Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor

Abstract

Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80؇C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 ؋ 10 ؊10 M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same as that toward an exogenous target enzyme, subtilisin BPN.Streptomyces spp. are filamentous gram-positive soil bacteria widely known as producers of a variety of hydrolytic enzymes as well as of commercially important antibiotics. A commercial crude sample, pronase (13), prepared from Streptomyces griseus is a mixture of several extracellular proteolytic enzymes and has been widely used in biological studies for a long time. Serine proteases produced by Streptomyces lactamdurans (5) and Streptomyces peucetius (4) were reported to coordinately regulate the cellular protein turnover associated with secondary metabolism and morphogenesis. This organism is also well known to produce protease inhibitors, including low-molecular-mass (26) and proteinaceous (6) compounds. A protease inhibitor might be considered a modulator molecule for the proteolytic activities of some enzymes involved in physiological processes, such as cell differentiation and secondary metabolism, as suggested by Aoyagi (1). However, the biological roles of the interactions between protease and protease inhibitors in the producer strains have not been well identified. Streptomyces su...

A novel member of the subtilisin-like protease family from Streptomyces albogriseolus

“…As described in our previous paper (34), three SSI-interacting proteases were isolated by affinity chromatography using a Sepharose column to which SSI was bound. The major protease among them, SAM-P20, was clearly demonstrated to be an endogenous target of SSI by complex formation analysis (35).…”

“…In addition, to clarify the physiological role of SIL proteins, we identified three endogenous target proteases in the culture supernatant of an SSI-nonproducing mutant strain using an affinity column to which SSI was bound. One of the target proteases, termed SAM-P20, was classified as a novel member of the chymotrypsin superfamily (25), and its proteolytic activity was strongly inhibited by tight complex formation with SSI (34,35). All of the SSI-nonproducing mutant strains derived from Streptomyces albogriseolus S-3253 exhibited several common pleiotropic properties: slightly slow growth even in a rich medium, a marked decrease in aerial mycelium-forming ability, and a remarkable increase in the extracellular activity and/or productivity of proteases (34).…”

Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiaevar.k11

“…Intense studies have been done with various strains, and results indicated that many enzymes from Streptomycetes can be applied in biotechnological processes (Horikoshi 1995). Serine proteases (Chandrasekaran and Dhar 1987;Kitadokoro et al 1994;Yum et al 1994;Taguchi et al 1995) and other proteases have been purified and characterized in Streptomycetes. Some serine proteases derived from Streptomycetes (Sinha et al 1991;Moormann et al 1993;Böckle et al 1995;Suzuki et al 1997;Bressollier et al 1999) or other microorganisms (Takiuchi et al 1984;Lin et al 1992;Kulakova et al 1999) were identified as keratinolytic proteases.…”

Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases

“…Some homologues of these proteases have been cloned from different Streptomyces strains. Among them, SAM-P20 (Taguchi et al, 1995b), SAM-P26 (Taguchi et al, 1998) and SAM-P45 (Suzuki et al, 1997) from Streptomyces albogriseolus have been studied. Pure enzymes (a SAM-P20 homologue isolated from S. coelicolor Müller, SAM-P26 and SAM-P45 from S. albogriseolus), provided by Dr S. Taguchi, were used to study the processing effects on Xys1L in in vitro experiments.…”