International Journal of Biological Macromolecules 114 (2018) 1295–1304

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

A novel serine protease from strawberry (Fragaria ananassa): Purification

and biochemical characterization
Esma Hande Alici, Gulnur Arabaci ⁎
Department of Chemistry, Faculty of Science and Arts, Sakarya University, Serdivan-Sakarya 54187, Turkey

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

Article history: In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-L-tyrosine-p-amino
Received 7 March 2018 benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by
Received in revised form 21 March 2018 SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and
Accepted 27 March 2018
was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold in-
Available online 27 March 2018
crease in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 °C, respec-
Keywords:
tively. The protease was stable at a wide temperature range of 40 to 70 °C and a pH range of 3.0 to 9.0. Co2+ ions
Serine protease stimulated protease activity very strongly. Cu2+, Hg2+, Cd2+ and Mn2+ ions significantly inhibited protease
Strawberry activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the
Fragaria ananassa presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin
Purification among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also in-
Affinity chromatography vestigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this pu-
Characterization rified protease was highly-likely a serine protease and its activity was significantly affected by the presence of
metal ions.
© 2018 Published by Elsevier B.V.

1. Introduction
Nowadays, technology is growing day by day and biotechnology is
attracting great interest by the scientists because of their broad applica-
tion areas in various industries. In particular, the use of environmentally
friendly enzymes and the acquisition of their patents based on their
easy and large amount of the purification from different sources are
very important areas of biotechnological applications. It is now possible
to find and obtain these enzymes commercially because of their
commercial patent [1,2]. Among these enzyme groups, proteases
representing one of the largest groups of commercial enzymes are im-
portant commercial enzymes that accounting for 60% of total global en-
zyme sales. Due to its versatility, proteases are mainly used in the
cleaning, cosmetics, leather and textile industries as well as food pro-
cessing in the food industry [3,4].
Proteases are group of enzymes that catalyze the hydrolytic reac-
tions leading to degradation of protein molecules into peptides and
amino acids. These enzymes are common in all organisms (plants,
animals, as well as most microorganisms) [3,5]. Six protease families
⁎ Corresponding author.
E-mail addresses: ealici@sakarya.edu.tr (E.H. Alici), garabaci@sakarya.edu.tr
(G. Arabaci).
for protease enzymes were identified as cysteine proteases, serine pro-
teases, serine carboxy proteases, metallo proteases, metallo carboxy
proteases and aspartic proteases. Serine proteases are the most com-
mon family of proteases and are followed by metallo, cysteine and
aspartic proteases [3,6].
Proteases can be obtained from animals and plants, but because of
their economic, technical, biochemical and genetic advantages, the
vast majority of industrial enzymes are derived from microorganisms.
However, with industrial processes, pharmacology and biotechnology
applications, plant-derived enzymes become increasingly important
[4,7]. Despite the higher production costs compared to microbial origin
proteases, plant proteases have been used in food, drug and detergent
industries for many years. The plant proteases have some advantages
over microbial proteases especially in meat tenderization process in
terms of safety problems such as pathogenicity due to use of microbial
proteases [8]. Various commercially available cysteine proteases such
as papain, bromelain and ficin [9–11] are commonly used as additives
in the food industry for processes such as fermentation, milk precipita-
tion and meat smoothing, as well as preparation of highly soluble and
aromatic protein hydrolysates for the cheese and beverage industry,
for the production of emulsifiers. Various other plant protease applica-
tions are known such as culture medium formulation, isolation of ge-
netic material, production of essential amino acids, dehairing process
in the leather industry and prevention of clogging of wastewater sys-
tems. Proteases have an important place not only in industry but also

https://doi.org/10.1016/j.ijbiomac.2018.03.165
0141-8130/© 2018 Published by Elsevier B.V.
1296 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304

in pharmaceutical applications; plant extracts containing high amounts
of proteolytic enzymes have been used for a long time in conventional
medicine.
Protease enzymes have been identified and investigated from the
latex of various plant families such as Asclepiadaceae, Moraceae,
Asteraceae, Caricaceae, Euphorbiaceae and Apocynaceae. In recent years,
plant proteases have attracted attention due to their large substrate
specificity and activity over a wide temperature and pH range [7,12].
The discovery of new enzymes with different properties has always
been demanded for the purpose of development of various industrial
processes. Because of this great demand for new enzymes, researchers
are constantly exploring new sources of enzymes and also discovering
the different aspects of proteases [3]. Despite the widespread use of
plant-derived proteases in industry, research on the potential for prote-
ase production from a variety of fruits and vegetables is inadequate. In
order to determine new plant-derived proteases and their properties,
the protease distribution among various plant species need to be inves-
tigated [12]. From this point of view; the isolation, purification and char-
acterization of protease enzyme from strawberry fruit were performed
in this study.

2. Materials and methods

2.1. Plant material and chemicals

Fresh strawberry fruit was obtained from a local grocery at Sakarya, north Turkey and kept frozen at −20 °C. Coomassie Brilliant blue R-
250, EDTA (Ethylene diamine tetraacetic acid, disodium salt), Bromophenolblue and tri-Sodium citrate dihydrate were purchased from
Merck (Darmstadt, Germany). N,N′-methylenebisacrylamide, Tris (hydroxymethyl) aminomethane hydrochloride, and Sodium
dihydrogen phosphate monohydrate were obtained from Alfa Aesar (Thermo Fisher Scientific, Waltham, MA, USA). Ammonium Persul-
fate (APS) and TEMED (Tetramethylethylenediamine) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). All other
chemicals used were obtained from Sigma Aldrich (St. Louis, MO, USA). All the chemicals used were of analytical grade.

2.2. Preparation of crude enzyme extract

50 g of strawberry fruit that stored in the freezer was sliced thinly. Sliced fruit was homogenized with 100 mL of 0.1 M cold phosphate buffer (pH
6.0) for 5 min using a blender. The homogenate obtained was stirred at 4 °C for 30 min using a magnetic stirrer, then filtered through three layers of
cheesecloth and centrifuged at 5000 rpm for 15 min. The resulting supernatant was used as crude enzyme extract.

2.3. Purification of protease by affinity chromatography

Sepharose-4B matrix was used for the affinity gel preparation. After activation of Sepharose-4B with CNBr (Cyanogen bromide), L-tyrosine was
covalently attached. Then, the p-amino benzoic acid was diazotized and L-tyrosine clamped. While the p-amino benzoic acid forms the part to be spe-
cifically bound to the enzyme, L-tyrosine forms the spacer arm of the affinity gel. The column matrix material was slowly filled into a glass column
(1.3 × 15 cm) and incubated for a while at room temperature so that the matrix material was well collapsed and packed. After packing, the affinity
column was equilibrated with passage of the equilibration buffer (0.05 M Na3PO4 buffer containing 10% DMSO (dimethyl sulfoxide), 0.5% n-butanol,
0.1 M KCl and 1 mM EDTA, pH 6.6). After equilibration, the crude enzyme extract was loaded onto the column. After loading, the column was washed
with washing buffer (0.05 M Na3PO4 buffer containing 10% DMSO, 0.5% n-butanol and 0.1 M KCl, pH 6.6) to allow binding of the enzyme to the col-
umn matrix material and removal of other impurities. After washing, the protease enzyme was eluted by passage of the elution buffer (0.05 M
Na3PO4 buffer containing 0.5 mM MnCl2, 10% DMSO, 0.5% n-butanol, 0.1 M KCl and 1 mM EDTA, pH 6.6). The eluates were collected in fractions of
3 mL at a flow rate of 36 mL/h and the tubes were quickly moved on ice to remain cold. Fractions were assayed for protease activity and protein con-
centration. Specific activity was calculated for purity control for each purification step. For this purpose, the amount of protein was determined ac-
cording to Bradford method [13] and BSA (bovine serum albumin) was used as standard. All treatments after the column equilibration were carried
out at 4 °C. Fractions containing pure protease were pooled and stored at −20 °C. Finally, the purity of the enzyme was evaluated by SDS–PAGE
(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using 5% stacking gel and 15% resolving gel according to the method of Laemmli [14].

2.4. Protease activity assay

The activity of the strawberry protease was measured according to Takami et al., with some modifications [15]. BSA (100 mg/mL) was used as
substrate for the determination of proteolytic activity. The substrate solution was prepared by dissolving BSA in 0.1 M pH 6.0 phosphate buffer.
The sample tube contained substrate solution in buffer and enzyme solution, and the total volume of this mixture was 3 mL. The mixture of the buffer
and substrate solution was used as blank sample. The reaction starts as soon as the enzyme solution is added to the substrate solution. The sample
tubes were then immediately placed in a water bath at 40 °C and incubated for 1 h. After 1 h, 10% (w/v) TCA (trichloroacetic acid) solution was added
to the reaction mixture to stop the reaction. After 15 min of incubation at room temperature, the mixture was centrifuged at 5000 rpm for 10 min.
After filtration of the centrifugate through a filter paper, the concentration of the degraded products in the supernatant was determined by spectro-
photometric measurement. For this purpose, the supernatant was mixed with 0.4 M Na2CO3 solution and Folin-Ciocalteu reagent (diluted twice with
distilled water), and then allowed to stand at room temperature for 30 min. The intensity of the resulting blue color was determined by measuring
the absorbance at 660 nm against the blank sample. Protease activity was calculated using the standard curve of tyrosine with the following formula:

OD660
   volume of reaction mixture ðmLÞ  dilution factor
−1 −1 slope
Activity U mL min ¼
volume of enzyme solution ðmLÞ  time of reaction ð minÞ

One unit of protease activity (1 U) is defined as the amount of enzyme required to produce 1 μg of tyrosine per min at pH 6.0 and 40 °C.
 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304 1297

2.5. Enzyme kinetics

Different concentrations of BSA solution ([S]: 0.03–0.5 mM) were used in the kinetic studies performed to find the maximum velocity (Vmax) and
the Michaelis-Menten constant (Km) kinetic values of the protease. When the maximum velocity and Km values of the enzyme were determined, the
absorbance increment at 660 nm was used. The enzyme activity was measured under standard conditions and calculated as unit activity (U). The
kinetic values (Km and Vmax) were calculated by using the Lineweaver-Burk plot [16].

2.6. Determination of the specificity of purified protease towards natural substrates and a modified substrate azocasein

In order to determine the activity of the protease purified from strawberry in the presence of some natural substrates; casein, BSA, gelatin and
hemoglobin were used as substrates. Azocasein is a chemically modified protein that was designed as a substrate for proteolytic enzymes. The pro-
tease activity towards azocasein was also determined and compared with natural substrates activities. They were dissolved in 0.1 M pH 6.0 phos-
phate buffer (6 mg/mL). Substrate solution was mixed with diluted enzyme solution and the activity was determined under standard conditions.
The activity of the substrate that showed the highest activity among all substrates was accepted as 100% and the activities of the other substrates
were calculated as relative (%) activity according to this value.

2.7. Determination of the specificity of the purified protease towards p-nitroanilide (p-NA) conjugated synthetic substrates

Synthetic peptide substrates conjugated with p-nitroanilide were used for determining the specificity of the protease purified from strawberry
towards some synthetic substrates. These substrates were L-Leu-pNA, N-Suc-Ala-Ala-Ala-pNA, Nα-Benzoyl-DL-Arg-pNA (DL-BAPNA) and N-Suc-L-
Phe-pNA. The DMSO stock solution of each substrate was freshly prepared before use.
The substrate solution was mixed with pH 6.0 phosphate buffer solution (0.1 M) and pre-incubated for 10 min at 40 °C. At the end of the incuba-
tion, the enzyme solution was added to the reaction mixture. After that the reaction mixture was incubated at 40 °C for 20 min. Phosphate
buffer solution (pH 6.0, 0.1 M) was used instead of the enzyme solution for blank sample preparation. After 20 min, acetic acid solution (0.35 M)
was added to the reaction mixture to terminate the reaction. The intensity of the resulting yellow color was determined by measuring the absor-
bance against the blank sample at 410 nm. The protease activity was calculated by using the following formula (Ԑ: molar absorption coefficient,
9780 M−1 cm−1):

volume of reaction mixture ðmLÞ  absorbance

Activity ðUÞ ¼  
volume of enzyme solution ðmLÞ  Ԑ M−1 cm−1  optical path length ðcmÞ  time ð minÞ

One unit of protease activity (1 U) is defined as the amount of enzyme required to produce 1 μmol of p-NA per min at pH 6.0 and 40 °C.

2.8. Determination of optimum temperature of protease

So that we could determine the optimum temperature of the strawberry protease, enzyme activity was measured at ten different temperature
values (30–75 °C). For this purpose, the BSA solution (6 mg/mL) prepared in 0.1 M pH 6.0 phosphate buffer was mixed with diluted enzyme solution
and allowed to incubate for 1 h in a 30–75 °C water-bath. The activity was determined under standard conditions for each temperature value. The
highest activity value was accepted as 100% and the other activity values were calculated as the relative activity (%) according to the highest activity
value. The optimum temperature graph was obtained by plotting the relative activity (%) value calculated for each temperature versus related tem-
perature (Fig. 1).

2.9. Determination of optimum pH of protease

So as to determine the optimum pH of the strawberry protease, enzyme activity was measured at seven different pH values (pH 3.0–9.0). Citrate
buffers were used for pH 3.0–5.0; phosphate buffers were used for pH 6.0–7.0; Tris-HCl buffers were used for pH 8.0–9.0. The BSA solution (6 mg/mL,
in distilled water) was mixed with diluted enzyme solution and related buffer (reaction total volume: 3 mL) and allowed to incubate for 1 h in a 40 °C
water-bath. The activity was determined under standard conditions for each pH value. The highest activity value was accepted as 100% and the other
activity values were calculated as the relative activity (%) according to the highest activity value. The optimum pH graph was obtained by plotting the
relative activity (%) value calculated for each pH versus related pH (Fig. 2).

2.10. Determination of thermal stability of protease

For the purpose of determining the thermal stability of the purified enzyme, the diluted enzyme solution was incubated at a temperature value
ranging from 40 to 70 °C for a length of time and a constant volume of incubated enzyme was removed. After that the residual activity (%) of the en-
zyme was determined under standard conditions. The residual activity calculated for each temperature was plotted versus related temperature and
the thermal stability profile of the enzyme was determined.
The enzyme solution was allowed to incubate at 40 °C for 3 h, 50 °C for 2 h, 60 °C for 1 h and 70 °C for 30 min and the residual activity was deter-
mined by removing a constant volume of incubated enzyme every 60 min for 40 °C, 30 min for 50 °C, 15 min for 60 °C, 5 min for 70 °C for thermal
stability test.

2.11. Determination of pH stability of protease

The enzyme solution was incubated in different buffers at various pH values in the range of 3.0–9.0 and then the residual activity (%) was mea-
sured for each pH for determination of the pH stability of protease enzyme purified from strawberry. The buffers used in the experiment were
1298 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304

Fig. 1. (A) Purification profile of the serine protease from strawberry by affinity chromatography on a sepharose-4B-L-tyrosine-p-aminobenzoic acid affinity column. (B) Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis of strawberry protease (Fragaria ananassa) (Lane M, molecular weight marker; Lane P, purified enzyme eluate).

described in Section 2.9. For this purpose, the diluted enzyme solution and the buffer solution were mixed pre-incubated for 3 h at room temperature.
At the end of 3 h, the activity determination for each pH was carried out under standard conditions. The enzyme activity that was not incubated was
accepted as 100% and the activity measured after incubation was calculated as the residual activity (%) for each pH.

2.12. Effects of some metal ions on purified protease activity

This assay was conducted to examine the activity of protease purified from strawberry in the presence of some metal ions. The monovalent metal
ions (K+ and Na+), divalent metal ions (Ca2+, Co2+, Cd2+, Mn2+, Pb2+, Hg2+, Cu2+, Ni2+) and also Fe3+ and Al3+ ions were used for this purpose.
The sulfate salt of Ni2+, the nitrate salt of Al3+, the chloride salt of all other metals were dissolved in 0.1 M pH 6.0 phosphate buffer to give final con-
centrations of 2 and 5 mM.
The diluted enzyme solution was mixed with the prepared metal solutions and allowed to incubate for 1 h at room temperature. A control sample
contained only enzyme solution was incubated under the same conditions. The activity determination after incubation was carried out under stan-
dard conditions. So as to calculate the enzyme activity after interaction with the metal ions, the activity of the control sample was accepted as 100%
and the activities of the other samples were calculated relative to the activity of the control sample and finally the residual activity (%) of the enzyme
was calculated for each metal ion.

2.13. Effects of some organic solvents on purified protease stability

This assay was performed to examine the stability of the protease purified from strawberry in the presence of some organic solvents. DMSO, eth-
anol, methanol, acetone, acetonitrile, ethyl acetate and 2-propanol were used as organic solvents. In the experiment, each organic solvent was
 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304 1299

B
Fig. 2. (A) Optimum temperature of the strawberry (Fragaria ananassa) protease: Effect of temperature on purified serine protease activity. (B) Thermal stability results of the strawberry
(Fragaria ananassa) protease: Effect of temperature on purified serine protease stability.

incubated with diluted enzyme solution for 1 h at 40 °C. A sample without organic solvent was incubated under the same conditions as the control
experiment. After incubation, the enzyme activity was determined under standard conditions. The activity of the control sample was assumed to be
100% and the residual activities (%) of other samples were calculated accordingly.

2.14. Effects of some inhibitors on purified protease activity

In an attempt to determine which group is involved in the catalytic domain of the protease purified from the strawberry fruit, the enzyme
was treated with serine protease inhibitors Phenylmethylsulfonyl fluoride (PMSF, 1 and 5 mM), tosyl-L-lysine chloromethyl ketone (TLCK, 1 and
3 mM) and tosyl-L-phenylalanine chloromethyl ketone (TPCK, 1 and 3 mM); the metalloprotease inhibitors EDTA (1 and 5 mM) and 1,10-
1300 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304

Table 1
Overall purification of the strawberry protease by affinity chromatography.

Step V (mL) Total activity (U) Total protein (mg) Specific activity (U/mg) Fold purification Activity yield (%)

Crude extract 100 3800 ± 14.14 417.5 ± 2.69 9.1 ± 0.02 1 100
Pure protease 15 1458 ± 1.41 0.405 ± 0.002 3600 ± 15.27 395.6 38.37
⁎One unit of protease activity (1 U) is defined as the amount of enzyme required to produce 1 μg of tyrosine per min at pH 6.0 and 40 °C.
a
± value is standard deviation. All experiments were conducted in triplicate.

phenanthroline (1 and 5 mM); the cysteine protease inhibitor iodoacetamide (IAA, 1 and 5 mM) and the aspartic protease inhibitor Pepstatin A (1
and 5 mM). Stock solutions of EDTA and IAA inhibitors were prepared in 0.1 M pH 6.0 phosphate buffer solution, stock solutions of TLCK and TPCK
inhibitors were prepared in methanol, stock solutions of PMSF and 1,10-phenanthroline inhibitors were prepared in pure ethanol and stock solution
of Pepstatin A was prepared in DMSO. In the inhibitor effect study, the enzyme solution was mixed with the prepared inhibitor solutions and incu-
bated at 40 °C for 1 h. The control sample without inhibitor (for each solvent) was also incubated under the same conditions and after incubation the
activity was determined under standard conditions. The activity of the control sample was accepted as 100% and the activities of other samples were
calculated as the residual activity (%).

2.15. Statistical analysis

All experiments were conducted in triplicate. All results were expressed as the average ± standard deviation of the measurements. Excel
(Microsoft Co, Redmond, WA, USA) was used to calculate the standard deviation.

3. Results and discussion et al. found a Km value of 330.4 mg/L (5 × 10−3 mM) and a Vmax value of
2.539 mg BSA/L.min of the Bromelain for BSA substrate in the kinetic
3.1. Protease purification and determination of molecular weight study that they performed [23].

The protease that was isolated from strawberry fruit was purified by
using the Sepharose-4B-L-tyrosine-p-amino benzoic acid affinity chro-
matography one step procedure (Fig. 1A). The p-amino benzoic acid
constitutes the part to be specifically bound to the enzyme while tyro-
sine forms the extension arm of the affinity gel. The advantage of the
method used was that the enzyme could be purified in one step depend-
ing on the specificity of the enzyme. The disadvantage was that a very
low amount of protein could be obtained. Table 1 summarizes the puri-
fication results of the strawberry protease. The protease was eluted
from the column with 38.37% recovery and 395.6-fold increase in spe-
cific activity. The 38.35 kDa Aeribacillus pallidus C10 protease is a serine
protease purified by affinity chromatography with a 19.56% recovery
[17]. In addition, Tenodera sinensis (Chinese mantis) serine protease
was purified by affinity chromatography with 5120-fold increase in spe-
cific activity [18].
The purity of the protease eluted from the affinity column was
checked using SDS-PAGE method and the molecular weight was calcu-
lated by comparing with the protein marker. After destaining, a single
band was observed on the gel and it was concluded that the enzyme
had monomeric character. The molecular weight of the strawberry pro-
tease was determined to be 65.8 kDa (Fig. 1B). Some serine proteases
possess close molecular weight to strawberry protease have also been
reported [19,20]. Serine protease sources that have larger molecular
weight (Mytella charruana, 83.1 kDa, dark-induced senescent wheat
leaves, 98.9 kDa) are also available in the literature [21,22].
3.2. Kinetic parameters of BSA hydrolysis of protease purified from
strawberry
3.3. The specificity results of strawberry protease towards natural and
modified protein substrates
Activity of protease purified from strawberry fruit was determined
in the presence of casein, hemoglobin, BSA, gelatin, and azocasein pro-
tein substrates. The enzyme reacted with all the substrates tested. It is
determined that the highest protease activity was obtained with hemo-
globin and the lowest protease activity was obtained with casein
(59.3%) among all protein substrates tested (Table 2). The serine prote-
ase purified from the leaves of Abrus precitorius was also tested for spec-
ificity towards natural substrates. The same natural substrates were
used to determine the specificity, only azocasein modified substrate
was not tested in that study. According to that study's findings, the
Abrus precitorius serine protease showed activity with all tested natural
substrates, but the specificity results were nearly the opposite of the
findings of strawberry protease in this study (except gelatin) [24]. A hal-
ophilic protease from Bacillus sp. EMB9 demonstrated similar natural
substrate specificity results, except casein. The highest protease activity
was obtained with casein contrary to this study's results [25].
3.4. Specificity results of strawberry protease towards p-nitroanilide
(p-NA) conjugate synthetic substrates
Synthetic peptide substrates conjugated with p-NA (final concentra-
tion: 5 mM) were also used to determine the substrate specificity of the
strawberry protease. The specificity of the strawberry protease towards
p-NA conjugated synthetic substrates was given in (Table 2). Straw-
berry protease reacted with all synthetic substrates tested. The highest

In this experiment, protease reaction velocities were measured Table 2

under standard conditions with the aim of determining the kinetic pa- Substrate specificity of the strawberry protease.
rameters (Km and Vmax) of pure strawberry protease for BSA hydrolysis Protein Relative activity Synthetic peptide Relative activity
by using the Lineweaver-Burk plot. BSA substrate solution was used substrate (%)a substrate (%)a
in the concentration range of 0.0304 mM and 0.532 mM. The Hemoglobin 100 N-Suc-L-Phe-pNA 100
Lineweaver-Burk plot was created by plotting the reciprocal values of Azocasein 84.6 ± 0.65 Nα-Benzoyl-DL-Arg-pNA 82.0 ± 0.33
the measured velocities (1/V) against the reciprocal values of the initial BSA 76.1 ± 0.42 N-Suc-Ala-Ala-Ala-pNA 61.5 ± 0.87
substrate concentrations (1/[S]). The Km and Vmax values of the Gelatin 63.8 ± 0.91 L-Leu-pNA 58.7 ± 0.51
pure strawberry protease for BSA at 40 °C and pH 6.0 were found to Casein 59.3 ± 1.01

be 0.4 mM and 3333.3 μmol tyrosine mL−1 min−1, respectively. Ferreira a

±value is standard deviation. All experiments were conducted in triplicate.
 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304
synthetic substrate specificity of the protease was observed towards
N-Suc-L-Phe-pNA followed by Nα-Benzoyl-DL-Arg-pNA (DL-BAPNA)
substrate (Table 2). The higher protease activity with N-Suc-L-Phe-
pNA and Nα-Benzoyl-DL-Arg-pNA (DL-BAPNA) substrates shows that
the strawberry protease specifically prefers Phe- and Arg-residues
for hydrolysis, respectively. Singh et al. reported that a serine protease,
Crinumin, showed activity towards N-Suc-L-Phe-pNA and L-Leu-pNA
similarly but did not show activity towards Nα-Benzoyl-DL-Arg-pNA
and N-Suc-Ala-Ala-Ala-pNA [20]. Strawberry protease exhibited
broader substrate specificity than Crinumin. In contrast to strawberry
protease, another serine protease, Wrightin, did not show amidolytic ac-
tivity towards N-Suc-L-Phe-pNA, Nα-Benzoyl-DL-Arg-pNA and L-Leu-
pNA substrates, it preferred only -Ala residues for hydrolysis [26].
3.5. Optimum temperature and thermal stability results of the strawberry
protease
So as to determine the temperature at which the protease showed
optimum activity, the protease activity was measured at various tem-
peratures (the temperature range: 30–75 °C). According to the test re-
sults the enzyme showed activity at all of the tested temperature
values, and the maximum activity was obtained at 40 °C (Fig. 2A).
Other serine protease enzymes with an optimum temperature of 40 °C
have been reported in the literature [21,24,27–29].
The strawberry protease was allowed to incubate at temperatures
between 40 and 70 °C as described in Section 2.10 with the intention
of investigating the thermal stability of the pure protease. It was deter-
mined that the enzyme didn't show significant activity loss at the end of
the 1 h of incubation, a slight decrease in activity was determined after
2 h of incubation, and the protease activity was reduced by half after 3 h
of incubation at 40 °C. In addition to this, it was observed that the en-
zyme retained almost completely its activity after 30 min of incubation
and lost 79% of its activity after 120 min of incubation at 50 °C. On the
other hand, at 60 °C, the enzyme lost 20% of its activity at the end of
the 15 min of incubation and lost more than half of its activity during
the longer incubation period. Finally, it is an important finding that
the protease retained 70% of its activity at the end of 15 min of incuba-
tion and still retained 48% of its activity after 30 min of incubation at a
high temperature value such as 70 °C (Fig. 2B). According to the thermal
stability results of the study that was performed by Hemici et al., the
Fig. 3. pH profile of strawberry protease (Fragaria ananassa): Effect of pH on purified serine protease activity and stability.
1301
strawberry protease was much more stable at 50 and 60 °C than the cys-
teine protease purified from Fasciola hepatica [30]. Also, the results
of the thermal stability of Aeribacillus pallidus C10 serine protease at
40 °C were similar to those of the strawberry protease at 40 °C, but at
higher temperature values the Aeribacillus pallidus C10 serine protease
was more stable [17].
3.6. The pH profile of the strawberry protease
The pH effect on the strawberry protease activity was determined in
the range of pH 3.0 to 9.0. The enzyme exhibited activity at all of the
tested pH values. Enzyme showed the highest activity at pH 6.0
(100%) and the lowest activity at pH 9.0 (85.25%) (Fig. 3). Other prote-
ase enzymes with mildly acidic or neutral optimum pH values were re-
ported in the literature [31–34]. Unlike the strawberry protease, the
activity of the dark-induced senescent wheat leaves protease was mark-
edly reduced at pH 4.0 and lower pH values, as well as at pH 8.0 and
higher pH values [22].
According to the results obtained from the stability studies of the pu-
rified enzyme, it was observed that when the enzyme was incubated for
3 h with buffers of pH 3.0–9.0, it retained most of its activity at all pH
values (Fig. 3). The maximum activity loss was calculated as 18.7% at
pH 9.0 compared to the control at the end of the incubation period. As
a result, it can be said that the stability of the strawberry protease is
very high in a wide pH range, which is a great advantage in terms of pos-
sible industrial applications. The strawberry protease gave similar re-
sults with Phanerochaete chrysosporium aspartic peptidase, Mytella
charruana serine protease and Aeribacillus pallidus C10 serine protease
in terms of pH stability [17,21,35]. The pH stability of the strawberry
protease was much higher than that of the Abrus precitorius serine pro-
tease [24].
3.7. Effects of some metal ions on strawberry protease activity
In this experiment, the effect of metal ions on the activity of pure
protease was investigated. It has been determined that Co2+ increased
the enzyme activity among all the metal ions tested and all other metals
reduced the enzyme activity. Since Co2+ increased the enzyme activity
by 172% at 2 mM and by 237% at 5 mM. It can be said that Co2+ strongly
activated the strawberry protease activity. However, Cu2+, Hg2+, Cd2+
1302 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304

and Mn2+ ions were found to strongly inhibit the strawberry protease.
In particular, Cu2+ and Mn2+ ions completely inhibited the enzyme ac-
tivity at 5 mM. The toxic heavy metals Hg2+ and Cd2+ also reduced the
enzyme activity to 23% and 36%, respectively. While monovalent metal
ions reduced the enzyme activity to about 60%, Al+3 reduced the en-
zyme activity to 74% and Fe+3 reduced the enzyme activity to 49%
(Fig. 4A). Similar to the results of this work, Cu2+ ions produced a signif-
icant inhibitory effect on the alkaline protease purified from Micrococcus
sp. In addition, the results obtained with Ca2+ and Ni2+ ions were sim-
ilar to those of strawberry protease metal effect. On the contrary, Mn2+
activated Micrococcus sp. protease [36]. While Cd2+, Cu2+ and Mn2+
ions strongly inhibited the serine protease which was purified from
Bacillus sp. by Suwannaphan et al., Co2+ ions that strongly activated
strawberry protease in this study significantly reduced the activity of
this enzyme [37]. According to the report of Yadav et al., Aspergillus
flavus MTCC 9952 serine protease was completely inhibited by Cu2+
and Hg2+ ions, but unlike strawberry protease, Ca2+ and Ni2+ ions in-
creased protease activity [38]. The Cu2+ and Hg2+ ions showed similar
effects on the serine protease purified from Bacillus sp., however Co2+
ions strongly inhibited the enzyme [39].

B
Fig. 4. (A) Effect of metal ions on the strawberry (Fragaria ananassa) serine protease activity. (B) Effect of organic solvents on the strawberry (Fragaria ananassa) serine protease activity.
Protease activity was detected in the presence of 30% organic solvent.
 E.H. Alici, G. Arabaci / International Journal of Biological Macromolecules 114 (2018) 1295–1304
3.8. Effects of some organic solvents on the stability of strawberry protease
In this study, it was investigated to what extent strawberry protease
could retain its activity when incubated with various organic solvents.
All solvents tested reduced the enzyme activity. Among all of them,
the solvents which had the least effect on the enzyme activity were eth-
anol and methanol. The enzyme was more stable to these solvents. On
the other hand, 2-propanol completely inhibited the enzyme activity.
Activity measurements with ethyl acetate, acetonitrile, acetone and
DMSO solvents gave similar results with each other. When the results
are examined, it can be said that the stability of the strawberry protease
was at an average level in the presence of organic solvents (Fig. 4B). The
stability results of the alkaline protease purified from Aspergillus oryzae
CH93 showed similarity in the presence of DMSO. In the presence of the
2-propanol solvent, stability of this enzyme was higher than that of the
strawberry protease [40]. The alkaline protease purified by Enling et al.
from Micrococcus sp. gave similar results to the strawberry protease in
the presence of 2-propanol. This enzyme was more resistant to DMSO
than the strawberry protease. Also, the strawberry protease was more
resistant to ethanol and methanol [36]. The stability of Aeribacillus
pallidus C10 protease, another serine protease, in the presence of or-
ganic solvents was generally higher than that of strawberry protease
[17]. According to the results of Wang et al.'s work, there was a consid-
erable decrease in the activity of serine protease purified from dark-
induced senescent wheat leaves in the presence of methanol while
maintaining activity in the presence of ethanol and DMSO [22].
3.9. Effects of some inhibitors on the strawberry protease activity
Serine protease inhibitors, TPCK, TLCK and PMSF; aspartic protease
inhibitor Pepstatin A; cysteine protease inhibitor IAA and metallo-
protease inhibitors EDTA and 1,10-phenanthroline active site-directed
irreversible inhibitors' inhibition effects on strawberry protease were
investigated and given as the remaining (%) enzyme activities in
Table 3. Among these inhibitors, it was found that serine protease inhib-
itors strongly inhibited the strawberry protease and even TPCK
completely inhibited the enzyme activity at 3 mM. It has been shown
that Pepstatin A did not inhibit the enzyme activity at 1 mM, while it
caused an inhibitory effect that is negligible at 5 mM. Metalloprotease
inhibitors did not inhibit the enzyme at 1 mM, but the protease activity
was reduced by half at 5 mM concentration level. The IAA had consider-
able inhibitory effect on the strawberry protease at both 1 and 5 mM.
These results suggest that strawberry protease could be a serine prote-
ase and is generally affected by the presence of active site-directed irre-
versible inhibitors. Similarly, a serine protease purified from Bacillus sp.
was strongly inhibited by both PMSF and EDTA, while the inhibition ef-
fect by 1,10-phenanthroline was lower [37]. In addition, a serine
Table 3
Effect of some inhibitors on the activity of the strawberry protease.
Inhibitor Concentration (mM) Residual activity (%)a
Control 0 100
PMSF 1 83.5 ± 0.77
PMSF 5 30.7 ± 0.45
TPCK 1 10.5 ± 0.59
TPCK 3 0
TLCK 1 101.1 ± 1.22
TLCK 3 24.7 ± 0.88
EDTA 1 101.2 ± 1.04
EDTA 5 50.8 ± 0.95
1,10-PHENANTHROLINE 1 124.9 ± 0.56
1,10-PHENANTHROLINE 5 48.0 ± 0.73
IAA 1 66.8 ± 0.23
IAA 5 54.5 ± 0.39
PEPSTATIN A 1 124.1 ± 0.95
PEPSTATIN A 5 95.9 ± 0.87
a
±value is standard deviation. All experiments were conducted in triplicate.
1303
protease purified from dark-induced senescent wheat leaves by Wang
et al. was strongly inhibited by PMSF and EGTA [22]. Besides, according
to the reports of Enling et al., Micrococcus sp. alkaline protease was also
highly influenced by the presence of both serine and metalloprotease
inhibitors. Although the enzyme was described as serine protease,
EDTA showed higher inhibitory effect than PMSF [36].
4. Conclusion
A protease enzyme (65.8 kDa) was firstly purified and characterized
from the strawberry fruit. Purification of the protease from strawberry
by affinity chromatography resulted in a 395.6-fold increase in specific
activity. The measurable activity of the strawberry protease in the pres-
ence of all tested protein substrates, and synthetic substrates, indicates
that the enzyme has broad substrate specificity. Increment of protease
activity by Co2+ ions by 172% at 2 mM and by 237% at 5 mM is a rare
and important finding about proteases. The strong activation of the
protease by Co2+ ions and also the apparent inhibitions by EDTA and
1,10-phenanthroline suggest that the strawberry protease might be a
metalloprotease. However, it can be concluded that the purified straw-
berry protease enzyme could be a serine protease due to its broad sub-
strate specificity and strongly inhibition by PMSF, TLCK and TPCK.
Maintaining almost half of its activity even at high temperatures such
as 60 and 70 °C, and stability at a wide pH range are promising for var-
ious industrial applications of the strawberry protease.
Acknowledgement
This work was supported by the Sakarya University research founda-
tion under Grant (FBYLTEZ (2017-50-02-029)).
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