title: A fusion protein consisting of the exopeptidases PepN and PepX-production, characterization, and application.
abstract: 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)).
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany. t.stressler@uni-hohenheim.de.
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Pfahler
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Merz
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Larissa
authors.initials: L
authors.lastname: Hubschneider
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Lutz-Wahl
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Claaßen
authors.affiliation: Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism; D000975:Antioxidants / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002364:Caseins / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019649:Food Industry; D006868:Hydrolysis; D052198:Lactobacillus helveticus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008623:Mercaptoethanol / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D001426:Bacterial Proteins; D002364:Caseins; D011993:Recombinant Fusion Proteins; C075633:pepN protein, Bacteria; D008623:Mercaptoethanol; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C098392:PepX dipeptidyl aminopeptidase
keywords: Antioxidative activity; Casein hydrolysis; Fusion protein; Lactobacillus helveticus; PepN; PepX