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Separation technology for gene recombinant heat-resistant manganese superoxide dismutase

A superoxide and dismutase technology, applied in the direction of enzymes, biochemical equipment and methods, enzymes, etc., can solve the problems of complex protein types and few functional proteins, and achieve controllable product purity, small activity loss, and high separation efficiency. Effect

Inactive Publication Date: 2011-01-26
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the field of bioprocessing, ultrafiltration technology is often used for protein purification and concentration, and has been widely used in industry. There are very few successful examples of isolating high-purity functional proteins

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1, the method for extracting gene recombinant heat-resistant manganese superoxide dismutase, steps:

[0015] Take 200ml of the fermentation broth of genetically engineered bacteria E.coli / p28ASOD, centrifuge for 15min (centrifugation condition: 4°C, 1000g), wash the bacteria with 100ml of 1M TE Buffer (pH 8.0), centrifuge for 15min, and discard the supernatant , add 20ml of 1M TEBuffer (pH 8.0), mix evenly; use an ultrasonic cell pulverizer to break up the bacteria (ultrasonic conditions: sonication time 2s, interval 3s, number of times 99 times); after ultrasonication, centrifuge for 15min, and take the supernatant ( 16ml).

[0016] Utilize a flat ultrafiltration membrane with a molecular weight cut-off of 30kDa (polyethersulfone material, 0.09m 2 ) to separate the supernatant, separation conditions: solution pH value 6.0, NaCl concentration 30mM, flow velocity 0.2ml / min, ultrafiltration time 2 hours, get retentate (16ml); Sulfone material, 0.09m 2 ) carry o...

Embodiment 2

[0017] Embodiment 2, due to the separation and purification process of recombinant heat-resistant manganese superoxide dismutase, steps:

[0018] Take 100ml of the fermentation broth of genetically engineered bacteria E.coli / p28ASOD, centrifuge for 15min (centrifugation condition: 4°C, 1000g), wash the bacteria with 100ml of 1M TE Buffer (pH 8.0), centrifuge for 15min, and discard the supernatant , add 20ml of 1M TEBuffer (pH 8.0), mix evenly; use an ultrasonic cell pulverizer to break up the bacteria (ultrasonic conditions: sonication time 2s, interval 3s, number of times 99 times); after ultrasonication, centrifuge for 15min, and take the supernatant ( 18ml).

[0019] Utilize a flat ultrafiltration membrane with a molecular weight cut-off of 50kDa (polyethersulfone material, 0.09m 2 ) to separate the supernatant, separation conditions: solution pH value 6.8, NaCl concentration 50mM, flow velocity 0.2ml / min, ultrafiltration time 2 hours, get retentate (16ml); Sulfone materi...

Embodiment 3

[0020] Example 3, the method for extracting gene recombinant heat-resistant manganese superoxide dismutase, steps:

[0021] Take 250ml of the fermentation broth of the genetically recombined engineered bacteria E.coli / p28ASOD, centrifuge for 15min (centrifugation condition: 4°C, 1500g), wash the bacteria with 150ml of 1M TE Buffer (pH 8.0), centrifuge for 15min, and discard the supernatant , add 30ml of 1M TEBuffer (pH 8.0), and mix evenly; use an ultrasonic cell pulverizer to crush the bacteria (ultrasonic conditions: sonication time 2s, interval 3s, number of times 99 times); after ultrasonication, centrifuge for 15min, and take the supernatant ( 25ml).

[0022] Utilize a flat ultrafiltration membrane with a molecular weight cut-off of 30kDa (polyethersulfone material, 0.09m 2 ) to separate the supernatant, separation conditions: solution pH value 6.5, NaCl concentration 10mM, flow velocity 0.2ml / min, ultrafiltration time 4 hours, get retentate (16ml); Sulfone material, 0....

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PUM

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Abstract

The invention provides separation technology for a gene recombinant heat-resistant manganese superoxide dismutase and belongs to the technical field of bioseparation engineering. By mainly applying the ultrafiltration separation technology, high-purity heat-resistant manganese superoxide dismutase can be separated from recombinant Escherichia coli (E. coli) by three simple steps of crushing, centrifuging and ultrafiltration; and in the separation preparation process, chromatography and electrophoresis centrifugal technology are not used, the extraction period is greatly shortened, and the rapid large-scale preparation of the recombinant heat-resistant manganese superoxide dismutase is realized.

Description

technical field [0001] The invention belongs to the field of biological separation engineering and technology, in particular to a method for isolating heat-resistant manganese superoxide dismutase from recombinant escherichia coli by using ultrafiltration technology, and the product purity reaches more than 92%. Background technique [0002] Superoxide dismutase (Superoxide Dismutase, SOD) is an important oxygen free radical scavenger in organisms. It is an important line of defense to protect the body from these free radicals. The adverse reactions caused by the excessive concentration of superoxide anion free radicals in organisms show unique functions in radiation protection, anti-aging, anti-inflammation, tumor and cancer suppression, autoimmune therapy, etc. Therefore, SOD is widely used in medicine, food and cosmetics. and other fields have broad application prospects. Due to the broad application prospect, the research on SOD has always been a hot spot. Mainly in tw...

Claims

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Application Information

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