Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide.

Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBM(MT) with improved capacity for extracellular production when expressed in E. coli. Two mutations, L(-57)Q and E(-10)D, were identified within the N-terminal propeptide. The amount of WBM(MT) in the culture medium was found to be about three times higher than that of wild type. Besides, the introduction of mutations L(-57)Q/E(-10)D into the N-terminal propeptide also accelerated the maturation of the enzyme. Biochemical analysis indicated that the thermostability and the catalytic activity of mature WBM(MT) were similar to those of wild type. Far-UV CD spectra analysis and limited proteolysis experiments suggested that the mutations L(-57)Q/E(-10)D resulted in a structural change in the N-terminal propeptide of the proform, and the N-terminal propeptide became more flexible, which might be beneficial for the proform to keep in a translocation-competent state. Our result indicates that N-terminal propeptide engineering may be a valuable approach for improving extracellular production of recombinant subtilases expressed in E. coli.