Unlock instant, AI-driven research and patent intelligence for your innovation.

A kind of purification method of ATP sulfurylase

A purification method and sulfurylase technology, applied in the biological field, can solve the problems of low product purity, inability to meet genome sequencing, affecting the accuracy of sequencing reaction, etc., and achieve the effects of high purity, high activity and increased stability.

Inactive Publication Date: 2016-05-25
BEIJING ZHONGKEZIXIN TECH
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing ATP sulfurylase purification technology, the purity of the obtained product is low, which easily affects the accuracy of the sequencing reaction and cannot meet the needs of genome sequencing

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of purification method of ATP sulfurylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Adjust the pH value: adjust the pH value of the crude enzyme solution to 7.6;

[0028] (2) Take 1L of crude luciferase enzyme solution and add 10mmol of magnesium chloride, 15mmol of magnesium sulfate and 16mmol of calcium chloride;

[0029] (3) Add 6g of soybean polysaccharide and 3g of dimercaptothreitol;

[0030] (4) Organic solvent precipitation: slowly add 15ml acetone and 20ml acetonitrile, stir, let stand for 10 minutes, centrifuge at 13000rpm, discard the supernatant, add 10ml Tris-HCl buffer, stir, centrifuge at 13000rpm, and collect the supernatant;

[0031] (5) Use 0.22μm filter membrane for sterile filtration;

[0032] (6) Purify on nickel column, use pH 8.0 Tris-HCl buffer for column equilibration and elution, and use 20-500 mM imidazole for gradient elution;

[0033] (7) Use Tris-HCl buffer for protein dialysis;

[0034] (8) Use GE molecular exclusion prepacked column superdex200 for molecular exclusion, and use pH 8.0 Tris-HCl buffer for column equilibration and ...

Embodiment 2

[0038] (1) Adjust the pH value: adjust the pH value of the crude enzyme solution to 8.0;

[0039] (2) Take 1L of crude luciferase enzyme solution, add 20mmol magnesium nitrate, 12mmol calcium sulfate, and 8mmol calcium chloride;

[0040] (3) Add 5g of glycerin;

[0041] (4) Organic solvent precipitation: slowly add 20ml methanol and 12ml acetonitrile, stir, let stand for 10 minutes, centrifuge at 13000rpm, discard the supernatant, add 10ml Tris-HCl buffer, stir, centrifuge at 13000rpm, and collect the supernatant;

[0042] (5) Use 0.22μm filter membrane for sterile filtration;

[0043] (6) Purify on nickel column, use pH8.0 Tris-HCl buffer for column equilibration and elution, and use 20-500mM imidazole for gradient elution during elution;

[0044] (7) Use Tris-HCl buffer for protein dialysis;

[0045] (8) Use GE molecular exclusion prepacked column superdex200 for molecular exclusion, and use pH 8.0 Tris-HCl buffer for column equilibration and elution to obtain 100 ml of enzyme solution;...

Embodiment 3

[0049] (1) Adjust the pH value: adjust the pH value of the crude enzyme solution to 7.5;

[0050] (2) Take 1L of crude luciferase enzyme solution and add 30mmol of magnesium chloride, 2mmol of calcium sulfate, 4mmol of calcium chloride and 4mmol of calcium nitrate;

[0051] (3) Add 8g of glycerin, 2g of ethylene glycol and 5g of soybean polysaccharide;

[0052] (4) Organic solvent precipitation: slowly add 30ml acetonitrile, stir, let stand for 10 minutes, centrifuge at 13000rpm, discard the supernatant, add 10ml Tris-HCl buffer, stir, centrifuge at 13000rpm, and collect the supernatant;

[0053] (5) Use 0.22μm filter membrane for aseptic filtration;

[0054] (6) Purify on nickel column, use pH8.0 Tris-HCl buffer for column equilibration and elution, and use 20-500mM imidazole for gradient elution during elution;

[0055] (7) Use Tris-HCl buffer for protein dialysis;

[0056] (8) Use GE molecular exclusion prepacked column superdex200 for molecular exclusion, and use pH 8.0 Tris-HCl buffe...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for purifying ATP sulfurylase. The method comprises the following steps: adjusting pH value; adding metal salt; adding stabilizer; organic solvent precipitation; sterile filtration; nickel column purification; using superdex? 200 for molecular exclusion; for lyophilization. The product obtained by the method of the invention has high purity and is suitable for pyrosequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for purifying ATP sulfurylase. Background technique [0002] ATP sulfurylase (ATPS) widely exists in plants, animals and microorganisms, but its biological role in various organisms is slightly different. In plants and some microorganisms, it is a key enzyme that catalyzes the first step of the sulfate assimilation reaction. Catalyzed by ATPS, sulfate ions react with ATP to produce adenine-5'-phosphosulfate ATS and pyrophosphate PPi. In some chemoautotrophic bacteria and chemoinorganotrophic bacteria, ATPS is the enzyme that catalyzes the oxidation of hydrogen sulfide to generate inorganic sulfate in the last step, and its function is to catalyze the reaction of ATS and PPi to generate ATP and sulfate ions, that is, the reverse reaction of the above reaction . ATPS has been extracted and purified from Penicillium chrysogenum, mouse liver, cabbage and other org...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12
CPCC12N9/1241C12Y207/07004
Inventor 高静蔡亦梅徐潇吴超张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH