An extracellular proteolytic enzyme from a strain of Arthrobacter II. Purification and chemical properties of the enzyme

Abstract

Summary 1. A new extracellular proteolytic enzyme has been isolated from the culture medium of a strain of Arthrobacter grown in the presence of gelatin. The fractionation procedure makes use of precipitation with ammonium sulfate in the presence of cellulose, treatment with DEAE-Sephadex, gel filtration on Sephadex G-100 and crystallisation with ammonium sulfate or acetone. 2. Ultracentrifugation of the crystallized enzyme showed only one sedimenting component and its molecular weight determined by equilibrium centrifugation was approx. 22 ooo. Free electrophoresis revealed only one ultraviolet-absorbing peak with a mobility of 2.57 · 10−5 cm2 · V−1 · sec−1 at pH 8.6. 3. Twice-crystallized enzyme may contain traces of peptides which can be removed by gel filtration in aqueous acetic acid. Some of the pitfalls encountered in studies on the amino acid composition of the enzyme are discussed. 4. The enzyme contained 221 amino acid residues with a formula weight of 23 041. The amino-terminal residue was valine. The protein contains 2 disulfide bridges, a total of 12 basic amino acid residues and 29 amide groups. The molar extinction coefficient is 44.4 · 103 reflecting a high content of tyrosine and tryptophan.