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Purification and characterization of proteolytic enzymes from bacteroides amylophilus H-18 Lesk, Earl Michael


This study purposes to examine extracellular proteases of the anaerobic rumen bacterium, Bacteroides amylophilus H-18. An enzyme was isolated and purified from 29 litres of 23 hr cell-free culture supernatant using DEAE Sephadex A-50, Sephadex G-200 and isoelectrofocusing techniques. Although proteolytic activity in the supernatant had a peak of activity at pH 6.7, there was activity at pH values from 4.5 to 11.5. Therefore, an attempt was made to purify the pH 6.7 activity and to follow the activity at other pH values as an index of purity. It was found that separation of the activities at different pH values was not achieved, even though the enzyme was purified 1265 times. Gel filtration of this purified material revealed the presence of two proteases, one of 60,000 and the other of 30,000 molecular weight. Since these enzymes were otherwise identical, they could have represented the monomeric and dimeric forms of a single protein. If the protease of 30,000 molecular weight was separated and resubjected to gel filtration, protease activity of molecular weight 60,000 reappeared. Ultracentrifugation of the 30,000 molecular weight protease demonstrated only one component. Therefore, if the two forms were in equilibrium, it appeared that the dimer was the more stable form of the enzyme. The purified protease did not contain cysteine, so that any tertiary structure in the enzyme could not involve disulfide bridges. All attempts to dissociate the dimeric into the monomeric form were unsuccessful. Examination of the inhibition of Nα benzoyl-L-arginine methyl ester esterase and protease activities with Nα tosyl-L-chloromethane revealed a complete inhibition of esterase activity at pH 8.0 but only a 30% inhibition of protease activity at the same pH, suggesting that more than one enzyme was responsible for the proteolytic activity exhibited by the purified enzyme. Because it was not possible to achieve separation of proteolytic activities at different pH values after a 1265 times purification, it must be assumed that if there are actually different proteases present they must have very similar structures.

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