Simultaneous Presence of PrtH and PrtH2 Proteinases in Lactobacillus helveticus Strains Improves Breakdown of the Pure α _s1 -Casein

Abstract

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified ␣ s1 -and ␤-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure ␤-casein was observed for all strains, the hydrolysis kinetics of ␣ s1 -casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from ␣ s1 -casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM

“…Gene deletion studies of serine proteases in other Lactobacillus species, including PrtB in L. delbrueckii subsp. bulgaricus and PrtH in L. helveticus , have not reported a similar morphological or autoaggregation phenotypes (Gilbert et al, 1996; Pederson et al, 1999; Genay et al, 2009; Sadat-Mekmene et al, 2011). However, this difference in observed phenotypes may be due to the subcellular localization of the referenced serine proteases.…”

Lactobacillus helveticus: the proteolytic system

“…PrtH1 and PrtH2 present in L. helveticus differ from the CEPs of other LAB because they lack the anchor domain (AN), which is located at the C-terminus of the CEP sequence. Some authors consider that there are at least two other types of CEP in L. helveticus , namely PrtH3 and PrtH4 (Yamamoto et al, 1998; Savijoki et al, 2006; Broadbent et al, 2011; Sadat-Mekmene et al, 2011a). In L. helveticus CNRZ32, two genes encoding the CEPs, prtH , and prtH2 have been reported (Sadat-Mekmene et al, 2011b).…”

“…So far, no specific physiological property has been proposed for the whole casein system, whereas various peptides that are inactive in the amino-acid sequence have been proven to possess bioactivities (Pihlanto-Leppala et al, 1999; Florisa et al, 2003; Kilara and Panyam, 2003). CEPs of L. helveticus are highly specific since the sites of cleavage on the caseins differ from one strain to another depending on the type of casein (α- or β-casein) (Zevaco and Gripon, 1988; Jensen et al, 2009; Sadat-Mekmene et al, 2011a). A study conducted on the proteolytic system of L. helveticus Zuc2, reported the potential presence of two CEPs (Scolari et al, 2006).…”

Strain-to-strain differences within lactic and propionic acid bacteria species strongly impact the properties of cheese–A review

“…For example, a unique CEP, PrtP, is present in L. lactis (Monnet et al 1987), PrtS in S. thermophilus (Fernandez-Espla et al 2000), PrtB in Lactobacillus delbrueckii (Laloi et al 1991), and PrtR in L. rhamnosus (Pastar et al 2003), whereas up to four CEPs are present in Lactobacillus helveticus, referred to as PrtH to PrtH4 (Broadbent et al 2011;Sadat-Mekmene et al 2011b). Moreover, the activity and specificity of CEP can also vary within a given species.…”

“…The strain-dependency of casein hydrolysis is still higher within the L. helveticus species. The specificity of cleavage of β-or α s1 -caseins varies from strain to strain and also depends on the substrate (purified caseins or casein micelles in milk) (Sadat-Mekmene et al 2011b). The 15 strains studied in vitro rapidly hydrolysed pure β-casein, but differed in the hydrolysis kinetics of α s1 -casein, depending on their number of CEPs (Sadat-Mekmene et al 2011a).…”