Purification and Characterization of an Intracellular Dipeptidase from Mycobacterium phlei

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Abstract
A dipeptidase has been isolated from cell extracts of Mycobacterium phlei 689 in a highly purified form, by a five‐step procedure comprising streptomycin sulfate precipitation, hydroxyapatite adsorption, ammonium sulfate fractionation, gel‐filtration and preparative acrylamide gel electro‐phoresis. The final product exhibited only one band on acrylamide gel, one arc in immunodiffusion and one amino acid in end‐group determination; it was at least 1000‐times more active than the crude material. The enzyme thus purified specifically hydrolyzed typical dipeptide such as l‐leucyl‐glycine, and does require metal ions for its activity. The molecular weight of the enzyme was found to be 88000 by gel‐filtration and 45000 by gel‐electrophoresis. Effects of enzyme concentration and substrate concentration on the rate of catalysis have been respectively obtained for dipeptides alanyl‐glycine, leucyl‐glycine and d‐leucyl‐glycine as substrates. Different inhibitors were assayed toward enzyme. The effect of different metal ions on the enzyme were also considered. The enzyme possesses a leucine residue at the N‐terminal position. Amino acid composition reveals a high content of aspartic and glutamic acids, of leucine and alanine and a low content of cysteine, methionine, tryptophan and histidine residues.