LYTIC ENZYMES OF SORANGIUM SP.: ISOLATION AND ENZYMATIC PROPERTIES OF THE α- AND β-LYTIC PROTEASES

Abstract

Procedures are described for the isolation of two lytic enzymes from culture filtrates of a species of Sorangium. The enzymes, designated α-lytic protease and β-lytic protease, are responsible for most of the filtrate's lytic activity towards Arthrobacter globiformis cells. The enzymes were adsorbed from the filtrate by Amberlite CG50, separated by displacement from the resin with citrate buffer containing a gradient of sodium citrate concentration, and refractionated on columns of the same resin. Trace impurities in the β-enzyme were removed by precipitation of the enzyme with ammonium sulfate. The β-enzyme has been crystallized.On electrophoresis in Tris buffer of pH 8.0, the α-enzyme migrates slightly faster than egg-white lysozyme, the β-enzyme slightly slower. The absorptivity of the α-enzyme at 280 mμ was estimated to be 0.89; that of the β-enzyme 2.05.Low concentrations of the β-enzyme lyse suspensions of Arthrobacter globiformis cells completely, and moderately higher concentrations lyse suspensions of Micrococcus lysodeikticus cells completely; corresponding concentrations of the α-enzyme lyse the suspensions incompletely. The concomitant changes in A660of the suspensions are consistent with zero-order kinetics for the β-enzyme and first-order kinetics for the α-enzyme. Untreated and partially lysed suspensions of Arthrobacter cells show little difference in the dependence of their absorbances on wavelength; corresponding suspensions of Micrococcus cells show marked differences in this respect, and the nature of the change suggests that the breakup of clumps of cells is responsible for a substantial part of the change in absorbance measured during lysis of suspensions of Micrococcus cells. Phase-contrast photomicrographs of cells undergoing lysis show that individual cells vary greatly in their rates of lysis; swelling and decreases in the refractive index precede fragmentation of the cell and may contribute appreciably to the change in absorbance of the suspension.Both enzymes hydrolyze casein. The α-enzyme has the greater activity towards this substrate.