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Preparation method of purified coupling immobilized adenylate cyclase

An adenylate cyclase and immobilization technology, which is applied in the field of preparation of purified coupled immobilized adenylate cyclase, can solve the problems of protein contamination, increased industrial production cost, weak binding between IDA and metal ions, etc.

Inactive Publication Date: 2017-03-08
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of course, LX-1000IDA also has its disadvantages. The price of IDA is more expensive than other commonly used resins, which increases the cost of industrial production.
At the same time, there is still a problem that cannot be ignored in the adsorbent using IDA as the chelating agent. If IDA is not firmly combined with metal ions, it will cause protein pollution or even denaturation.

Method used

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  • Preparation method of purified coupling immobilized adenylate cyclase
  • Preparation method of purified coupling immobilized adenylate cyclase
  • Preparation method of purified coupling immobilized adenylate cyclase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The metal chelating resin carrier of surface modified iminodiacetic acid is prepared as follows:

[0039] (1a) Agarose is dissolved in water by 6% to 12% to make a water phase, slowly poured into the oil phase while hot, and stirred evenly, the composition of the oil phase is as follows: toluene, chloroform, Span-80 The volume ratio is 72-80:28-40:1-2, keep the mixture at 50-60°C for 3-5 minutes, screen out the agarose microspheres with a particle size of 60 mesh, and wash them;

[0040] (2a) Weigh 15-20g of agarose microspheres into 30-40mL, 2mol / L NaOH aqueous solution, add 100-110mg of sodium borohydride, 3-4mL of epoxychloroethylene, shake at 30-35°C, Add 15-20mL of NaOH and 9-12mL of chloroethylene oxide dropwise within 12 hours, then continue the shaking reaction for 10-12 hours, and wash the carrier with water;

[0041] (3a) Add the carrier obtained in step (2a) to 40-50mL, 2mol / L Na 2 CO 3 Add 4-5 g of IDA to the solution, shake it in a shaker at 30-35° C. fo...

Embodiment 2

[0042] Example 2: Ni 2+ Determination of adsorption capacity

[0043] Weigh 2.38g of NiCl 2 .6H 2 O was fixed to a 100mL volumetric flask, and Ni in the solution 2+ The concentration is 0.1mol / L. Take 5mL and dilute to a 500mL volumetric flask, then take 10mL from the 500mL solution and dilute to a 200mL volumetric flask, at this time the Ni in the solution 2+ The concentration is 5×10 -5 mol / L, finally take out 5mL, 10mL, 20mL, and 50mL from the 200mL solution in turn, and set the volume to a 100mL volumetric flask, the corresponding Ni 2+ The concentration is 2.5×10 -5 mol / L, 1×10 -5 mol / L, 5×10 -6 mol / L and 2.5×10 -6 mol / L. Determination of Ni in Solution by Diacetyl Oxime Spectrophotometry 2+ content, each concentration of prepared Ni 2+ Take 10mL of each solution into a 25mL volumetric flask, and take 10mL of pure water for the blank control, add 2mL of ammonium citrate solution and 1mL of iodine solution to the sample, add water to 20mL and shake well, add 2m...

Embodiment 3

[0044] The Ni adsorbed by 1g resin can be calculated from the standard curve 2+ The concentration is 4.68×10 -5 mol / L, about 0.278mg. Embodiment 3: the determination of enzyme protein adsorption amount

[0045] First prepare a 1g / L standard protein solution, take 7 test tubes and label them separately, add each reagent according to the following table, measure the absorbance at 595nm, and use A 595 is the ordinate, the standard protein concentration is the abscissa, and the standard curve is drawn on the axis. Adsorb the enzyme solution with the obtained affinity adsorption medium, use 15mL of the enzyme solution, shake at 25°C and 150r / min for 2 hours, and measure the A 595nm , and then use the standard curve to calculate the protein content. Get the original enzyme solution, the supernatant after adsorption and the eluate after elution of immobilized enzyme with 500mmol imidazole to carry out gel electrophoresis experiment, the results are as follows figure 1 and figu...

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Abstract

The invention discloses a preparation method of purified coupling immobilized adenylate cyclase. The preparation method comprises the following steps: (1) preparing an affinity adsorption medium: adding 5-100g of a metal chelating resin carrier with surface modified by iminodiacetic acid to 10-200mL of 0.01-1mol / L of an NiCl2.6H2O solution, conducting oscillating at 25-30 DEG C and under 150-200rpm for 2-4h, and conducting filtration, so that the affinity adsorption medium is obtained; (2) immobilizing enzyme: adding 1.5-15ml of an adenylate cyclase solution which is 0.1-2mg / mL in concentration to 0.1-1g of the affinity adsorption medium obtained in the step (1), conducting oscillating at 25-30 DEG C and under 150-200rpm for 2-4h, and conducting centrifuging, so that the immobilized adenylate cyclase is obtained, wherein the adenylate cyclase is provided with a histidine tag. The immobilized enzyme prepared by the invention has the advantages of being high in enzymatic activity, low in enzymatic activity loss and the like, and the immobilized enzyme can be repeatedly used.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and specifically designs a preparation method of a purified coupling immobilized adenylate cyclase. Background technique [0002] Enzymes are a class of biomolecules with catalytic functions. They are closely related to human production and life. All organisms in the world cannot survive without enzymes. The characteristics of enzymes include high efficiency, specificity, and mild reaction conditions. However, the free enzyme has poor stability, is easily inactivated and cannot be reused, making it difficult to apply to large-scale industrial production. In order to solve this problem, immobilized enzyme technology came into being. Compared with free enzymes, immobilized enzymes have many advantages, such as high stability, processability, and reusability. The commonly used enzyme immobilization methods include adsorption method, cross-linking method, covalent method and embedding method...

Claims

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Application Information

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IPC IPC(8): C12N11/08C12N15/70C12P19/40C12R1/19
CPCC12N9/88C12N11/08C12P19/40C12Y406/01001
Inventor 应汉杰朱倩倩庄伟牛欢青吴菁岚周精卫陈勇陈晓春朱晨杰柳东
Owner NANJING UNIV OF TECH
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