Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus

The thermostable cellulase Cell2A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 degreesC, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydro phobic putative signal peptide. Two deletion mutants lacking this hydrophob ic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for t he toxicity of the full-length enzyme in the host organism. This was furthe r corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, m ade up of either the catalytic module of Cell2A or the catalytic module and the putative linker sequence, were characterised and their properties comp ared to those of the full-length enzyme. The specific activity of the mutan ts was approximately threefold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 degreesC and unfolding temperatures up to 103 degreesC.