Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide

Abstract

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-Darginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (DBAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. Most proteolytic enzymes are capable of discriminating between the D and L forms of molecules. Naturally occurring amino acids found in proteins are generally L stereochemical isomers. Synthetic protease substrates were used as L forms also. One of these substrates, benzoyl-L-arginine-p-nitroanilide (L-BAPA), was found to be hydrolyzed by trypsin (10), trypsinlike enzymes (7), various peptidases of plants (6,7,9,15,18,21,26,28), and the peptidase of Corynebacterium matruchotii (11). The D isomer, D-BAPA, is known as a competitive inhibitor for trypsin (10) and plant peptidases (5, 7). It has been shown that germinated spores of Bacillus cereus T hydrolyzed the racemate DL-BAPA more effectively than the L isomer of BAPA alone (3). This result suggested hydrolysis of the D isomer of BAPA. The present investigation revealed that the D-BAPA-hydrolyzing enzyme is more active in vegetative cells than in germinating spores of bacilli. As no peptide-hydrolyzing enzyme stereospecific to the D isomer of BAPA has been described in the literature, we decided to study the prevalence and some properties of this enzyme in a variety of bacteria. In the following paper we refer to the D-BAPA-hydrolyzing enzymes as D-BAPAase. MATERIALS AND METHODSBacterial strains and culture conditions. Bacillus subtilis 168 trpC2 and B. subtilis 43.4 (spoOA43 leu-8) were used throughout this study. Additional strains tested were B. cereus T; B. thuringiensis var. israelensis; 6A10, an aerobic sporeforming marine bacterium (31) The majority of the work was carried out with B. subtilis 168 and B. cereus T. B. subtilis 168 cells were grown at 30°C in LB medium (11) and B. cereus T cells were grown at 37°C in brain heart infusion (BHI) medium (Difco Laboratories), both with shaking. Starter inocula were prepared from individual colonies on nutrient agar followed by cultivation of B. subtilis 168 in nutrient broth (Difco Laboratories) medium at 30°C and cultivation of B. cereus T in BHI medium at 37°C. The log-phase starters were used as 1% (vol/vol) of the final cultivation medium. To obtain semisynchronized cultures, cu...

Hydrolysis of ? by members of the Haloanaerobiaceae: additional evidence that Haloanaerobium praevalens is related to endospore-forming bacteria

“…As reported earlier [2] all non-sporulating bacteria tested, aerobic as well as anaerobic, and Gram-positive as well as Gram-negative, hydrolyze L-BAPA, while showing negligible D-BAPAase activity. We included in our study the Gram-negative, moderately halophilic facultative anaerobe V. costicola, which is unrelated to the Haloanaerobiaceae, and does not form endospores, and as expected this organism showed a high L-BAPA-ase activity; after 2 days incubation at 45 o C, however, also a minor D-BAPA hydrolyzing activity was detected.…”

“…If D-BAPA-ase activity in bacteria can be used as a chemotaxonomic marker for endospore-forming bacteria, as suggested before [2], the activity should also be found in endospore-forming bacteria unrelated to the Bacillus or the Clostridium group. We recently characterized a group of obligately anaerobic, moderately halophilic eubacteria, the Haloanaerobiaceae [7,8], part of which are able to produce endospores [9,10]• It is thus of interest to test the members of this group for D-BAPA-ase activity, not only to confirm the hypothesis that all endospore-forming bacteria possess this activity, but also to determine whether members of the group in which endospore formation has not (yet) been detected hydrolyze D-BAPA, A positive result would then confirm their presumed relationship with the endospore-forming moderately halophilic anaerobes.…”

“…It was recently shown that endospore-forming bacteria of the genera Bacillus and Clostridium possess a stereospecific enzymatic activity cleaving the D-isomer of N '-benzoyl-arginine-p-nitroanihde (D-BAPA) [1,2]. The ability to cleave D-BAPA was detected only in endospore-forming bacteria, while all other bacteria tested (Gram-positive as well as Gram-negative, aerobes as well as anaerobes) readily hydrolyzed L-BAPA, but did not show significant hydrolyzing activity towards the Disomer [2].…”

“…The function of the D-BAPA hydrolyzing activity in endospore-forming bacteria is as yet un-known• No direct involvement of the D-BAPA-ase in the sporulation process was postulated, as its activity was higher during the exponential growth phase of Bacillus cultures than during germination, and a non-sporulating mutant of B. subtilis also showed high D-BAPA-ase activity [2].…”

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168

“…This was done as described before (Gofshtein-Gandman et al, 1988). The stacking gel (2 cm long) contained 5% (w/v) acrylamide.…”