Production and purification of alkaline protease inhibitors of Streptomyces sp.

Abstract

A large number ofprotease inhibitor have been found in animals, plants and microorganisms.1} Some of them were reported to be inhibitors of alkaline proteases such as subtilisin BPN', S-SI,2) AP-I3) and Plasminostreptin4) as proteineous inhibitors, and MAPI5) as a peptidic inhibitor. We isolated Streptomyces sp. AS 631, showing high productivity of plural inhibitors against subtilisin BPN' from soil in GunmaPrefecture. In this paper, we report the cultivation, purification and some properties of the inhibitors from strain AS 631. The inhibitory activity against subtilisin BPN' was measured by the modified method of Murao and Sato.2) One unit of inhibitory activity is equivalent to 50% inhibition of the caseinolytic activity of subtilisin BPN' (10^g). Identification of the strain was carried out with reference to the descriptions of Bergey's Manual of Determinative Bacteriology, 8th Ed.,6) and according to the procedures in the reports of the International Streptomyces Project.7) This strain was Gram-positive and formed aerial mycelia with gray colored spores, in straight and small branching chains, with a smooth surface. The cell wall contained LL-2,6-diaminopimeric acid and glycine as the major constituents, i.e., cell wall type I. The strain produced melanoid pigments, hydrolyzed gelatin, starch and skim milk, and utilized D-glucose and D-galactose. The optimum temperature and pH were 28°C and 7.0, respectively. On the basis of these characteristics, the strain should belong to the genus Streptomyces.The cultural conditions for the production of an alkaline protease inhibitor (named SAPI) by this strain were investigated. Glucose and soluble starch were good carbon sources for growth. SAPI was produced abundantly at the soluble starch concentration of 1%. Polypeptone was effective for SAPI production as a nitrogen source at the concentration of 3%. The addition of0.5% bonito extract enhanced the productivity 3 times, the maximumproduction of 5400units/ml being attained at 28 hr. Thus the optimum conditions for the production of SAPI were 841 determined to be follows; 1% soluble starch, 3% polypeptone, 0.5% bonito extract and 0.1% NaCl (pH 7.0), at 27°C. SAPI in the culture filtrate (3.31) was adsorbed on Amberlite XAD-2and then eluted with 50% «-propanol. The active fraction was evaporated in vacuo at 35°C. The dry material was dissolved in water and then extracted with fl-butanol. The dry active material was dissolved in a 10% acetic acid-methanol solution, applied to a Toyopearl HW-40Fcolumn (2.5 x 80cm) and then eluted with the solvent. The molecular weight of SAPI was estimated to be 600~700 on this gel filtration. The dry active material was dissolved in ethylacetate-rc-butanolacetic acid (5:5: 1), applied to a silica gel prepacked column (30/mi, 2.5 x 30cm) and then eluted with a linear gradient from the same solvent mixture (200ml) to nbutanol-H2O (9 : 1, 200ml) at the flow rate of 3.5ml/min. The main fraction was evaporated, dissolved in a 10% acetic acid-50% methanol solution and applied to a reverse...