STUDIES ON MASIKINOSIN,A NOVEL TYPE OF PROTEINASE

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The stock strains in this laboratory were screened using succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide as a substrate. Of more than 2,800 strains screened, we found ability to release p-nitroaniline in the culture filtrate of Streptomyces griseoloalbus SN-22. The culture filtrate was fractionated with ammonium sulfate and column chromatographies on DEAE-Cellulose, Butyl-Toyopearl 650S,and Sephadex G-75. By these procedures, SN-22 proteinase (named Masikinosin) was purified about 92.7-fold from culture filtrate with an activity recovery of 10.3%. The molecular weight was estimated to be 26,000 by SDS polyacrylamide gel electrophoresis and by gel filtration on a TSKgel G2000SW column. The isoelectric point was 6.4. The optimum pH was pH 9.0. The enzyme retained more than 80% of the original activity between 8.0 and 11.0. The optimum temperature was 45ﾟC and 80% of the initial activity was observed after incubating at 40ﾟC,for 30 min, and at pH 9.0. The enzyme was markedly inhibited by DFP and PMSF.However, it was not affected by SSI,MAPI,elastatinal, TLCK,TPCK,antipain, and EDTA.From the cleavage sites of the insulin B-chain and lysozyme, Masikinosin specifically hydrolyzed the carboxyl side of alanine and valine residues. The enzyme could also hydrolyze elastin, elastin-orcein, and casein. In conclusion, masikinosin is a unique serine proteinase with specificity for cleavage at the carboxyl side of alanine and valine residues.