Purification and properties of an acid proteinase from Choanephora cucurbitarum

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Abstract
An acid proteinase has been purified from mycelial extracts of Choanephora cucurbitarum by treatment with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The enzyme hydrolysed haemoglobin rapidly compared with casein, bovine albumin, cytochrome c, and hide powder azure, but failed to hydrolyse any of the synthetic peptides tested. The acid proteinase is a glycoprotein with an apparent molecular weight of 12 700. Optimal hydrolysis of haemoglobin by the proteinase was observed at 20 °C, pH 3.0, and has a Km value of 2.8 mg∙mL−1. Heavy metallic ions, such as Hg2+, Fe2+, and Zn2+, inhibited the hydrolytic activity, whereas Ca2+ and Cu2+ enhanced the enzyme activity by two- and four-fold, respectively, as compared with the controls. Benzamidine and phenylmethanesulfonyl fluoride inhibited severely the enzyme activity, while diisopropyl fluorophosphate, antipain, N-α-p-tosyl-L-lysine chloromethyl ketone inhibited moderately. Phenylmethanesulfonyl fluoride inhibition could be reversed by 2-mercaptoethanol. Reducing agents such as cysteine and dithiothreitol did not enhance the enzyme activity. The data presented suggest that the enzyme has the characteristics of serine proteinases.