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)

et al.

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. © 2014 American Chemical Society.

This work was supported by Project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by Projects AGL2009-08348 and AGL2011-24643 from the Ministerio de Ciencia e Innovación (MICINN), by a CENIT project (2006−2009) from the MITC-CDTI, by Grant GR35-10A from the BSCH-UCM, and by Grants S2009/AGR-1489 and P2013/ABI-2747 from the Comunidad de Madrid (CAM). L.G. holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), J.B. held a research contract from the CAM, and J.J.J. was the recipient of a fellowship from the MICINN, Spain.

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