Primary structure of subtilisin DY

Abstract

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis, strain DY, is presented. The enzyme's primary structure was elucidated using peptides obtained by tryptic hydrolysis and peptides released from BrCN, tryptophan and Asn-Gly cleavage (using hydroxylamine). The peptides were isolated by gelfiltration and by reversed phase high performance liquid chromatography and were degradated automatically in the sequenator. The complete sequence has been verified by peptide overlapping. The subtilisin DY polypeptide chain, like that of subtilisin Carlsberg, consists of 274 amino-acid residues. 32 Amino-acid replacements were found between these two molecules (37 nucleotide mutations, 5 of them two-point mutations). Between the subtilisins DY and Novo 82 amino-acid residue replacements (106 nucleotide mutations, 24 two-point mutations) and one deletion were found. The polypeptide chains of the three subtilisins mentioned were compared and some differences discussed.