Method for extracting and purifying superoxide dismutase (SOD) from saccharomyces cerevisiae

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae cells, which is applied in the field of extracting and purifying SOD, which can solve the problems of unsuitability for large-scale industrial production, SOD enzyme activity damage of Saccharomyces cerevisiae, and undisclosed SOD purification method, so as to achieve high purity of SOD products and keep the original Activity, the effect of loss of environmental enzyme activity

Inactive Publication Date: 2015-03-25
XINGTAI YITONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the patent No. 2011100503115 reports a method for producing recombinant human SOD with Saccharomyces cerevisiae, but this patent focuses on the construction of recombinant Saccharomyces cerevisiae, and does not disclose the SOD purification method, and ultrasound is used to obtain SOD from cells method, which is not suitable for large-scale industrial production; patent number 201310103919.9 reports a strain of Saccharomyces cerevisiae and its application in the preparation of heat-resistant superoxide dismutase. This patent mainly focuses on the screening and fermentation of Saccharomyces cerevisiae strains , the purification of SOD has not been studied in depth; in the patent No. 2011103369936, a method for simultaneously extracting S-adenosyl-L-methionine and SOD from Saccharomyces cerevisiae has been announced, but the purified by this method The purity of SOD is relatively low, and the organic solvent method is adopted when breaking the wall, and the acetone precipitation method is also adopted during purification, which has certain pollution to the environment and also makes the SOD enzyme activity have a certain loss; the patent No. is 200810061168.8 in the patent, reports A method for extracting high-temperature resistant superoxide dismutase from recombinant Saccharomyces cerevisiae has been proposed, but this method requires an 80°C water bath, which will inactivate SOD in Saccharomyces cerevisiae. In addition, this patent requires precipitation with an organic solvent-ethanol, which There is also damage to the enzyme activity of SOD in Saccharomyces cerevisiae, and finally dialysis will also affect the amplification of the industrialization of the process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] After filtering the fermentation liquid of Saccharomyces cerevisiae fermented in the 200L fermenter with a plate-and-frame filter, add about 24kg of bacteria sludge into the stirring tank, add 60 liters of deionized water at 4~15°C to it and stir to make the yeast cells uniform To disperse, add deionized water to a final volume of 90 L. The above Saccharomyces cerevisiae suspension was homogenized with a pressure of 1000 bar, and the homogenized product was homogenized again with a pressure of 1000 bar. After repeated 3 times, the yeast cell breakage rate was greater than 95%.

[0035]Add 10 liters of 250g / L polyetherimide (PEI) solution with a pH of 7.9 to the homogenate product, so that the final concentration of PEI in the homogenate product is 25g / L, stir at a speed of about 50rpm for 10min, and Add 30 kg of diatomaceous earth and mix evenly, then filter again with a plate and frame filter to collect the filtrate. In order to increase the SOD yield, the filter cake...

Embodiment 2

[0040] After filtering the fermentation liquid of Saccharomyces cerevisiae fermented in the 200L fermenter with a plate-and-frame filter, add about 24kg of bacteria sludge into the stirring tank, add 48 liters of deionized water at 4~15°C and stir to make the yeast cells uniform dispersion. Homogenize the Saccharomyces cerevisiae suspension with a pressure of 500 bar, and homogenize the homogenate product again with 500 bar. After repeated 8 times, the yeast cell breakage rate is greater than 95%. When the homogenization pressure is low, the homogenization rate is faster, the flux is large, and the temperature of the homogenization product is lower. When there is no cooling equipment, the low-pressure multiple homogenization method can be used.

[0041] The homogenized product was centrifuged at 14000rpm with a tubular centrifuge, and centrifuged at a discharge rate of about 30L / h. The centrifuged liquid is filtered with a ceramic membrane from Nanjing Aiyuqi Membrane Technol...

Embodiment 3

[0045] After filtering the fermentation liquid of Saccharomyces cerevisiae fermented in the 100L fermenter with a plate-and-frame filter, add about 10kg of bacteria sludge into the stirring tank, add 80 liters of deionized water at 4~15°C to it and stir to make the yeast cells uniform Disperse and add deionized water to a final volume of 99 L. The above Saccharomyces cerevisiae suspension was homogenized with a pressure of 1000 bar, and the homogenized product was homogenized again with a pressure of 1000 bar. After repeated 3 times, the yeast cell breakage rate was greater than 95%.

[0046] Add 1 liter of 200g / L polyetherimide (PEI) solution with a pH of 7.9 to the homogenate product, so that the final concentration of PEI in the homogenate product is 2g / L, stir at a speed of about 50rpm for 10min, and 10 kg of diatomaceous earth was added therein and mixed evenly, and then the filtrate was collected by filtering again with a plate and frame filter. In order to increase the...

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Abstract

The invention relates to a method for extracting and purifying superoxide dismutase (SOD), and in particular relates to a method for extracting and purifying SOD from saccharomyces cerevisiae. The method comprises the following operation steps: collecting bacteria, homogenizing bacteria, preparing a crude enzyme fluid, carrying out ultrafiltration, implementing chromatography and desalting. The method disclosed by the invention can be used for effectively purifying non-modified SOD protein and keeping the original activity of the SOD; and through a pyrogallol oxidation method, the specific activity of the purified high-purity SOD can reach up to 15000U / g and the entire process is relatively high in yield; and the method is simple to operate and capable of achieving industrial mass production.

Description

technical field [0001] The invention relates to a method for extracting and purifying SOD, in particular to a method for extracting and purifying SOD from Saccharomyces cerevisiae. Background technique [0002] o 2 -· It is called superoxide anion free radical, which is an intermediate product naturally generated in various physiological reactions of organisms. Superoxide anion is both an anion and a free radical, both an oxidizing agent and a reducing agent. It has been confirmed that there are as many as 60 kinds of diseases caused by oxygen free radicals. In the body, superoxide anion is mainly scavenged by superoxide dismutase. [0003] Superoxide dismutase (Superoxide Dismutase, SOD) is widely distributed in the biological world, almost from animals to plants, and even from humans to single-celled organisms. SOD is an important antioxidant enzyme in organisms, has special physiological activity, and is the primary enzyme for removing superoxide anions in organisms....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12R1/865
CPCC12N9/0089C12Y115/01001
Inventor 马功臣
Owner XINGTAI YITONG BIOLOGICAL TECH
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