second nanofiltration retentate volume is reduced to 25% of the original volume.
[0042] In the 4th step, the volume ratio...
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Description
Claims
Application Information
- IPC
- C12N9/88
- Inventors
- 张杰; 周小华