Purification and characterization of a nuclease(3'-nucleotidase) from a Penicillium sp.

Abstract

A nuclease (3'-nucleotidase) similar to P1 nuclease from Penicillium citrinum was purified from a commercial digestive from a Penicillium sp. The activity of the nuclease (PA) was separated to three fractions by diethylaminoethyl-Toyopearl 650M column chromatography, in total yield of 10%. The apparent molecular weight of these three nucleases, PA1, PA2 and PA3 was 35000, 33000, and 32000, respectively. All of them were homogeneous so far as checked by sodium dodecyl sulfate slab gel electrophoresis. The three nucleases differed in carbohydrate content, but their amino acid composition was practically the same, and very similar to that of P1 nuclease. The molecular weight of nuclease PA3, the major component of nuclease PA, was approximately 27000 after digestion by endoglycosidase F. The N-terminal and C-terminal amino acid sequences of nuclease PA3 were determined by Edman degradation and carboxypeptidase(s) digestion, respectively. The nuclease PA3 was inactivated in the presence of 10 mM ethylenediamine tetraacetic acid (EDTA) and 65% of its native enzyme activity restored by the addition of 20 mM ZnCl2. The pH-dependent photooxidative inactivation of nuclease PA3 was accelerated by removal of Zn ion by EDTA or trishydroxymethyl aminomethane, indicating the possible chelation of Zn2+ with some histidine residues.