Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

Article abstract of DOI:10.1021/jf201879e

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co²⁺ and Mn²⁺, and completely inhibited by Cu ²⁺. Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.

Full text of DOI:10.1021/jf201879e

ARTICLE
pubs.acs.org/JAFC
Purification and Characterization of a Novel Extracellular Tripeptidyl
Peptidase from Rhizopus oligosporus
Jia-Shin Lin,†,‡,§ Shuo-Kang Lee,†,‡ Yeh Chen,‡ Wei-De Lin,||,^ and Chao-Hung Kao*,‡
‡Department of Biotechnology, and §Department of Food Science and Technology, Hungkuang University, 34 Chung-Chie Road,
Taichung 43302, Taiwan
Department of Medical Research, China Medical University Hospital, 2 Yuh-Der Road, Taichung 40447, Taiwan
^School of Post-Baccalaureate Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan
ABSTRACT: A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of
Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of
identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by
divalent cations, such as Co2+ and Mn2+, and completely inhibited by Cu2+. Enzyme activity was optimal at pH 7.0 and 45 ꢀC with a
specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic
tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and
tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.
KEYWORDS: Tripeptidyl peptidase, Rhizopus oligosporus, metalloprotease, serine protease
’ INTRODUCTION
R. oligosporus appears to be a critical factor in the production of
high-quality tempeh.26 Furthermore, R. oligosporus has potential as a
protease producer because it does not produce toxins.27 The regula-
tion and production of secretory acid proteases by R. oligosporus
during fermentation have been extensively studied.28,29 However,
the biochemical properties of this specific protease produced by
this fungus have not been reported. Therefore, our objective was
to purify and characterize the proteases secreted by R. oligosporus.
Tripeptidyl peptidases (TPPs) cleave peptide bonds to liber-
ate tripeptides sequentially from the free N terminal of poly
peptides. TPPs are classified as TPP I (EC 3.4.14.9) and TPP II
(EC 3.4.14.10) based on their catalytic mechanisms, molecular
weights, and cellular localization.1 TPP I is an acidic lysosomal
protease that exhibits broad substrate specificity; its inhibitory
profiles indicate that it may represent an unusual serine protease.2À4
TPP II is a high-molecular-weight extralysosomal serine protease
with a neutral pH optimum that exhibits both exo- and endo-
proteolytic activities.5À8 In mammals, TPPs may play important
roles in intracellular protein turnover,1 antigen presentation,9
peptide hormone and neuropeptide production,10 bacteria-
induced apoptosis,11 and neurodegenerative disorders.2
In addition to the mammalian TPPs, a secreted tripeptidyl
aminopeptidase (TAP) from Streptomyces lividans12 and three
secreted sedolisins (SedB, SedC, and SedD) from Aspergillus
fumigatus13 with TPP activity have been identified. Moreover, a
protease classified as a prolyl tripeptidyl peptidase (PTP) specific
for peptides with a proline residue in the P1 position has also
been characterized.14À17 TPPs in microorganisms may play
substantial roles in provision of nutrients and may also contribute
to pathogenesis.13,15,16,18 TPPs are used in industrial hydrolysis
of protein sources13 and in activating zymogens.14,17
’ MATERIALS AND METHODS
Materials. The chromogenic substrates listed in Table 4 were
purchased from Bachem AG (Bubendorf, Switzerland). The peptides
Ala-Ala-Phe-Tyr-Tyr (AAFYY) and Ala-Ala-Phe (AAF) were synthe-
sized at Mission Biotech Co., Ltd. (Taipei, Taiwan) using a Symphony
Multiple Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ).
Pepstatin A, phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC, E-64,
N-ethylmaleimide, iodoacetic acid, β-mercaptoethanol, and dithiothrei-
tol were purchased from Sigma-Aldrich (St. Louis, MO), and Ala-Ala-
Phe-chloromethyl ketone was obtained from New England Biolabs
(Ipswich, MA). All other chemicals and solvents were of analytical
grade and were purchased from Merck (Darmstadt, Germany) and
Sigma-Aldrich.
Strains, Media, and Culture Conditions. Rhizopus microsporus
var. oligosporus BCRC31750 (R. oligosporus) was obtained from the
Bioresource Collection and Research Center (Hsinchu, Taiwan) and
grown in DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract,
0.5% KH2PO4, 0.05% MgSO4 7H2O, and 0.001% FeSO4 7H2O) under
Rhizopus oligosporus, a filamentous fungus belonging to the
class zygomycetes, is economically and medically important. It
has been used for production of industrial enzymes,19À21 treat-
ment of wastewater,22 and production of antibiotics that inhibit
Gram-positive bacteria.23 In the food and fermentation indus-
tries, R. oligosporus has been used in east and southeast Asia to
produce soybean tempeh since ancient times, and there is increas-
ing interest in this food worldwide24 as a low-fat, high-protein
food with high antioxidant activity.25 Secretion of proteases from
3
3
aerobic conditions at 30 ꢀC for 36 h on an orbital shaker at 150 revolutions
Received: May 12, 2011
Revised:
September 9, 2011
Accepted: September 10, 2011
Published: September 11, 2011
r
2011 American Chemical Society
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Journal of Agricultural and Food Chemistry
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per minute (rpm). TPP medium (1% glucose, 11.2 mM KH2PO4,7mMKCl,
2.1 mM MgSO4 7H2O, 0.04 mM FeCl2 4H2O, 0.04 mM CoCl2 6H2O,
and 0.07 mM ZnCl2 at pH 7.0) supplemented with different nitrogen
sources, i.e., 2% casein, skim milk, beef extract, bovine serum albumin
(BSA), Soytone, or NH4Cl, was used to induce TPP activity.
and subjected to N-terminal amino acid sequencing. The N-terminal
sequence was determined by the Edman degradation method using
an ABI Procise 494 protein sequencer (Applied Biosystems, Foster
City, CA).
3
3
3
Enzyme Activity Assay. The activity of purified TPP was deter-
mined spectrophotometrically as described by Exterkate.31 The reaction
mixture, containing 100 mM sodium phosphate buffer (pH 7.0), 1 mM
CoCl2, 1 mM Ala-Ala-Phe-pNA as the substrate, and the purified
enzyme (20 ng), was incubated at 45 ꢀC for 30 min. The reaction was
terminated by the addition of acetic acid to a final concentration of 30%,
and the absorbance at 405 nm was measured using a Hitachi U3000
spectrophotometer (Hitachi, Tokyo, Japan). One unit of enzyme
activity was defined as the amount of enzyme that catalyzed the release
of 1 μM pNA per minute at 45 ꢀC, with pNA as the standard.
Enzyme Characterization. The optimal temperature for TPP was
determined at temperatures ranging from 15 to 65 ꢀC for 30 min in
100 mM sodium phosphate buffer (pH 7.0) containing 1 mM CoCl2 and
1 mM Ala-Ala-Phe-pNA as the substrate. The optimal pH for TPP was
determined by measuring its activity at 45 ꢀC for 30 min over a pH range
of 4.0À9.0 using the following buffers: 100 mM glycine-HCl (pH 4À6),
sodium phosphate (pH 6À8), potassium phosphate (pH 6À8), and
Tris-HCl (pH 7À9).
Purification of Proteases. R. oligosporus (107 spores) was cul-
tured in 400 mL of TPP medium containing 2% Soytone as the sole
nitrogen source at 30 ꢀC for 3 days. Cells were removed by centrifuga-
tion (8000g at 4 ꢀC for 30 min), and the supernatant was collected for
enzyme purification. All purification steps were performed using an AKTA
purifier 10 system (GE Healthcare Biosciences, Uppsala, Sweden) at 4 ꢀC.
The culture supernatant was concentrated by ultrafiltration using
an Amicon Ultra-15 10K Centrifugal Filter Device (10 kDa cutoff;
Millipore, Bedford, MA) and dialyzed overnight against 50 mM sodium
phosphate buffer (pH 7.0). The dialyzed supernatant was loaded onto a
HiTrap Q Sepharose Fast Flow column (GE Healthcare Biosciences),
previously equilibrated with 50 mM sodium phosphate buffer (equilibration
buffer; pH 7.0). TPP was eluted at a flow rate of 0.2 mL/min using the
equilibration buffer with a NaCl stepwise gradient of 0À55 mM
(10 mL), 55 mM (12 mL), 55À155 mM (10 mL), 155 mM (12 mL),
and 155 mMÀ1.0 M (5 mL). TPP activity in the eluted fractions was
measured using Ala-Ala-Phe-p-nitroanilide (Ala-Ala-Phe-pNA) as the
substrate. TPP-containing fractions were pooled and dialyzed against
50 mM sodium phosphate buffer (pH 6.5) in an Amicon Centrifugal
Filter Device.
The pooled fractions from the HiTrap Q Sepharose Fast Flow
column were further purified on a MonoQ HR 5/5 high-performance
column (GE Healthcare Biosciences) equilibrated with 50 mM sodium
phosphate buffer (pH 6.5). TPP was eluted using a NaCl gradient of
0À120 mM (5 mL), 120À270 mM (15 mL), and 270 mMÀ1.0 M
(3 mL) in 50 mM sodium phosphate buffer (pH 6.5) at a flow rate of
0.15 mL/min. The active fractions were pooled, concentrated, and
dialyzed against 50 mM sodium phosphate buffer (pH 6.5). The pooled
sample was then subjected to gel filtration through a HiLoad 16/60
Superdex 200 prep-grade column (GE Healthcare Biosciences) equili-
brated with 50 mM sodium phosphate buffer (pH 6.5). Elution of TPP
was performed using a 50 mM sodium phosphate buffer (pH 6.5) at a
flow rate of 0.4 mL/min. Fractions with the highest TPP activity were
pooled, concentrated by ultrafiltration, and then stored at À20 ꢀC.
Electrophoresis and Protein Determination. The proteins
obtained from each chromatography step were separated using 12% sodium
dodecyl sulfateÀpolyacrylamide gel electrophoresis (SDSÀPAGE) accord-
ing to standard protocols.30 After electrophoresis, the gel was stained
with 0.1% silver nitrate. Broad-range protein markers were used to estimate
protein size (6.5À200 kDa; Bio-Rad Laboratories, Richmond, CA). Protein
concentrations were quantified using a Bio-Rad Protein Assay Kit with
BSA as the standard.
Estimation of the Molecular Mass. The molecular mass of the
purified enzyme was estimated by both size-exclusion chromatography
using a Superdex 200 column and SDSÀPAGE. The purified enzyme
and a solution of standard molecular-weight markers, such as β-amylase
(200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), and
carbonic anhydrase (29 kDa), were loaded separately onto a Superdex
200 column, using 50 mM sodium phosphate (pH 6.5) as the mobile
phase at a flow rate of 0.4 mL/min. Blue Dextran (2000 kDa) was used to
determine the void volume. Protein bands on the polyacrylamide gels
were analyzed by densitometry, and protein sizes were estimated using
Kodak 1D Image Analysis Software (Windows, version 2.0.3; Eastman
Kodak, Rochester, NY).
N-Terminal Amino Acid Sequencing. The purified enzyme on
the polyacrylamide gel was electrophoretically transferred to a poly-
vinylidene fluoride membrane (PVDF, Millipore) in 3-(cyclohexylamino)-
1-propanesulfonic acid buffer at pH 11.0 and stained with Coomassie
Blue R-250. The stained protein band was excised from the membrane
To determine the effects of protease inhibitors and reducing agents
on TPP activity, the purified enzyme (20 ng) was incubated in 100 mM
sodium phosphate (pH 7.0) with each of the chemical agents at 30 ꢀC for
30 min, followed by measurement of its activity under the standard assay
conditions described above. To investigate the effects of metal ions on
TPP activity, the purified enzyme was incubated at 30 ꢀC for 30 min in
100 mM sodium phosphate buffer (pH 7.0) with 10 mM ethylenedia-
minetetraacetic acid (EDTA) or 5 mM dipicolinic acid. After the chelating
agent was removed by centrifugal ultrafiltration (Amicon Ultra-15 10K
Centrifugal Filter Device), the divalent metal salt solution was added and
incubation was continued for an additional 30 min before the enzyme
assay. TPP activity was assayed using the standard procedure in the absence
of metal ions and chelating agents. To avoid interference by the anions, all
metal ions were added as chlorides (Table 3). Substrate specificity was
examined using a range of synthetic tripeptidyl-pNAs, dipeptidyl-pNAs,
and monopeptidyl-pNAs at a final concentration of 1 mM (Table 4)
under the standard assay conditions.
TPP Activity of the Enzyme. The TPP activity was investigated
using the AAFYY synthetic pentapeptide. The peptide was dissolved in
100 mM sodium phosphate buffer (pH 7.0) containing 1 mM CoCl2 to a
final concentration of 1 mM, and purified TPP (20 ng) was added and
incubated at 45 ꢀC for 30 min. The reaction was terminated by the
addition of acetic acid to a final concentration of 30%, and the samples
were then analyzed by high-performance liquid chromatography/
tandem mass spectrometry (HPLC/MS/MS). The HPLC/MS/MS system
consisted of two PerkinElmer Series 200 LC micro pumps, a Series 200
Autosampler (PerkinElmer Co., Waltham, MA), and an AB Sciex API 2000
triple quadrupole mass spectrometer with a TurboIonSpray probe (Applied
Biosystems). The data were processed using AB Sciex software (Analyst,
version 1.3.2; Applied Biosystems).
HPLC analysis was conducted using a reversed-phase Polaris C18-A
column (2 mm inner diameter  50 mm; particle size, 3 μm; Varian,
Inc., Palo Alto, CA). The mobile phases were (A) 0.1% formic acid and
(B) acetonitrile, and the flow rate was 0.1 mL/min. The linear gradient
was initiated with 10% B and was increased to 95% B within 10 min. For
MS/MS detection, the TurboIonSpray, orifice voltages, temperature,
collision energy, and entrance potential were set to 5500 V, 60 V, 200 ꢀC,
16 eV, and À9.5 V, respectively. The collision gas (nitrogen) was
maintained at a pressure of 2.3 Â 10À5 Torr. The positive-ion mode and
multiple-reaction monitoring (MRM) mode were used to detect AAFYY
(m/z 634.3À453.2) and AAF (m/z 308.2À166.1), respectively. Total
analysis time was 10 min for each sample.
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Journal of Agricultural and Food Chemistry
ARTICLE
Figure 1. Effects of nitrogen sources on TPP production of R. oligosporus.
Cells were cultivated in TPP medium containing 2% casein (0), 2%
Soytone (9), 2% BSA (4), 2% skim milk (b), 2% beef extract (O), or
NH4Cl (2) as the sole nitrogen source at 30 ꢀC with shaking at 150 rpm
for 7 days. The data are means of three independent experiments, and the
bars indicate standard deviations.
’ RESULTS AND DISCUSSION
Production of R. oligosporus TPP. TPP production from
R. oligosporus reached a maximum of ∼1.0 unit/mL after 48 and
72 h of incubation when the cells were cultivated in medium
containing skim milk or Soytone as the sole nitrogen source,
respectively (Figure 1). When the cells were cultured in TPP
medium containing casein, BSA, or beef extract as the sole
nitrogen source, the maximum activity of TPP was ∼0.5, 0.6,
and 0.2 unit/mL, respectively. No detectable activity was found
when NH4Cl was used as the sole nitrogen source. TPP enzyme
activity was induced by organic nitrogen sources, which may
indicate that this enzyme plays a role in the provision of nutrients.
Proteolytic enzymes are secreted by a variety of fungi and likely
supply nitrogen for cell survival when inorganic nitrogen in the
environment is depleted.28,32 The extracellular carboxyl protei-
nase of R. oligosporus is also suppressed when amino acids are
used as a nitrogen source, while the enzyme activity is enhanced
when the medium contains protein as the sole nitrogen source.28
Purification of R. oligosporus TPP. The concentrated super-
natant of R. oligosporus BCRC31750 exhibiting TPP activity
(18.5 units/mg) was subjected to chromatography using a
HiTrap Q Sepharose Fast Flow column and eluted with a
stepwise NaCl gradient. The chromatographic profile is shown
in Figure 2A. The column yielded four peaks, of which that
eluting at 155 mM NaCl was active. The specific activity of the
collected active fractions was 24.2 units/mg, with a yield of
22.3%. The active fractions were fractionated on a MonoQ HR
5/5 column. All of the proteins present in fractions 25À31 were
active (Figure 2B), and the target enzyme, with specific activity of
29.5 units/mg and a yield of 7.1%, was obtained. All active
fractions were further subjected to chromatography on a HiLoad
16/60 Superdex 200 prep-grade column, which resulted in
elution of five protein peaks, among which only the third peak
showed proteolytic activity (Figure 2C). A summary of the
purification steps for TPP produced by R. oligosporus is shown
in Table 1. The enzyme was purified 15.4-fold from the
culture supernatant, with a yield of 0.9% and a specific activity
of 285.4 units/mg.
Figure 2. Elution profiles for chromatographic purification of TPP from
the culture supernatant of R. oligosporus. (A) Anion-exchange chroma-
tography on a Q Sepharose column. (B) Anion-exchange chromatog-
raphy on a MonoQ column. (C) Gel filtration chromatography on a
Superdex 200 column. The experimental conditions are described in the
Materials and Methods. (O) Absorbance at 280 nm, (b) proteolytic
activity, and (—) NaCl gradient.
respectively.7,8 The yields of the enzyme purified from Prevotella
nigrescens and Streptomyces mobaraensis were also low, ∼8.3 and
4%, respectively.15,17 In our previous work, ammonium sulfate
precipitation was used to concentrate TPP, which resulted in
almost the complete loss of TPP activity. Whether the low yield
of the R. oligosporus enzyme reflects greater initial heterogeneity
or greater instability of the enzyme remains to be determined.
Purity and Molecular Mass. The purity of the enzyme was
confirmed by SDSÀPAGE after silver staining (Figure 3). The
purified protein migrated as a single band on the denaturing gel
with an apparent molecular mass of ∼70 kDa (lane 4 of Figure 3).
The molecular mass of the native enzyme determined by gel
filtration chromatography was ∼136.5 kDa, indicating that
purified TPP consisted of two subunits with similar molecular
masses.
PTP from Porphyromonas gingivalis is a homodimeric cell
surface-associated serine protease with two 77 kDa subunits,33
while the enzyme from P. nigrescens is a 56 kDa intracellular
protease.15 TAPs from S. lividans and S. mobaraensis have
molecular masses of 55 and 53 kDa, respectively.12,17 A. fumigatus
was reported to secrete the proteases SedB (60À90 kDa), SedC
(65 kDa), and SedD (70À100 kDa), which possess TPP
Low purification yields were also observed with TPP II purified
from rat liver and from Arabidopsis thaliana, with 2 and 5% yields,
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Table 1. Purification of TPP from R. oligosporus BCRC31750
purification step
total protein (mg)
total activity (units)
specific activity (units/mg)
purification (fold)
yield (%)
supernatant
Q Sepharose
MonoQ
17.0
2.90
0.76
0.01
314.78
70.13
22.42
2.85
18.5
24.2
1
100
22.3
7.1
1.3
1.6
15.4
29.5
Superdex 200
285.4
0.9
Figure 3. SDSÀPAGE analysis of TPP purification from R. oligosporus.
After electrophoresis, the gel was stained with silver nitrate. Lane M,
molecular mass markers; lane 1, supernatant of the R. oligosporus culture
broth; lane 2, eluted fraction with TPP activity from the Q Sepharose
column; lane 3, eluted fraction with TPP activity from the MonoQ
column; lane 4, eluted fraction with TPP activity from the Superdex 200
column. The arrow indicates the position of the monomer at 70 kDa.
activity.13 The molecular mass of rat spleen TPP I was estimated
to be 47 kDa by SDSÀPAGE and 64 kDa by gel filtration
chromatography.3 TPP II from rat liver, Drosophila melanogaster,
and A. thaliana is composed of a single 135À150 kDa subunit
that forms an oligomeric complex with a native molecular mass of
g1000 kDa.5À8 Unlike the known TPPs from prokaryotic and
eukaryotic organisms, R. oligosporus TPP represents a novel
homodimeric extracellular TPP.
N-Terminal Amino Acid Sequence of TPP. The N-terminal
amino acid sequence of purified TPP was identified as “S-K-I-Q-
V-K-Y-A-T-T-P-K-M-S-T”. Protein homology searches revealed
no significant similarity with published TPP amino acid sequences
in the National Center for Biotechnology Information (NCBI)
GenBank database. The highest similarity was observed to Polys-
phondylium pallidum aminopeptidase N (GenBank EFA77142.1)
spanning amino acid residues 297À307, indicating that the purified
enzyme possesses aminopeptidase activity. For protein identifi-
cation, the N-terminal sequence was also searched for within the
genome database for Rhizopus oryzae RA 99-880 (http://www.
broadinstitute.org/annotation/genome/rhizopus_oryzae/Blast.
html); however, no detectable similarity was observed.
Figure 4. Effects of the (A) temperature and (B) pH on the activity of
purified TPP. (A) Temperature dependence was determined in 100 mM
sodium phosphate buffer (pH 7.0) at various temperatures for 30 min.
(B) The effect of pH on TPP activity was determined at 45 ꢀC for 30 min
in a mixture containing purified TPP (20 ng) in 100 mM glycine-HCl
buffer at pH 4À6 (0), 100 mM sodium phosphate buffer at pH 6À8
(b), 100 mM potassium phosphate buffer at pH 6À8 (O), and 100 mM
Tris-HCl buffer at pH 7À9 (9). The enzyme activity was measured as
described in the Materials and Methods. The data are means of three
independent experiments, and the highest activity was defined as 100%.
The bars indicate standard deviations.
Ala-Ala-Phe-pNA as the substrate at 45 ꢀC. TPP was found to be
active between pH 6.0 and 8.0, with maximum activity at pH 7.0
in sodium phosphate buffer (Figure 4B). The relative activities
at pH 6.0 and 8.0 in sodium phosphate buffer were about 63
and 39%, respectively, of that at pH 7.0. Only about 10% of
the optimum activity was observed at pH 5.5 and 8.5. These
results are similar to those for PTP from P. gingivalis 16 and TAP
from S. mobaraensis,17 which had an optimum pH of 7.0À7.5.
An optimum alkaline pH (7.5À8.5) was reported for TAP from
S. lividans,12 while an optimum acidic pH (5.0À6.0) was observed
for SedB, SedC, and SedD from A. fumigatus.13 Mammalian TPP
II has an optimum pH of 7.5,6,8 while an acidic environment is
preferable for TPP I.3,34
Effects of the Temperature and pH on TPP Activity. The
optimum temperature for TPP activity was determined at pH
7.0 using the substrate Ala-Ala-Phe-pNA; TPP was found to be
active between 35 and 55 ꢀC, with maximum activity at 45 ꢀC
(Figure 4A). The effect of pH on TPP activity was studied using
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Journal of Agricultural and Food Chemistry
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Table 2. Effects of Protease Inhibitors and Reducing Agents
on TPP Activity
Table 3. Effects of Chelating Agents and Various Metal Ions
on TPP Activitya
concentration
(mM)
relative
activity (%)a
concentration
(mM)
relative
activity (%)b
inhibitor
compound
none
100 ( 2.5
97.1 ( 2.3
95.4 ( 2.2
65.9 ( 1.3
39.1 ( 1.1
43.2 ( 0.9
40.9 ( 1.1
70.6 ( 1.4
97.0 ( 2.0
89.0 ( 1.7
102.0 ( 2.3
103.9 ( 2.6
94.0 ( 2.2
95.2 ( 2.0
none
0
10
100.0 ( 3.3
10.3 ( 1.0
19.1 ( 0.9
66.1 ( 2.5
0
0.1
1
EDTA
pepstatin A
EDTA + MgCl2
10 + 1
10 + 1
10 + 1
10 + 1
10 + 1
10 + 1
10 + 1
5
0.1
1
EDTA + CaCl2
phenylmethylsulfonyl fluoride
EDTA + ZnCl2
0.1
1
EDTA + NiCl2
52.9 ( 1.9
0
Ala-Ala-Phe-
EDTA + CuCl2
chloromethylketone
Pefabloc SC
1
EDTA + MnCl2
233.0 ( 4.2
269.3 ( 4.2
3.1 ( 0.2
26.2 ( 1.1
77.9 ( 3.5
7.2 ( 0.2
101.0 ( 3.8
0
0.1
1
EDTA + CoCl2
E-64
dipicolinic acid
N-ethylmaleimide
iodoacetic acid
β-mercaptoethanol
dithiothreitol
1
dipicolinic acid + MgCl2
dipicolinic acid + CaCl2
dipicolinic acid + ZnCl2
dipicolinic acid + NiCl2
dipicolinic acid + CuCl2
dipicolinic acid + MnCl2
dipicolinic acid + CoCl2
5 + 1
5 + 1
5 + 1
5 + 1
5 + 1
5 + 1
5 + 1
1
1
1
a TPP activity assayed in the absence of inhibitors was defined as 100%.
288.7 ( 6.0
312.3 ( 5.8
Effects of Enzyme Inhibitors and Metal Ions on TPP
Activity. To determine the nature of the peptidase, the effects
of various inhibitors on enzyme activity were examined (Table 2).
TPP was inhibited by the serine protease inhibitors PMSF
(1 mM) and Pefabloc SC (1 mM), with ∼60 and 30% inhibition,
respectively. A tripeptidyl chloromethyl ketone analogue, Ala-
Ala-Phe-chloromethyl ketone, was also inhibitory. The aspartyl
protease inhibitor pepstatin A had no effect on enzyme activity.
E-64 (1 mM) inhibited activity slightly, while other cysteine
protease inhibitors (N-ethylmaleimide and iodoacetic acid) had
no effect on enzyme activity. The cysteine protease activator
agents dithiothreitol (1 mM) and β-mercaptoethanol (1 mM)
were also ineffective.
These inhibition results are similar to those for TPP from
P. nigrescens. Enzyme activity was inhibited by diisopropylfluor-
ophosphate (DIFP) by 60% at 1 mM and 70% at 10 mM and was
reduced to about 40% in the presence of Pefabloc SC (1 mM).15
PTP from P. gingivalis was strongly inhibited by the serine
protease inhibitors DIFP and Pefabloc SC, while PMSF had no
effect.16 Low effectiveness of PMSF and differing degrees of
inhibition have also been observed for dipeptidyl peptidases II
from various organisms and tissues.35 Variations in inhibition
profiles for different serine protease families remain poorly
understood. Our results indicate that purified TPP was inhibited,
if not strongly, by serine protease inhibitors and may be classified
as a serine protease.
The effects of chelating agents and various metal ions on the
activity of TPP were examined at pH 7.0 and 45 ꢀC with Ala-Ala-
Phe-pNA (Table 3). TPP was strongly inhibited by chelating
agents, such as 10 mM EDTA (∼90%) and 5 mM dipicolinic acid
(∼97%), suggesting that it is a metalloenzyme. After EDTA was
removed by ultrafiltration, the enzyme activity could be strongly
reactivated and enhanced to ∼233 and 269.3% of the original
activity by 1 mM Mn2+ or Co2+, respectively. The addition of
1 mM Mg2+, Ni2+, or Ca2+ caused slight recovery of TPP activity
to about 19.1, 52.9, and 66.1%, respectively, whereas the addition
of 1 mM Zn2+ or Cu2+ inhibited the activity. Similarly, upon
treatment of the enzyme with dipicolinic acid, ∼288.7, 312.3, and
101% of the original activity could be reactivated by 1 mM Mn2+,
a The purified enzyme was incubated at 30 ꢀC in 100 mM sodium
phosphate buffer (pH 7.0) with 10 mM EDTA or 5 mM dipicolinic acid
for 30 min. After the chelating agent was removed by centrifugal
ultrafiltration (Amicon Ultra-15 10K Centrifugal Filter Device), 1 mM
of the indicated divalent metal salt solution was added and incubation
was continued for an additional 30 min before enzyme assay. b TPP
activity assayed in the absence of metal ions and inhibitors was regarded
as 100%.
Co2+, or Ni2+, respectively. The addition of 1 mM Zn2+, Mg2+, or
Ca2+ increased TPP activity to about 7.2, 26.2, and 77.9%,
respectively, whereas the addition of 1 mM Cu2+ inhibited
residual enzyme activity. When TPP was not treated with
chelating agents, 1 mM Co2+ or Mn2+ stimulated activity by
∼294.5 and 162.8%, respectively. Zn2+ reduced TPP activity by
up to 50%. Moreover, 0.1 mM CuCl2 inhibited 88.2% of the
original activity and completely inhibited activity at 1 mM (data
not shown).
On the basis of the above results, the peptidase purified from
R. oligosporus was identified as a metallo-dependent TPP, which
has not previously been reported in eukaryotes. The enzyme
activity of TAP from S. mobaraensis was found to be enhanced by
Ca2+ and reduced by up to half in the presence of chelating
agents.17 However, in contrast to TPP, TAP from S. mobaraensis
is a proline-specific tripeptidyl and tetrapeptidyl peptidase.14,17
Dipeptidyl peptidase III, belonging to the serine protease family,
is also a metalloprotease harboring a conserved HEXXXH motif
as a Zn-binding site, which provides a rationale for substrate
recognition.36 The role of metal ions in proteolytic activity of
TPP from R. oligosporus remains to be determined, including the
exact function of metal ions in catalysis and the enzyme
proteolytic mechanism.
Proteolytic Activity of TPP. Specificities of purified TPP for
various chromogenic substrates are summarized in Table 4. The
enzyme most actively cleaved Ala-Ala-Phe-pNA, and hydrolysis
of Ala-Ala-Ala-pNA was also efficient (about 60% of the relative
activity toward Ala-Ala-Phe-pNA). About 10% relative activity was
detected for Phe-Pro-Ala-pNA and Pro-Leu-Gly-pNA. No activity
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toward Ala-Phe-Pro-pNA was observed in the purified enzyme.
Hydrolysis of dipeptidyl-pNA, monopeptidyl-pNA, and N-term-
inally blocked tripeptidyl-pNA substrates was also not detected.
These results indicated that purified TPP had no endoproteolytic
activity and catalyzed most tripeptidyl-pNA substrates, except
when proline occupied the P1 position. Moreover, these data
also indicated that purified TPP was free of any contamination
with aminopeptidase, dipeptidyl peptidase, and endopeptidase
activities.
Substrate specificity of R. oligosporus TPP was similar to that
reported for mammalian TPP I and TPP II, which show the
highest activity toward Ala-Ala-Phe-pNA.3,8 SedB, SedC, and
SedD from A. fumigatus also efficiently catalyze Phe-Pro-Ala-
pNA and Ala-Ala-Phe-pNA.13 The TPPs described above show
no catalytic activity toward the P1-Pro tripeptide substrates. In
contrast, PTPs isolated from S. mobaraensis,14,17 P. nigrescens,15
and P. gingivalis16 specifically hydrolyze tetra-, tri-, and/or
dipeptide substrates with a P1-proline residue.
To further demonstrate the proteolytic activity of TPP, the
synthetic peptide AAFYY was used as the substrate and the
resulting reaction products were analyzed by HPLC/MS/MS.
Using the full-scan mode for MS analysis, the most abundant
[M + H]+ ions for AAFYY and AAF standards were m/z 634.3
and 308.2, respectively; these were selected as the precursor ions
for MS/MS analysis. Figure 5 shows the product ion spectra for
AAFYY and AAF obtained by infusion in the positive-ion mode.
The primary product ions for AAFYY were m/z 290.2, 345.2, and
453.2 (Figure 5A). The primary product ions for AAF were m/z
143.1, 166.1, and 237.1 (Figure 5B). The MS/MS transitions
m/z 634.3À453.2 and m/z 308.2À166.1 were then selected for
AAFYY and AAF analysis, respectively, using MS/MS in the
MRM mode. To determine AAFYY and AAF using HPLC/MS/
MS analysis, the standard peptides (10 μM each) were separated
by a Polaris C18-A column and subjected to MS/MS. AAFYY
and AAF were eluted at retention times of 6.65 and 5.60 min,
respectively, in the MRM chromatograms (panels C and D of
Figure 5). When purified TPP was incubated with the peptide
Table 4. Substrate Specificity of Purified TPP
specific activity
relative
activity (%)a
substrate (1 mM)
Ala-Ala-Phe-pNA
(units/mg)
281.9 ( 8.1
100
60.2
0
Ala-Ala-Ala-pNA
Ala-Phe-Pro-pNA
Phe-Pro-Ala-pNA
Pro-Leu-Gly-pNA
N-succinyl-Ala-Ala-Phe-pNA
N-succinyl-Ala-Ala-Ala-pNA
Gly-Pro-pNA
169.8 ( 6.1
0b
31.6 ( 3.8
11.2
10.1
0
28.7 ( 2.2
0
0
0
0
0
0
0
0
0
Gly-Arg-pNA
0
Ala-pNA
0
Phe-pNA
0
Pro-pNA
0
a The rate of hydrolysis is expressed as a percentage of the activity
compared to that obtained using Ala-Ala-Phe-pNA as the substrate.
Values are means of three independent experiments, with a maximum
sample mean deviation of (3%. b No detectable activity observed under
the assay conditions.
Figure 5. HPLC/MS/MS analysis of the proteolytic activity of TPP. Positive electron spray ionization product ion mass spectra of (A) AAFYY and (B)
AAF. The MS/MS transitions of m/z 634.3À453.2 and m/z 308.2À166.1 were selected for detection of AAFYY and AAF, respectively. Mass
chromatograms for (C) AAFYY and (D) AAF. Standard solutions (10 μM) of AAFYY and AAF in 100 mM sodium phosphate buffer (pH 7.0) were
separated using a Polaris C18-A column and detected under MRM mode. (E) Mass chromatogram for AAFYY cleaved by purified TPP. The catalytic
conditions are described in the Materials and Methods. RA = relative abundance.
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AAFYY, a mass peak consistent with the degradation product
AAF was detected, confirming the TPP activity of the enzyme
(Figure 5E).
(11) Hilbi, H.; Puro, R. J.; Zychlinsky, A. Tripeptidyl peptidase II
promotes maturation of caspase-1 in Shigella flexneri-induced macro-
phage apoptosis. Infect. Immun. 2000, 68, 5502–5508.
(12) Krieger, T. J.; Bartfeld, D.; Jenish, D. L.; Hadary, D. Purification
and characterization of a novel tripeptidyl aminopeptidase from Strepto-
myces lividans 66. FEBS Lett. 1994, 352, 385–388.
’ AUTHOR INFORMATION
(13) Reichard, U.; Lechenne, B.; Asif, A. R.; Streit, F.; Grouzmann,
E.; Jousson, O.; Monod, M. Sedolisins, a new class of secreted proteases
from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase
activity at acidic pHs. Appl. Environ. Microbiol. 2006, 72, 1739–1748.
(14) Umezawa, Y.; Yokoyama, K.; Kikuchi, Y.; Date, M.; Ito, K.;
Yoshimoto, T.; Matsui, H. Novel prolyl tri/tetra-peptidyl aminopepti-
dase from Streptomyces mobaraensis: Substrate specificity and enzyme
gene cloning. J. Biochem. 2004, 136, 293–300.
Corresponding Author
*Telephone: +886-4-2631-8652, ext. 5611. Fax: +886-4-2652-
5386. E-mail: chkao@sunrise.hk.edu.tw.
Author Contributions
†These authors contributed equally to this work.
Funding Sources
(15) Fujimura, S.; Ueda, O.; Shibata, Y.; Hirai, K. Isolation and
properties of a tripeptidyl peptidase from a periodontal pathogen
Prevotella nigrescens. FEMS Microbiol. Lett. 2003, 219, 305–309.
(16) Banbula, A.; Mak, P.; Bugno, M.; Silberring, J.; Dubin, A.;
Nelson, D.; Travis, J.; Potempa, J. Prolyl tripeptidyl peptidase from
Porphyromonas gingivalis. A novel enzyme with possible pathological
implications for the development of periodontitis. J. Biol. Chem. 1999,
274, 9246–9252.
(17) Zotzel, J.; Pasternack, R.; Pelzer, C.; Ziegert, D.; Mainusch, M.;
Fuchsbauer, H. L. Activated transglutaminase from Streptomyces mobar-
aensis is processed by a tripeptidyl aminopeptidase in the final step. Eur. J.
Biochem. 2003, 270, 4149–4155.
(18) Oda, H.; Saiki, K.; Tonosaki, M.; Yajima, A.; Konishi, K.
Participation of the secreted dipeptidyl and tripeptidyl aminopeptidases
in asaccharolytic growth of Porphyromonas gingivalis. J. Periodontal Res.
2009, 44, 362–367.
(19) Takaya, N.; Yamazaki, D.; Horiuchi, H.; Ohta, A.; Takagi, M.
Intracellular chitinase gene from Rhizopus oligosporus: Molecular cloning
and characterization. Microbiology 1998, 144, 2647–2654.
(20) Casey, A.; Walsh, G. Identification and characterization of a
phytase of potential commercial interest. J. Biotechnol. 2004, 110,
313–322.
This work was supported by Grants 98-2313-B-241-001 and 98-
2313-B-241-006-MY3 from the National Science Council of
Taiwan.
’ ABBREVIATIONS USED
AAF, Ala-Ala-Phe; AAFYY, Ala-Ala-Phe-Tyr-Tyr; BSA, bovine
serum albumin; DIFP, diisopropylfluorophosphate; DPY, dex-
trin, polypeptone, and yeast extract; EDTA, ethylenediaminete-
traacetic acid; HPLC/MS/MS, high-performance liquid chroma-
tography/tandem mass spectrometry; MRM, multiple-reaction
monitoring; PMSF, phenylmethylsulfonyl fluoride; pNA, p-nitroani-
lide; PTP, prolyl tripeptidyl peptidase; PVDF, polyvinylidene
difluoride; SDSÀPAGE, sodium dodecyl sulfateÀpolyacrylamide
gel electrophoresis; TAP, tripeptidyl aminopeptidase; TPP, tripeptidyl
peptidase
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