Method for quickly preparing recombinant heat-resistant manganese superoxide dismutase

A superoxide and dismutase technology, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of less super-large pore media, limitations, and no reports of high-temperature-resistant SOD enzymes, and achieve fast separation speed and recovery The effect of high efficiency and small loss of SOD enzyme activity

Inactive Publication Date: 2011-03-16
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since agarose microspheres themselves belong to polysaccharide soft gels, mechanical strength is a limiting factor for the application of the microspheres
At present, in the field of separation and purification of biological macromolecules such as proteins, there are few reports on the application of super-macroporous media, or limited to the separation of model proteins on a laboratory scale, and there are no reports on the application of separation and purification of high-temperature-resistant SOD enzymes.

Method used

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  • Method for quickly preparing recombinant heat-resistant manganese superoxide dismutase
  • Method for quickly preparing recombinant heat-resistant manganese superoxide dismutase
  • Method for quickly preparing recombinant heat-resistant manganese superoxide dismutase

Examples

Experimental program
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Embodiment 1

[0025] Take 500ml of fermentation broth, centrifuge for 15mm (centrifugation condition is 4°C, centrifugal force is 1000×g), wash the bacteria with 5-10ml of 1M TE Buffer (pH 8.0), centrifuge for 15min, discard the supernatant, add 1M TE Buffer (pH 8.0) TE Buffer (pH 8.0) 5ml, mix well; use an ultrasonic cell pulverizer to crush the bacteria (ultrasonic conditions: ultrasonic time 2s, interval 3s, frequency 99 times); after ultrasonication, centrifuge for 15min, the supernatant is the crude enzyme liquid. Get 5ml of crude enzyme solution, utilize sample loader to load the sample, in the semi-preparative grade ultra-large hole nickel column (300 * 10mm I.D.) that is housed Chromatographic separation on purifier 100system, separation conditions: mobile phase, 20mM phosphate buffer + 0.5M NaCl + 0.12M imidazole, pH 7.4; elution phase, 20mM phosphate buffer + 0.5M NaCl + 0.5M imidazole, pH 7.4; Elution mode, step elution; operating flow rate, 2528cm / h. Collect the elution peak ...

Embodiment 2

[0027] Take 500ml of fermentation broth, centrifuge for 15min (centrifugation condition is 4°C, centrifugal force is 1000×g), wash the bacteria with 5-10ml of 1M TE Buffer (pH 8.0), centrifuge for 15min, discard the supernatant, add 1M TE Buffer (pH 8.0) TE Buffer (pH 8.0) 5ml, mix well; use an ultrasonic cell pulverizer to crush the bacteria (ultrasonic conditions: ultrasonic time 2s, interval 3s, frequency 99 times); after ultrasonication, centrifuge for 15min, the supernatant is the crude enzyme liquid. Get 5ml of crude enzyme solution, utilize sample loader to load the sample, in the semi-preparative grade ultra-large hole nickel column (300 * 10mm I.D.) that is housed Chromatographic separation on purifier 100system, separation conditions: mobile phase, 20mM phosphate buffer + 0.5M NaCl + 0.1M imidazole, pH 7.4; elution phase, 20mM phosphate buffer + 0.5M NaCl + 0.5M imidazole, pH 7.4; Elution mode, step elution; operating flow rate, 3251cm / h. Collect the elution peak ...

Embodiment 3

[0029] Take 500ml of fermentation broth, centrifuge for 15min (centrifugation condition is 4°C, centrifugal force is 1000×g), wash the bacteria with 5-10ml of 1M TE Buffer (pH 8.0), centrifuge for 15min, discard the supernatant, add 1M TE Buffer (pH 8.0) TE Buffer (pH 8.0) 5ml, mix well; use an ultrasonic cell pulverizer to crush the bacteria (ultrasonic conditions: ultrasonic time 2s, interval 3s, frequency 99 times); after ultrasonication, centrifuge for 15min, the supernatant is the crude enzyme liquid. Get 5ml of crude enzyme solution, utilize sample loader to load the sample, in the semi-preparative grade ultra-large hole nickel column (300 * 10mm I.D.) that is housed Chromatographic separation on purifier 100system, separation conditions: mobile phase, 20mM phosphate buffer + 0.5M NaCl + 0.1M imidazole, pH 7.4; elution phase, 20mM phosphate buffer + 0.5M NaCl + 0.1M imidazole, pH 5; Elution mode, step elution; operating flow rate, 1806cm / h. Collect the elution peak ( ...

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Abstract

The invention provides a method for quickly preparing recombinant heat-resistant manganese superoxide dismutase and belongs to the technical field of bioseparation engineering. In the method, the metal-chelating chromatographic separation technology and the self-prepared ultra-macroporous metal-chelating medium are adopted, high-purity high temperature-resistant manganese superoxide dismutase can be quickly separated from recombinant Escherichia coli by four steps, namely centrifugation of fermentation liquor, cell breakage, centrifugation of breaking liquid and chromatography. The separation rate is over 5 times higher than the conventional chromatographic separation rate, the loss of SOD enzymatic activity is low, the recovery is high, the purity is over 85 percent, and the method makes mass production of low-cost high-purity SOD enzyme possible.

Description

technical field [0001] The invention belongs to the field of bioseparation engineering and technology, in particular to a method for rapidly separating and purifying recombinant high-temperature-resistant manganese superoxide dismutase (SOD) expressed in Escherichia coli by using a super-macroporous metal chelating chromatography medium, and the product purity reaches 85%. above. Background technique [0002] SOD is an important oxygen free radical scavenger in the body, which can balance the oxygen free radicals in the body, so as to avoid the adverse reactions caused by the excessive concentration of superoxide anion free radicals in the body. At the same time, SOD is a very useful medicinal enzyme. The medical field has proved that it has the functions of anti-aging, anti-tumor, anti-radiation, anti-ischemia, and improving human immunity (Bioehem J, 1991, 274: 153-158). At present, SOD enzyme has been widely used in medicine, food, beverage, cosmetics and other industri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08
Inventor 曲剑波朱虎刘建国吕建仁
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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