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Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis.

Stressler T, Eisele T, Schlayer M, Lutz-Wahl S, Fischer L - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany.

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 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 Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). 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.

Show MeSH
Activation effects of various divalent metal ions on apo-PepN.The enzymatic activity of the native PepN was considered 100% (each point represents the average of triplicate measurements; the standard deviation was <5%).
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pone-0070055-g005: Activation effects of various divalent metal ions on apo-PepN.The enzymatic activity of the native PepN was considered 100% (each point represents the average of triplicate measurements; the standard deviation was <5%).

Mentions: PepN is known to be a neutral zinc metallopeptidase [23], [24]. We prepared the apo-enzyme (apo-PepN) and investigated the reactivating effect of divalent metal ions. Apo-PepN was incubated with different concentrations of divalent cations (Fig. 5). Co2+ was found to enhance PepN activity to 180% at a concentration of 0.25 mM CoCl2, And even higher concentrations of the latter, up to 1 mM, preserved this high activity. The “natural” Zn2+ ion at a concentration of 0.1 mM restored the PepN activity to 99% compared with the activity of native holo-PepN. However, higher concentrations of ZnCl2 decreased the PepN activity, which was consistent with the inhibition studies (Table 2). Only slightly lower recovery yields of up to 94% were obtained with MnCl2 in the broad range of 0.1–1 mM.


Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis.

Stressler T, Eisele T, Schlayer M, Lutz-Wahl S, Fischer L - PLoS ONE (2013)

Activation effects of various divalent metal ions on apo-PepN.The enzymatic activity of the native PepN was considered 100% (each point represents the average of triplicate measurements; the standard deviation was <5%).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716637&req=5

pone-0070055-g005: Activation effects of various divalent metal ions on apo-PepN.The enzymatic activity of the native PepN was considered 100% (each point represents the average of triplicate measurements; the standard deviation was <5%).
Mentions: PepN is known to be a neutral zinc metallopeptidase [23], [24]. We prepared the apo-enzyme (apo-PepN) and investigated the reactivating effect of divalent metal ions. Apo-PepN was incubated with different concentrations of divalent cations (Fig. 5). Co2+ was found to enhance PepN activity to 180% at a concentration of 0.25 mM CoCl2, And even higher concentrations of the latter, up to 1 mM, preserved this high activity. The “natural” Zn2+ ion at a concentration of 0.1 mM restored the PepN activity to 99% compared with the activity of native holo-PepN. However, higher concentrations of ZnCl2 decreased the PepN activity, which was consistent with the inhibition studies (Table 2). Only slightly lower recovery yields of up to 94% were obtained with MnCl2 in the broad range of 0.1–1 mM.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany.

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 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 Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). 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.

Show MeSH