Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 I. Purification and some enzymatic properties

Abstract

Achromobacter lyticus M497-1 produces three kinds of alkaline proteases (protease I, II and III) in culture medium along with the bacteriolytic enzyme (Masaki, T., Nakamura, K., Isono, M. and Soejima, M. (1978) Agric. Biol. Chem. 42, 1443–1445). Among these three proteases, Achromobacter protease I (EC 3.4.21.-) shows strict splitting for lysine residues at the carboxyl side of the splitting point. This enzyme was purified through a sequence of benzalkonium chloride treatment, acetone fractionation, CM-cellulose and DEAE-cellulose treatment, chromatography on AH-Sepharose 4B and isoelectric focusing method. This form was shown to be homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation analysis. The physicochemical properties of the enzyme were: M r 30 500; partial specific volume ( ν ), 0.717 ml/g; intrinsic viscosity (η), 0.0385 dl/g; isoelectric point (p I) 6.9; and E 1cm 1% at 280 nm, 18.77. The enzyme was composed of 294 residues of amino acid per molecule, with glycine as NH 2-terminal and lysine as COOH-terminal amino acids. The optimum pH values with casein, Bz-Lys-pNA and Tos-Lys-OMe were 8.5–10.7, 9.0–9.5 and 7.8–8.2, respectively. The enzyme was inhibited by iPr 2P-F, PhCH 2SO 2F and Tos-LysCH 2Cl but not by Tos-ArgCH 2Cl, EDTA, o-phenanthroline and PCMB.