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Preparation and application of nylon affinity membrane with reactive blue 4 as ligand

A nylon membrane and affinity membrane technology, applied in the field of nylon affinity membrane, can solve the problems of complicated operation, unsatisfactory purification effect, low sample activity recovery rate, etc., and achieves simple operation, convenient large-scale extraction and purification, and convenient sample source. Effect

Inactive Publication Date: 2010-06-02
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods have certain advantages, they all have disadvantages such as cumbersome operation, low recovery rate of sample activity, and unsatisfactory purification effect, especially when extracting a small amount of biologically active substances from a large volume of dilute solution.

Method used

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  • Preparation and application of nylon affinity membrane with reactive blue 4 as ligand
  • Preparation and application of nylon affinity membrane with reactive blue 4 as ligand

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The activation of the nylon membrane and the modification of the bonded chitosan, the specific steps are as follows:

[0028] (1) The nylon membrane was first hydrolyzed in 1mol / L HCl at 25°C for 24 hours, and then activated with formaldehyde. Ten hydrolyzed nylon membranes were immersed in 20mL formaldehyde solution (>36.5wt.%), added with 0.2mL phosphoric acid (85wt.%), reacted at 60°C for 7h, and washed with hot water at 40-50°C for several times.

[0029] (2) Immerse the above-mentioned formaldehyde-activated nylon membrane into 10 mL of chitosan solution (dissolved with 1 vol.% acetic acid) with a mass fraction of 2%, react at room temperature for 1 h, then transfer to an oven at 80 ° C, take it out after 1 h, and use 1 vol. .% acetic acid and deionized water to wash away unreacted chitosan. The content of chitosan on the nylon film can be tested according to the ninhydrin method.

Embodiment 2

[0031] The specific steps for the preparation of a nylon affinity membrane with Reactive Blue 4 as a ligand are as follows:

[0032] The above-mentioned nylon membrane modified by chitosan was immersed in the Reactive Blue 4 dye solution with a concentration of 10mg / mL, and kept at 60°C for 40 minutes, then added a 20% NaCl solution by mass fraction, reacted with constant temperature oscillation for 30 minutes, and then raised the temperature of the water bath to 80°C, add a mass fraction of 25% Na 2 CO 3 solution, continue to react at constant temperature for 4 hours, and wash with hot deionized water, methanol solution with a volume fraction of 10%, 2mol / L NaCl solution, 6mol / L urea solution and deionized water until the washing solution is colorless , to obtain a nylon affinity membrane with Reactive Blue 4 as a ligand, and store it in double distilled water.

Embodiment 3

[0034] The characterization of the enzyme activity by ultraviolet spectrophotometry using casein as a substrate, the specific steps are as follows:

[0035] Take 0.1mL enzyme solution, add 0.7mL 0.05mol / L Tris-HCl buffer solution (pH 8.0), then add 0.2mL papain activator (the above buffer solution contains 0.5mol / L L-cysteine ​​and 0.02mol / L of EDTA, pH 8.0) kept constant at 35°C, added 1mL of the same preheated 1% (W / V) casein solution (prepared by the above buffer), reacted at 35°C for 15mim, added 3mL of 5% trichloroacetic acid ( TCA) solution to terminate the reaction (in the control group, TCA was added first and then the substrate was added). Let it stand still, centrifuge at 8000 rpm for 20 minutes, and take the filtrate for colorimetry at a wavelength of 280nm.

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Abstract

The invention relates to a preparing method and the application of a nylon affinity membrane which takes Reactive Blue 4 as a petunidin. The preparation includes: the nylon membrane after being activated is modified by chitosan; the modified nylon membrane is dipped into a Reactive Blue 4 dye and added with NaCL solution after heat preservation to react, added with Na2CO3 solution for reacting continuously after temperature rising, respectively washed by hot de-ionized water, methanol solution, NaCl solution, urea solution and de-ionized water to obtain the nylon affinity membrane which takesReactive Blue 4 as a petunidin. The application includes that pawpaw powders are sampled on the nylon affinity membrane which takes Reactive Blue 4 as a petunidin, washed by Tris-HCL buffer solution and eluted by NaSCN solution to obtain pawpaw prolease; the activity of the prolease and the protein content are tested; the purification times are calculated. The method is fast, simple and convenientand has large separation capacity; the prolease extracted has the advantages of good activity, high purity as well as being suitable for scale production.

Description

technical field [0001] The invention belongs to the field of nylon affinity membranes, in particular to a preparation method and application of a nylon affinity membrane with Reactive Blue 4 as ligand. Background technique [0002] Papain (EC3.4.22.2) is a kind of sulfhydryl protease, which widely exists in the root, stem, leaf and fruit of papaya (Carica papaya), and is most abundant in immature milk. Widely used in food industry, such as clarification of beer and tenderization of meat, as well as leather, textile, daily chemical and pharmaceutical industries. The purification of papain mostly adopts the precipitation method, but this method is still mixed with other proteases, which cannot meet the needs of the pharmaceutical industry. [0003] At present, the methods for separating and purifying papain include rough separation and refined separation, wherein rough separation includes salting out method, isoelectric point precipitation method, organic solvent fractionatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/286C12N9/50
Inventor 朱利民苏赛男聂华丽陈天翔
Owner DONGHUA UNIV
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