title: Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis.
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.
authors.affiliation: Department of Biotechnology, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.forename: Thomas
authors.initials: T
authors.lastname: Eisele
authors.forename: Michael
authors.initials: M
authors.lastname: Schlayer
authors.forename: Sabine
authors.initials: S
authors.lastname: Lutz-Wahl
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D052198:Lactobacillus helveticus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C075633:pepN protein, Bacteria; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C098392:PepX dipeptidyl aminopeptidase
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