Purification and properties of the chymotrypsin-like serine proteinase overproduced by Stremptomyces sp. strain C5-A13

Abstract

The major serine proteinase of Streptomyces sp. strain C5-A13, a proteinase-overproducing mutant strain, was purified to homogeneity. It has a relative molecular mass (Mr) of 19500 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Superose-6 gel filtration chromatography, a low isoelectric point, optimal activity at pH 8.5-9.5, and an optimal temperature of approx. 55 ° C. This purified enzyme has a high specific activity on azocasein of 11000 units'rag -1 of protein, a Km of 2.7 X 10 -5 M and a Vmax of 2.0 gmol. rain -1. m g -1 of protein when succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide was used as substrate. It was inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but not by ethylenediaminetetraacetate (EDTA) or N-tosyl-L-phenylalanine chloromethyl ketone. Thus, this serine proteinase appears to be a chymotrypsin-like enzyme and represents nearly 90% of the total extracellular azocasein-hydrolyzing activity produced by strain C5-A13. Since CaCO3 did not stimulate activity of the purified enzyme itself, the carbonate effect may be a transcriptional or translational rather than a post-translational one.

Purification and characterization of alkaline protease from Alcaligenes faecalis

“…KSM‐K16 and Streptomyces sp. strain C5‐A13 [22,23]. A. faecalis protease was stable up to 50 °C, as apparent from Figure 5, but was rapidly inactivated at higher temperatures.…”

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253

“…4), with the already well-characterized proteins SGPA and SGPB from Streptomyces griseus (8). Also, to date, production of ␣-chymotrypsin-type serine proteases with molecular sizes ranging from 14 to 20 kDa from several Streptomyces species has been reported, although their structures have not been well characterized (1,8,19,42). These findings strongly suggest that a variety of proteases of this type might be distributed ubiquitously, just like SSI-like inhibitor proteins, among the genus Streptomyces, although some might be genetically masked as, e.g., in S. albogriseolus S-3253.…”