Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70°C, with half-lives of 13 h and 19 min at 80 and 90°C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro- Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln- 4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75°C. The enzyme is expressed in E. coli as an inactive proproteinase at 37°C and is converted to the mature enzyme by heating the cell-free media to 60°C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH₂- terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH₂-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme. SDS-PAGE was shown to produce anomalous results with the proenzyme, in contrast to those obtained by isoelectric focusing, which resulted in the conversion of the proproteinase to the mature enzyme in SDS sample buffer, after acid precipitation and in the presence of the serine proteinase inhibitor phenylmethanesulfonyl fluoride.