Cloning,Expression,Purification And Characterization Of Serine Protease From Dictyoglomus Thermophilum H-6-12

Serine proteases are an extremely large family and are therefore used in a wide variety of applications.They are widely used in the production of foodstuffs,nutraceuticals,pharmaceuticals,cosmetics,detergents and the leather and textile industry,as well as waste treatment.Serine protease is a kind of enzyme with the active center for serine,which widely exists in animals,plants,bacteria,viruses,fungi.And they play a variety of physiological and pathological roles in organism through activating or inhibiting the pro-enzyme.For example,it plays important roles in the process of embryonic development,tissue reconstruction,cellular differentiation,blood vessel formation,pathogen infection,host defense,degradation and aging cell death and so on.Due to low production cost,high yield and economic feasibility,microbial sources serine protease has been used widely recent years.In this study,the dt1871 protease gene was cloned from the genome of Streptococcus thermophilus,amplified the gene by PCR,the target gene was cut with restriction enzyme,and ligated overnight with DNA ligase to construct the recombinant plasmid and transformed into Escherichia coli for expression,due to the misfolding during protein folding,Dt1871 was expressed in inclusion bodies,the inclusion bodies were then denatured and renaturation treatment to obtain soluble expression of protease.The protease can be over expressed in Escherichia coli trasetta(DE3).The optimal temperature and concentration of the protease were 80°C and pH 9.0,which is an alkaline protease.The half-life of Dt1871 at 80°C was over 4 hours,So it has very good thermostability.