PROTEOLYTIC ENZYMES FROM PSEUDOMONAS PUTREFACIENS. II. CHARACTERISTICS OF AN ENDOCELLULAR PROTEOLYTIC ENZYME SYSTEM^a

Abstract

A previous investigation ( 7 ) showed that the cell-free growth medium of a milk culture of Ps. pzitrefaciens contained a heat-labile proteolytic enzyme system which attacked casein and lactalbumin. Subsequent experiments failed to show any peptidase activity in the extracellular preparation. The endocellular extract, however, was active against various di-and tripeptides. The literature reveals many reports on microbial peptidases ; few, however, have been studied in detail. More recently Dudani (I), Zimmerman (8), and van der Zant and Nelson (6) reported on the peptidases of some of the lactic streptococci. The optimum p H for activity usually was between p H 7.0 and 8.5. I n many instances metallic ions were necessary for optimum activity of microbial peptidases. The present study was undertaken to investigate the peptidases of Ps. putref aciens. EXPERIMENTAL METHODSThe culture Ps. putrefaciens 12 was the same organism as used in a previous study (7). The stock culture was handled as described in that paper. The culture was transfered daily for 3 transfers prior to each experiment in the culture medium employed in the trial. One percent inoculum was used with incubation at 25'' C.Preparation of cell-free extract. Ps. putrefariens 1 2 was grown in a medium of the following composition: 1 g. glucose, 5 g. yeast extract, 15 g. casamino acids, and 1 1. water. The above medium was autoclaved a t 15 Ib. steam pressure for 15 min., cooled to 25" C., adjusted to p H 7.0, and inoculated. The inoculated medium was incubated for 2 days a t 25" C. The cells were collected by centrifugation in a Sharpless super centrifuge at 30,000 r.p.m. The cells were washed twiee in ice cold phosphate buffer ( p H 7.0, M/15) and collected by centrifugation i n a refrigerated centrifuge and stored in a freezer unit. Cell-free extracts were prepared by disintegration of the cells by sonic vibration. A detailed procedure of this process was presented i n a previous publication (5). The cell-free extract was stored in a freezer unit and used within a few days.Enzyme-substrate mixture. Buffered M/30 solutions of glycyl-L-tyrosine, L-leucyl-L-tyrosine, glycylglycine, glycylglycylglycine, glycyl-DL-valine, glycyl-DL-phenylalanine, glycyl-L-tryptophan, and Y/15 solutions of DL-alanyl-DL-alanine, DL-alanylglycine, DLalanyl-DL-asparagine, and DL-leueylglycine were used as substrates. The substrates were prepared as reported in a previous publication (6). Hydrolysis of the peptides was determined by titration of the carboxyl groups with ethanolic KOH with thymolphthalein as indicator (6). Unless stated otherwise, 0.2 ml. of cell-free extract was added to 3 ml. of substrate and incubated at p H 7.0 and 35" f o r 2 hours.