Michael Merz

Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC 3.4.11.2) and the proline-specific peptidase PepX (EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30 °C for the single enzyme to 35 °C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50 °C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent β-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the K _M values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC₅₀ value: 5.81 μg mL⁻¹).

Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl₂, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.