Molecular cloning and expression of the alkaline protease gene from an alkaline -resistant bacterial strain

Abstract

An alkaline-resistant bacterial strain (gram-negative), which exhibited protease activity at alkaline condition (pH 11), was isolated. A gene (aprJ) responsible for the alkaline protease activity of the bacterial strain was cloned into Escherichia coli JM83 and expressed. DNA sequencing found an open reading frame of 1,266 nucleorides which could potentially encode a precursor of the alkaline protease (AprJ) comprised 422 amino acids. The N-terminus of the open reading frame had a putative signal sequence. The C-terminal portion (283 residues), which probably corresponded to the mature form of the AprJ, showed high sequence homology (31-46\%) with the subtilisin-type serine proteases. Extremely high sequence identity was observed in the regions containing the active-site residues, $Asp^{169}$, $His^{202}$, and $Ser^{358}$. The synthesis of AprJ in E. coli was started in the stationary phase of growth and the produced Aprj was released to the extracellular medium immediately without any detectable intracellular accumulation. The AprJ was most active around pH 12.0 and stable in the pH range of 6-11. $Ca^{2+}$ stablized AprJ to het treatment. The maximum activity was observed at 60$^\circ$C and its stability was maintained below 55$^\circ$C in the presence of 1mM $CaCl_2$. AprJ was completely inactivated by 1mM PMSF, an inhibitor of serine enzyme. The results of the homology analysis for the predicted amino acid sequence and the study for the effect of inhibitor (PMSF) suggest that AprJ is a subtilisin-type serine protease (EC 3.4.21.14).