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

Pritchard, G. G.; Coolbear, Tim

doi:10.1111/j.1574-6976.1993.tb00018.x

Cited by 274 publications

(177 citation statements)

References 154 publications

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“…Table I reveal the presence of activity on aminopeptidase substrates in the crude cell free extract of most of the Enterococcus strains tested. Enterococci are in that respect comparable to other lactic acid bacteria [41,42,46,60]. The enzyme system of the Enterococcus strains studied was most active on leu-4NA when compared to the other 4-nitroanilide derivatives.…”

Section: Analysis Of Cd-ninhydrin Reactive Amino Groupsmentioning

confidence: 71%

Proteolytic, lipolytic and autolytic activities of enterococci strains isolated from Egyptian dairy products

El-Din¹,

El‐Soda

Ezzat

2002

Lait

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-Enterococci were isolated from Egyptian Domiatti and Mish cheeses made from raw milk. The proteolytic, lipolytic and autolytic activities of the isolated strains were studied to select the most appropriate cultures to be used for the enhancement of the organoleptic properties of cheese. The intracellular peptide hydrolase activities of enterococci strains were determined using various synthetic aminopeptidase and dipeptidyl-aminopeptidase substrates. 4-nitrophenyl and 2-nitrophenyl derivatives of fatty acids were used to measure the esterolytic activity of the different strains while emulsions of various triglycerides and butter fat were used to evaluate their lipolytic activities.The autolytic properties of the cultures were also considered. Three strains of Enterococcus faecium were freeze-shocked and evaluated as adjunct cultures in a cheese slurry system. All the strains exhibited an aminopeptidase activity hydrolysing leucyl 4-nitroanilide faster than the arginyl, alanyl, prolyl or glycyl derivatives. An active dipeptidyl-aminopeptidase activity was also detected. The Enterococcus faecium strains showed an active esterase system. The 4-nitrophenyl derivatives of the fatty acids were hydrolysed faster than the 2-nitrophenyl. The lipase substrates were hydrolysed according to the following order: tributyrin > tricaproin > tricaprylin > butterfat. The autolytic properties varied according to the strains tested. Cheese slurries ripened with adjunct Enterococcus faecium exhibited considerably higher levels of water-soluble nitrogen (WSN) and total free amino acids (FAA) when compared to the control after incubation for 5 d at 32 o C. With this finding, a high variability of some technologically relevant properties was found and could be the basis for the selection of specific cultures to be used as adjunct cultures in the production of traditional cheeses. Enterococcus / aminopeptidase / esterase / autolyse / levain atténué

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Section: Analysis Of Cd-ninhydrin Reactive Amino Groupsmentioning

confidence: 71%

Proteolytic, lipolytic and autolytic activities of enterococci strains isolated from Egyptian dairy products

El-Din¹,

El‐Soda

Ezzat

2002

Lait

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“…Since the concentrations of small peptides and amino acids in milk are insufficient to support their growth to high density, these bacteria are dependent on a complex proteolytic system for the liberation of amino acids from transport systems, and numerous cytoplasmic peptidases. Recently, extensive biochemical and genetic analyses of individual components of the proteolytic system of lactic acid bacteria and their role in cheese ripening and flavour formation has been conducted (Kok, 1990;Pritchard & Coolbear, 1993 ;Tan et a/. , 1993 ;Visser, 1993 ;Kok & De Vos, 1994).…”

Section: Introductionmentioning

confidence: 99%

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

Vesanto¹,

Savijoki²,

Rantanen³

et al. 1995

Microbiology

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The X-prolyl dipeptidyl aminopeptidase gene (pepx) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 906 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with AG of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 906 kDa protein showed 49*3,494 and 777% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Ladobacillus delbnreckii subsp. Iactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6 5 and 45 OC. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and l,l0-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding Dll, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.

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“…The hydrolysis of milk proteins by dairy starter lactic acid bacteria involves the action of a cell-envelope-associated proteinase, various peptidases and transport systems for peptides and amino acids (Kok, 1990; Smid e t al., 1991;Pritchard & Coolbear, 1993;Tan e t al., 1993). The cellenvelope-associated proteinase catalyses the partial hydrolysis of one or more of the casein components of bovine milk to a series of oligopeptide products, the nature of which has been well characterized (Monnet et a/., , 1992Visser e t al., 1988Visser e t al., , 1991Reid et al, 1991a, b).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

Pritchard

Freebairn²,

Coolbear³

1994

Microbiology

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An endopeptidase has been purified from Lactococcus lactis subsp. crernoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,l O-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor plchloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl f luoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. T w o of the oligopeptides produced during the early stages of & casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic s ys tem .

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

Cited by 274 publications

References 154 publications

Proteolytic, lipolytic and autolytic activities of enterococci strains isolated from Egyptian dairy products

Proteolytic, lipolytic and autolytic activities of enterococci strains isolated from Egyptian dairy products

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

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