Abstract
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 µkatH-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 µkatH-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.
Highlights
Enzymatic protein hydrolysis is a complex process
The nucleotide sequence of the pepX gene obtained was deposited in the GenBank database and exhibited 100% identity to that of Lb. helveticus CNRZ 32
The reducing agents DTT and b-mercaptoethanol did not demonstrate an inactivating effect on proline-specific X-prolyl dipeptidyl aminopeptidase (PepX) from Lb. helveticus ATCC 12046, which is similar to the results described for PepX from Lb. helveticus ITG LH1 [5] and LHE-511 [12]
Summary
Enzymatic protein hydrolysis is a complex process. The synergistic effects of various peptidases are necessary to achieve a reasonable degree of hydrolysis. Lactic acid bacteria (LAB) are one potential source of these proteolytic enzymes. Based on their extensive proteolytic systems, these auxotrophic microorganisms are perfectly adapted to grow in milk [4,5]. The synergistic effect of X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) is important to achieve a high degree of hydrolysis. Hydrolysis is terminated and/or the velocity of hydrolysis is reduced when proline residues are present [7,8]. This mechanism of proline inhibition is compensated by the action of PepX, which hydrolyzes X-Pro dipeptides from the N-terminus of peptides. The native aminopeptidase PepN from Lactobacillus helveticus CNRZ 32 [7] and Lb
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