Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 μkat mg(-1) protein. The enzyme exhibited a Mr of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of Mr 43,000. Monospecific antibodies were raised against the homogeneous protein.

Aminoacylase (EC 3.5.1.14) from Aspergillus oryzae was purified from a commercially available crude material by heat treatment, precipitation by polyethyleneimine and ammoniumsulfate, gel chromatography and preparative disc-gel-electrophoresis. The purified product was homogenous as judged by polyacrylamide gel electrophoresis. SDS-gel electrophoresis, polyacrylamide-gel-gradient electrophoresis, gel chromatography and amino acid analysis demonstrated the enzyme to be composed of two subunits with Mr of 36 600. The kinetic properties of the enzyme were studied with chloroacetyl derivatives of alanine, phenylalanine, methionine, leucine, norleucine and tryptophan. The pH optimum of the acylase activity with chloroacetyl-alanine as substrate is at pH 8.5. Acyl derivatives of hydrophobic amino acids are preferred substrates. The enzyme has no dipeptidase activity. Aminoacylase is not inhibited by SH-blocking agents and no SH-groups could be detected with Ellman’s reagent in the native and denatured enzyme. The enzyme activity is insensitive to phenylmethylsulfonyl fluoride and N-α-p-tosylʟ-lysine chloromethyl ketone. The microbial acylase is a zinc metallo enzyme. Metal chelating agents are strong inhibitors; it is further inhibited by Cd2+, Mn2+, Ni2+, Cu2+ and activated by Co2+. The properties of pig kidney and Aspergillus acylase are compared.

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