Preparation method of recombinant halophilic archaea protease

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a preparation method of recombinant halophilic archaea protease. The method comprises the following steps: constructing a recombinant plasmid by taking a genome of Halostella sp. DL-M4 as a template and taking a pET28a plasmid as an expression vector; transforming escherichia coli cell by recombinant plasmid, inducing expression, centrifugally collecting thalli for cell resuspension and crushing, centrifuging again, taking a supernatant, adding beta-mercaptoethanol to obtain a mixed solution, enabling the mixed solution to pass through a nickel column, combining zymogen with a nickel medium for renaturation, and finally performing secondary impurity protein removal and target protein elution, performing ultrafiltration centrifugal concentration and performing gel filtration chromatography to obtain the recombinant halophilic archaea protease. The method provided by the invention has good universality, simpler purification steps and shorter production cycle, the obtained protease has high purity, and finally the halophilic archaea protease with specific enzyme activity of 1159.15 U / mg is obtained.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a preparation method of recombinant halophilic archaea protease. Background technique [0002] Protease is the most sold enzyme preparation in the world's industrial production, and it is widely used in the washing textile industry, leather industry, food processing, waste treatment, and biotechnology. In high-salt industrial production processes such as high-salt food processing and high-salt wastewater treatment, commercially available proteases are easily inactivated, which limits the use of proteases, and the halophilic archaeal proteases secreted by extreme halophilic archaea conditions to maintain high hydrolytic activity. Halophilic archaeal proteases are members of the subtilisin-like S8 family of serine proteases, and most of them have the characteristics of high salt resistance, organic solvent resistance, tolerance to various metal ions and strong stability, ...

AI Technical Summary

Problems solved by technology

However, the disadvantages of slow growth of halophilic archaea, high cost of culture medium and high-salt-tolerant fermentation equipment, low expression of extracellular enzymes, and cumbersome purification steps increase the production cost and production cycle of pure enzymes.

Halophilic archaeal proteases are often irreversibly denatured and inactivated under low-salt and no-salt conditions, and the purification process mostly relies on a high-salt environment, which limits the use of many protein purification methods, such as salting out, ion exchange chromatography and hydrophobic chromatography Wait

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