Cloning and characterization of debittering peptidases, PepE, PepO, PepO2, PepO3, and PepN, of Lactobacillus helveticus WSU19 (original) (raw)
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International Dairy Journal, 2008
This study determined specificities of aminopeptidase N (PepN), endopeptidase E (PepE), endopeptidase O (PepO), endopeptidase O2 (PepO2), and endopeptidase O3 (PepO3), from Lactobacillus helveticus WSU19 on the a s1-CN f1-23 peptide, formed by residual chymosin during cheese ripening. Cell-free extracts (CFEs) were prepared from Escherichia coli DH5a derivatives expressing peptidase genes of Lb. helveticus WSU19. The a s1-CN f1-23 peptide was digested by CFEs under cheese ripening conditions. Degradation pattern was analyzed qualitatively using MALDI-TOF mass spectrometry. PepN exhibited activity on a s1-CN f1-23 only in the presence of an endopeptidase, particularly PepO-like endopeptidases. PepO, PepO2, and PepO3 cleaved a s1-CN f1-23 predominantly at Glu 14-Val 15 , forming the bitter peptide a s1-CN f1-14. PepE cleaved a s1-CN f1-23 primarily at Lys 3-His 4 , suggesting a role for PepE in degrading bitter peptides from the N-terminus of a s1-CN f1-23. Combinations of PepE/PepO and PepE/PepO2 were determined to have the potential to decrease the accumulation of a s1-CN f1-14.
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.
Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants, due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mecha-nisms. This review focuses on the post-proline cleaving enzymes (PPCEs), especially the prolyl endoprotease/oligopeptidase (PEP/POP). To date, most PPCEs studied are of microbial and ani-mal origins. Recently, there are reports of new plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes reaction at the carboxyl-terminal of proline residues. PPCEs have diverse applications, are widely used in the beer brewing industry, and have potential as therapeutic a...
Applied and Environmental Microbiology, 2003
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Deletion of the four C-terminal residues of PepC converts an aminopeptidase into an oligopeptidase
Protein Engineering Design and Selection, 1999
The aminopeptidase PepC is a cysteine peptidase isolated from lactic acid bacteria. Its structural and enzymatic properties closely resembles those of the bleomycin hydrolases, a group of cytoplasmic enzymes isolated from eukaryotes. Previous biochemical and structural data have shown that the C-terminal end of PepC partially occupies the active site cleft. In this work the substrate specificity of PepC was engineered by deletion of the four C-terminal residues. The mutant PepC∆ ∆432-435 cleaved peptide substrates as an oligopeptidase while the aminopeptidase specificity was totally abolished. The substrate size dependency indicated that PepC∆ ∆432-435 possesses an extended binding site able to accommodate four residues of the substrate on both sides of the cleaved bond. The activity of PepC∆ ∆432-435 towards tryptic fragments of casein revealed a preference for peptides with hydrophobic amino acids at positions P2 and P3 and for Gly, Asn and Gln at position P1. PepC∆ ∆432-435 was shown to be highly sensitive to the thiol peptidase inhibitors leupeptin or E64 which are inefficient towards the wild-type PepC. In conclusion, deletion of the four C-terminal residues in PepC produces a new enzyme with properties resembling those of an endopeptidase from the papain family.
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.
The Analyst, 2003
There are enzymes that specifically recognise the amino acid proline within peptides and proteins that are called post-proline cleaving enzymes. Many of them are implicated in neurodegenerative disorders and psychiatric diseases. ZIP is a newly-discovered one of these peptidases. In this work, it has been purified from bovine serum and subjected to various analytical studies in order to characterise it. A series of reactions between synthesised peptides and ZIP were carried out in order to elucidate the size and specificity of the active site of the enzyme. On-line LC-MS was carried out on samples before and