Formation of a dipeptidyl arylamidase by Bacteroides splanchnicus NCTC 10825 with specificities towards glycylprolyl-x and valylalanine-x substrates.

Bacteroides splanchnicus in common with several members of the B. fragilis group constitutively produced a number of protein and peptide hydrolysing enzymes. Amongst the most active was an arylamidase, which specifically hydrolysed the dipeptidyl chromogenic substrates glycylprolyl p-nitroanilide (GPRPNA), glycylprolyl beta-naphthylamide (GP beta NA) and valylalanine p-nitroanilide (VAPNA), and had some proteolytic activity towards azocasein. No activity was detected against proline beta-naphthylamide, glycine, valanine or alanine p-nitroanilides. Physiological studies showed that the enzyme was largely cell-associated during exponential growth in batch culture, but was progressively released by the bacteria before the cells entered stationary phase. Glycylprolyl arylamidase (GPA) was completely cell-bound during growth in continuous culture, where synthesis increased concomitantly with dilution rate (specific growth rate) in both carbon- and nitrogen-limited chemostats. Gel-filtration chromatography of B. splanchnicus cell extracts yielded a single peak of GPA activity, with an apparent molecular mass of c. 160 kDa, while one peak of enzyme activity was eluted by 0.3 M NaCl during cation-exchange chromatography. Activity staining of SDS polyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded during preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possible existence of GPA isoenzymes. GPRPNA, VAPNA and azocasein were hydrolysed by the major fraction (fraction 3), while only the p-nitroanilide substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relatively broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated that while aspartic (pepstatin A), thiol (iodoacetate) and metalloprotease (EDTA, cysteine) inhibitors had little effect on hydrolysis of glycylproline p-nitroanilide, GPA was strongly inhibited (c. 80%) by 5 mM phenylmethylsulphonyl fluoride (PMSF), indicating it to be a serine enzyme.