2015 Volume 21 Issue 3 Pages 445-451
An X-prolyl-dipeptidyl aminopeptidase has been purified from Lactobacillus gasseri by lysozyme treatment and seven chromatographic steps. The yield was 1.4% and the specific activity increased 265-fold over the crude enzyme extract. SDS-PAGE provided a single band with a MW of approximately 82 kDa. Gel filtration chromatography showed that the native enzyme was approximately 173 kDa, suggesting that the protein is a dimer. The amino acid sequences of the peptide fragments obtained after tryptic treatment of the purified enzyme were determined and found to be consistent with that of an X-prolyl-dipeptidyl aminopeptidase from L. gasseri ATCC 33323 (LGAS_0712, coverage: 31.74%). Optimal activity was observed at pH 7.0 and 55°C. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonylfluoride, and by divalent cations (Cu2+, Hg2+, and Zn2+). From these inhibition characteristics, the enzyme is likely a serine proteinase. Beta-casomorphin 7 (BCM-7, Tyr-Pro-Phe-Pro-Gly-Pro-Ile) was hydrolyzed into X-proline fragments from the N-terminus of the peptide. The characteristics of the enzyme are very similar to those of the enzyme from Lactobacillus sakei. However, as free amino acids were detected among the degradation products of BCM-7, some aspects of substrate specificity require further clarification.
The proteolytic systems of various lactic acid bacteria are very similar and involve the following steps: i) proteolysis by extracellular serine-proteinases, ii) translocation of extracellularly-produced oligopeptides into the cell by specific di-tripeptide and oligopeptide transport systems, and iii) hydrolysis of the intracellularly-transported oligopeptides to amino acids by a multitude of intracellular peptidases such as endopeptidases, aminopeptidases, dipeptidases, tripeptidases, and dipeptidyl peptidases (Kunji et al., 1996; Christensen et al., 1999).
Casein accounts for 80% of the protein in cow's milk, and β-casein makes up about 30% of casein. The amino acid composition of β-casein is approximately 17% proline (35 residues out of 209 amino acids) (Kiefer-Partsch et al., 1989). Proline influences the conformation of peptides and restricts the cleavage of proline-containing peptides by common exo- and endo-peptidases. Therefore, in order to efficiently digest this major protein component in milk, the presence of a proline-specific peptidase, which can cleave peptides containing proline, is important.
One such important enzyme is X-prolyl-dipeptidyl aminopeptidase. The characteristic enzyme activity of X-prolyl-dipeptidyl aminopeptidases is as follows: an N-terminal dipeptide is released from an oligopeptide, which has proline as the second amino acid from the N-terminus. At the end of digestion, the dipeptide, which is released from the oligopeptide by enzymatic action, is easily broken down into its component amino acids by various peptidases.
Several lactic acid bacteria strains have been used to study X-prolyl-dipeptidyl aminopeptidase, including Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus (Bockelmann and Fobker, 1991), Lactococcus lactis subsp. lactis (Zevaco et al., 1990; Lloyd and Pritchard, 1991), Lactococcus lactis subsp. cremoris (Yan et al., 1992), Lactobacillus helveticus (Khalid and Marth, 2001), Streptococcus thermophilus (Tsakalidou et al., 1998) and Lactobacillus sakei (Sanz and Toldra, 2001). While other lactic acid bacteria have also been studied, there are no published studies of the purification and characterization of the X-prolyl-dipeptidyl aminopeptidase from Lactobacillus gasseri. The genomic analysis of L. gasseri ATCC 33323 (www.uniprot.org/uniprot/Q044L8) suggests the existence of an X-prolyl-dipeptidyl aminopeptidase with 794 amino acid residues (LGAS_0712, molecular weight: 90,940); however, the biochemical analysis of this enzyme has not been reported. In this study, we aimed i) to prove that X-prolyl-dipeptidyl aminopeptidase is present in L. gasseri and ii) to analyze its enzymatic properties.
Bacterial strain and growth conditions Lactobacillus gasseri ME-284 (Meiji Co. Ltd., Kanagawa, Japan) was stored at −80°C in 10% sterile skim milk. The culture was subcultured once in MRS broth (Oxoid, Basingstoke, England) for 24 h at 37°C, then used as the starter culture (inoculation size: 2%). For purification of the enzyme, the strain was grown in MRS broth (6 L). The medium was autoclaved at 121°C for 15 min.
Measurements of enzyme activity and protein concentration Enzyme activity was routinely determined using Gly-Pro p-nitroanilide (Peptide Inst., Osaka, Japan) as the substrate. Enzyme solution (50 µL) and substrate solution (10 mM, 50 µL) were incubated in 400 µL of phosphate buffer (20 mM, pH 7.0, hereafter called PB) at 37°C for 30 min. The reaction was stopped with 100 µL of 12% tricarboxylic acid (TCA), then, after 15 min, the reaction mixture was centrifuged (10,000×g, 10 min, 15°C; KR-1500, Kubota, Tokyo, Japan). The absorbance of the supernatant was measured at 410 nm (UV mini-1240, Shimadzu, Kyoto, Japan) to determine the degree of substrate hydrolysis. Protein content was determined from the OD of the protein solution at 280 nm using a standard solution of bovine serum albumin (Wako Pure Chemical, Osaka, Japan).
Enzyme purification All purification steps were carried out at 4°C. Cells were harvested by centrifugation (8,000×g, 30 min; CR22G, Hitachi, Tokyo, Japan), suspended in Tris-HCl buffer (50 mM, pH 8.5, 300 mL), and treated with lysozyme (final concentration 2 mg/mL, from egg white, Wako Pure Chemical) for 2 h at 37°C. The crude enzyme extract was collected by centrifugation in the supernatant, then dialyzed against PB. The dialyzed enzyme solution was loaded onto a DE-52 column (2.5 × 30 cm, Whatman, Tokyo, Japan) equilibrated with PB, then the column was washed with PB and the enzyme was eluted using 0.1 M NaCl in PB. The active fraction was dialyzed against PB, and then applied again to the DE-52 column. The enzyme was eluted using a linear gradient of 0 to 0.2 M NaCl in PB and 5 mL fractions were collected. The active fractions were dialyzed against PB and loaded onto a Blue-Sepharose column (1.8 × 30 cm, Sigma-Aldrich, Tokyo, Japan) equilibrated with PB. The active fraction passed through the column and was collected, dialyzed against PB containing 1.5 M ammonium sulfate, then loaded onto a Phenyl-Sepharose column (1.8 × 30 cm, Sigma-Aldrich) equilibrated with PB containing 1.5 M ammonium sulfate. The enzyme was eluted using a linear gradient of 1.5 to 0 M ammonium sulfate in PB and 2.5 mL fractions were collected. The active sample was concentrated (membrane filter; MW cut-off: 20 kDa, ADVANTEC, Tokyo, Japan) and dialyzed against PB containing 0.15 M NaCl, then loaded onto a Sephacryl S-200 HR column (2.5 × 87.5 cm, Sigma-Aldrich) equilibrated with the same buffer. The enzyme was eluted and 5 mL fractions were collected. The active fractions were re-chromatographed by gel filtration and 2.5 mL fractions were collected. Finally, the active fractions were dialyzed against PB and applied to a Heparin-Sepharose column (1.8 × 30 cm, Sigma-Aldrich) equilibrated with PB. Elution was performed using a linear gradient of 0 to 0.2 M NaCl in PB and 2.5 mL fractions were collected. The active fractions were pooled, dialyzed against PB, and stored at −30°C.
Polyacrylamide gel electrophoresis SDS-PAGE in the presence of beta-mercaptoethanol (β-ME) was carried out using 10 – 20% polyacrylamide gels (90 × 90 × 1 mm, Super Sep™ Ace, Wako Pure Chemical). The gels were run at 100 V, 50 mA, for 2 h. The proteins were visualized with silver stain as described in the manufacturer's manual (Wako Pure Chemical).
Molecular weight determination of the enzyme The molecular weight (MW) of the purified enzyme under reducing conditions was estimated by SDS-PAGE. WIDE-VIEW™ Prestained Protein Size Marker III (molecular size range: 11 ∼ 245 kDa, Wako Pure Chemical) was used as the molecular marker. The MW of the native enzyme was estimated by gel filtration on a Sephacryl S-200 HR column using the following molecular marker proteins (Bio-Rad Laboratories, Munich, Germany): thyroglobulin (670,000), γ-globulin (158,000), ovalbumin (44,000), myoglobin (17,000) and Vitamin B12 (1,350). Blue dextran 2000 (GE Healthcare UK, Little Chalfont, England) was used to determine the void volume of the Sephacryl S-200 HR column. The MW was calculated from a regression expression according to the manufacturer's instructions (GE Healthcare Biosciences).
In-gel digestion with trypsin The enzyme was subjected to SDS-PAGE and stained with MS-compatible silver staining (Shevchenko et al., 1996). A gel slice was cut into small pieces and put into a 1.5 mL tube. The gel pieces were destained by rinsing in destaining solution (15 mM K3[Fe(CN)6], 50 mM Na2S2O3). Cysteine disulfide bonds were reduced by incubating the gel pieces with 10 mM dithiothreitol in 25 mM NH4HCO3 at 56°C for 1 h, then alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 at room temperature for 45 min in the dark. Gel pieces were washed with 50% acetonitrile and dried in a vacuum concentrator (CVE200D, Tokyorikakikai, Tokyo Japan). Gel pieces were re-hydrated with 30 µL of trypsin solution (10 µg/mL in 50 mM NH4HCO3). In-gel digestion was performed at 37°C overnight. The resulting peptides were extracted with 50% acetonitrile containing 5% trifluoroacetic acid (TFA), then dried in a vacuum concentrator and re-dissolved in 0.1% formic acid. The peptide mixtures thus obtained were subjected to nanoLC-ESI-MS/MS analysis.
Mass Spectrometric analysis NanoLC-ESI-MS/MS analysis was carried out using a Q Exactive mass spectrometer (Thermo Scientific, USA) equipped with a Captive spray ionization source (Michrom Bioresources, USA) and interfaced on-line with an Avance UHPLC system (Michrom Bioresources). Samples were introduced onto the analytical column using an autosampler (HTC-PAL system, CTC Analytics AG, Zwingen Switzerland). One µL peptide extract samples were transferred onto a C8 cartridge (Peptide Captrap, Michrom Bioresources) and the eluted peptides were then transferred onto the analytical column (L-column 2 Micro C18, 0.2 mm × 50 mm, CERI, Tokyo Japan) using a switching valve. Following an initial wash with buffer A (0.1% formic acid in water), peptides were eluted using a linear gradient from 5 – 65% buffer B (acetonitrile) over a 30 min interval. The HPLC column eluent was introduced directly into the Captive spray ionization source of the Q Exactive mass spectrometer. Automated peak recognition, dynamic exclusion, and daughter ion scanning of the 10 most intense ions were performed using Xcalibur software (Thermo Scientific). Spectra were scanned over the range 350 – 2000 mass units.
Database searching and data interpretation The raw files were analyzed with Sequest HT proteome discoverer 1.4 I (Thermo Scientific) with 1% False Discovery Rate (Elias and Gygi, 2007) using Percolator (Käll et al., 2007). All matched peptides were confirmed by visual examination of the mass spectra, and all spectra were searched against the latest version of the public non-redundant protein database of the National Center for Biotechnology Information (NCBI).
Dependence of enzyme activity on pH and temperature The dependence of enzyme activity on pH was examined in the range pH 4.0 to 9.0 using the following buffers: acetate buffer (50 mM, pH 4.0∼5.0), phosphate buffer (50 mM, pH 6.0∼8.0), and Tris-HCl buffer (50 mM, pH 9.0). The temperature dependence was examined in the range 30 to 60°C at pH 7.0 using 50 mM phosphate buffer.
Effects of chemical agents on enzyme activity The effects of various chemical agents on enzyme activity were assayed by the addition of the chemical agent to the reaction mixture at a final concentration of 0.1 mM or 1 mM. Enzyme activity was measured and expressed as a percentage of the control.
Effect of peptide inhibitor on enzyme activity The effect of a specific inhibitor of X-prolyl-dipeptidyl aminopeptidase, diprotin A (Peptide Inst.), on enzyme activity was determined using diprotin A concentrations between 0 to 200 µM in PB. The inhibition assay was carried out as follows: 350 µL of phosphate buffer (50 mM, pH 7.0), 50 µL of diprotin A solution, and 50 µL of purified enzyme solution were pre-incubated for 5 min at 37°C, then 50 µL of substrate was added. Enzyme activity was measured following a 30 min incubation at 37°C.
Hydrolysis of beta-casomorphin 7 and analysis of the fragment peptides Hydrolysis of beta-casomorphin 7 (BCM-7, Peptide Inst.) was carried out as follows: 50 µL of 6.0 mg/mL BCM-7 solution in PB was added to 50 µL of purified enzyme solution (corresponding to an OD at 410 nm of approximately 1 when enzyme activity was determined as described above) and incubated at 37°C for various times up to 120 min. The reaction solution was then heated at 100°C for 10 min to inactivate the enzyme. After cooling and filtering (0.45 µm), fragment peptides were separated on a Shodex Asahipak ODP-50 6D column (5 µm, 6.0 × 150 mm, Showa Denko, Tokyo, Japan) using gradient elution with acetonitrile (0 – 100% for 60 min) containing 0.1% TFA in water. The flow rate was set at 1.0 mL/min and the column temperature was maintained at 30°C. The injection volume was 30 µL, and the monitoring detection wavelength of the UV-vis detector was 214 nm. The fragment peptides in each elution peak were identified by UPLC-TQD-MS using a Waters Acquity Ultra Performance LC system with a PDA detector and a Quattro Premier XE MS system (Waters, Milford, MA) equipped with a Waters Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 50 mm). The mobile phase was composed of solvent A (0.1% formic acid in water) and solvent B (acetonitrile). The gradient increased from 5% to 35% solvent B over 3.5 min. The flow rate was 0.3 mL/min, and the column temperature was maintained at 40°C. The injection volume was 2 µL, and the monitoring wavelength of the PDA detector was 214 nm. MS analysis was performed using positive ion-mode ESI, a capillary voltage of 2.5 kV, and a cone voltage of 10 to 40 V. The source temperature was set at 120°C and the cone gas flow rate was 50 L/h. Nitrogen was used as the desolvation gas at a flow rate of 700 L/h and at a temperature of 400°C.
Enzyme purification Results of the purification procedure are summarized in Table 1. The recovery yield was 1.4% and the specific activity increased 265-fold over the enzyme extract. SDS-PAGE under reducing conditions provided a single band with a molecular weight of approximately 82 kDa (Fig. 1).
| Purification Step | Total Volume (ml) | Total Activity* (OD 410 nm) | Total Protein** (mg) | Yield (%) | Purification (fold) |
|---|---|---|---|---|---|
| Crude extract | 300 | 765.7 | 6824.9 | 100.0 | 1.0 |
| DEAE-cellulose (1st) | 200 | 396.5 | 1118.2 | 51.8 | 3.5 |
| DEAE-cellulose (2nd) | 92 | 267.7 | 369.3 | 35.0 | 7.2 |
| Blue-Sepharose | 172 | 125.3 | 113.8 | 16.4 | 11.0 |
| Phenyl-Sepharose | 37 | 93.0 | 20.2 | 12.1 | 46.0 |
| Sephacryl S-200 HR (1st) | 20 | 29.9 | 3.2 | 3.9 | 93.4 |
| Sephacryl S-200 HR (2nd) | 10 | 18.5 | 0.9 | 2.4 | 205.6 |
| Heparin-Sepharose | 2 | 10.6 | 0.4 | 1.4 | 265.0 |
SDS-PAGE analysis of the X-prolyl-dipeptidyl aminopeptidase from Lactobacillus gasseri ME-284.
SDS-PAGE under reducing conditions was carried out using 10 – 20% polyacrylamide gels. The gels were run at 100 V, 50 mA, for 2 h. Proteins were visualized after silver staining as described in the manufacturer's manual. Lane 1: 5 µL of WIDE-VIEW™ Prestained Protein Size MarkerIII (molecular size: 11 – 245 kDa), Lane 2: 1 µg of purified enzyme after Heparin-Sepharose column chromatography.
Molecular weight determination of the enzyme The MW was estimated to be about 173 kDa by gel filtration (data not shown) and about 82 kDa by SDS-PAGE (Fig. 1). These results suggest that the enzyme consists of two subunits.
Amino acid sequence of the enzyme The amino acid sequences of the peptide fragments obtained after tryptic digestion were analyzed. Comparison of the obtained sequences with the deduced amino acid sequence (794 residues) of L. gasseri ATCC 33323 reported in www.uniprot.org/uniprot/Q044L8 (October 29, 2014) showed that the obtained sequences were consistent with the deduced amino acid sequence of X-prolyl-dipeptidyl aminopeptidase of L. gasseri ATCC 33323 (LGAS_0712, coverage: 31.74%, 252 residues) (Fig. 2). The positions of the consistent parts of the sequence were: residues 60–72, 78–84, 114–124, 144–155, 174–182, 240–252, 258–265, 268–308, 315–326, 337–342, 350–362, 437–442, 589–605, 654–675, 684–711, 718–729, 759–772, and 787–794 (C-terminus). Thus, the data indicate that the purified enzyme is an X-prolyl-dipeptidyl aminopeptidase.
Sequences of peptide fragments obtained after tryptic treatment of the purified enzyme were analyzed and compared to the deduced amino acid sequence of X-prolyl-dipeptidyl aminopeptidase (www.uniprot.org/uniprot/Q044L8, 794 residues) of L. gasseri ATCC 33323. The obtained sequences were consistent with the 18 sections of the deduced sequence shown in black (coverage: 31.74%, 252 residues). The database search and data interpretation methodology are described in the Materials and Methods and the consistent parts of the sequence are presented in the Results.
Dependence of enzyme activity on pH and temperature Optimum hydrolysis of Gly-Pro p-nitroanilide was observed at pH 7.0 (Fig. 3-A) and 55°C (Fig. 3-B).
Effects of pH (A) and temperature (B) on the activity of the X-prolyl-dipeptidyl aminopeptidase from Lactobacillus gasseri ME-284.
Enzyme activity was assayed by monitoring the hydrolysis of Gly-Pro p-nitroanilide at 410 nm. The activity at the optimal pH of 7.0 or at the optimal temperature of 55° was given a value of 100%, corresponding approximately to an OD of 1 at 410 nm.
Effects of chemical agents on enzyme activity Concentrations of 0.1 mM or 1 mM Ca2+, Co2+ or Mg2+ did not affect enzyme activity significantly (4 – 11% inhibition), but Cu2+ and Hg2+ inhibited enzyme activity severely, even at a concentration of 0.1 mM. Zn2+ moderately inhibited activity at a concentration of 1 mM. Ethylenediaminetetraacetic acid (EDTA) and β-ME had no effect, whereas diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) inhibited enzyme activity significantly at a concentration of 1 mM (Table 2).
| Metal salt and Chemical agent | Relative activity(%)* | |
|---|---|---|
| 0.1 mM | 1.0 mM | |
| CaCl2 | 95.9 | 96.1 |
| CoCl2 | 90.9 | 88.8 |
| CuCl2 | 20.5 | 14.6 |
| HgCl2 | 5.2 | 2.0 |
| MgCl2 | 95.3 | 91.2 |
| ZnCl2 | 72.2 | 44.2 |
| PMSF | 81.8 | 11.5 |
| DFP | 85.7 | 13.1 |
| EDTA | 99.1 | 106.7 |
| β-ME | 98.2 | 97.7 |
Effect of peptide inhibitor on enzyme activity Activity decreased in a concentration-dependent manner in the presence of diprotin A: activity was inhibited by 50% (IC50) at about 24.4 µM diprotin A, as calculated from the regression formula (Eq. 1)
 |
Hydrolysis of beta-casomorphin 7 and analysis of the fragment peptides Fig. 4 shows the HPLC chromatogram of the reaction solution after 120 min hydrolysis of BCM-7 (1Tyr-2Pro-3Phe-4Pro- 5Gly-6Pro-7Ile). The BCM-7 peak and five new peaks (Peaks A-E) were detected. The MS spectra indicated that the compounds corresponding to each peak were as follows: Peak A, 1Tyr; Peak B, 1Tyr-2Pro, 3Phe; Peak C, 5Gly-6Pro-7Ile; Peak D, 3Phe-4Pro; and Peak E, 3Phe-4Pro-5Gly-6Pro-7Ile. Time courses of the peak areas measured during a 120 min incubation are shown in Fig. 5. The peak area of BCM-7 decreased and that of Peak B (1Tyr-2Pro, 3Phe) increased throughout the reaction period. Peak D (3Phe-4Pro) and Peak E (3Phe-4Pro-5Gly-6Pro-7Ile) increased during the first 60 min of reaction, then gradually decreased between 60 and 120 min. The peak area of Peak C (5Gly-6Pro-7Ile) increased up to 120 min incubation. In the HPLC analysis, Pro, 5Gly, 7Ile, and 5Gly-6Pro were not observed.
HPLC chromatogram of the reaction solution after 120 min incubation of BCM-7 with the X-prolyl-dipeptidyl aminopeptidase from Lactobacillus gasseri ME-284.
The fragment peptides were separated on a Shodex Asahipak ODP-50 6D column and identified by MS analysis using positive ion-mode ESI. Details are described in Materials and Methods. Peak A, 1Tyr; Peak B, 1Tyr -2Pro, 3Phe; Peak C, 5Gly-6Pro-7Ile; Peak D, 3Phe-4Pro; Peak E, 3Phe-4Pro-5Gly-6Pro-7Ile.
Time course of the peak area of the peptides released during BCM-7 digestion by the X-prolyl-dipeptidyl aminopeptidase from Lactobacillus gasseri ME-284.
The solutions were analyzed after 30, 60 and 120 min incubation. For details regarding the separation and identification of each peak, refer to Materials and Methods.
○ – ○, BCM-7
(1Tyr-2Pro-3Phe-4Pro-5Gly-6Pro-7Ile); ● -- ●, Peak A (1Tyr); △ – △, Peak B (1Tyr -2Pro, 3Phe); ▲ -- ▲, Peak C (5Gly-6Pro-7Ile); □ – □, Peak D (3Phe-4Pro); ■ -- ■ , Peak E (3Phe-4Pro-5Gly-6Pro-7Ile).
Investigation of the MW of X-prolyl-dipeptidyl aminopeptidases from lactic acid bacteria showed that the enzymes from lactococci are mostly dimers with molecular weights of 160 to 180 kDa. On the other hand, the enzymes from lactobacilli exhibit a wide range of MWs depending on the strain, as well as diversity in the number of subunits (from 1 to 3) (Christensen et al., 1999). The MW of the enzyme described here, which comprises two 82 kDa subunits, is 173 kDa. Amino acid sequence analysis showed that the sequence of the protein was consistent with that of X-prolyl-dipeptidyl aminopeptidase of L. gasseri ATCC 33323 (LGAS_0712, coverage : 31.74%) indicating that the purified enzyme is an X-prolyl-dipeptidyl aminopeptidase. The enzyme characteristics of the purified protein are similar to those of enzymes from lactococci. The optimal pH for the enzymatic reaction is 7.0. While the optimal temperature of the enzyme for many lactic acid bacteria (lactococci and lactobacilli) is 40 – 50°C (Bockelmann and Fobker, 1991; Zevaco et al., 1990; Khalid and Marth, 2001; Tsakalidou et al., 1998), the optimal temperature for the enzyme described here is slightly higher, at 55°C. Enzyme inhibition studies indicated that the enzyme from L. gasseri ME-284 is neither a metalloenzyme nor a SH-enzyme, as it was not inhibited by EDTA or β-ME. We concluded that the enzyme was a type of serine proteinase, as it was strongly inhibited by DFP and PMSF (87 to 88% inhibition).
The peak area of BCM-7 decreased between 60 and 120 min incubation with the purified enzyme, while that of Peak B (1Tyr-2Pro, 3Phe) increased slightly. Peak E (3Phe-4Pro-5Gly-6Pro-7Ile) reached its maximum value after 60 min incubation, and then decreased. The concurrent decrease and increase in the peak areas of Peak E and Peak C (5Gly-6Pro-7Ile), respectively, between 60 and 120 min incubation indicate a gradual degradation of Peak E and a more gradual degradation of Peak C. Peak D (3Phe-4Pro) is potentially also a degradation product of Peak E; however, the area of Peak D was smaller at 120 min compared to at 60 min (approximately 64%). This decrease may be explained by the slight increase in Peak B up to 120 min: degradation of Peak D would release 3Phe, resulting in an increase in Peak B with a corresponding decrease in Peak D. The area of Peak A, which contains 1Tyr, increased up to 60 min-incubation and then remained constant. 1Tyr is derived from the 1Tyr -2Pro of Peak B, indicating that 1Tyr -2Pro was also degraded. The present findings clarified that the enzyme purified from Lactobacillus gasseri ME-284 hydrolyzes the peptide bond on the C-terminal side of Pro residues and releases the dipeptide from the N-terminus. Furthermore, a difference in the degradation rate of the peptide bonds of -2Pro-3Phe-, -4Pro-5Gly-, and -6Pro-7Ile- is likely, with the peptide bond C-terminal to the Pro residue being degraded more slowly in the X-Pro-Ile sequence compared to the X-Pro-Phe and X-Pro-Gly sequences. This difference in degradation rate has also been reported for the degradation of BCM-7 by the enzymes from Lc. lactis subsp. lactis (Zevaco et al., 1990; Lloyd and Pritchard, 1991) and Lc. lactis subsp. cremoris (Yan et al., 1992). The enzymes from both these subspecies of Lc. lactis are believed to degrade 5Gly-6Pro-7Ile slowly. In addition, 1Tyr and 3Phe were detected as free amino acids in this study. 1Tyr is believed to have been released from BCM-7 and/or 1Tyr-2Pro, and 3Phe from Peak E (3Phe-4Pro-5Gly-6Pro-7Ile) and/or 3Phe-4Pro; however, the derivative peptides could not be identified. HPLC analysis of the degradation products after 120 min incubation (Fig. 4) detected no peaks for Pro, Gly, Ile, and Gly-Pro; however, this may have been due to the non-optimal wavelength used (214 nm). HPLC analysis under the same conditions showed no peaks for Pro, Gly and Ile as amino acids at concentrations equivalent to those calculated for complete degradation of BCM-7 by the purified enzyme. The present findings regarding physical properties and substrate specificity suggest that the newly-isolated enzyme belongs to the same family as X-prolyl-dipeptidyl aminopeptidases. However, as free amino acids were detected among the degradation products, some aspects of substrate specificity require further clarification.
L. gasseri is a common bacterium in the mammalian gastrointestinal tract, especially in the lower section of the small intestine. This study showed that L. gasseri possesses an X-prolyl-dipeptidyl aminopeptidase. The enzyme likely supports bacterial cell growth by releasing amino acids and peptides from proline-rich proteins like caseins. These amino acids and peptides also act as nutrients for the host's intestinal cells, and the conditioned medium of L. gasseri was recently reported to reduce apoptosis caused by TNFα and cell proliferation in HCT116 cell culture (Di Luccia et al., 2013). Their findings indicate that it is very important to stimulate the growth of lactobacilli colonized in the intestine. Further research will likely reveal many symbiotic relationships between lactobacilli and intestinal cells.
