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Localization and function of proteolytic enzymes in Bacteroides amylophilus H-18 Hullah, William Arthur

Abstract

Bacteroides amylophilus produces a proteolytic enzyme of which 20% is liberated into the medium and 80% is bound.to the cell. Treatment .of the .cells with toluene or mechanical disinteration does not increase the proteolytic activity, indicating that all the protease is superficially located at the bacterial surface. Less than 1% of the total protease activity is released from the cells by osmotic shock procedures which indicated that the protease is not free in the periplasmic space. Speroplast formation liberates 33% of the cell bound protease 40% of which is sedimentable by prolonged high speed centrifugation. Sonic disruption of spheroplasts releases 72% of the protease. After gentl osmotic rupture 48% of the enzyme activity, remained bound to the spheroplast envelope. Prolonged high speed centrifugation results in the sedimentation of all but 16% of the total enzyme. The results give further evidence to the particle bound nature of the protease of Bacteroides amylophilus. Bacteroides amylophilus has a faster rate of growth, with a reduced lag phase and produces a greater cell yield when tryptic peptides are included in the basal medium. Radioactive amino acids are incorporated into cells in significant amounts, indicating that they were not excluded from the cell by a permeability barrier. The amount of incorporation of ¹⁴C amino acids is found to vary for different amino acids and is shown to be concentration dependent. The exogenous ¹⁴C amino acids were incorporated into the cell protein either directly or after an interconversion step. The inhibition of ¹⁴C amino acids uptake by peptides and the direct uptake of ¹⁴C olig-peptides demonstrated peptide uptake by Bacteroides amylophilus. The results suggest that organic nitrogen contributes to the nutrition of Bacteroides amylophilus.

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