A kind of preparation method of recombinant halophilic archaeal protease

Abstract

The invention belongs to the field of bioengineering, in particular to a method for preparing a recombinant halophilic archaeal protease; the steps are as follows: using the genome of Halostella sp.DL-M4 as a template and pET28a plasmid as an expression vector to construct a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli The cells were induced to express, and the cells were collected by centrifugation for cell resuspension and fragmentation. The supernatant was obtained by centrifugation again, and β-mercaptoethanol was added to obtain a mixed solution, which was passed through a nickel column, and the zymogen was combined with the nickel medium to renature. Protein and target protein elution, ultrafiltration centrifugal concentration, gel filtration chromatography to obtain recombinant halophilic archaeal protease; the invention has good versatility, simpler purification steps, shorter production cycle, and high purity of the obtained protease. The halophilic archaeal protease with a specific enzyme activity of 1159.15 U / mg was finally obtained.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a method for preparing a recombinant halophilic archaeal protease. Background technique [0002] Protease is the enzyme preparation with the highest sales volume in global industrial production, and is widely used in the fields of washing and textile industry, leather industry, food processing, waste treatment, and biotechnology. Commercial proteases are easily inactivated in high-salt industrial production processes such as high-salt food processing and high-salt wastewater treatment, which limits the use of proteases. conditions maintain high hydrolytic activity. Halophilic archaealase is a member of the subtilisin-like S8 family of serine proteases. Most of them have the characteristics of high salt resistance, organic solvent resistance, resistance to various metal ions and strong stability, and have broad application prospects. Similar to most serine proteases, halophilic arc...

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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