Secretory overproduction of the aminopeptidase from Bacillus licheniformis by a novel hybrid promoter in Bacillus subtilis

Abstract

Bacillus licheniformis aminopeptidase (BAP) was overexpressed in B. subtilis using a novel expression vector carrying a hybrid promoter, BJ27UP, which was constructed from a strong promoter BJ27Δ88 and a fragment of the tac promoter. When added upstream of the BJ27Δ88 promoter, the tac fragment (including the -10 box) improved the promoter activity of the BJ27Δ88 promoter by approximately threefold. The hybrid promoter, BJ27UP, allowed overexpression of BAP in B. subtilis, and over 95% of the produced BAP was secreted into the culture medium, whereas in E. coli, BAP was poorly expressed, despite the use of the T7 expression system. The volumetric production of BAP mediated by the hybrid promoter BJ27UP was reproducibly over 9.0 U/ml in Luria–Bertani medium after cultivation for 12 h, representing a 20-fold increase over that of the endogenous promoter of the bap gene. Due to its high-yield secretion, the recombinant BAP was purified using a simple inexpensive purification method consisting of ammonium sulfate fractionation and Q-Sepharose column chromatography.