ABSTRACT
The procedure of isolation and purification of lipases from the fungus Oospora lactis have been developed. The existence of two different lipases has been demonstrated, one of which (Mr = 43000) is localized in the periplasmic space and can be liberated into the external medium in an unchanged form, while the other (Mr = 40000) is tightly bound to the membranes and can be solubilized by detergent treatment. The most essential properties of the lipases are discussed and a detailed analysis of the functional and physicochemical properties of extracellular lipase is given.
Subject(s)
Lipase/isolation & purification , Mitosporic Fungi/enzymology , Cell Membrane/enzymology , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Lipase/metabolism , Molecular WeightABSTRACT
The paper describes how to prepare osmotically sensitive forms from the fungus Oospora lactis. The lytic enzyme from actinomycetes at a concentration of 25 mg/ml causes abundant formation of protoplasts at 36 degrees C and pH 7.5 within 10--15 min. The best stabilizing agent is 1 M NaCl solution or a mixture of 1 M NaCl with 1 M mannitol solution (1:1). In order to induce lysis of cell walls by the enzyme from Helix pomatia, the cells must be pretreated with a solution containing 40 mM tris (hydroxymethyl)-aminomethane, 5 mM EDTA and 0.2 M cysteine. The production of Oospora lactis protoplasts was accompanied with the liberation of lipase from the periplasmic space. The activity of lipase was 90% of the overall intracellular activity as was confirmed by the results of differential centrifugation of a homogenate prepared by mechanical disintegration of the cells. Since the Rf in disc electrophoresis, the pH optimum and the substrate specificity of exocellular lipase were identical with those of periplasmic lipase, the enzyme must be liberated without a change in the molecular weight.
