Controlled enterolysin A-mediated lysis and production of angiotensin converting enzyme-inhibitory bovine skim milk hydrolysates by recombinant Lactococcus lactis

et al.

Cloning of the enterolysin A (EnlA) gene (enlA) from Enterococcus faecalis DAC9 into the pMSP3545-derived pMLG2-protein expression vector encoding EnlA under control of the inducible PnisA promoter permitted the controlled release and heterologous expression of mature EnlA by Lactococcus lactis subsp. cremoris NZ9000 and Lactococcus lactis subsp. lactis IL1403. The nisin-induced expression of enlA by L.lactis NZ9000 (pMLG2) and L.lactis IL1403 (pMLG2), grown in GM17 or bovine skim milk (BSM), caused a noticeable reduction of the optical density (OD600) of the cultures and death of the growing cells. However, a high angiotensin converting enzyme (ACE)-inhibitory activity (ACE-IA) was only observed in the BSM-derived hydrolysates of L.lactis IL1403 (pMLG2) after 48h-induction with nisin. Analysis of these hydrolysates by reversed phase high performance liquid chromatography-tandem mass spectrometry permitted the identification of major peptide fragments with known ACE-IA or sharing at least three C-terminal residues with those displaying ACE-IA. © 2013 Elsevier Ltd.

This work was partially 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), and by the CENIT project (2006–2009) from the MITC-CDTI. Funding by grants GR35-10A from the BSCH-UCM and S2009/AGR-1489 from the Comunidad de Madrid (CAM) are also acknowledged. L. Gútiez holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), Spain.

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