Cloning and characterization of debittering peptidases,PepE, PepO,PepO2, PepO3, and PepN,of Lactobacillus helveticus WSU19 |
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| Affiliation: | 1. Department of Food Science and Human Nutrition, Washington State University, PO BOX 646376, Pullman, WA 99164–6376, USA;2. Department of Food Science and Toxicology, Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Agricultural Biotechnology Laboratory 205, Moscow, ID 83844–2312, USA;1. Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Zografou 15780, Greece;2. Biomolecular Physics Laboratory, National Center for Scientific Research “Demokritos”, 15310 Aghia Paraskevi, Greece;3. Laboratory of Biotechnology, Department of Biological Applications & Technology, University of Ioannina 45110 Ioannina, Greece;4. Institute of Technology of Agricultural Products, Hellenic Agricultural Organisation-DEMETER, Lykovrissi 14123, Attica, Greece;1. School of Agriculture and Biology, Shanghai Jiao Tong University, China;2. Zhejiang Go Peptides Life Science and Healthcare Technology Co., Ltd., China;3. School of Systemic Biology and Medicine, Shanghai Jiao Tong University, China;4. Zhejiang Panda Dairy Group Co. Ltd., China;1. CIPROVE-Centro Asociado CICPBA, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, UNLP, Calle 47 y 115 S/N, B1900AJL La Plata, Argentina;2. CONICET, Argentina;3. CICPBA, Argentina;4. Centre for Neuroscience and Cell Biology (CNC), University of Coimbra, 3004-517 Coimbra, Portugal;5. Biocant, Biotechnology Innovation Centre, Núcleo 04, Lote 3, 3060-197 Cantanhede, Portugal;6. Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, Coimbra 3000-456 Portugal;2. Agrocampus Ouest, UMR1253, Science et Technologie du Lait et de l''?uf, 65 rue de Saint Brieuc, F-35042 Rennes, France;3. Actilait, BP 50915, 35009 Rennes Cedex, France;4. INRA, UMR 782, Génie et microbiologie des procédés alimentaires, Site de Grignon, Bâtiment CBAI, F-78850 Thiverval-Grignon, France;1. Dipartimento di Scienze degli Alimenti e del Farmaco, University of Parma, Parco Area delle Scienze 47/A, 43100, Parma, Italy;2. Centro di Ricerca Zootecnia e Acquacoltura (CREA-ZA), via Lombardo 11, 26900, Lodi, Italy |
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| Abstract: | Peptidases of Lactobacillus helveticus WSU19 are important for debittering aged Cheddar–type cheese. Our objective was to determine specificities of aminopeptidase N (PepN) and endopeptidases E, O, O2, and O3 (PepE, PepO, PepO2, and PepO3) of Lb. helveticus WSU19 on the bitter peptide, β–CN f193–209. Aminopeptidase and endopeptidase genes of Lb. helveticus WSU19 were cloned in Escherichia coli DH5α. The β–CN f193–209 peptide was digested by cell–free extracts from peptidase–positive clones under cheese ripening conditions. The degradation pattern was analyzed qualitatively using matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. Proline residues precluded PepN activity on β–CN f193–209. Complete degradation of β–CN f193–209 by PepN required post–proline endopeptidases, particularly PepO and PepO3. PepO–like endopeptidase activities on Pro206–Ile207 prevented formation of bitter peptides from the C–terminus of β–CN f193–209. PepE cleaved β–CN f193–209 only when combined with PepN or PepO–like endopeptidases. Aminopeptidase and post–proline endopeptidase activities contributed to the initial degradation of β–CN f193–209. |
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