A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.
A preparative hydrophobic interaction chromatography for purification of recombinant nucleocapsid protein of Nipah virus from clarified Escherichia coli homogenate
