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.

Bacillus lentus alkaline peptidase (BLAP) was used for casein (CN) hydrolysis in an enzyme membrane reactor (EMR) because it was found that BLAP was competitively inhibited by its products. The employed membranes had different molecular weight cut-offs (MWCO 1, 5 and 10 kDa). It was shown that the productivity of the EMR could be significantly improved (28 %) in comparison with batch hydrolysis under the same conditions after 20 h. All resulting EMR peptide mixtures showed a homogenous peptide pattern in HPLC–UV analysis. The obtained peptide mixtures exhibited Angiotensin-I-converting enzyme (ACE) inhibitory and antioxidative activity. The ACE inhibition of the peptide mixtures was dependent on the MWCO of the membranes. The resulting apparent IC₅₀ values were 115, 131 and 420 μg ml⁻¹ for the 1, 5 and 10 kDa MWCO membranes, respectively. In kinetic studies, a mixed-type inhibition was observed for the three peptide mixtures. The radical scavenging activity was determined with the ABTS assay, and IC₅₀ values between 20 and 25 μg ml⁻¹ were obtained for the generated peptide mixtures. In addition, the identified VYPFPGPIPN peptide exhibited ACE inhibition and antioxidant activity with IC₅₀ values of 325 and 6.2 μM, respectively. The peptide YQEPVLGPVRGPFPIIV exhibited radical scavenging activity with an IC₅₀ value of 5.2 μM.