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

Inactive Publication Date: 2012-02-29
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The main disadvantages of this method are: ① there is no pretreatment of xylanase raw materials, the utilization rate is low, the produced product has low purity, and cannot meet the requirements of preparing hi

Method used

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Examples

Experimental program
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Example Embodiment

[0021] Example 1

[0022] A method for purifying nitrilase, the specific method steps are as follows:

[0023] (1) Preparation of pretreatment liquid

[0024] Using commercially available crude nitrilase as raw material, according to the ratio of crude nitrilase mass (g): volume (mL) of buffer solution (mL) of 1:80, first add the crude nitrilase to the buffer solution and stir After mixing well, stir and dissolve for another 20 minutes. Then the dissolving liquid is pumped into a vacuum suction filter to perform vacuum suction filtration, and the suction filtration filtrate and the suction filtration residue are respectively collected. The collected suction filtration filtrate (that is, the preparation of the pretreatment liquid) is used for the next treatment; the collected suction filtration residue, after drying and crushing, can be used as a feed additive. Among them, the buffer solution is a borax-calcium chloride buffer solution with a pH of 8.0.

[0025] (2) Preparation of u...

Example Embodiment

[0031] Example 2

[0032] A method for purifying nitrilase is the same as in Example 1, wherein:

[0033] In step (1), the mass ratio of the crude nitrilase: the volume ratio of the buffer solution is 1 g: 100 mL, and the solution is stirred and dissolved for 25 minutes, wherein the buffer solution is a borax-potassium chloride buffer solution with pH 9.0.

[0034] In step (2), the molecular weight cut-off of the ultrafilter is 55KDa, the ultrafiltration pressure is 0.1MPa, the first ultrafiltration is performed until the volume of the ultrafiltrate is reduced to 10% of the original volume, and the second ultrafiltration is performed until the ultrafiltration The liquid retention volume is reduced to 10% of the original volume.

[0035] In step (3), the molecular weight cut-off of the nanofilter is 500Da, the pressure of the nanofiltration is 0.3MPa, the volume of the first nanofiltration retentate is reduced to 35% of the original volume, and the volume of the second nanofiltration r...

Example Embodiment

[0037] Example 3

[0038] A method for purifying nitrilase is the same as in Example 1, wherein:

[0039] In the step (1), the mass ratio of the crude nitrilase: the volume ratio of the buffer solution is 1 g: 120 mL, and the solution is stirred and dissolved for 30 minutes, wherein the buffer solution is a borax-water buffer solution with pH 9.18.

[0040] In step (2), the molecular weight cut-off of the ultrafilter is 60KDa, the ultrafiltration pressure is 0.2MPa, the first ultrafiltration is performed until the volume of the ultrafiltrate is reduced to 12% of the original volume, and the second ultrafiltration is performed until the ultrafiltration The liquid retention volume is reduced to 12% of the original volume.

[0041] In step (3), the molecular weight cut-off of the nanofilter is 600Da, the pressure of the nanofiltration is 0.4MPa, and the volume of the retentate of the first nanofiltration is reduced to 40% of the original volume, and the volume of the retentate of the sec...

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Abstract

The invention relates to a method for purifying nitrilase and belongs to a preparation method for the nitrilase. By the method, the purified nitrilase product is prepared from commercial coarse nitrilase by the simple process of preprocessing, ultrafiltration and classification, and freeze drying. The method is simple; the conventional simple equipment is adopted; the method is easy to operate and control, and convenient to popularize and apply, and the production is safe; in the production process, the reaction condition is mild, energy consumption is low, material resources are utilized completely, emission is avoided, and environmental protection is facilitated; and the produced product has high purity, activity and stability and can meet the requirement on preparation of products with high additional value. The product prepared by the method can be widely applied to the industries such as medicines, chemical industry, materials, agriculture and the like.

Description

1. Technical field [0001] The invention belongs to the technical field of purifying nitrilase, and in particular relates to a method for obtaining high-purity nitrilase by using commercially available crude nitrilase as a raw material. 2. Background technology [0002] Nitrilases are biocatalysts with broad substrate adaptability and are widely used in chemical industry, food and agriculture. The advantage of using nitrilase to hydrolyze nitrile groups is not only mild reaction conditions and environmental friendliness, but also chiral selectivity that is not available in general chemical reactions. However, the commercially available nitrilase has low purity and contains a large amount of impurities such as polysaccharides and miscellaneous proteins. It is often used as a feed additive and food additive, and it is difficult to meet the conditions for preparing high value-added products (such as medicine, chemical industry, etc.) that require high-purity enzymes. Seriously ...

Claims

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Application Information

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IPC IPC(8): C12N9/88
Inventor 张杰周小华康宏宽徐溢王丹周兴王永红
Owner CHONGQING UNIV
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