Absorption micro-filtration separation purification method for producing zytase

A technology for separation and purification of xylanase, which is applied in the field of separation and purification of xylanase by affinity microfiltration, can solve problems such as low efficiency and complicated process, and achieve high separation efficiency, simple separation process, and high recovery rate of enzyme activity Effect

Inactive Publication Date: 2008-07-02
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the separation and purification process of xylanase is based on the difference in physical and chemical properties between xylanase and coexisting miscellaneous proteins, such as the difference in molecular size, surface charge, etc. The separation techniques used are mainly It is ammonium sulfate precipitation, ultrafiltration, gel filtration and ion exchange chromatography, etc. Usually, it is necessary to adopt a multi-step (generally two to five steps) series separation and purification process, and the recovery rate of xylanase is generally 0.2 Between % and 52%, the process is complicated and the efficiency is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Use 0.05mol / L citric acid solution to adjust the pH value of 1L xylanase crude enzyme solution to pH3.0, place it in a jacketed reactor, cool the xylanase crude enzyme solution to 4°C, add An appropriate amount of oat xylan, so that the xylan content is 10g / L, is stirred (250r / min) at 4°C for 3 hours for adsorption.

[0011] With a pressure of 30kPa and a feed rate of 200mL / min, an effective membrane area of ​​140cm 2 The 0.2 μm microfiltration membrane intermittently diafiltration (diafiltration) the suspension in the reactor. During diafiltration, the suspension was first concentrated 4 times, and then the citric acid-sodium citrate buffer solution of pH 3.0 was added to make the reactor The volume of the material in the reactor was restored to the original volume (1 L). This was done 3 times, and the replacement volume was 2.0 L. At the end of the washing, the volume of the suspension in the reactor was about 200 mL.

[0012] Use sodium dihydrogen phosphate-disodium...

Embodiment 2

[0015] Use 0.05mol / L citric acid solution to adjust the pH value of 1L xylanase crude enzyme solution to pH3.0, place it in a jacketed reactor, cool the xylanase crude enzyme solution to 4°C, add An appropriate amount of oat xylan, so that the xylan content is 100g / L, is stirred (250r / min) at 4°C for 3 hours for adsorption.

[0016] Under the condition that the pressure is 30kPa and the feed rate is 200mL / min, the suspension in the reactor is intermittently diafiltration (diafiltration) with a 0.2 μm microfiltration membrane with an effective membrane area of ​​140cm2. During diafiltration, the suspension is first concentrated 5 times, then replenish the citric acid-sodium citrate buffer solution of pH3.0, make the material volume in the reactor return to original volume (1L), carry out like this 3 times, replacement volume is 2.5L, when washing finishes, reactor The inner suspension volume is about 250 mL.

[0017] Use sodium dihydrogen phosphate-disodium hydrogen phosphate ...

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PUM

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Abstract

The invention relates to a method for producing xylanase by microfiltration separation and purification using specific affinity of biomolecules. The method has the following steps of: (1) mixing coarse xylanase solution (4 DEG C) and oat-spelt xylan, and adsorbing for 3h; (2) microfiltration concentrating to 1/3 volume of the original volume, feeding citric acid-sodium citrate (pH 3.0), and performing diafiltration and washing to remove impure proteins; and (3) eluting the microfiltration cake in step (2) with disodium hydrogen phosphate-sodium dihydrogen phosphate (pH 7.0) as eluent, and collecting the eluate, which is xylanase. The inventive method can obtain electrophoresis-grade xylanase by one-step separation, and has the advantages of simple separation, high separation efficiency, and high recovery of enzyme activity.

Description

technical field [0001] The invention relates to a technical method for separating and purifying xylanase by affinity microfiltration. The invention belongs to the technical field of combining biological separation and membrane separation. Background technique [0002] Endo-β-1,4-D-xylanase (EC 3.2.1.8) can efficiently degrade xylan by cleaving the β-1,4 glycosidic bond in the xylan backbone. In food, feed, medicine, energy, papermaking, textile and many other industries, xylanase has broad application prospects, especially in the papermaking industry, the enzyme can be used for pulp bleaching, reducing the amount of chemicals used in the pulp bleaching process , so as to effectively reduce the environmental pollution caused by the paper industry. Therefore, in order to use xylanase on a large scale and efficiently, it is necessary to separate xylanase from complex fermentation media; at the same time, in order to study the biological characteristics and mechanism of xylana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42
Inventor 江华薄开静周振
Owner NANJING FORESTRY UNIV
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