title: Genetic and biochemical evidence that recombinant Enterococcus spp. strains expressing gelatinase (GelE) produce bovine milk-derived hydrolysates with high angiotensin converting enzyme-inhibitory activity (ACE-IA).
abstract: In this work, genes encoding gelatinase (gelE) and serine proteinase (sprE), two extracellular proteases produced by Enterococcus faecalis DBH18, were cloned in the protein expression vector pMG36c, containing the constitutive P32 promoter, generating the recombinant plasmids pCG, pCSP, and pCGSP encoding gelE, sprE, and gelE-sprE, respectively. Transformation of noncaseinolytic E. faecalis P36, E. faecalis JH2-2, E. faecium AR24, and E. hirae AR14 strains with these plasmids permitted detection of caseinolytic activity only in the strains transformed with pCG or pCGSP. Complementation of a deletion (knockout) mutant of E. faecalis V583 for production of gelatinase (GelE) with pCG unequivocally supported that gelE is responsible for the caseinolytic activity of the transformed strain grown in bovine skim milk (BSM). RP-HPLC-MS/MS analysis of hydrolysates of transformed Enterococcus spp. strains grown in BSM permitted the identification of 38 major peptide fragments including peptides with previously reported angiotensin converting enzyme-inhibitory activity (ACE-IA), antihypertensive activity, and antioxidant activity.
authors.affiliation: Departamento de Nutrición, Bromatologı́a y Tecnologı́a de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM) , 28040 Madrid, Spain.
authors.forename: Loreto
authors.initials: L
authors.lastname: Gútiez
authors.forename: Juan
authors.initials: J
authors.lastname: Borrero
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Jiménez
authors.forename: Beatriz
authors.initials: B
authors.lastname: Gómez-Sala
authors.forename: Isidra
authors.initials: I
authors.lastname: Recio
authors.forename: Luis M
authors.initials: LM
authors.lastname: Cintas
authors.forename: Carmen
authors.initials: C
authors.lastname: Herranz
authors.forename: Pablo E
authors.initials: PE
authors.lastname: Hernández
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D013293:Enterococcus faecalis / Q000201:enzymology / Q000235:genetics*; D018093:Gelatinases / Q000235:genetics* / Q000378:metabolism; D008892:Milk / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011492:Protein Hydrolysates / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D001426:Bacterial Proteins; D011492:Protein Hydrolysates; D011994:Recombinant Proteins; D057057:Serine Proteases; D018093:Gelatinases