Molecular characterization of a novel subtilisin inhibitor protein produced by Streptomyces venezuelae CBS762.70

We report here on the isolation and identification of a gene coding for a n ovel subtilisin inhibitor (VSI) isolated from Streptomyces venezuelae CBS76 2.70. The asi gene was isolated on a 5-kb chromosomal PvuII fragment as ide ntified by DNA sequencing and inhibitor activity testing of the gene produc t. Primer extension studies revealed that the mRNA transcriptional start po int was situated at -37 and -36 relatively to the ATG start codon assuming the presence of solely one promoter. Vsi promoter strength was about double of those of ermE-P1a and aph-P1, as tested with the mRNA production of the aphII gene preceded by the respective promoters. Translation of the vsi co ding sequence revealed a 28 amino acids long signal peptide. The mature VSI protein consists of 118 amino acids of which 87% was verified by N-termina l amino acid sequence analysis. Compared with the already known Streptomyce s proteinase inhibitors, VSI shows a relatively high amino acid identity in the conserved domains. Nevertheless, only a maximum amino acid identity of 56.1% was noticed and some highly conserved residues were substituted in V SI. As a consequence, VSI could be classified within a separate group of St reptomyces subtilisin inhibitors.