Purification, kinetic and functional characterization of membrane bound dipeptidyl peptidase-III from NCDC 252: a probiotic lactic acid bacteria (original) (raw)
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International Journal of Biological Macromolecules, 2016
Plant based diet such as vegetables, cereals, legumes and oilseeds contain 80% of total phosphorous in the form of phytic acid-cation complexes, bound phosphorous being excreted in manure due to unavailability of phytate degrader in the gastrointestinal tract of monogastric animals (Ashraf et al., 2013). The undegraded phytate leads to phosphorous deficiency in animals, elevated levels of phosphorous in soil and eutrophication of water bodies and renders phytic acid as the anti-nutritive factor by decreasing the bioavailability cations such as iron, calcium, magnesium, phosphorous, zinc, iodine, etc (Madsen, 2019; Singh et al., 2013). Most of these cations are involved in various physiological functions as their deficiency may lead to conditions such as anemia,
International Journal of Peptide Research and Therapeutics, 2021
An arginine aminopeptidase (EC 3.4.11.6) called aminopeptidase B was purified to apparent homogeneity from membrane extract of a potential probiotic Pediococcus acidilactici NCDC 252 using successive chromatographies on sephadex G-100 and phenylsepharose CL-4B. Purified enzyme was a heterotrimer with molecular mass of ~ 101.36 kDa. Predicted molecular weight of the enzyme from its gene (93.9 kDa) was close to the calculated molecular weight. The enzyme was optimally active at pH 7.5 and 40 °C. It was strongly inhibited by metal chelating agent and thiol protease inhibitors suggesting that enzyme is a metalloprotease with involvement of thiol. The K m and V max of enzyme for Arg-4mβNA were calculated to be 26 μM and 19.9 nmol/ml/min respectively. Its 3-D structure was modeled and validated using in-silico approach. In-silico analysis revealed Ser, His, Phe, Tyr and Thr to be present at active site of aminopeptidase B. Docking studies revealed that Arg-4mβNA binds with high affinity to the enzyme followed by Lys-4mβNA. The enzyme also hydrolyzed dipeptide-4mβNA derivatives containing hydrophobic amino acids and diaminocarboxylic acids (Arg, Lys and Asp) at the N-termini but not tripeptides, endopeptidase substrates and-βNA derivatives or peptides with proline and phenyl at their N-termini or C-termini.
Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei
Applied and Environmental Microbiology, 2001
An X-prolyl-dipeptidyl peptidase has been purified fromLactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hy...
Proline-Specific Peptidases of Lactobacillus casei Subspecies
Journal of Dairy Science, 1994
Proline iminopeptidase was purified 76-fold from crude cell-free extracts of Lactobacillus casei ssp. cmei LLG by ion-exchange chromatography (preparative and analytical) and gel filtration chromatography using fast protein liquid chromatography. The purified enzyme appeared as a single band on native-and SDS-PAGE and had a molecular mass of 46 kDa. Enzyme activity was maximal at pH 7.5 and 40°C with proline aminomethyl coumarin as substrate. The activity was inhibited by Fe3+ and Hg2+ ions. This enzyme evidently was sulfhydryl; p-chloromercuribenzoate caused complete inhibition at 10 mM. The Michaelis-Menten constant and maximum velocity were .6 mM and 1.7 nM/ mg per min respectively, using the same substrate. This enzyme showed the ability to cleave the Pro-Pro bond, which is of significant importance in cheese ripening.
International Journal of Peptide Research and Therapeutics, 2020
Pediococcus acidilactici NCDC 252 is a Gram-positive lactic acid bacteria (LAB) that is being studied to develop it as probiotic and starter culture for dairy and meat industry. Peptidoglycan hydrolases (PGHs) are also gaining interest because of their antibacterial activity. NCDC 252 genome was searched for PGH encoded genes and product of these genes were in silico characterized for different parameters. Extracellular PGHs of NCDC 252 were precipitated and characterized. PGHs were screened turbidometrically with Bacillus cereus and Staphylococcus albus as substrates. Spectrum of PGH(s) was studied in renaturing SDS-PAGE by varying substrates and renaturing conditions. PGH(s) band intensity decreased in the presence of NaCl whereas some additional secondary lytic bands appeared in the presence of EDTA. Autolysis of LAB in cheese and other fermented food products is associated with release of oligopeptides by proteolysis and other enzyme activities. Autolytic phenotype of P. acidilactici was evaluated and characterized. Lytic activity studies revealed 45-60% activity against Micrococcus lysodeikticus and B. cereus greater than for P. acidilactici. PGH of P. acidilactici exhibited broad growth inhibiting spectrum of antibacterial activity against studied Gram positive and Gram negative bacteria and thus can be promising in controlling pathogenic bacteria. This also confers probiotic attribute to the studied strain.
Proline-specific peptidases of Lactobacillus helveticus CNRZ32
1998
Proline iminopeptidase was purified 76-fold from crude cell-free extracts of Lactobacillus casei ssp. cmei LLG by ion-exchange chromatography (preparative and analytical) and gel filtration chromatography using fast protein liquid chromatography. The purified enzyme appeared as a single band on native-and SDS-PAGE and had a molecular mass of 46 kDa. Enzyme activity was maximal at pH 7.5 and 40°C with proline aminomethyl coumarin as substrate. The activity was inhibited by Fe3+ and Hg2+ ions. This enzyme evidently was sulfhydryl; p-chloromercuribenzoate caused complete inhibition at 10 mM. The Michaelis-Menten constant and maximum velocity were .6 mM and 1.7 nM/ mg per min respectively, using the same substrate. This enzyme showed the ability to cleave the Pro-Pro bond, which is of significant importance in cheese ripening. proline iminopeptidase, purification, cheese ripening, lactobacilli)
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, 1999
Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (P(nisA)). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of P(nisA)::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of indi...
Biochimie, 2017
Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly(673) in DPP7 and Arg(673) in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly(673) exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with...
FEBS Open Bio, 2013
Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gramnegative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with k cat /K m of 10.62 ± 2.51 μM −1 s −1 , while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2-as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1-and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing k cat and lowering K m values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/ DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2-and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria.