Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp lactis DSM 20481

The aminopeptidase P (PepP, EC 3.4.11.9) gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 degrees C, resulting in PepP activity of 90 mu kat(LPP) L-culture(-1). After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 mu kat(LPP) L-culture(-1) was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be similar to 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters K-m and V-max were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50 degrees C and 60 degrees C and exhibited reasonable stability at 0 degrees C, 20 degrees C and 37 degrees C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA.