Characterisation of the aminopeptidase from non-germinated winter rape (Brassica napus L.) seeds

Article abstract of DOI:10.1016/j.foodchem.2016.03.097

Rapeseed plays a crucial role in food and fuel industry. Since aminopeptidases take part in many physiological processes in all organisms, it is important to learn their role and characteristics in economically relevant plants. Extracts of non-germinated winter rape seeds were screened for aminopeptidase activity. Substrate specificity, the influence of pH and temperature, as well as effect of protease inhibitors and chosen metal ions on the aminopeptidase activity were determined. The approximate molecular weight estimated by NATIVE-PAGE and SDS-PAGE electrophoresis was ～60 kDa. The partially purified enzyme as well as the aminopeptidases present in crude extract cleaved preferentially Phe-pNA. The activity profiles toward several substrates were also determined. Maximum activity was observed at pH 6.5 and temperature of 40 °C for Phe-pNA as a substrate. Two visible picks in the pH profile toward Phe-pNA, together with other results (IEF) suggest the presence of more than one aminopeptidase, having similar molecular mass. Much lower activity and broad pH profiles were observed for Leu- and Ala-pNA as substrates.

Full text of DOI:10.1016/j.foodchem.2016.03.097

Food Chemistry 207 (2016) 180–186
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Characterisation of the aminopeptidase from non-germinated winter
rape (Brassica napus L.) seeds
⇑
Joanna Kania, Danuta M. Gillner
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, 44-100 Gliwice, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Rapeseed plays a crucial role in food and fuel industry. Since aminopeptidases take part in many physi-
ological processes in all organisms, it is important to learn their role and characteristics in economically
relevant plants. Extracts of non-germinated winter rape seeds were screened for aminopeptidase activity.
Substrate specificity, the influence of pH and temperature, as well as effect of protease inhibitors and cho-
sen metal ions on the aminopeptidase activity were determined. The approximate molecular weight esti-
mated by NATIVE-PAGE and SDS-PAGE electrophoresis was ꢀ60 kDa. The partially purified enzyme as
well as the aminopeptidases present in crude extract cleaved preferentially Phe-pNA. The activity profiles
toward several substrates were also determined. Maximum activity was observed at pH 6.5 and temper-
ature of 40 °C for Phe-pNA as a substrate. Two visible picks in the pH profile toward Phe-pNA, together
with other results (IEF) suggest the presence of more than one aminopeptidase, having similar molecular
mass. Much lower activity and broad pH profiles were observed for Leu- and Ala-pNA as substrates.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 7 September 2015
Received in revised form 29 February 2016
Accepted 28 March 2016
Available online 30 March 2016
Keywords:
Aminopeptidase
Non-germinated rape seeds
Characterisation
Substrate specificity
Inhibition
1. Introduction
most preferred substrate). The enzyme was also active in hydroly-
sis of 4-nitroanilides of other L-amino acids such as Ala, Val, Ile,
Brassicaceae is an economically important family of plants con-
taining rapeseed, cabbage, broccoli, brussels, radish and mustard.
Among them rapeseed plays a crucial role, because of its applica-
tion in food and also in fuel industry. According to the USDA report
(January 2015) (United States Department of Agriculture, 2015),
rapeseed is the second largest oilseed crop worldwide (production
of 71.94 mln MT) and the third largest source of vegetable oil.
Additionally, the post-extraction protein meal is widely used as a
source of proteins for animal feed (production of 40.02 mln MT).
Plant aminopeptidases take part in many crucial physiological
processes such as protein turnover, maturation and degradation.
It has been proven that they are involved in processes of amino
acids turnover, germination, aging, plant defense, stress response
and meiosis (Walling, 2006).
Several information concerning aminopeptidases isolated from
important oilseed crops have been published. Aminopeptidase iso-
lated from sunflower seeds (Helianthus annus L.) (Tishinov,
Stambolieva, Petrova, Galunsky, & Nedkov, 2009) was a 80 kDa,
monomeric enzyme, with broad substrate specificity, and prefer-
ence for hydrophobic, bulky side chains (e.g. phenylalanine – the
Leu. It is interesting that Pro-pNA was also cleaved by that enzyme.
Substrates with positively charged side chains of amino acids were
not hydrolysed. The aminopeptidase showed optimum activity
within the pH range of 7.5–8.0 and at the temperature of
45–50 °C. The other aminopeptidase, isolated from expanded
soybean cotyledons, was a 85 kDa monomer, with high affinity
for hydrophobic terminal amino acids. Increased activity was
observed during seedlings growth (Couton, Sarath, & Wagner,
1991). Peanut cotyledons were also screened for aminopeptidase
activity. Five aminopeptidases were identified (Isola & Franzoni,
1996). Among them, one was an iminopeptidase with only Pro-
pNA activity, while other revealed broad activities toward many
amino acids p-nitroanilides. High aminopeptidase activity was
observed in resting seeds, with decreasing tendency after imbibi-
tion. The molecular mass of the aminopeptidase active toward
Leu-pNA (APa) was in the range of 55–60 kDa. Leucine aminopep-
tidase was also identified in cotyledons of germinating peanuts
(Basha & Cherry, 1978).
Several plants of Brassicaceae family (cabbage, brussels, kohlrabi,
broccoli, cauliflower and chinese cabbage) were screened for
aminopeptidase and iminopeptidase activities (Marinova
&
Tchorbanov, 2008; Marinova et al., 2008). The molecular mass of
studied enzymes was similar for all studied plants: aminopepti-
dases ꢀ60 3 kDa and iminopeptidases 200 3 kDa. The pH
⇑
Corresponding author at: Department of Organic Chemistry, Bioorganic Chem-
istry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, ul.
Krzywoustego 4, 44-100 Gliwice, Poland.
E-mail address: Danuta.Gillner@polsl.pl (D.M. Gillner).
http://dx.doi.org/10.1016/j.foodchem.2016.03.097
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

J. Kania, D.M. Gillner / Food Chemistry 207 (2016) 180–186
181
optimum of 7.2–7.5 for aminopeptidases and of 8.0–8.5 for
2.2. Equipment
iminopeptidases was determined. Aminopeptidases predominantly
hydrolyzed phenylalanine- and leucine-4-nitroanilides, with little
or no activity toward alanine-4-nitroanilide. Proline iminopepti-
dase hydrolysed only proline-4-nitroanilide. Both peptidases were
successfully applied in the production of soy protein hydrolysates,
and high degree of hydrolysis (36–38%) was obtained (Marinova,
Thi Kim Cun, Tchorbanov, 2008).
Spectrophotometer UV/VIS Jasco V-650; centrifuge 5804R
Eppendorf, Mini-PROTEANÒ Tetra Cell vertical electrophoresis sys-
tem Bio Rad, BioLogic LP and BioFrac fraction collector from BIO-
RAD, vortex PV-1 from Grant-bio, homogenizer ErgoMix Bosch.
2.3. Synthesis of chosen amino acids p-nitroanilides
There are only few information about rapeseed (Brassica napus
L.) aminopeptidases in the literature. Alanine specific aminopepti-
Boc-protected amino acids (glycine,
L-phenylalanine,
L-
dase, with the highest activity toward L-alanine-4-(phenylazo)-
phenylamide, was isolated from germinated rapeseed (Barth &
Hermann, 1974; Hermann, Hermann, Neubert, Huebner, & Barth,
methionine) (4 mmol) were coupled with p-nitroaniline using
phosphoryl chloride in pyridine, according to the procedure
described in the literature (Rijkers et al. 1995). N-Boc amides were
deprotected with HCl in EtOAc, at room temperature. Obtained
solid was recrystallized from 2-propanol. Pure compounds (based
on NMR spectroscopy and melting points) were obtained with
1979).
L-leucine, glycine- and L-lysine derivatives were also hydro-
lyzed. The molecular weight was estimated for 79 kDa and the pH
optimum for 8.0–8.5. Addition of metal ions such as Hg2+, Zn2+
,
Cu2+,Co2+, Mn2+ and Mg2+ (Hermann & Barth, 1976) decreased
the activity of the enzyme.
the yields of 64% (0.843 g)
12% (0.130 g) -Met-pNA respectively.
-Phenylalanine-p-nitroanilide: 1H NMR (400 MHz, CDCl3): d
L-Phe-pNA, 58% (0.453 g) Gly-pNA, and
L
Herein, we report for the first time characterisation of the
enzymes with aminopeptidase activity, present in extract of
non-germinated winter rape seeds cv. Bellevue, as well as in
partially purified sample. Substrate specificity, pH and tempera-
ture optimum, as well as thermal stability are widely discussed.
The activities of aminopeptidases treated with several metal
salts and protease inhibitors are compared. Isoelectric focusing
(IEF) study as well as estimation of molecular mass are also
presented.
L
9.88 (s, 1H, NHCO), 8.19 (d, 2H, ArH, ortho to NO2), 7.74 (d, 2H,
ArH), 7.27 (m, 5H, Ph), 3.77 (dd, 1H, CHCO), 3.35 (dd, 1H, CH2Ph),
2.81 (dd 1H, CH2Ph); 13C NMR (100 MHz, CDCl3): d 172.9 (CO),
143.5 (CNO2 (ipso)), 143.4 (CNHCO (ipso)), 137.1, 129.2, 128.9,
127.18 (Ph), 125.1 (ArC (ortho to NO2)), 118.8 (ArC), 56.7 (CHCO),
40.4 (CH2Ph).
Glycine-p-nitroanilide: 1H NMR (400 MHz, CDCl3): d 11.45 (s,
1H, NHCO), 8.17 (d, 2H, ArH, ortho to NO2), 7.94 (d, 2H, ArH),
3.91 (s, 2H, CH2CO), 13C NMR (100 MHz, CDCl3): d 164.9 (CO),
143.6 (CNO2 (ipso)), 142.2 (CNHCO (ipso)), 124.0 (ArC (ortho to
NO2)), 118.4 (ArC), 41.0 (CHCO).
2. Experimental
L
-Methionine-p-nitroanilide: 1H NMR (400 MHz, CDCl3): d 10.08
2.1. Materials and reagents
(s, 1H, NHCO), 8.21 (d, 2H, ArH, ortho to NO2), 7.77 (d, 2H, ArH),
3.70 (dd, 1H, CHCO), 2.69 (m, 2H, CH2S), 2.34 (m 1H, CH2CH),
2.14 (s, 3H CH3S), 1.86 (m 1H, CH2CH), 13C NMR (100 MHz, CDCl3):
d 173.2 (CO), 143.5 (CNO2 (ipso)), 143.4 (CNHCO (ipso)), 125.1 (ArC
(ortho to NO2)), 118.8 (ArC), 54.5 (CHCO), 33.4 (CHCH2), 30.7
(CH2S), 15.3 (CH3S).
L
-leucine p-nitroanilide (Leu-pNA),
L-alanine p-nitroanilide
(Ala-pNA), bestatin, 1,10-phenanthroline, ethylenediaminete-
traacetic acid tetrasodium salt dihydrate (EDTA), ethylene
glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA),
phenylmethanesulfonyl fluoride (PMSF) L-3-carboxy-trans-2,3-
epoxy-propionyl-L-leucine-4-guanidinobutyl-amide (E-64), Sepha-
dex G-25, dithiothreitol (DTT), DEAE-Sepharose, Sephacryl HR 300
were purchased from Sigma-Aldrich (St Louis, MO, USA). Bovine
serum albumin fraction V and glycine were obtained from Merck
(Darmstadt, Germany). Tris(hydroxymethyl)aminomethane (Tris),
2-mercaptoethanol (BME), polyvinylpyrrolidone (PVP) were pur-
chased from Acros Organics (Geel, Belgium). Dimethyl sulfoxide
(DMSO), ammonium sulfate, sodium chloride, and n-hexane were
from POCH (Gliwice, Poland). Chemicals for electrophoresis,
protein molecular weight markers and ready IEF gel, pH 5–8 were
purchased from Bio Rad (Hercules, CA, USA). IEF standard was from
Sigma Aldrich (St. Louis, MO, USA), Coomassie R-350 was pur-
chased from GE Healthcare (Upsala, Sweden) and trichloroacetic
acid (TCA) from Carl Roth (Karlsruhe, Germany). Glycine
L
-proline p-nitroanilide was obtained using procedure described
by Pansare and Kirby (Pansare & Kirby 2009). Briefly, Boc- -proline
L
(8 mmol) was coupled with p-nitroaniline using isobutyl chlorofor-
mate and 4-methylmorpholine in THF. The obtained N-Boc-
protected amide was deprotected with trifluoroacetic acid (TFA)
in CH2Cl2, at room temperature. The resulting solid was recrystal-
lized (EtOAc/hexane) to give 0.380 g (21% yield) of
L-Pro-pNA.
L
-Proline-p-nitroanilide: 1H NMR (400 MHz, CDCl3): d 10.18 (s,
1H, NHCO), 8.20 (d, 2H, ArH, ortho to NO2), 7.78 (d, 2H, ArH), 3.90
(dd, 1H, CHCO), 3.13 (dt, 1H, CH2N), 3.00 (dt 1H, CH2N), 2.24 (m,
1H, CH2CH), 2.04 (m, 2H, NH, CH2CH), 1.77 (m, 2H, CH2CH2NH).13
C
NMR (100 MHz, CDCl3): d 174.1 (CO), 143.6 (CNO2 (ipso)), 143.3
(CNHCO (ipso)), 125.0 (ArC (ortho to NO2)), 118.7 (ArC), 61.0
(CHCO), 47.4 (CH2NH), 30.7 (CH2CH), 26.3 (CH2CH2NH).
p–nitroanilide (Gly-pNA),
L
-methionine p-nitroanilide (Met-pNA),
L
-phenylalanine p–nitroanilide (Phe-pNA),
L
-proline p-nitroanilide
(Pro-pNA) were synthesized according to the known procedures
(Pansare & Kirby 2009; Rijkers, Adams, Hemker, & Tesser 1995).
2.4. Crude extract preparation and purification
Gly-L-Phe p-nitroanilide (Gly-Phe-pNA), L-Phe-Gly p-nitroanilide
(Phe-Gly-pNA), Gly-Phe-Gly-Phe p-nitroanilide (Gly-Phe-Gly-Phe-
pNA) were kindly provided by Dr. Maciej Makowski from the
University of Opole. Inorganic salts AlCl3, BaCl2, CaCl2, CdCl2, CuCl2,
FeSO4, MgCl2, MnCl2, NaCl, NiCl2, ZnCl2 were purchased from
Sigma-Aldrich (St. Louis, MO, USA) or POCH (Gliwice, Poland) in
highest available purity. All other chemicals were of high analytical
grade and used as purchased. Winter rapeseed cv. Bellevue was
obtained from Bayer CropScience, Poland.
200 g of rape seeds (winter rapeseed cv. Bellevue) were soaked
in 750 cm3 of 50 mM Tris-HCl buffer (pH 8.0), containing 50 mM
NaCl and 1% PVP, for 24 h. The resulting mixture was homogenized
(homogenizer ErgoMix Bosch, 10 min) and the obtained slurry was
centrifuged at 4 °C, at 10 000 rpm (12 208 rcf), for 30 min. In order
to separate the oil fraction, the supernatant was carefully decanted
and extracted several times with cold n-hexane. Organic fraction
was rejected. Aqueous fraction was saturated with ammonium

182
J. Kania, D.M. Gillner / Food Chemistry 207 (2016) 180–186
sulfate up to 35%. The mixture was centrifuged (4 °C, 12 208 rcf,
30 min), and the precipitate was rejected (inactive toward Leu-
pNA). Additional portion of ammonium sulfate was added to the
supernatant, to give 65% saturation. The mixture was centrifuged
again (under the same conditions), the supernatant was rejected,
while the active precipitate was resuspended in 150 cm3 of
50 mM Tris-HCl buffer (pH 8.0), containing 50 mM NaCl and
10 mM BME. The obtained crude extract was desalted on the
Sephadex G-25 column (2.5 ꢁ 30 cm; flow rate 2.0 cm3/min).
Active fractions were pooled, concentrated and loaded onto DEAE
Sepharose column (1.5 ꢁ 20 cm; flow rate 1.0 cm3/min; 50 mM
Tris-HCl buffer; pH 8.0; NaCl gradient from 50 mM to 250 mM;
1 cm3 fractions were collected). The aminopeptidase active frac-
tions were eluted at NaCl concentration of ꢀ130 mM. They were
collected, concentrated and loaded on the gel filtration Sephacryl
HR300 column (1.5 ꢁ 50 cm; 50 mM Tris-HCl buffer; pH 8.0, con-
taining 50 mM NaCl and 10 mM BME; flow rate 0.2 cm3/min).
1 cm3 fractions were collected and the hydrolytic activity toward
Leu-pNA was determined. The active fractions were pooled, con-
centrated, desalted (buffer exchange in order to remove NaCl)
Concentrated protein eluates were subjected to SDS-PAGE elec-
trophoresis. The molecular weight was finally estimated in 12%
polyacrylamide gel using Precision Plus Protein Dual Xtra Stan-
dards (Bio-Rad) as a molecular weight standards (250-10 kDa).
Proteins in the gel were stained with Coomassie Brilliant Blue.
2.7. Isoelectric focusing (IEF) procedure
Purified protein sample was diluted with 1 part of 50% glicerol
and applied on the ready IEF gel pH 5–8. IEF standard was also
applied. NaOH solution (50 mM) was applied as a cathode buffer,
and 7 mM H3PO4 as an anode buffer. IEF running conditions: 100
V for 1 h, 250 V for 1 h and 500 V for 30 min. IEF gel staining
was performed using the following stock solutions: A- 0.2% w/v
CuSO4 + 20% acetic acid; B – 60% v/v methanol; C – 0.4% w/v solu-
tion of Coomassie R-350 in 60% methanol. Conditions of IEF gel
staining procedure: fixing (10 min in 20% w/v TCA); washing
(2 min with the mixture of A + B 1:1 v/v); staining (15 min, 50 °C,
A + C 1:1 v/v); destaining (A + B 1:1); impregnating (5% glicerol
and 10% acetic acid).
and loaded onto ion exchange column Macro Prep High
Q
(1.5 ꢁ 20 cm; 50 mM Tris-HCl buffer pH 8.0, NaCl gradient from
0 mM to 300 mM; flow rate 1 cm3/min; 1 cm3 fractions). The
aminopeptidase active fractions were eluted at NaCl concentration
of ꢀ180 mM. Active fractions (toward Leu-pNA as a substrate)
were collected and concentrated. Protein concentration was deter-
mined using Bradford assay (Bradford 1976).
2.8. Determination of Km and Vmax values
Kinetic parameters were determined using either Leu-pNA or
Phe-pNA as substrates. Eight concentrations of each substrate were
used (0.026–1.30 mM, of Leu-pNA, and 0.026–0.780 mM of Phe-
pNA respectively). Kinetic parameters were calculated using the
software Hyper32.
2.5. Aminopeptidase activity assay
2.9. Thermal stability and temperature optimum
Partially purified extracts were assayed for catalytic activity
using L-leucine p-nitroanilide as a substrate, according to the
Thermal stability of the studied enzyme was measured in
50 mM PBS buffer, at several temperatures (25, 30, 35, 37, 40, 45,
50, 55 °C). Enzyme was incubated at each temperature for 15, 30,
45, and 60 min. The aminopeptidase residual activity was deter-
mined with Phe-pNA as a substrate. Temperature optimum was
determined in 50 mM PBS buffer (pH 7.0), after 10 min of incuba-
tion at chosen temperature (from the range of 25–65 °C), using
Phe-pNA as a substrate.
slightly modified method described by Appel et al. (Appel, Van
Oudheusden, & Bergmeyer, 1974). In this assay, the hydrolysis of
Leu-pNA was measured spectrophotometrically (37 °C, 10 min),
by monitoring the formation of p-nitroaniline at 405 nm. Briefly,
enzyme extract (25
950 l of 50 mM potassium phosphate buffer (PBS) (pH 7.0),
25 l of 52 mM Leu-pNA in DMSO (final concentration of substrate
ll) was added to the mixture containing
l
l
in the sample – 1.3 mM). The measured value was converted to
enzymatic units, where one unit was defined as the conversion
of 1 lmol of substrate per 1 min, at 37 °C. The hydrolytic activity
2.10. pH dependence
of studied enzyme toward Ala-pNA, Gly-pNA, Met-pNA, Pro-pNA,
Phe-pNA, Gly-Phe-pNA, Phe-Gly-pNA and Gly-Phe-Gly-Phe-pNA
as substrates was determined by the same method. In all cases
The effect of pH on the rapeseed aminopeptidase activity was
determined at 37 °C, using Phe-pNA, Leu-pNA or Ala-pNA as sub-
strates, in 50 mM buffers: sodium acetate (pH 5.0–5.5), PBS (pH
6.0–7.0), Tris-HCl (pH 7.5–8.5) and Na2CO3-NaHCO3 (pH 9.0–10.0).
L-amino acid derivatives were used as substrates.
2.6. Estimation of molecular mass
2.11. Effect of metal ions
Polyacrylamide gel electrophoresis (PAGE) was performed
under non-denaturing conditions in 7.0% polyacrylamide gels in
the absence of SDS. 10 identical samples of extract were loaded
on the gel. After electrophoresis, two strips of the gel were cut
off and incubated in 50 mM Tris–HCl buffer (pH 8.0), containing
5 mM Leu-pNA or Phe-pNA, for 30 min, at room temperature. Third
strip was stained with Coomassie Brilliant Blue for 1 h, and
destained in methanol-acetic acid-water mixture. The rest of the
unstained gel was cut into strips according to the bands stained
with Coomassie Brilliant Blue. Unstained gel strips were placed
in microcentrifuge tubes and immersed in 1 ml of 50 mM Tris–
HCl buffer (pH 8.0). Gel pieces were fragmented using clean blade
and stirred overnight at room temperature, using magnetic stirrer.
Proteins extracted from the gel strips were then centrifuged. Elu-
tion was repeated by adding fresh portion of buffer. Filtrates from
each unstained gel strip were combined. Each protein eluate was
concentrated separately on Amicon Centrifugal Unit (Merck).
The influence of metal salts of Al3+, Ba2+, Ca2+, Cd2+, Cu2+, Fe2+
,
Mg2+, Mn2+, Na+, Ni2+ and Zn2+ on the activity of rapeseed
aminopeptidase was determined using two concentrations of each
salt (1 mM and 0.1 mM). The enzyme was incubated with each salt
solution for 2 h at 4 °C. Control sample without any metal salt was
also prepared. Enzyme activity was determined using the assay
previously described, with Phe-pNA as a substrate.
2.12. Effect of proteolytic enzymes inhibitors
In order to determine the influence of standard protease inhibi-
tors, the enzyme was incubated with various concentrations of
EDTA, EGTA, 1,10-phenantroline, bestatin, E-64 and PMSF, at
30 °C for 30 min. Control measurement in the absence of inhibitors
was also performed. The residual enzyme activity was determined
using the assay previously described.

J. Kania, D.M. Gillner / Food Chemistry 207 (2016) 180–186
183
ular mass, estimated by Native-gel electrophoresis (Supplementary
materials, Fig. SI 2) was about 60 kDa. It revealed, that there is only
one clear protein band showing the hydrolytic activity toward both
Phe-pNA as well as Leu-pNA. The estimated molecular mass was
lower than that described for aminopeptidase isolated by A. Barth
from germinated rapeseed (79 kDa) (Barth & Hermann, 1974). Sim-
ilar molecular mass was determined for several cereal aminopepti-
dases isolated from seeds, e.g. barley (58 kDa) (Oszywa, Makowski,
& Pawełczak 2013). The molecular mass of aminopeptidase within
the same range was also determined in other oilseed plants e.g.
3. Results and discussion
3.1. Characterisation of crude extract and partially purified
aminopeptidase
Preliminary experiments revealed that the crude extract
obtained from dry rape seeds, after separation of oil fraction and
precipitation with ammonium sulfate, showed the highest specific
activity (65.1 mU/mg) toward Phe-pNA as a substrate, but was also
active in hydrolysis of Leu-pNA (25.0 mU/mg), Ala-pNA (15.1 mU/
mg), and Pro-pNA (7.1 mU/mg). The activity profiles obtained dur-
ing gel filtration of crude extract are depicted in Fig. 1. As can be
peanut cotyledons (55–60 kDa) (Isola
&
Franzoni 1996).
Aminopeptidases isolated from soybean cotyledons as well as sun-
flower seed had higher molecular weights – 85 kDa and 80 kDa
respectively (Couton et al., 1991; Tishinov et al., 2009). Isoelectric
focusing showed that at least 4–5 visible protein bands with pI val-
ues of 5.3–6.7 can be observed in the partially purified aminopep-
tidase (Supplementary materials, Fig. SI 3). The aminopeptidase
that gives the most visible band has slightly acidic pI of 6.25. Sim-
ilar pI values were obtained for aminopeptidases in wound-
induced aminopeptidase proteins of tomato (pI 6.2–6.4 for
60 kDa LAP-A and pI 5.6–5.8 for 55 kDa aminopeptidases) (Gu,
Chao, & Walling 1996), as well as LAP from Solanum tuberosum
tubers (pI 5.45) (Vujcic, Dojnov, Milovanovic, & Bozic 2008). The
observed, the hydrolytic activity toward several L-amino acids
accumulates in the same region. Three peaks (with the middle
peak partially separating into three additional parts) can be
observed in Phe-pNA activity profile. Also in Leu- and Ala-pNA pro-
files 4–5 signals can be noticed, but they are not separated. The
Native-gel electrophoresis of crude extract showed that the Rf val-
ues of bands corresponding to all substrates hydrolysed (Ala-, Phe-,
Leu-, Pro-pNA) (Supplementary materials, Fig. SI 1) were the same
suggesting, that either there is one enzyme with broad activity or
more aminopeptidases with the similar molecular mass.
Partial purification of extract (131.17-fold purification)
increased the specific activity of studied enzyme to 788 mU/mg
toward Leu-pNA (Table 1). The highest activity was obtained for
Phe-pNA as a substrate (4944.2 mU/mg). The approximate molec-
K
m value of 334.3
as 247.5 M and 403
higher affinity of the enzyme toward phenylalanine substrate.
l
M and Vmax of 76.82
lM/s for Leu-pNA, as well
l
lM/s for Phe-pNA were determined, showing
Fig. 1. The profile of aminopeptidase activity during gel filtration of winter rape seeds cv. Bellevue crude extract. Column: Sephacryl HR 300 (BioRad) 1.5 ꢁ 50 cm; flow rate
0.20 cm3/min, volume of fractions 1 cm3. Buffer Tris-HCl 50 mM, pH 8.0; Substrates: Phe-pNA, Ala-pNA, Leu-pNA and Pro-pNA.
Table 1
Partial purification of aminopeptidase isolated from winter rape seeds cv. Bellevue.
Purification step
Total activity* [U]
Total protein [mg]
Specific activity* [U ꢁ mgꢂ1 protein]
Purification (-fold)
Recovery [%]
Crude extract
63.33
51.46
25.60
25.56
12.76
5.38
10089.0
3976.0
1031.0
955.0
65.6
0.006
0.013
0.025
0.027
0.195
0.268
0.788
1.0
2.17
4.16
4.50
32.33
44.67
131.17
100.0
81.3
40.4
40.4
20.2
8.5
35% (NH4)2SO4
65% (NH4)2SO4
Sephadex G-25
DEAE – Sepharose
Sephacryl HR 300
MacroPrep High Q
20.1
2.5
1.97
3.1
*
The activity was assayed with Leu-pNA as a substrate, at pH 7.0 (PBS buffer).

184
J. Kania, D.M. Gillner / Food Chemistry 207 (2016) 180–186
Fig. 2. The influence of temperature on the activity of partially purified aminopep-
tidase from winter rape seeds cv. Bellevue. Incubation time 10 min.; pH 7.0; the
residual aminopeptidase activity was determined using Phe-pNA as a substrate.
Fig. 4. The pH dependence of partially purified rapeseed aminopeptidase activity
from winter rape seeds cv. Bellevue. Enzyme activity was assayed with Phe-pNA,
Leu-pNA and Ala-pNA as substrates.
3.2. Temperature optimum and thermal stability
Table 2
Substrate specificity of aminopeptidase from winter rape seeds cv. Bellevue in crude
extract and partially purified enzyme at the optimum pH 6.5. Final substrate
concentration – 1.3 mM; PBS buffer pH 6.5; temperature 37 °C.
The influence of temperature on the activity and stability of par-
tially purified aminopeptidase was determined in the range of 25–
65 °C, using Phe-pNA as a substrate (Fig. 2). The maximum activity
of the aminopeptidase toward Phe-pNA can be observed at 40 °C.
The enzyme remains over 80% of activity over the temperature
range of 35–45 °C, and at 25 °C it shows ꢀ52% of its maximum
activity toward Phe-pNA. It is completely inactive over 55 °C. Sim-
ilar results were obtained for Leu-pNA (maximum activity at
40 °C), but for Ala-pNA the maximum activity was shifted to 45–
50 °C. Both temperatures correlate well with the optima of
aminopeptidases from other species: 52 °C for aminopeptidase
from barley seeds (Oszywa et al., 2013), and 35–45 °C for
aminopeptidases from several Brassicaceae plants (Marinova &
Tchorbanov, 2008). The thermal stability of the aminopeptidase
was determined by incubation for 15, 30, 45 and 60 min at the
temperatures of 25, 30, 35, 37, 40, 45, 50 and 55 °C (Fig. 3). The
study revealed, that the enzyme is stable in the temperature range
of 25–40 °C. It remains over 40% of activity after incubation for
60 min at 45 °C. The dramatic loss of activity is observed at the
temperatures above 45 °C.
Substrate
Relative activity* [%]
Purified enzyme
Crude extract
Phe-pNA
Leu-pNA
Ala-pNA
Gly-pNA
Met-pNA
Pro-pNA
Gly-Phe-pNA
Phe-Gly-pNA
Gly-Phe-Gly-Phe-pNA
100
16.0
6.7
2.7
8.3
9.0
0.2
0
3.4
0.7
0.4
0.1
0.3
0.2
0.1
0.01
0
0
*
The activity of purified enzyme with Phe-pNA was taken as 100%.
seeds as well as chosen Brassicaceae plants aminopeptidases (pH
7.2–8.0), Solanum tuberosum tubers LAP (pH 9.0) and iminopepti-
dases (pH 8.0–8.5) (Marinova & Tchorbanov, 2008; Marinova
et al., 2008; Tishinov, Petrova, & Nedkov, 2010; Vujcic et al. 2008).
Substrate specificity of partially purified enzyme was also
determined at optimum pH (pH 6.5 for Phe-pNA cleveage), using
4-nitroanilides of chosen amino acids. Two dipeptides and one
tetra-peptide were also applied as potential substrates of studied
enzyme (Table 2). Additionally, substrate specificity of crude
extract at pH 6.5 was determined, in order to compare results with
partially purified sample. The obtained data indicate, that the
enzyme catalyzes preferentially the hydrolysis of Phe-pNA and also
Leu-pNA. This indicates that amino acids with larger hydrophobic
side chains are much better substrates. Specific activities toward
Ala- and Gly-pNA are much lower. It is also worth mentioning, that
the enzyme is active in cleavage of Met-pNA and Pro-pNA. Among
the studied dipeptides only Gly-Phe-pNA was hydrolysed. No
detectable activity of partially purified enzyme was observed for
Gly-Phe-Gly-Phe-pNA.
3.3. pH optimum and substrate specificity
The pH dependence of aminopeptidase activity in the range of
pH 5.0–10.0, for Phe-, Leu- and Ala-pNA as substrates, was deter-
mined (Fig. 4). The obtained curve is bell shaped for Phe-pNA, with
the maximum at pH 6.5, but smaller peak can be observed at pH
9.0, suggesting the presence of more than one enzyme isoforms
in the sample. Similar pH optima were determined for sunflower
3.4. Protease inhibitors
The influence of the most efficient protease inhibitors on
the rapeseed aminopeptidase was determined in the presence of
Phe-pNA as a substrate (Supplementary materials, Table SI 1).
We found that the inhibitory effect of chelating agent EDTA
was observed after 30 min of incubation (50%, 73%, 84% of
residual activity, for the concentrations of 33 mM, 10 mM and
1 mM, respectively). The results indicate, that the studied
enzyme requires some metal ions for full activity. The other
Fig. 3. Thermal stability of partially purified aminopeptidase from winter rape
seeds cv. Bellevue. Enzyme was incubated at different temperatures (25–55 °C) for
15, 30, 45, and 60 min. The residual aminopeptidase activity was determined using
Phe-pNA as a substrate.
chelating agents did not show such
a significant influence,
and 1,10-phenantroline had even slightly activating effect.

J. Kania, D.M. Gillner / Food Chemistry 207 (2016) 180–186
185
Phenylmethylsulfonyl fluoride known serine proteases inhibitor
also lowered the enzyme activity (46% and 64% of residual activity
for 4 mM and 2 mM concentration of PMSF, respectively). Bestatin,
which is a typical leucine aminopeptidase inhibitor, showed only
minimum inhibitory effect (at the concentration of 0.1 mM, the
residual aminopeptidase activity was 94%). Inhibitor of cysteine
proteases (E-64) did not show any significant inhibition (96% of
residual activity for 0.1 mM concentration). Proteases can be clas-
sified according to the mechanism of hydrolysis of peptide bond.
Therefore four groups of peptidases can be distinguished: metallo-
proteases, serine/threonine proteases, cysteine and aspartyl pro-
teases. The obtained results suggest that serine plays a crucial
role in the activity of rapeseed aminopeptidase and that metal ions,
required for full enzymatic activity, are tightly bound to the active
site. Similar influence of typical protease inhibitor was also
observed in other plants. PMSF was one of most effective inhibitors
towards purified Phe-AP from shoots of 3-week-old pea plants,
however EDTA and 1,10-phenantroline had slightly activating
influence on this aminopeptidase (Pyrzyna, Szawłowska,
Bielawski, & Zdunek-Zastocka, 2011). In the case of crude extract
of the whole fruit of A. deliciosa (kiwifruit), the presence of 1 mM
of 1,10-phenanthroline, EDTA and iodoacetamide inhibited
aminopeptidase activity while the 1 mM PMSF had no effect
(Premarathne & Leung, 2010). 1,10-Phenantroline was also effi-
cient inhibitor of leucine aminopeptidase from Solanum tuberosum
tuber, while EDTA, EGTA and sodium citrate had no effect on
aminopeptidase activity (Vujcic et al., 2010).
ions are required for full enzymatic activity. The enzyme was
slightly activated by Ba2+, Na+ ions and no influence of Ca2+, Mg2+
or Al3+ ions was observed. The addition of Cu2+ Zn2+ and Ni2+ had
significant inhibitory effect. The pI values as well as pH and tem-
perature optima were quite similar to other plant aminopeptidases
and iminopeptidases. The obtained results suggest that there are
few aminopeptidases (including prolyl aminopeptidase) present
in the partially purified sample, having similar molecular weight
and slightly different pI values.
Acknowledgments
We would like to thank Dr. Maciej Makowski from the Univer-
sity of Opole for providing us with dipeptide and tetrapeptide
substrates.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
03.097.
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