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Purification, characterization and the use of

recombinant prolyl oligopeptidase from
Myxococcus xanthus for gluten hydrolysis

Article in Protein Expression and Purification · January 2017

DOI: 10.1016/j.pep.2016.09.016

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 Protein Expression and Purification 129 (2017) 101e107

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Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Purification, characterization and the use of recombinant prolyl

oligopeptidase from Myxococcus xanthus for gluten hydrolysis
Ebru Kocadag Kocazorbaz*, Figen Zihnioglu
_
Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova, Izmir, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing
Received 21 July 2016 small peptides. The members of prolyl oligopeptidase family play important roles in many physiological
Received in revised form processes such as neurodegenerative diseases, maturation and degradation of peptide hormones. Thus
23 September 2016
the enzyme has been purified and characterized from various sources to elucidate the potential use as
Accepted 27 September 2016
therapeutics. In this study recombinant Myxococcus xanthus prolyl oligopeptidase expressed in E. coli was
Available online 28 September 2016
purified 60.3 fold, using metal-chelate affinity and gel permeation chromatography. The recombinant
enzyme had a monomeric molecular weight of 70 kDa. Isoelectric point of the enzyme was found to be
Keywords:
Serine protease
approximately 6.3 by two-dimensional polyacrylamide gel electrophoresis. The optimum pH and tem-
Prolyl oligopeptidase perature was estimated as 7.5 and 37
C, respectively. The purified enzyme was stable in a pH range of 6.0
Enzymatic hydrolysis e8.5 and thermally stable up to 37
C. The Km and Vmax values were 0.2 mM and 3.42 mmol/min/mg. The
proteolytic activity was inhibited by active-site inhibitors of serine protease, Z-Pro-Prolinal, PMSF, and
metal ions, Cd2þ, and Hg2þ. Furthermore, the hydrolysis efficiency of the recombinant prolyl oligo-
peptidase was investigated with wheat gluten.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction yeasts and actinomycetes, prolyl oligopeptidase activity has not

Prolyl oligopeptidase (POP; E.C.3.4.21.26) is an enzyme that is
among serine protease family, with an approximate molecular
weight of 80 kDa [1]. POP family includes prolyl oligopeptidase,
dipeptidyl peptidase IV (E.C.3.4.14.5), oligopeptidase B (3.4.21.83)
and acylaminoacyl peptidase (3.4.19.1) enzymes. This enzyme
family differs from trypsin-subtilisin-like serine proteases with
their catalytic triad region arrangement and specificities for small
peptide substrates. POP hydrolyses peptides consisting of less than
30 residues from carboxy terminal of proline. The role of POP
enzyme in hydrolysing bioactive peptides such as thyrotropin
releasing hormone, substance P, arginine-vasopressin, neurotensin,
angiotensin is known [2,3].
Prolyl oligopeptidase was discovered by the release of leucyl-
glycinamide peptide via hydrolysis of oxytocin (Cys-Tyr-Ile-Gln-
Asn-Cys-Pro-Leu-Gly-NH2) from C-terminus (COO) in human
uterus homogenate [4,5]. Prolyl oligopeptidase enzyme is wide-
spread such as bacteria, plant, arceae and human [6e14]. Only in
* Corresponding author.
E-mail address: kocadage@hotmail.com (E. Kocadag Kocazorbaz).
been observed [1,15,16]. Although membrane bound form of prolyl
oligopeptidase enzyme has been characterized, it is generally re-
ported to be cytosolic [17,18].
Recent studies showed that POP enzyme has an important role
in the treatment of diseases such as Alzheimer's, amnesia,
depression, cancer and celiac disease. Discovering the potential role
of POP in pathophysiology of some diseases has led to many re-
searches for the development and identification of POP inhibitors
[2,19e21]. POP enzyme thought to be participating in cheese
ripening acceleration and used for production of recombinant
functional peptides, gluten degradation for celiac disease cases and
antibody directed enzyme prodrug therapy [22].
Expression of prolyl oligopeptidase from Sphingomonas capsu-
late, Flavobacterium meningosepticum and Myxococcus xanthus have
been reported (9, 14, 23). However, functional characterization of
recombinant Prolyl oligopeptidase activity have not been fully
investigated up to now. Therefore, in this study recombinant pro-
duction of Myxococcus xanthus prolyl oligopeptidase enzyme in
E. coli and purification of the enzyme with Nickel-Chelate Affinity
and gel filtration chromatography were carried out. Characteriza-
tion of the purified enzyme included explorations of pH and tem-
perature optimum, isoelectric point, molecular weight, substrate

http://dx.doi.org/10.1016/j.pep.2016.09.016
1046-5928/© 2016 Elsevier Inc. All rights reserved.
102 E. Kocadag Kocazorbaz, F. Zihnioglu / Protein Expression and Purification 129 (2017) 101e107
specificity, kinetic parameters, effects of metal ions, effects of
various chemicals and inhibitors; pH, thermal, and storage
stabilities.
2. Materials and methods
2.1. Cloning of POP gene
A strain of Myxococcus xanthus ATCC 25232 was obtained from
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkultu-
ren GmbH, Germany). The bacteria was grown and maintained on
liquid CTT medium (1% Casitone, 10 mM Tris-HCl [pH 8.0], 8 mM
MgSO4, 10 mM KPO4 [pH 7.4]). The culture was incubated at 30 >
C
for 6 days on an orbital shaker (225 rpm). Genomic DNA from
Myxococcus xanthus ATCC 25232 extraction was carried with the
Invitrogen genomic DNA kit as described (Invitrogen, France). For-
ward and reverse primers were used based on the POP gene
sequence of Myxococcus xanthus according to the method of Shan
et al., 2004 [23]. The cycling conditions were as follows: an initial
denaturation of 95
C for 5 min; 30 cycles of 95
C for 45 s, 55
C for
45 s, 72
C for 45 s; and a final extension of 72
C for 5 min. The PCR
product and pET21a (þ) were digested with NdeI and HindIII re-
striction endonucleases. PCR products were separated on 1%
agarose gels, extracted and purified using the GeneJET Gel Extrac-
tion Kit (Thermo Fisher Scientific) and then ligated together by
T4DNA ligase. Competent Escherichia coli cells were transformed
with the ligation mixture to obtain the required recombinants.
2.2. Expression and purification of recombinant POP gene
Expression was carried out in E. coli BL21(DE3) strain. Cultures
transformed with recombinant plasmid were grown in Luria e
Bertani medium containing 50 mg/mL chloramphenicol and 150 mg/
mL ampicillin. Cultures were induced with 0.3 mM isopropyl thio-
b-D-galactoside (IPTG). Before induction, cultures were grown at
37
C to an A600 of 0.6e0.8. The induced cultures were centrifuged
at 4.000 rpm for 30 min at 4
C. The cell pellets were suspended in
extraction buffer (50 mM sodium-phosphate buffer, pH 7.4 con-
taining 500 mM NaCl and 5 mM imidazole) per 1 g cell pellet and
disrupted by sonication. The lysate was centrifuged at
13,000 rpm at 4
C for 30 min. The soluble protein was filtered
through a 0.45-mm syringe filter (Millex, Millipore), and loaded
€ €
onto the AKTA FPLC system (AKTA Pharmacia GE FPLC) equipped
with a HisTrap HP column (5 mL, GE Healthcare), with Ni2þ ions
immobilized on Sepharose. The purification procedure was carried
out under increasing gradient of elution buffer (phosphate buffer,
50 mM, pH 7.8; NaCl, 500 mM; imidazole, 250 mM) at the flow rate
of 2.5 mL/min. The protein was eluted with a linear gradient of
imidazole (0 to 250 mM) in the buffer. Fractions were immediately
analyzed for the prolyl oligopeptidase activity. Gel filtration was
performed on an Ultragel AcA 44 column (1.8  30 cm) attached to
a Biorad MPLC system. Runs were performed at a flow rate of
1.0 mL/min and protein elution was monitored at a wavelength of
280 nm. The column was calibrated with bovine serum albumin
(66 kDa), peroxidase (40 kDa), proteinase K (28 kDa) and lysozyme
(14 kDa). Protein concentration was determined by the Bradford
method [24], using bovine serum albumin (BSA, Sigma®) as
standard.
The purity of the enzyme was assessed by SDS-PAGE with 4%
concentrating gel and 12% separating gel using a Mini PROTEAN II
gel system (Bio-Rad Laboratories), according to the method of
Laemmli [25]. The isoelectric point of the enzyme was determined
using ampholytes with pH 3.0e10.0 (Ampholyte; Bio-Rad) in a 2D-
PAGE system (Bio-Rad). A set of standards with range of isoelectric
points of 3e10 (Amersham) was used.
2.3. Biochemical characterization
2.3.1. Substrate specificity and kinetic analysis
Prolyl oligopeptidase activity was determined using synthetic
and natural substrates. The mixture (0.2 mL) contained 10 mL
enzyme solution, 2 mM Suc-Ala-Pro-pNA (Bachem AG, Bubendorf,
Switzerland) as chromogenic substrate and 100 mM Tris-HCl buffer
(pH 7.4). After incubation for 5 min at 37
C, the amount of liberated
p-nitroaniline (pNA) was calculated by spectrophotometric ab-
sorption at 405 nm. One unit of prolyl oligopeptidase activity (U)
was expressed as micromoles of substrate hydrolyzed per minute
by the enzyme [25]. The POP activity was also assayed on other
chromogenic substrates (Leu-pNA, Pro-pNA, Glu-pNA, Glu-Ala-
pNA, H-Ala-Pro-pNA, H-Arg-Pro-pNA, H-Val-Ala-pNA, H-Gly-Pro-
pNA, Suc-Ala-Pro-pNA, Z-Gly-Pro-pNA, and Suc-Gly-Pro-pNA). Ac-
tivity of recombinant POP preparate from Myxococcus xanthus
tested against natural substrates IGF-1 (H-Gly-Pro-Glu-OH) and
Substance P (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-
NH2) by RP-HPLC analysis. Kinetic parameters estimated by the use
of Suc-Ala-Ala-Pro-pNA as substrate (0.05e2 mM). Assays carried
out under standard conditions (37
C, 5 min) and replicated 3 times.
GraphPrism 6 used for evaluation of Km, Vmax, and Kcat.
2.3.2. Effect of temperature and pH on activity and stability of
recombinant POP
The optimal pH was determined by measuring the activity be-
tween pH 6.0 and 10.0 (100 mM) using sodium-phosphate (pH
6.0e7.5), Tris-HCl (pH 6.0e7.5) and sodium-borate buffer (pH
9.0e10.0) at 37
C. The optimum temperature for enzyme activity
was determined by performing the enzymatic reaction in temper-
atures ranging from 4
C to 80
C. The pH stability was studied by
pre-incubating the enzyme for 30 min at 37
C at different pH
values ranging from 3.0 to 10.0 using buffered substrate. In addition
to that, thermostability studies of the enzyme investigated by
preincubating the enzyme solution at different temperatures
ranging from 4 to 55
C for 30 min at constant pH 7.4. Finally, the
storage stability of the enzyme was tested at 4
C.
2.3.3. Effect of various chemicals, inhibitors and metal ions on
recombinant POP
In order to determine the effects of metal ions, chemical re-
agents and classical protease inhibitors on the enzyme activity with
inhibitors were pre-incubated at 37
C for 30 min. The relative
activity, defined as the activity value relative to the activity of
control (without any effector), was also estimated. IC50 values were
only determined for POP inhibitors by nonlinear regression analysis
from the residual activity versus inhibitor concentrations curve.
2.4. Hydrolysis of gluten with recombinant POP
The gluten 3% (w/v) was suspended in 0.1 M sodium-phosphate
(pH 7.4) and heated at 90
C for 10 min. Recombinant POP was
added at an enzyme-to-substrate ratio of 4.6 U/mg and hydrolysis
was carried out at 37
C for 8 h with constant agitation. Reaction
was terminated for the samples that were taken from the media at
defined time intervals by heating in 90
C water bath for 15 min.
The hydrolysates were centrifuged at 9000 rpm for 15 min to
remove the unhydrolyzed residue, followed by lyophilisation and
storage at 20
C until further use. Free amino groups were
measured using TNBS and the degree of hydrolysis (DH) was
calculated as:
DHð%Þ ¼ ½ðh  h0 Þ=htot   100
where, htot is the total number of peptide bonds in the protein
 E. Kocadag Kocazorbaz, F. Zihnioglu / Protein Expression and Purification 129 (2017) 101e107
substrate (8.38 meqv/g gluten protein).; h is the number of peptide
bonds cleaved during hydrolysis, and h0 is the content of free amino
groups of substrate.
3. Results and discussion
3.1. Recombinant POP expression and purification
Myxococcus xanthus strain was chosen for production and pu-
rification of recombinant POP in E. coli. cDNA of Myxococcus xanthus
strain obtained POP enzyme coding gene fragment amplified with
appropriate primers via PCR. After amplification of the Myxococcus
xanthus POP gene from the Myxococcus xanthus by PCR, obtained
fragments were cloned into the pET-21a vector as described in the
Experimental section. Plasmid was transformed into competent
E. coli BL21(DE3). The Myxococcus xanthus POP was purified in two
steps by using 6xHis affinity chromatography and gel filtration
according to the purification procedure from the soluble fraction in
E. coli. The results of purification procedure are summarized in
Table 1. The purified enzyme preparation exhibited a specific ac-
tivity of 226 mmol/min/mg protein, while purification fold and yield
were 60.3 and 38.5%, respectively.
Prolyl oligopeptidase was further purified to homogeneity and
to confirm its purity the purified enzyme was resolved on 4%
stacking and 12% running gel and found to be a homogenous
monomeric protein as evident by single band corresponding to
70 kDa on SDS-PAGE (Fig. 1). Structure of recombinant POP en-
zymes obtained and purified from Myxococcus xanthus strain and
other microbial resources determined to be monomer in most
studies, dimer in a few studies, and trimer in another few studies
with SDS-PAGE, gel filtration, and native-PAGE [26e30]. Molecular
weight of POP from different species is known to be varying be-
tween 57 and 81 kDa Molecular weight of POP purified with metal
affinity and gel filtration chromatography was estimated to be
70 kDa with SDS-PAGE, which is consistent with literature [30e32].
On the other hand relative molecular weight of POP purified with
gel filtration chromatography is estimated to be 42.6 kDa. This
finding is smaller than that of SDS-PAGE, which is also consistent
with literature since most of other studies shown POP as a mono-
mer [33e38]. However determination of molecular weight of POP
requires advanced techniques since two different results obtained
from different methods. When DNA sequence of POP is considered,
molecular weight is estimated to be 71 kDa, which is consistent
with the result obtained from SDS-PAGE but not with that of gel
filtration. Similar differences between results of two different
methods for molecular weight estimation of POP reported before
[21,31]. Difference between results of SDS-PAGE and gel filtration
was attributed to possible interactions between gel matrix and the
enzyme (see Fig. 2).
Isoelectric point (pI) of the enzyme was estimated as 6.3 by two-
dimensional polyacrylamide gel electrophoresis (2D-PAGE). pI
values for POP obtained from different resources varies, such as pI
value of animal derived POP is between pH 4.5e5.5 and that of
microbial POP is between 5.5 and 6.2. Estimated pI value of POP
obtained and purified from Myxococcus xanthus strain is consistent
with those of other microbial POP enzymes [39].
Table 1
Purification of Prolyl oligopeptidase from Myxococcus xanthus.
Total activity (U) Total protein (mg)
Homogenate 792 211.2
Niþ2-NTA 329.4 20.8
Ultragel AcA 44 305 1.4
103
3.2. 3 substrate specificity and kinetic analysis
Both synthetic (Suc-Ala-Pro-pNA, Z-Gly-Pro-pNA, H-Gly-Pro-
pNA, H-Ala-Pro-pNA, H-Arg-Pro-pNA, H-Val-Ala-pNA, H-Glu-Ala-
pNA, H-Leu-pNA, H-Pro-pNA, H-Glu-pNA) and natural substrates
(IGH-1 and Substance P) are used for substrate specificity studies.
Peptides with high proline content were hydrolyzed faster and
peptides with high alanine content were hydrolyzed slower. Ac-
tivities of POP for synthetic dipeptides (Suc-Ala-Pro-pNA, Z-Gly-
Pro-pNA, H-Gly-Pro-pNA, H-Ala-Pro-pNA, H-Arg-Pro-pNA, H-Val-
Ala-pNA, H-Glu-Ala-pNA, H-Leu-pNA, H-Pro-pNA and H-Glu-pNA)
are given in Table 2 for each substrate. Recombinant POP purified
from Myxococcus xanthus could not hydrolze dipeptides with pro-
line content but shown activity for proteins with proline content.
This validates that POP is an endopeptidase that hydrolyzes from
the carboxyl terminus of the proline amino acid. Y-Pro-X bond is
hydrolyzed in sequence. X symbolizes an amino acid aside from
proline and Y symbolizes a preserved residue or peptide. POP also
can hydrolyze Ala-X bond but with a 100e1000 fold decrease in
affectivity. Activity increases for hydrophobic amino acids rather
than acidic or basic ones in the place of X [13]. POP also specifically
hydrolyzes from amino acids aside from proline such as alanine,
serine, a-aminobutril, hydroxyproline and cysteine, in the P1 po-
sition of peptide. Hydrolysis efficiency of POP according to amino
acid residue can be given in the order of Pro > Ala > Cys > Ser
[40e42] which shows the relation of increased hydrolytic activity
with existence of hydrophobic and basic amino acids in P3 position.
The data also showed that synthetic substrates with proline
attached to a hydrophobic N-benzyloxycarbonyl (Z) of Y are hy-
drolyzed faster and importance of preserved regions of natural
substrates such as IGF-1 and Substance P. As shown in Table 2 Suc-
or Z-content has a little effect on activity. Due to low solubility of
peptides starting with Z-, Suc-Ala-Pro-pNA was used during puri-
fication and characterization steps (see Table 3).
Comparison of substrate specificities of POP purified from
different resources indicates the requirement of hydrophobic
amino acids in N-terminus of the substrates and vice versa. Exis-
tence of alanine or especially proline at P2 position effects the ac-
tivity positively [43].
Effect of substrate concentration on POP activity was tested with
Suc-Ala-Pro-pNA. Substrate saturation concentration, Km, Vmax, kkat
values for POP purified from Myxococcus xanthus strain was
calculated as 0.20 ± 0.014 mM, 3.42 ± 0.07 mmol/min and 244
min1 with GraphPad Prism 6, respectively.
Effect of substrate concentration have been studied with chro-
mogenic and fluorogenic substrates for various species. In com-
parison with the related studies, Z-Gly-Pro-pNA found to be the
spesific substrate for POP with higher affinity. Due to low solubility
of Z-Gly-Pro-pNA as stated before, Suc-Ala-Pro-pNA is preferred in
this study. Km value of recombinant purified POP for Suc-Ala-Pro-
pNA is found to be coherent with those of literature.
3.3. Effect of temperature and pH on activity and stability of
recombinant POP
Optimum temperature for POP from Myxococcus xanthus strain
Specific activity (U/mg) Purification fold Recovery (%)
3.8 1 100
15.8 4.2 42
226 60.3 38.5
104 E. Kocadag Kocazorbaz, F. Zihnioglu / Protein Expression and Purification 129 (2017) 101e107

Fig. 1. Electrophoretic analysis of purified prolyl oligopeptidase from Myxococcus xanthus. A) (a) SDS-PAGE of purified Lane 1, standard protein markers: a-Lactalbumin, bovine milk
(14.2 kDa); Trypsin inhibitor (20 kDa); Trypsinogen (24 kDa); Carbonic anhydrase (29 kDa); glyceraldehyde-3-phosphate (36 kDa); Albumin egg (45 kDa); Albumin bovine (66 kDa)
Lane 2, crude protein Lane 3, protein from purified pop of Ni-NTA column Lane 4, protein from purified POP of gel filtration. B) two-dimensional electrophoretic resolution of
purified prolyl oligopeptidase.

Fig. 2. Gluten hydrolysis with recombinant POP. a) Effect of reaction time b) Effect of E/S ratio on the hydrolysis degree.

Table 2
Substrate specificity of recombinant Prolyl oligopeptidase.

Substrates Activity (U/mg) Time (min)

Suc-Ala-Pro-pNa 1.43 5
Z-Gly-Pro-pNa 1.56 5
Suc-Gly-Pro-pNa 0.52 5
H-Ala-Pro-pNa e 5
H-Arg-Pro-pNa e 10
H-Val-Ala-pNa e 10
H-Gly-Pro-pNA 15
H-Leu-pNa e 15
H-Pro-pNa e 15
H-Glu-pNA e 15
H-Glu-Ala-pNA e 15
IGF-1 (H-Gly-Pro-Glu-OH) 0.34  10-4 60
Substance P (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) 0.23  103 60

observed to be 37
C. For temperatures higher than 45
C, loss of
activity was increased. The enzyme is stable over broad range of
temperature (4e37
C) retaining 100% activity where 50% at 50
C.
There are consistent with thermal stability values for POP purified
from different resources in literature [30,34,44].
Optimum pH for POP estimated to be 7.5. For POP purified from
different species and sources, optimum pH estimated to be in be-
tween pH 3.7e10.0 [45e47]. It is well known that serine proteases
have maximum activities around alkali and neutral pHs. The puri-
fied enzyme was most stable between pH 7.0, and at pH 8.5, there
was only 12e14% reduction in the activity. This data is also well
agreed with the recent studies [31,48].
Recombinant enzymes are also known for their low storage
stability. The data obtained from this study showed that recombi-
nant POP lost 50% of its activity at 4
C after 6 weeks. There were no
data found in literature for recombinant POP storage stability.
 E. Kocadag Kocazorbaz, F. Zihnioglu / Protein Expression and Purification 129 (2017) 101e107 105

Table 3 This inhibition is reversible in the long term. Identification of pu-

Effect of some chemicals on the activity of recombinant Prolyl oligopeptidase. rified protease as serine protease is accomplished by using spesific
Metal ions or reagents Concentration (mM) Relative activitya (%) inhibitors. 0.38 mM of PMSF, a serine protease inhibitor caused 50%
MnCl2 1.0 87 ± 1.8
inhibition, which validates catalytic classification of the purified
CuCl2 1.0 57 ± 4.0 enzyme as serine protease. Other studies have shown that 1 mM of
KCl 1.0 63 ± 1.8 PMSF can cause 20% inhibition. p-aminobenzamidine (pABA) is a
BaCl2 10.0 48 ± 2.7 competitive inhibitor for trypsin-like serine proteases such as
MgCl2 1.0 87 ± 3.1
thrombin, plasmin, kallikrein, urokinase, enterokinase. As a result
NaCl 1.0 104 ± 3.5
LiCl2 1.0 96 ± 4.2 of being positively charged, pABA blocks S1 negative charge pocket
CaCl2 1.0 104 ± 2.5 of enzyme, thus resulting in inhibition [50]. 1e7.5 mM of pABA did
NiCl2 0.05 58 ± 2.5 not have significant effect on activity. 15% inhibition observed with
CdSO4 0.05 e
10 mM pABA. Low effect of pABA on POP attributed to differences
HgCl2 0.05 e
Z-proprolinal 0.1 30 ± 1.6
between trypsin-like proteases and POP. EDTA and 1e10 phenan-
PMSF 1.0 18 ± 2.5 throline are known metallo protease inhibitors. These inhibitors
pABA 1.0 100 ± 0.5 did not inhibit the activity which indicates that this enzyme is not a
1,10-Phenanthroline 1.0 98 ± 1.5 metalloprotease. Most of other studies have shown similar results,
EDTA 1.0 94 ± 8.2
however there are few studies stated EDTA as an effective inhibitor
b-Mercaptoethanol 1.0 98 ± 3.8
DTT 5.0 86 ± 1.9 for serine proteases (10 mM EDTA shows 70% inhibition) [51]. Effect
of reducing agents on POP activity investigated with the use of b-
Control - 100 ± 2.1
mercaptoethanol and DTT. 1 mM of b-mercaptoethanol and
a
Values are means ± S.D. (n ¼ 3). The activity measured at 37
C, pH 7,4, any 1e2.5 mM of DTT did not inhibit activity. Higher concentrations for
addition, was considered as 100%.-No activity detected.
b-mercaptoethanol (2.5e10 mM) inhibited 12e20% of activity and
5e10 mM DTT inhibited 14e24% of activity. Inhibition attributed to
3.4. Effect of various chemicals, inhibitors and metal ions on short distance between Cys (255) residue and S1eS3 binding re-
recombinant POP activity gions. For homologs of POP, cysteine residue is further from these
regions and ineffectiveness of reducing agents shown [33,35]. POP
1-10 mM concentrations of Mnþ2 and Liþ did not show a sig- purified from F. Meningsepticum also resistant against reducing
nificant inhibition. Hgþ2 and Cdþ2 strongly inhibited POP. Inhibition agents which is a result of exchange of cysteine (255) residue with a
of POP occured with Cuþ2, Baþ2 and Niþ2 linearly proportional to threonine residue [4].
concentrations of ions and IC50 values are respectively 3, 10,
0.16 mM. Strong inhibition of POP by Hgþ2 and Cd2þ ions attributed 3.5. Hydrolysis of gluten with recombinant POP
to linkages between these ions and imidazole ring of catalytically
active histidine [49]. Apart from their applications in biotechnology, POPs play an
Z-Pro-Prolinal is an effective POP inhibitor. Even at low con- important role in biomedical research. Diseases like Parkinson, type
centrations (10 mM), 77% of activity inhibited and IC50 value was 2 diabetes, depression or celiac disease are associated with proline-
calculated to be 70 mM. P1 functional group is important for inhi- rich proteins. Due to the cyclic structure of proline, these proteins
bition, an aldehyde group that forms hemiacetal linkage with are well protected from enzymatic degradation by typical digestive
catalytically active serine (554) residue, thus inhibiting the enzyme. enzymes.

Fig. 3. HPLC profile of gluten hydrolysates. a) gluten before hydrolysis b) gluten hydrolysate after 8 h hydrolysis (Nucleosil RP-C18; A: % 0.1 TFA, B:% 0.1 TFA:Acetonitrile;
gardient:0e20 min %5 B, 20e25 min % 40 B, 25e30 min % 50, 30e35 min % 5 B; Flow rate; 0.9 mL/min; 45
C; UV: 210 nm).
106 E. Kocadag Kocazorbaz, F. Zihnioglu / Protein Expression and Purification 129 (2017) 101e107
In this study the potential application of recombinant POP in the
field of protein hydrolysis was searched by means of gluten hy-
drolysis. Hydrolysis conditions such as, enzyme/substrate ratio and
hydrolysis time to obtain a high degree of hydrolysis was optimized
[52]. It is known that POP effectively hydrolyses peptides consists of
up to 30 residues so that to increase the effect of hydrolysis, gluten
solution (3% w/v) was thermally denatured as explained in section
2.4. The degree of hydrolysis was assayed by using TNBS method.
Optimization of E/S ratio is the most important parameter for
hydrolysis studies. Degree of hydrolysis linearly correlated with E/S
ratio up to 4.6 U/mg gluten. Optimization for incubation time of
hydrolysis carried out by testing degree of hydrolysis of samples
taken from a reaction mixture with specified time intervals up to
8 h. According to this result, the most appropriate E/S proportion is
found to be 4.6 U/mg gluten for preparation of gluten hydrolysates
with POP with 24% degree of hydrolysis.
Furthermore to check the hydrolysing ability and the peptide
complexity of the hydrolysate produced with recombinant enzyme,
RP-HPLC was used. For this purpose gluten peptide before and after
hydrolysis were dried under vacuum and dissolved in initial mobile
phase (% 0.1 TFA) for separation (Fig. 3).
Variety of peptides can be seen clearly when two chromato-
grams are compared. The intensity of some peaks decreased and
new peaks are observed after hydrolysis, showing new peptides are
released by the enzymatic hydrolysis of pop. In the context of
evaluation all data, recombinant POP has an effective hydrolysing
ability of proline rich protein. However to increase the degree of
hydrolysis and the complexity of the peptides dual enzyme diges-
tion should be suggested. On the other hand it was observed that
the peptide hydrolysate showed some biological activity (data not
shown) that further studies should be necessary for purification
structure analysis of peptides from gluten hydrolysate prepared
with recombinant purified POP.
4. Conclusions
In this study POP was expressed, purified and characterized
from Myxococcus xanthus strain, Results of the preliminary work for
the application of recombinant enzyme for gluten hydrolysis seems
promising for the production of bioactive peptides. Results of this
study can be further evaluated for the medical or industrial use of
the resulting bioactive peptides with different bioactivities.
Acknowledgements
This study was financially supported by the Ege University Sci-
entific Research Fund (Project No.: 2011/FEN/041).
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