Processing of the lactococcal extracellular serine proteinase

Haandrikman, Alfred J.; Meesters, Rene; Laan, Harry; Konings, Wil N.; Kok, Jan; Venema, Gerard

doi:10.1128/aem.57.7.1899-1904.1991

Cited by 61 publications

(25 citation statements)

References 36 publications

(37 reference statements)

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“…In other words, free a S1 -casein could be less susceptible to the action of proteinase than when it is in a complex with other casein fractions. Release of lactococcal proteinase from the cell envelope was found to be an autoproteolytic event (Laan and Konings 1989;Haandrikman et al 1991). It was also found that Ca 2+ ions play an important role in maintaining the attachment of the enzyme to the cell.…”

Section: Discussionmentioning

confidence: 97%

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

Fira

Kojić

Banina

et al. 2001

Journal of Applied Microbiology

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S O J E V I C , I . S T R A H I N I C A N D L . T O P I S I R O V I C . 2001.The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6á5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both a S1 -casein and b-casein, showing very low activity towards j-casein. The BGPF1 proteinase completely hydrolysed only b-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA±DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.

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Section: Discussionmentioning

confidence: 97%

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

Fira

Kojić

Banina

et al. 2001

Journal of Applied Microbiology

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“…200 kDa), which involves removal of an N-terminal signal sequence of 33 amino acid residues and a 154-residue pro-sequence [32,34,35], results in a mature proteinasc of approximately 180 kDa. The product of a second closely linked gene (prtM) encoding a membrane-bound lipoprotein is also essential for maturation of the proteinase [33,36,37]. Most of what we know about the action of the cell envelope proteinase from lactococci in hydrolysing milk proteins has been learnt from studies of the enzyme after its release from the cell.…”

Section: The Cell Ent'elope Proteinasementioning

confidence: 99%

“…The other mechanism [42] involves an autoproteolytic cleavage at a site close to the C-terminal wall-associated sequence occurring as a result of a conformational change induced by the removal of calcium. Recent studies [36,43], in which release of the proteinase was monitored by a monoclonal antibody ELISA assay, provide compelling evidence for the involvement of an autoproteolytic event in release. Release was prevented (even in the presence of EDTA) by treatment with proteinase inhibitors [43] or by abolishing proteolytic activity by site-directed mutagenesis of a single active site residue [36].…”

Section: Nature Of the Proteinase Release Mechanismmentioning

confidence: 99%

“…Recent studies [36,43], in which release of the proteinase was monitored by a monoclonal antibody ELISA assay, provide compelling evidence for the involvement of an autoproteolytic event in release. Release was prevented (even in the presence of EDTA) by treatment with proteinase inhibitors [43] or by abolishing proteolytic activity by site-directed mutagenesis of a single active site residue [36].…”

Section: Nature Of the Proteinase Release Mechanismmentioning

confidence: 99%

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The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Pritchard

Coolbear²

1993

FEMS Microbiology Reviews

270

155

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The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source. Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci. Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane. Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo. This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria.

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“…One should bear in mind that PrtP is being activated through the autoproteolytic removal of the N-terminal pro-region, during the maturation process. In addition, active enzymes are prone to additional auto-digestion, especially when no Ca 2+ ions are present to stabilize the enzyme on the cell surface (24). Finally, PrtP is capable of hydrolyzing autolysin AcmA, a protein responsible for cell separation and autolysis during the stationary phase of growth (25), as well as some extracellular proteins of unknown function (24).…”

Section: Discussionmentioning

confidence: 99%

Lactococcin B is inactivated by intrinsic proteinase PrtP digestion inLactococcus lactissubsp.lactisBGMN1-501

Vukotić

Polović

Mirković

et al. 2018

Preprint

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In our previous study, we showed that PrtP is able to impair bacteriocin LcnB activity despite being produced by the same organism, and even if they were encoded by the same plasmid. However, exact cleavage site within LcnB bacteriocin, as well as the activity of the resulting peptides remained unknown. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that the hydrolysis occurs between the sixth and seventh amino acid on the N terminus of LcnB. Although it was suspected that the cleaved form of LcnB could retain some level of activity, both chemically synthesized and recombinant variant of truncated LcnB exhibited no antimicrobial activity. Wild type form of LcnB was recombinantly overexpressed using the same expression system, its antimicrobial activity was tested before and after the treatment with PrtP proteinase, and the degradation products were analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin.ImportanceLactococcal enzyme PrtP, considered as a growth promoting factor, is involved in casein breakdown and enabling bacteria efficient growth in amino acids-poor, but protein-rich media. However, its interaction with bacteriocins was not known until recently. Bacteriocin LcnB can also be considered as growth promoting factor, since its known physiological role mirrors in preventing competing bacteria of reaching high growth densities. In this manuscript, we define the exact peptide bond inside the bacteriocin LcnB which is recognized by PrtP. This N-terminal removal of six amino acids completely inactivates the bacteriocin. The biological function of such action remains elusive. It is unexpected that in the same strain one enzyme inactivates a protein important for survival, unless it is some type of regulation.

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Processing of the lactococcal extracellular serine proteinase

Cited by 61 publications

References 36 publications

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Lactococcin B is inactivated by intrinsic proteinase PrtP digestion inLactococcus lactissubsp.lactisBGMN1-501

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