Expression Of Thermophilic Protease Tth0724 By Bacillus Subtilis And Its Application

Serine protease is a type of protease with serine residue as the catalytic active center,which can break the peptide bond by the His-Ser-Asp catalytic triplet.In addition,as the most extensively studied and applied enzyme in the protease family,serine protease has been widely used in industrial production.The thermophilic protease Tth0724 derived from Thermus thermophilus HB8 has been successfully expressed in E.coli.However,the limitation of this expression system is that most foreign proteins exist in the form of inclusion bodies,and it is easy to lead to bacterial autolysis after a large number of expressions.Therefore,improving the expression of enzymes plays a significant role in industrial application.Current studies have shown that the expression system of Bacillus subtilis has good exocrine ability.Exogenous proteins can be soluble expressed in the extracellular medium guided by signal peptides.It can effectively avoid the overexpression of heterologous proteins in E.coli expression system,resulting in the production of inclusion bodies and bacterial autolysis.In this paper,a signal peptide library was constructed through the expression system of Bacillus subtilis,and the signal peptides used for the extracellular expression of Tth0724 were optimized,in an attempt to screen a signal peptide that could efficiently express the Tth0724 protease.In addition,the applications of Tth0724 were studied and compared with other commercial proteases.The recombinant plasmid p BE-S-0724 was constructed by connecting Tth0724 gene with p BE-S shuttle vector.The signal peptide library was constructed by substituting apr E of recombinant plasmid p BE-S-0724 with SP DNA mixture containing 173 different signal peptide sequences.And the signal peptide library was transformed into protease-deficient strain RIK1285 of Bacillus subtilis by electrotransformation.The transformed cells containing recombinant plasmid were cultivated for 48 h to express Tth0724.It was found that the signal peptide yoa W expressed Tth0724 most efficiently by determination of the enzyme activity.In the same volume medium,the secretory expression of Tth0724 in the optimized Bacillus subtilis was increased by 16.72 fold compared to E.coli.The characterization of Tth0724 showed the same properties,such as optimum p H and temperature with E.coli.In addition,Tth0724 has the obvious advantages in high-temperature industrial applications due to its high thermal stability.The enzymatic hydrolysis reaction is rapid at high temperature,and it can improve protein solubility and inhibit the growth of polluting microorganisms.It was found that Tth0724 could effectively hydrolyze milk protein at 75?,improve the digestibility of milk protein and reduce milk allergy.Tth0724 can be used to produce heparin sodium with the function of anticoagulation and reducing blood fat by enzymolysis of casings,and the potency of heparin sodium was 10.82 U/m L.Tth0724 can also be used as a detergent additive at 60?,which can sterilize and eliminate mites and thoroughly clean protein stains.Furthermore,the enzymatic hydrolysis of soybean meals by Tth0724 at high temperature produced a higher content of antioxidant peptides compared to commercial enzymes.Antioxidant active peptides were isolated from soybean peptides with molecular weight of less than 3 k Da,which could be used as additives for anti-aging products.In conclusion,thermophilic protease Tth0724 can be highly expressed by yoa W signal peptide in the expression system of Bacillus subtilis.It can also be used in hydrolysis of defatted milk powder,production of heparin sodium,washing of protein stains,and production of antioxidant soybean polypeptide under high temperature.Due to the high expression of thermophilic enzyme Tth0724 in Bacillus subtilis,it is expected to be widely used in other high-temperature hydrolysis reactions.