Cloning of the gene responsible for the extracellular proteolytic activities of Bacillus licheniformis

Abstract

Effect of the cloned gene of Bacillus licheniformis on the extracellular proteolytic activities of B. subtilis was investigated. The gene was cloned onto the vector plasmid pUB110 (3.0 Md), and the introduction of the hybrid plasmid [pAN2 (5.4 Md)] into the cells of B. subtilis resulted in a marked increase of activities of the extracellular alkaline and neutral proteases, which had optimal pHs at 10.5 and 7.2, respectively. On DEAE-Sephadex column chromatography, the extracellular activity of B. subtilis with pAN2 was separated into two active fractions (a1 and b1). The activity in a1 was specifically inactivated by diisopropyl phosphorofluoridate (DFP) and tosyl fluoride (TSF), potent inhibitors of alkaline proteases, while, the activitiy in b1 was inhibited by ethylenediaminetetraacetate (EDTA), an inhibitor of neutral protease, but not by DEP or TSF. Sub-cloning with genes shortened to about 0.85 Md (pAN2-1) and 0.25 Md (pAN2-2) increased the activities of both alkaline and neutral proteases. The extracellular α-amylase and ribonuclease production was also increased when the host strain was transformed with these hybrid plasmids (pAN2, pAN2-1, pAN2-2). The increase in activity of proteases by the cloning was discussed in relation to regulation of the production and/or secretion of the enzyme.