Degradation of αs1-CN f1-23 by aminopeptidase N and endopeptidases E, O, O2, and O3 of Lactobacillus helveticus WSU19 under cheese ripening conditions (original) (raw)
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International dairy journal, 2007
Peptidases of Lactobacillus helveticus WSU19 are important for debittering aged Cheddar–type cheese. Our objective was to determine specificities of aminopeptidase N (PepN) and endopeptidases E, O, O2, and O3 (PepE, PepO, PepO2, and PepO3) of Lb. helveticus WSU19 on the bitter peptide, β–CN f193–209. Aminopeptidase and endopeptidase genes of Lb. helveticus WSU19 were cloned in Escherichia coli DH5α. The β–CN f193–209 peptide was digested by cell–free extracts from peptidase–positive clones under cheese ripening conditions. The degradation pattern was analyzed qualitatively using matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. Proline residues precluded PepN activity on β–CN f193–209. Complete degradation of β–CN f193–209 by PepN required post–proline endopeptidases, particularly PepO and PepO3. PepO–like endopeptidase activities on Pro206–Ile207 prevented formation of bitter peptides from the C–terminus of β–CN f193–209. PepE cleaved β–CN f193–209 only when combined with PepN or PepO–like endopeptidases. Aminopeptidase and post–proline endopeptidase activities contributed to the initial degradation of β–CN f193–209.
Applied and Environmental Microbiology, 2005
Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5␣ derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, -casein (-CN) (f193-209) and ␣ S1 -casein (␣ S1 -CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards ␣ S1 -CN (f1-9) and -CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze -CN (f193-209) and ␣ S1 -CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides ␣ S1 -CN (f1-9), ␣ S1 -CN (f1-13), and ␣ S1 -CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with -CN (f193-209) and ␣ S1 -CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.
Applied and …, 2005
Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5␣ derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, -casein (-CN) (f193-209) and ␣ S1-casein (␣ S1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards ␣ S1-CN (f1-9) and -CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze -CN (f193-209) and ␣ S1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides ␣ S1-CN (f1-9), ␣ S1-CN (f1-13), and ␣ S1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with -CN (f193-209) and ␣ S1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.
Food Research International, 1995
The effects of crude enzyme extract of Lactobacillus casei ssp. casei LLG on the water-soluble peptides of enzyme-modified cheese (EMC) were studied by reverse phase HPLC and amino acid analysis. A wide range of peptidolytic activities (aminopeptidase 161544 unit/ml; x-prolyldipeptidyl peptidase 66.73 unit/ml; proline-iminopeptidase 38.63 unit/ml) were detected in the crude enzyme extract. Bitter enzyme-modified cheese (EMC N24) was prepared with Neutrase@ 0.5L for 24 h at 45°C and treated with (EMC NL72) and without (EMC N96) the crude enzyme for 72 h at 35°C. The percent peak areas of two hydrophobic peptides (peak I and peak II) in EMC N24 were increased from 1.63% to 3.65% and from 088% to 3.23% in EMC N96, respectively, but decreased to below the detectable range in EMC NL72. The bitterness of EMC N96 may have been related to the increase in areas of these two peaks. Based on the amino acid compositions, peak I was identified as the qcasein fraction 26-31(Ala-Pro-Phe-Pro-Glu-Val), and peak II as the @casein fraction 190-192 (Phe-Leu-Leu), respectively. The results suggest that both aminopeptidase and proline-specific peptidases present in the crude extracts are responsible for degrading the hydrophobic peptides in bitter EMC.
Le Lait, 1995
The activity of the purified aminopeptidase from Pseudomonas f1uorescens ATCC 948 on synthetic bitter peptides, bitter hydrolysate of UHT milk proteins and on the ripening of Italian Caciotta type cheese was studied. The aminopeptidase almost completely hydrolyzed the bitter pentapeptide H-Leu-Trp-Met-Arg-Phe-OH and liberated Val from the bitter tetrapeptide H-Val-Pro-Leu-Leu-OH. No cleavage of the Pro-bound was observed. In UHT milk in which bitter protein hydrolysate was caused by externally added proteinase, the aminopeptidase produced a concentration of free amino acids about 8 times higher than those determined on UHT unhydrolyzed non-bitter milk (211.3 vs 25.3 Ilg/ml, respectively). Glu, Leu, Met, Trp and Val were the most abundant amino acids. The enzyme showed 44% of its maximum activity at the storage temperature of UHT milk (20°C). The aminopeptidase was stable during the ripening of Caciotta type cheese: no activity was lost during 2 months. Alter 45 days of ripening, the cheese containing aminopeptidase had a higher amino acid level (990.2 Ilg/ml) than the untreated-control (591.1 Ilglml). The amino acid profile of the treated cheese reflected the aminopeptidase activity. The specific hydrolysis of peptidic bounds involving amino acids (Leu, Trp and Val), usually identified as major components of bitter peptides, probably indicates a debittering activity of this aminopeptidase. aminopeptidase 1bitter peptide 1bitter UHT milk 1cheese ripening Résumé -Effet de l'aminopeptidase de Pseudomonas fluorescens ATCC 948 sur les peptides amers synthétiques, les hydrolysats de protéines du lait UHT et sur l'affinage du fromage italien de type caciotta. On a étudié l'activité de l'aminopeptidase purifiée de Pseudomonas fluorescens ATCC 948 sur les peptides amers synthétiques, les hydrolysats amers des protéines du lait UHT et sur l'affinage du fromage italien de type caciotta. L'aminopeptidase a presque totalement hydrolysé le pentapeptide amer H-Leu-Trp-Met-Arg-Phe-OH et libéré Val du tétrapeptide amer H-Val-Pro-Leu-Leu-OH. Aucun clivage de la liaison Pro n'a été observé. Dans le lait UHT, dans lequel M Gobbetti et al l'hydrolyse des protéines a été provoquée par addition d'une protéinase, l'aminopeptidase a produit une concentration d'acides aminés libres environ 8 fois plus élevée que celles déterminées sur le lait UHT non hydrolysé et non amer (211,3 contre 25,3~g1ml respectivement). Glu, Leu, Met, Trp et Val ont été les plus abondants. L'enzyme a présenté 44% de son activité maximale à la température de conservation du lait UHT (2(J'C). L'aminopeptidase a été stable pendant l'affinage de la caciotta: aucune activité n'a été perdue pendant 2 mois. Au terme de 45 jours d'affinage, le fromage contenant l'aminopeptidase présentait un niveau d'acides aminés plus élevé (990,2~g1ml) que le contrôle non traité (591, 1~glml). Le profil des acides aminés du fromage traité reflétait l'activité de l'aminopeptidase. L 'hydrolyse spécifique des liaisons peptidiques impliquant des acides aminés (Leu, Trp et Val) habituellement identifiés comme étant les principaux composants des peptides amers indique probablement une activité d'élimination de l'amertume de cette aminopeptidase. aminopeptidase 1 peptide amer lIait UHT amer 1 affinage
PLoS ONE, 2013
The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 mkat H-Ala-Pro-pNA L 21 , which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 mkat H-Ala-pNA L 21. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn 2+-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co 2+ .Zn 2+ .Mn 2+ .Ca 2+ .Mg 2+. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50uC and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.
European Journal …, 1995
The biosynthesis of Peps, a lanthionine-containing antimicrobial peptide, is directed by the 20-kbp plasmid pED503. We identified a 7.9-kbp DNA-fragment within this plasmid which covers the information for Peps synthesis in the homologous host Staphylococcus epidermidis S which has been cured of pED503. This fragment contained, in addition to the previously described structural gene pepA and the immunity gene pep1 [Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T. & Sahl, H. 4. (1994) Appl. Env. Microbiol. 60, 2876-28831, a genepepTcoding for a translocator of the ABC transporter family, a gene pepP coding for a serine protease and two genes pepB and pepC coding for putative modification enzymes ; the gene arrangement is pepTIAPBC. We analyzed the biosynthetic genes with respect to their function in Peps biosynthesis. Deletion of PepT reduced Pep5 production to about lo%, indicating that it can be partially replaced by other host-encoded translocators. Inactivation of PepP by site-directed mutagenesis of the active-site His residue resulted in production of incorrectly pi-ocessed Peps fragments with strongly reduced antimicrobial activity. Deletion of pepB and pepC leads to accumulation of Pep5 prepeptide in the cells without excretion of processed peptide. A pepC-deletion clone did not excrete correctly matured Peps but it did produce fragments from which serine and threonine were absent. Only one of these fragments contained a single lanthionine residue out of three expected while the remaining, unmodified cysteine residues could be detected by reaction with Ellman's reagent. These results demonstrate that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonine.
LWT - Food Science and Technology, 2004
Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on b-CN f193-209 under simulated cheese condition. Bovine b-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30 C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of b-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of b-CN f193-209 hydrolysis by CFE of
Food Research International, 2011
Hard cooked cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food. In this work, we assessed the proteolysis produced by enzymes of two strains of L. helveticus in a new cheese model, which consisted of a sterile substrate prepared with hardcooked cheeses, and identified the time of ripening when main changes in proteolysis are produced. The extract, a representative model of the aqueous phase of the cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90, and 180 days) made with starters composed of the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 μm). The substrates were incubated at 34°C during 21 days; samples were taken at 0, 3, 7, 14, and 21 days. Sterility was verified by plating samples on skim milk agar and incubating at 37°C for 48 h. Proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Great variation in peptide profiles was found as incubation progressed in cheese extracts, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the extracts. The extracts derived from cheeses with L. helveticus SF138 showed low production of peptides and a notable increase in free amino acids content during incubation. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, but the free amino acids accumulation was lower than in the first case, which suggested that L. helveticus SF209 had either a low peptydolitic activity or produced an intense amino acids breakdown. This trend was more evident for extracts prepared with 90-day-old cheeses. It was concluded that the strains of L. helveticus assayed showed potentially complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. The extract was an appropriate medium to model hard cooked cheese ripening in short periods of time.
Modulation of casein proteolysis by lactococcal peptidase gene inactivation
International Dairy Journal, 2000
Peptidases from lactic acid bacteria participate in cheese ripening by hydrolysing peptides and liberating free amino acids, which are precursors of aroma compounds. Using a pseudo-curd model and lactococcal mutants, negative for di!erent peptidases, as ripening agents, we determined some of the key peptidases in the ripening process. The total level of amino acids quanti"ed after 28 days of ripening was signi"cantly reduced when the general aminopeptidase PepN was absent. The situation was aggravated when the tripeptidase PepT was also missing. The de"ciency in the proline-speci"c aminopeptidase PepX more speci"cally decreased the level of free proline in the pseudo-curds after 28 days of ripening. These three peptidases, which are also necessary for optimal lactococcal growth in milk, can be considered as peptidases of technological importance. On the other hand, the suppression of the other peptidases tested (PepF1, PepF2, PepC, PepP) did not signi"cantly a!ect amino acid pools in the conditions of the test. 2001 Elsevier Science Ltd. All rights reserved.