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. frag ilis group constitutively produced a number of protein and peptide hyd rolysing enzymes. Amongst the most active was an arylamidase, which sp ecifically hydrolysed the dipeptidyl chromogenic substrates glycylprol yl p-nitroanilide (GPRPNA), glycylprolyl beta-naphthylamide (GP beta N A) and valylalanine p-nitroanilide (VAPNA), and had some proteolytic a ctivity towards azocasein. No activity was detected against proline be ta-naphthylamide, glycine, valanine or alanine p-nitroanilides. Physio logical studies showed that the enzyme was largely cell-associated dur ing exponential growth in batch culture, but was progressively release d by the bacteria before the cells entered stationary phase. Glycylpro lyl arylamidase (GPA) was completely cell-bound during growth in conti nuous culture, where synthesis increased concomitantly with dilution r ate (specific growth rate) in both carbon- and nitrogen-limited chemos tats. Gel-filtration chromatography of B. splanchnicus cell extracts y ielded 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 N aCl during cation-exchange chromatography. Activity staining of SDS po lyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded du ring preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possibl e existence of GP;I isoenzymes. GPRPNA, VAPNA and azocasein were hydro lysed by the major fraction (fraction 3), while only the p-nitroanilid e substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relativ ely broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated t hat while aspartic (pepstatin A), thiol (iodoacetate) and metalloprote ase (EDTA, cysteine) inhibitors had little effect on hydrolysis of gly cylproline p-nitroanilide, GPA was strongly inhibited (c. 80%) by 5 mM phenylmethylsulphonyl fluoride (PMSF), indicating it to be a serine e nzyme.