Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326

Abstract

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pU702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.

Purification and Properties of an Extracellular Aminopeptidase from Streptomyces Lividans 1326

“…Recently, a small, extracellular, neutral proteinase of S. Zividans has been identified through cloning experiments Butler et al, 1992). Under normal conditions, however, this milk-hydrolysing proteinase has very little activity (Butler et al, 1992;Lampel et al, 1992). Additionally, a gene encoding an intracellular aminopeptidase P has been isolated from S. Zividans and sequenced (Butler et al, 1991).…”

“…Powdered dry milk (Carnation Co.) was autoclaved separately as a 20 % (w/v) solution in double-distilled water and added to the medium after cooling to make a final concentration of 20 g I-'. Conditions used for growth and analysis of the primary and secondary seed cultures and 10 litre fermentation cultures of S. lividuns are described elsewhere (Lampel et al, 1992).…”

“…The enzymic hydrolysis of azocasein was assayed as described previously (Gibb et al, 1989). The milk hydrolysis activity of the proteinase was determined by the spectrophotometric assay described by Lampel et al (1992).…”

“…C5 expressed in S. lividans, we noticed that the major extracellular proteinase activity contributed by the host strain, s. lividans 1326, was an aminopeptidase (Lampel et al, 1992). Thus, we have examined S. lividans for its extracellular proteinase activities, and have purified and characterized the major extracellular proteinase of S. lividans, a leucine aminopeptidase (LAP; EC 3.4.11.…”

“…There has been significant interest in the use of Streptomyces lividans, the primary streptomycete recombinant DNA host strain, as a host strain for secretion of heterologous recombinant proteins into the culture broth (Lichenstein et al, 1988;Taguchi et al, 1989;Bender et al, 1990;Malek et al, 1990). Thus, it is important to elucidate the complement of extracellular proteinases which may degrade potentially valuable secreted recombinant products produced in fermentations with this strain.…”