A Proteolytic Enzyme of Streptococcus Zymogenes

Abstract

Dudani (1950) presented some evidence which indicated that casein hydrolysis and milk clotting in Streptococc liquefacien were due to the same enzyme. It is known that the closely related species, Streptococcus zymogens, can hydrolyze casein, clot milk, and liquefy gelatin. In the present work, culture filtrates of S. zymogenes containing the enzymatic fraction responsible for the three types of activity were purified and studied. In addition, an attempt was made to determine whether these three manifestations of enzymatic activity were due to one enzyme. METHODS The organism used in this work was a nonhemolytic mutant derived from S. tymogene8 strain 26C1 by ultraviolet irrdiation (Zimmerman, 1951). All stock cultures were maintained on litmus milk. Two drops of an actively growing litmus milk culture were inoculated into a tube of litmus milk, and the tube was frozen and stored at-17 C. The incubation temperature used throughout was 37 C. A ten per cent washed cell suspension was used to inoculate a casein semisynthetic medium from which active supernates were obtained after 24 hours' incubation. The broth for inoculum had the following composition: Sheffield N-Z-Case, 1.0 g; yeast extract, 1.0 g; K2HPO4, 0.2 g; glucose, 0.1 g; and distilled water to 100 ml. The pH was adjusted to 7.0. The casein medium had the following composition: vitamin-free casein, 0.2 g; KH2PO4, 1 This report is from a dissertation submitted by Frederick H. Grutter in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of The Pennsylvania State University.

PROPERTIES OF PROTEINASE FROMSTREPTOCOCCUS FAECALISVAR.LIQUEFACIENS

“…Proteinase purification. A modification of the method of Grutter and Zimmerman (1955) was employed to recover the proteinase. (This procedural change was necessary because of the change of induction media; however, the degree of recovery and purity of enzyme was similar to that reported in the original paper.)…”

“…Enzymatic activity was measured by the method of Anson (1938). In the present studies, 1.0 ml of enzyme sample was added to 5.0 ml of 1% vitamin-free casein solution, prepared as described previously (Grutter and Zimmerman, 1955). After 1 hr at 37 C, 10.0 ml of 0.6 M trichloroacetic acid were added, and the precipitate was removed by filtration through Whatman no.…”

“…Group D streptococci also produce an extracellular proteinase. The enzyme was partially purified by Grutter and Zimmerman (1955), the metabolic optima for enzyme biosynthesis were determined (Rabin and Zimmerman, 1956;Hartman, Zimmerman, and Rabin, 1957), and biosynthetic control mechanisms were studied (Hartman and Zimmerman, 1960). The present communication details initial chemical studies of the enzyme, and presents data that indicate basic dissimilarities with the group A proteinase.…”

Purification and Properties of Intracellular Proteinase from Streptococcus cremoris

“…This optimum pH range for intracellular proteinase was slightly on the acidic side from those of extracellular proteinases from S. lactis (21), S. zymogenes (28), and S. faecalis var. liquefaciens (7). However, the value by Westhoff et al (27) was slightly on the alkaline side from those of many other intracellular proteinase.…”

Milk-Clotting Enzymes From Microorganisms

“…This may confirm the finding of Bargoin (1963). Attempts to separate these activities have been reported in some plant materials, such as papaya latex (Jansen and Balls, 1941;Balls and Lineweaver, 1939), fig latex (Whitaker, 1959), and papain (Bahadur and Kumari, 1959); attempts with microbial preparations, such as Streptococcus zymogenes (Grutter and Zimmerman, 1955) and coccus P (Gorni and Lanzavecchia, 1954), have not been successful.…”