Determination of the kinetic parameters of PepN for the substrate H-Ala-<i>p</i>NA according to Michaelis-Menten analysis (A) and Lineweaver-Burk linearization (B) (each point represents the average of triplicate measurements; the standard deviation was <5%).

<p>Determination of the kinetic parameters of PepN for the substrate H-Ala-<i>p</i>NA according to Michaelis-Menten analysis (A) and Lineweaver-Burk linearization (B) (each point represents the average of triplicate measurements; the standard deviation was <5%).</p

Characterization of the purified PepX (A; C; E; G) and PepN (B; D; F; H) from <i>Lb.</i><i>helveticus</i> ATCC 12046.

<p>A and B: pH optimum; C and D: temperature optimum; E and F: thermal stability; G and H: influence of NaCl and KCl (each point represents the average of triplicate measurements; the standard deviation was <5%).</p

Kinetic parameters of PepX¹ using chromogenic <i>p</i>-nitroanilide substrates.

1<p>Protein concentration of the enzyme solution: 2.61 mg mL⁻¹.</p>2<p>Substrate for the standard assay. Triplicate measurements; the standard deviation was <5%.</p

Lineweaver-Burk linearization of PepX inhibition by H-L-Phe-L-Pro-OH (H-Ala-Pro-<i>p</i>NA as substrate; each point represents the average of triplicate measurements; the standard deviation was <5%).

<p>Lineweaver-Burk linearization of PepX inhibition by H-L-Phe-L-Pro-OH (H-Ala-Pro-<i>p</i>NA as substrate; each point represents the average of triplicate measurements; the standard deviation was <5%).</p

Increase of the relative degree of hydrolysis of a prehydrolyzed casein solution by PepX and/or PepN (each point represents the average of triplicate measurements; the standard deviation was <5%).

<p>Increase of the relative degree of hydrolysis of a prehydrolyzed casein solution by PepX and/or PepN (each point represents the average of triplicate measurements; the standard deviation was <5%).</p

Effect of various solvents, cations, inhibitors, reducing agents, metal chelators, and denaturing agents on the activity of PepX and PepN from <i>Lb. helveticus</i> ATCC 12046 (at 37°C in 50 mM Na₂HPO₄/KH₂PO₄ buffer, pH 6.5).

1<p>The value of 100% was determined in the presence of the corresponding solvent without the additional substance. The substance was dissolved in: ²H₂O_dd; ³DMSO; ⁴Acetone; ⁵EtOH. Triplicate measurements; the standard deviation was <5%; n.d., not determined.</p

Kinetic parameters of PepN¹ using chromogenic <i>p</i>-nitroanilide substrates.

1<p>Protein concentration of the enzyme solution: 2.16 mg mL⁻¹.</p>2<p>Substrate for the standard assay.</p>3<p>The maximum substrate solubility was 3.73 mM under the assay conditions. Triplicate measurements; the standard deviation was <5%.</p

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

<div><p>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 <i>Lactobacillus helveticus</i> ATCC 12046 were produced recombinantly in <i>E. coli</i> BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-<i>p</i>NA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in <i>E. coli</i> at high levels for the first time. The PepN activity reached 1,000 µkat_{H-Ala-<i>p</i>NA} 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.</p></div