Heterologous expression, purification, and biochemical characterization of protease 3075 from Cohnella sp. A01.

Proteases as one of the most significant categories of commercial enzymes, serve nowadays as the key ingredients in detergent formulations. Therefore, identifying detergent-compatible proteases with better properties is a continuous exercise. Accordingly, we were interested in the recombinant production and characterization of protease 3075 as a novel enzyme from thermophilic indigenous Cohnella sp. A01. The biochemical and structural features of the protease were probed by employing bioinformatic methods and in vitro studies. The bioinformatics analysis discovered that protease 3075 belongs to the C56-PfpI superfamily. The protease 3075 gene was cloned and heterologous expressed in Escherichia coli (E. coli) BL21. It was found that the enzyme contains 175 amino acids and 525 bp with a molecular weight of 19 kDa. Protease 3075 revealed acceptable activity in the range of 40-80°C and pH 5.5-8. The optimum activity of the enzyme was observed at 70°C and pH 6. The activity of protease 3075 increased about 4-fold in the presence of Tween 80 and acetone, while its activity attenuated in the presence of iodoacetic acid and iodoacetamide. Docking analyses revealed the dominant interaction between Tween 80 and protease 3075, mediated by hydrogen bonds and Van der Waals forces. Furthermore, molecular dynamics simulations (MDS) showed that Tween 80 increased the stability of the protease 3075 structure. Altogether, our data provided a novel enzyme by genetic manipulation process that could have significant industrial applications.