High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase

Abstract

The present invention provides a high density fermentation and a purification process of a recombination high temperature resistance superoxide dismutase, the construction method of the invention includes: using gene coded for SOD in a thermophilic bacteria as a template, designing specific primer amplification target gene having restriction enzyme sites, after double digestion, connecting to plasmid vector pET28a after the same double digestion, constructing a recombinant plasmid, named for pSOD, transforming plasmid pSOD to competence escherichia coli BL21(DE3) by chemical transformation method, obtaining strain having high SOD yield after screening, completing the construction of SOD engineering bacteria; the fermentation process includes four steps of first order seed culture, secondary order feed culture, batch fermentation and induced expression, fermentation product SOD is finally obtained; the fermentation process realizes high level expression of SOD, the expression of the target protein is more than 60% of the bacterial protein total; SOD has excellent thermal stability and heat resistance, the expression product accounts for more than 60% of the whole proteins, and fully soluble protein, avoiding any trouble in the course of inclusion body renaturation; the purification process is simple, having high yield, lower cost, the final product SOD has high purification, high activity and strength stability.

Description

Technical field [0001] The invention relates to a method for constructing a recombinant high-temperature-resistant superoxide dismutase engineering bacteria and a high-density fermentation and purification process, belonging to the technical field of genetic engineering pharmacy. Background technique [0002] Superoxide Dismutase (Superoxide Dismutase, hereinafter referred to as SOD) is a metalloenzyme widely present in organisms; it is named Cu / Zn-SOD, Mn-SOD, Fe-SOD according to the different metal ions to which it binds. And Ni-SOD. SOD is a highly effective scavenger of oxygen free radicals, which also has anti-tumor, anti-fatigue, anti-disease and anti-aging effects; the enzyme has important applications in the fields of food, cosmetics and medicine. [0003] At present, SOD is mainly obtained by extracting from animal blood or plant tissues, so the output of SOD is largely limited by raw materials. Many researchers have also explored the production of SOD through genetic en...

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Problems solved by technology

[0003] At present, SOD is mainly obtained by extracting from animal blood or plant tissue, so the output of SOD is largely limited by raw materials

Many researchers have also carried out a lot of explorations to produce SOD by genetic engineering methods, but the existing methods still have the following problems: 1) most of the recombinantly expressed SOD exists in the form of inactive inclusion bodies, which must be added in the purification process. Therefore, it is difficult to guarantee the yield of SOD; 2) The SOD produced has problems such as low activity, poor heat tolerance, and easy inactivation, and needs to be chemically modified before it can be used; 3) The amount of recombinant expression Relatively low, generally at the level of 20-40%

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