Cloning, sequencing, and some properties of a novel Bacillus amyloliquefaciens gene involved in the increase of extracellular protease activities

Abstract

A gene involved in the increase of extracellular protease activities from Bacillus amyloliquefaciens strain F was cloned into the EcoRI site of plasmid pUB110 (Gryczan et al., 1978). A plasmid obtained, designated as pNP718, contained a 4.0 kb insert. Extracellular activities of both alkaline and neutral proteases of B. subtilis were increased by introduction of pNP718, and the secreted neutral protease of the transformant was indistinguishable from that of B. subtilis on double immunodiffusion test. The stimulated secretion of other enzymes, such as α-amylase, alkaline phosphatase and ribonuclease, was not observed in the transformant. Four plasmids with smaller DNA inserts were constructed by removing ClaI fragments within the 4.0 kb EcoRI insert of pNP718. Transformation by one of them with a 2.25 kb EcoRI insert resulted in the same stimulation effect as that by pNP718. As this result suggested that an inner 1.75 kb ClaI fragment within the insert contained the putative gene responsible for the increase of protease activities, the nucleotide sequence of the fragment was determined. The putative gene also stimulated the extracellular production of the neutral protease derived from a cloned gene, when the 2.25 kb EcoRI fragment was ligated into the EcoRI site of the neutral protease clone, pNP150.