Date Published: August 1, 2012
Author(s): Timo Stressler, Thomas Eisele, Michael Schlayer, Lutz Fischer.
The aminopeptidase P (PepP, EC 18.104.22.168) 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 °C, resulting in PepP activity of 90 μkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 μkatLPP Lculture-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 ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax 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°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°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.
Aminopeptidase P (PepP; EC 22.214.171.124) is an exopeptidase that removes the N-terminal amino acid at the P1 position [nomenclature following [Schechter and Berger (1967])] from a peptide when proline is in the P1´ position ([Cunningham and O’Connor 1997]). Several enzymes with the specificity of PepP have been found in species such as Escherichia coli ([Yaron and Mlynar 1968]; [Yoshimoto et al. 1988b])Neisseria gonorrhoeae ([Chen and Buchanan 1980]), Thermococcus sp. strain NA1 ([Lee et al. 2006]), Lactococcus lactis ssp. lactis NCDO 763 ([Mars and Monnet 1995]), Lactococcus lactis ssp. cremoris AM2 ([McDonnell et al. 1997]) and tissues from several mammalian species ([Hooper et al. 1990]; [Harbeck and Mentlein 1991]). While the physiological role of PepP in bacteria is unclear, mammalian PepP is involved in processes such as the regulation of biologically active peptides, including substance P and bradykinin ([Cunningham and O’Connor 1997]; [Yaron and Naider 1993]).
The classical assay methods for PepP activity determination present some problems ([Yoshimoto et al. 1988b]; [McDonnell et al. 1997]; [Lasch et al. 1988]; [Fleminger et al. 1982]; [Holtzman et al. 1987]; [Hawthorne et al. 1997]; [Stöckel-Maschek et al. 2003]). A precise determination of PepP activity using ninhydrin is nearly impossible because crude extracts contain large amounts of ninhydrin-positive substances ([Yoshimoto et al. 1988b]). It is also difficult to measure an initial reaction velocity via fluorometric methods ([Doi et al. 1981]). The method employing Gly-Pro-pNA coupled with proline iminopeptidase ([Yoshimoto et al. 1988b]) can be used for routine assays, but not for screening the PepP enzyme activity in crude extracts from lactic acid bacteria without further pre-treatment because of the existence of parallel PepX activity (X-prolyl dipeptidyl aminopeptidase; EC 126.96.36.199), which is common in lactic acid bacteria. Our novel assay method using gas chromatography for detection of the release of L-leucine during hydrolysis of the substrate (LPP) could be used for routine analysis as well as for screening experiments. Even when free amino acids are present in the crude extract, or other aminopeptidases are present, such as PepX, which did not hydrolyze Xaa-Pro-Pro peptide sequences, our GC assay is applicable and specific for PepP activity.
The authors declare that they have no competing interests.