Formation of a dipeptidyl arylamidase by Bacteroides splanchnicus NCTC 10825 with specificities towards glycylprolyl-x and valylalanine-x substrates
Formation of a dipeptidyl arylamidase by Bacteroides splanchnicus NCTC 10825 with specificities towards glycylprolyl-x and valylalanine-x substrates
复制标题
DOI：
10.1099/00222615-46-7-547
复制
发表时间：
1997-07-01
影响因子：
3
通讯作者：
Macfarlane, GT
中科院分区：
文献类型：
--
作者：
Macfarlane, S;Macfarlane, GT
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 GP;I 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.
