An alkalophilic Bacillus sp., strain GX6638 (ATCC 5327O), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of -4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. ]Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity it pH 12 over a 24-h incubation period at 25°C; protease HS exhibited similar alkaline stability properties ¢o pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50°C and 25 min at 60°C. At pH above 9.5, protease HS readily lost enzymrtic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60°C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.Alkaline proteases secreted by both neutralophilic and alkalophilic bacilli are of interest because they represent a major source of commercially produced proteolytic enzymes (1; 11, 16). These proteases exhibit optimal activity at pHs of 9 to 11 and are inactivated by serine active-site inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In general, these enzymes have molecular weights ranging from 20,000 to 30,000, are stabilized by Ca2+, and have characteristically high isoelectric points (2,11,12,16,19,23). Alkaline proteases, or subtilisins, secreted by neutralophilic Bacillus spp. are stable from pH 5 to 10 at low temperatures, but are readily inactivated at higher temperatures and alkalinities in the absence of Ca2+ (1, 2). The subtilisins have been divided into two groups based on differences in amino acid composition and immunological and kinetic properties (12). Recent results, however, suggest that these proteases possess identical amino acid residues in 63% of their primary sequences (17). Alkaline proteases from alkalophilic bacteria have been studied in less detail, yet many similarities exist between subtilisins and alkaline proteases from alkalophilic bacilli (11). However, serine proteases from alkalophiles have superior alkaline...

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55°C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2C03 and thioglycollic acid, the total proteolytic activity and its stability at 55°C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8·8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8'8, suggest that this property may be used to distinguish virulent and benign strains.

The relationship between the serological classification system for serogroup B and for serogroup H of Bacteroides nodosus and cross-protection between subgroups within these serogroups was examined. Protection against ovine footrot following vaccination was achieved against other subgroup strains provided sufficient cross-reactive antibody was induced by shared pilus antigens. Within serogroup B, better cross-protection against one subgroup was obtained with a pili vaccine than a whole cell vaccine which correlated with higher pilus antibody titres induced by the former. For serogroup H, a lack of cross-protection and serological reactivity between subgroups was demonstrated, which indicates that the prototype strain of subgroup H2 should be designated a new serogroup.

Summary A total of 17 monoclonal antibodies (MoAb) were used to analyse the antigenic structure of pilus protein from three serogroups of Bacteroide.s nodosus. The four MoAb which agglutinated pili were serogroup (and subgroup) specific, and the agglutinating epitope was present on the pili monomer and dependent on the intra-chain disuifide bond.Non-agglutinating MoAb identified two further non-linear and serogroup-restrioted epitopes on strain 198 (Al) pili and two linear epitopes on 336 (Fl) and 265 (HI) pili. Three MoAb cross-reacted with pili from six of the eight major serogroups and recognized an epitope in the N-terminal region of the molecule. This panel of MoAb has therefore identified at least four epitopes on pilus protein and will facilitate serotopic analyses of the immunogenicity of each epitope in sheep during vaccination against footrot.