Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

Fira, Djordje; Kojić, Milan; Banina, A.; Spasojević, Ivan; Strahinić, Ivana; Topisirović, Ljubiša

doi:10.1046/j.1365-2672.2001.01226.x

Cited by 56 publications

(45 citation statements)

References 26 publications

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“…In the current study, the optimum pH was pH 7.0 (Table 4) and decreased towards the acidity. These findings agreed with Fira, [39], Kabadjova-Hristova [33], and El-Ghaish et al [38], with some variations but with the same trend. The variations might be due to the difference in bacterial species.…”

Section: E Phsupporting

confidence: 89%

Chemically Defined Medium for Optimization of Proteolytic Activity of Lactobacillus bulgaricus 761N

Nour¹,

Fattouh²,

El-Adawi³

2014

LSMR

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Abstract-Subcellular fractions of Lactobacillus bulgaricus 761N were investigated for their proteolytic activity that results in production of bioactive peptides in the cell-free supernatant. It was observed that the extracellular extract of L. bulgaricus 761N possessed the highest proteolytic activity of about 63.1 U/ml/min. Consequently, the extracellular fraction was chosen for assessing the proteolytic activity after each step of the optimization process was carried out. The factors of the optimization process involve the incubation period, temperature, pH, salinity (expressed by NaCl concentration), casein content, trace element concentration. The trace elements include cobalt chloride, copper sulphate, calcium chloride and ferrous sulphate. The basal medium used was skim milk agar. The other constituents of the chemically synthesized medium, that would optimize the proteolytic activity that in turn elevate the bioactive peptides production, are determined upon the results of the optimization process. The preferred conditions for maximal activity occurrence was after 48h incubation period, at pH 7, 37 o C, brackish environment at 5gl -1 NaCl content and 5gl -1 casein content. CaCl 2 and CoCl 2 were found to enhance the proteolytic activity at 1mM, while CuSO 4 and FeSO 4 were found to be needed in fewer amounts at 0.5mM and 0.1mM, respectively. The antibacterial activity of these bioactive peptides produced in the cell free supernatant was investigated against some human pathogenic bacteria. It was found that it impose a high antibacterial capacity on tested pathogens reaching 89% inhibition.

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Section: E Phsupporting

confidence: 89%

Chemically Defined Medium for Optimization of Proteolytic Activity of Lactobacillus bulgaricus 761N

Nour¹,

Fattouh²,

El-Adawi³

2014

LSMR

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“…The cell-enveloped proteases and different intracellular peptidases result in an efficient breakdown of casein, major milk protein, into different amino acids and peptides required for cell growth [17,28]. Several studies reported pH and temperature dependence of the protease activity of several thermophilic LAB species [1,4]. The high proteolytic activity of a β-gal preparation could be undesirable during lactose hydrolysis in milk since it may result in bitterness [19] as well as the digestion of the β-gal enzyme.…”

Section: Introductionmentioning

confidence: 99%

Oligosaccharide production and proteolysis during lactose hydrolysis using crude cellular extracts from lactic acid bacteria

Vasiljevic

Jelen

2003

Lait

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-The extent of the lactose hydrolysis and oligosaccharide formation through transgalactosyl reactions by crude cellular extracts (CCE) containing intracellular β-galactosidase from disrupted Lactobacillus delbrueckii subsp. bulgaricus 11842, Lactobacillus delbrueckii subsp. lactis DMF 3078 or Streptococcus thermophilus 143 was investigated in lactose-containing buffered solutions or skim milk. Lactose hydrolysis was performed with lactose concentration ranging from 5 to 30% (w/w) at 30, 40, 50 or 60°C and terminated after 120 min. The proteolytic activities of the CCEs in skim milk systems were also assessed. All CCEs produced di-, tri-and tetrasaccharides in addition to monosaccharides as major products of the lactose hydrolysis in buffered lactose solutions. However, only Lb. lactis produced tetrasaccharides at detectable levels in skim milk preparations. The amount and the rate of oligosaccharide formation were significantly (P < 0.05) affected by the origin of the enzyme, lactose concentration and temperature. The maximum oligosaccharide production by all CCEs was reached at 50°C in 30% (w/w) lactose solution. St. thermophilus CCE produced significantly higher (P < 0.05) amounts of oligosaccharides than the other two CCE preparations at all lactose concentrations studied. However, Lb. bulgaricus CCE showed better lactose-hydrolyzing ability and higher proteolytic activity than the other two CCEs. The lactose hydrolysis generally proceeded faster at 50-60°C than at 30-40°C as opposed to the proteolytic maxima reached at 40°C.Crude cellular extract / β-galactosidase / thermophilic dairy culture / lactose hydrolysis / transferase reaction / proteolytic activity Résumé -Production d'oligosaccharides et protéolyse lors de l'hydrolyse de lactose par extraits cellulaires bruts de bactéries lactiques. Le degré d'hydrolyse du lactose et la formation d'oligosaccharides par réaction de transgalactosylation sous l'action d'extraits cellulaires bruts (ECB) contenant de la β-galactosidase obtenue par rupture de Lactobacillus delbruekii subsp. bulgaricus 11842, Lactobacillus delbruekii subsp. lactis DMF 3078 et Steptococcus thermophilus 143 ont été étudiés sur des solutions tamponnées de lactose ou sur le lait écrémé. L'hydrolyse du lactose a été effectuée sur des concentrations de 5 à 30 % (masse) à des températures de 30, 40, 50 et 60°C pour une durée totale de 120 min. L'activité protéolytique des ECBs sur les systèmes à base de lait écrémé a également été évaluée. Tous les ECBs ont produit des di-, tri-et tetrasaccharides en plus des monosaccharides comme composants majeurs de l'hydrolyse du lactose contenu dans les solutions tamponnées. Toutefois, seul Lb. lactis a produit des tetrasaccharides à un niveau * Corresponding author: paul.jelen@ualberta.ca 454 T. Vasiljevic, P. Jelen

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“…However, constitutive and inducible expression systems generally led to the production of microgram-level amounts of recombinant protein per liter of culture (1,2,18). We examined the potential of L. bulgaricus PrtB proteinase to serve as a protein carrier for a foreign sequence because (i) it is a large protein (1,947 amino acids) most likely prone to sustain changes in its primary structure and (ii) it is constitutively expressed at high levels on the surface of L. bulgaricus (5). Successful expression was achieved in L. lactis, as shown by the surface display and release in the supernatant of the heterologous proteinase.…”

Section: Discussionmentioning

confidence: 99%

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

Bernasconi

Germond

Delley

et al. 2002

Appl Environ Microbiol

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Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine ␤-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding fulllength PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB٢, PrtB٢-BLG, and PrtB٢-T6) or 807 (PrtB⌬) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB٢-BLG yielded up to 170 g per 10 9 CFU in the culture supernatant and 9 g per 10 9 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.An inappropriate immunological response to bovine milk proteins, including ␤-lactoglobulin (BLG), has been put forward as a key factor in cow's milk allergy (34). Studies have shown that a reequilibration of this response toward a state of specific oral tolerance can be achieved upon oral administration of partially hydrolyzed cow's milk formula or BLG peptides obtained by tryptic hydrolysis (8,22). Interestingly, the degradation of cow's milk proteins by lactic acid bacteria (LAB) used in food fermentation was also found to generate tolerogenic peptides, with a suppression of the specific lymphoproliferative response (32). In addition, certain LAB strains are more and more widely used as a vehicle for the delivery of heterologous proteins to the mucosal immune system (13,20). Therefore, it can be hypothesized that the introduction of LAB capable of expressing full-length or BLG fragments may prove them to be a useful tool to aid in host protection against allergic sensitization to this dietary antigen.Lactococcus lactis is a gram-positive, nonpathogenic bacterium that is widely used in industrial food fermentation. A noncolonizing bacterium, L. lactis has been shown to be quite resistant to gastric acidity when administered together with food, remaining metabolically active all the way through the digestive tract (3). Its use as an antigen delivery vehicle for mucosal immunization has been reported (1,4,26,31). Induction of oral tolerance might require a large amount of target antigen, in contrast to immune modulators such as cytokines (30,31). Therefo...

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Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

Cited by 56 publications

References 26 publications

Chemically Defined Medium for Optimization of Proteolytic Activity of Lactobacillus bulgaricus 761N

Chemically Defined Medium for Optimization of Proteolytic Activity of Lactobacillus bulgaricus 761N

Oligosaccharide production and proteolysis during lactose hydrolysis using crude cellular extracts from lactic acid bacteria

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

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