GLUTAMYL ENDOPEPTIDASE OF BACILLUS-INTERMEDIUS STRAIN-3-19 - PURIFICATION, PROPERTIES, AND CRYSTALLIZATION

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutam ic and, rarely, of aspartic acid residues in peptides and proteins was isolated from Bacillus intermedius 3-19 culture filtrate using chroma tography on CM-cellulose and Mono S. The enzyme molecular mass is 29 k D and the pi is 8.4. The proteinase is inhibited by DFP. The enzyme, l ike other glutamyl endopeptidases, reveals two pH optima (pH 7.5 and 9 .0) for casein and one (pH 8.0) for Z-Glu-pNA hydrolysis. The K-m, for the hydrolysis of the latter substrate is 6 mM. The enzyme activity i s optimal at 55 degrees C. The enzyme is stable in the pH range 6.5-11 .0. Its N-terminal sequence shows 56% coinciding residues when compare d with that of Bacillus licheniformis glutamyl endopeptidase. Crystal prisms or plates 0.25-0.3 x 0.15 x 0.07-0.1 mm have been grown using t he vapor diffusion technique in a hanging drop followed by macroseedin g. The crystals belong to the space group B2 with the following unit c ell parameters: a = 69.59 Angstrom; b = 61.61 Angstrom; c = 56.11 A; g amma = 117.57 degrees. The X-ray data set to 1.7 Angstrom resolution h as been collected on an automatic synchrotron (EMBL Hamburg Station).