Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

Publication Type

Journal contribution (peer reviewed)

Authors

Stressler, T.; Eisele, T.; Schlayer, M.; Lutz-Wahl, S.; Fischer, L.

Year of publication

2013

Published in

PLOS ONE

DOI

10.1371/journal.pone.0070055

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-pNA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat_H-Ala-pNA L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.