Studies on a Novel Thermostable Acid Proteinase "Kumamolysin"

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In the course of our screening of an acid proteinase, insensitive to the known specific inhibitors, in thermophilic microorganism, we found a novel enzyme designated "Kumamolysin" in Bacillus novosp. MN-32. The present project is to elucidate the unique substrate specificity and primary structure of the enzyme from protein sequencing and/or coloning of the kumamolysin gene. The summary of experimental results are as follows.(1) The unique character of kumamolysin is its substrate specificity : kumamolysin specificaly hydrolyzed the peptide bond of Leu (15) -Tyr (16) in oxidized insulin B chain. The subsite requirement was disclosed from synthetic peptide substrate, mimic to the sccile site in oxidized insulin B chain. The minimum length of substrate was determined to be hexapeptide (VEALYL).(2) Primary structure : Partial amino acid sequence (total 100 residues) of kumamolysis was determined from tryptic, lysyl endopeptidase fragements by automated protein sequencer. From these results, DNA probe for cloning was designed. After hybridization, Sal I fragment of chromosomal DNA was deduced to contain kumamolysin gene. Insertion of this fragemnt in pUC18 to yield plasmid pK1. Subcloning of this plasmid was also carried out, and partial base sequence of kumamolysin gene was elucidated.