ST-PEPA, A STREPTOCOCCUS-THERMOPHILUS AMINOPEPTIDASE WITH HIGH SPECIFICITY FOR ACIDIC RESIDUES

The proteolytic system of lactic acid bacteria has been extensively st udied over the past 10 years and peptidases from lactococci are now we ll known. The situation is, however, different for Streptococcus therm ophilus from which only a few peptidases have been purified and charac terized. The present work was conducted to characterize an aminopeptid ase of S. thermophilus CNRZ 302, called St-PepA, with high specificity for acidic amino acids. St-PepA was purified by a three-step procedur e from a spheroblast extract of 5. thermophilus CNRZ 302. Its molecula r mass was estimated to be 360 kDa by gel filtration and 45 kDa by SDS -PAGE, indicating that it had an octameric structure. Its activity aga inst aspartate-p-nitroanilide was maximal at ph 8.5 and 62 degrees C a nd highly enhanced by Zn2+, Co2+ and Mg2+. St-PepA was inhibited by me tal-chelating reagents and, to a lesser extent, by agents modifying su lfhydryl groups. It showed an activity towards p-nitroanilide derivati ves, di- and tripeptides, and larger peptides such as fragment 43-58 o f alpha(s1)-casein, It had a high substrate specificity towards N-term inal acidic amino acid residues but it could also release serine and m alic acid, the alpha-hydroxy acid homologue of aspartic acid. Kinetic studies revealed that the affinity of St-PepA was more than Is-fold hi gher for aspartic acid-p-nitroanilide (K-m = 0.42 mM) than for glutami c acid-p-nitroanilide (K-m = 7.65 mM) with a similar V-max for both su bstrates [about 40 mu mol min(-1) (mg enzyme)(-1)]. St-PepA is heat st able, with a maximal loss of activity of 15% after incubation for 120 min at 50 degrees C and 60 degrees C. It is likely to be involved in t he nitrogen metabolism of 5. thermophilus and in the development of th e organoleptic characteristics of cheese and yoghurt.