Purification and Properties of Intracellular Proteinase from Streptococcus cremoris

Abstract

Proteolytic activity in the extract from the cells of Streptococcus cremoris increased in the presence of casein, lactose, glucose, and CaCl2 in the media but was negligibly detectable in the extract of the cells harvested from the culture containing succinate or citrate. The intracellular proteinase from S. cremoris harvested from tomato medium was purified 150-fold in this experiment. The enzyme had a molecular weight of 140,000, optimum pH at 6.5 to 7.0, and maximum activity at 30 C. The proteinase was activated by Ca2+ and inhibited by Zn2+, Cu2+, Hg2+, Fe2+, ethylenediaminetetraacetate, and sodium lauryl sulfate. The Km value of the enzyme towards each casein fraction was almost the same, and the Vmax of the enzyme towards a,-casein was smaller than those towards the other casein fractions.

Proteolytic enzymes of dairy starter cultures

“…In view of the possibility that polyclonal antibodies may bind to a variety of bacterial cell wall antigens, the inclusion of proteinase-negative (Prt-; see Section 7) controls treated in the same way would have improved this very useful and direct approach to the study of proteinase localization. The existence and possible functions of intracelhilar proteinases have been thoroughly reviewed [3] and the few recent studies have not clarified the uncertain status and relevance of earlier studies [35][36][37][38] on these enzymes. Two distinct types of intracellular proteinase activity, one with an optimal temperature at around 30-40°C, the other at 5-10°C, have been reported [39,40] from lactic streptococci and L. bulgaricus although the criteria establishing the intracellular location of these enzymes are not clear.…”

Cell Wall-Associated Proteases of Streptococcus cremoris Wg2

“…The collected data on the proteases of group N streptococci are mostly confusing. The molecular weights of the proteases have been found to vary from 130,000 to 180,000 (11,13,21), although lower molecular weights have been reported (5). The number of different proteases in one strain has been claimed to be 1 (11, 21) to 4 (3, 13), and the localization of the proteases in the cell has been found to be either extracellular (8), cell wall associated (9-11, 13, 20, 28), membrane bound (25), or cytoplasmic (1,2,5,21).…”

“…The molecular weights of the proteases have been found to vary from 130,000 to 180,000 (11,13,21), although lower molecular weights have been reported (5). The number of different proteases in one strain has been claimed to be 1 (11, 21) to 4 (3, 13), and the localization of the proteases in the cell has been found to be either extracellular (8), cell wall associated (9-11, 13, 20, 28), membrane bound (25), or cytoplasmic (1,2,5,21). With respect to one characteristic feature, most investigators seem to agree; Ca2+ ions are important for the activity of the proteases (9,11,13,20,21), although in one case, Mn2' and Co2+ ions were mentioned instead of Ca2' as cofactors (5).…”

Purification and Characterization of Intracellular Proteinase from Lactobacillus casei ssp. casei LLG

“…lactis ssp. cremoris (Ohmiya and Sato, 1975). This enzyme was also inactivated by metal chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonylfluoride) and SH-groups reagents (iodoacetamide, iodoacetic acid).…”

“…The cellenvelope proteinase genes have also been sequenced from Lactococcus lactis, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus pyogenes, and Streptococcus agalactiae (Siezen, 1999). However, the data on the intracellular proteinases from lactic acid bacteria (LAB) are very limited (Ohmiya and Sato, 1975;Muset et al, 1989;Akuzawa and Okitani, 1995), and their functions are not well known.…”