Glutamyl endopeptidase of Bacillus intermedius strain 3-19. Purification, properties, and crystallization

Abstract

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic and, rarely, of aspartic acid residues in peptides and proteins was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM-cellulose and Mono S. The enzyme molecular mass is 29 kD and the p/ is 8.4. The proteinase is inhibited by DFP. The enzyme, like 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 Km for the hydrolysis of the latter substrate is 6 mM. The enzyme activity is optimal at 55°C. The enzyme is stable in the pH range 6.5-11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase. Crystal prisms or plates 0.25-0.3 × 0.15 × 0.07-0.1 mm have been grown using the vapor diffusion technique in a hanging drop followed by macroseeding. The crystals belong to the space group B2 with the following unit cell parameters: a = 69.59 Å; b = 61.61 Å; c = 56.11 Å; γ = 117.57°. The X-ray data set to 1.7 Å resolution has been collected on an automatic synchrotron (EMBL Hamburg Station).