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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

AI Technical Summary

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 high value-added products; ② the produced product has not been added with protective agent in the later stage During drying, the loss of enzyme activity is large, and the catalytic efficiency of the product is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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PUM

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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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