International Journal of Biological Macromolecules 133 (2019) 971–979

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International Journal of Biological Macromolecules

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

Anionic trypsin from the spleen of albacore tuna (Thunnus alalunga):

Purification, biochemical properties and its application for proteolytic
degradation of fish muscle
Tanchanok Poonsin a, Benjamin K. Simpson b, Soottawat Benjakul c, Wonnop Visessanguan d, Asami Yoshida e,
Kyoshi Osatomi e, Sappasith Klomklao f,⁎
a
Biotechnology Program, Faculty of Agro- and Bio-Industry, Thaksin University, Phatthalung Campus, Phatthalung 93210, Thailand
b
Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec H9X 3V9, Canada
c
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
d
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
e
Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan
f
Department of Food Science and Technology, Faculty of Agro- and Bio-Industry, Thaksin University, Phatthalung Campus, Phatthalung 93210, Thailand

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

Article history: Anionic trypsin from albacore tuna spleen was purified by chromatographic separations on Q-Sepharose,
Received 17 November 2018 Superdex 75 and Arginine Sepharose 4B. The trypsin migrated as single bands in both SDS-PAGE and native-
Received in revised form 16 April 2019 PAGE. The molecular weight of purified trypsin was estimated to be 30 kDa using SDS-PAGE. The enzyme exhib-
Accepted 16 April 2019
ited maximal activity at pH 9.0 and 55 °C for hydrolysis of Boc-Val-Pro-Arg-MCA. pH and temperature stabilities
Available online 24 April 2019
of the trypsin were well maintained in the pH range of 6–11 and over a temperature range from 20 up to 50 °C.
Keywords:
The enzyme was effectively inhibited by soybean trypsin inhibitor, N tosyl L phenyl alanine chloromethyl ketone
Purification (TLCK) and Pefabloc SC. The N-terminal amino acid sequence of 20 residues of the purified enzyme was
Anionic trypsin IVGGYECQAHSQPHQVSLNA, which is highly homologous to other fish trypsins. The kcat/Km of the enzyme for
Albacore tuna Boc-Val-Pro-Arg-MCA was 2.60 ± 0.07 s−1 mM−1. Purified trypsin also hydrolysed fish muscle proteins, suggest-
Spleen ing its effectiveness in degradation of food proteins.
Degradation © 2019 Elsevier B.V. All rights reserved.

1. Introduction
Fish viscera are a source of digestive enzymes that may have some
unique properties of interest to both basic research and industrial appli-
cations [1]. Especially, proteinases from fish often have high activity
over a wide range of pH and temperature conditions. These characteris-
tics have made them suitable for several industrial applications, e.g., in
the detergent, food, pharmaceutical and agrochemical industries [2].
Moreover, fish proteinases are inactivated at relatively low tempera-
tures, which make such enzymes potentially useful in food applications
where ready and rapid denaturation is desirable [3].
In the digestive tract of fish, one of the main proteinases is trypsin
(EC 3.4.21.4), a serine proteinase that specifically hydrolyses peptide
bonds at the carboxyl sides of lysine and arginine residues. This enzyme
plays a key role in the digestion of dietary proteins and is also responsi-
ble for the activation of trypsinogen and other zymogens. Trypsin has
been used increasingly because it is both stable and active under
harsh conditions [4]. Due to its potential industrial usage, trypsins
⁎ Corresponding author.
E-mail address: sappasith@tsu.ac.th (S. Klomklao).
have been isolated and characterized from various species of fish, in-
cluding brownstripe red snapper (Lutjanus vitta) [1], Tunisian barbel
(Barbus callensis) [5], vermiculated sailfin catfish (Pterygoplichthys
disjunctivus, Weber, 1991) [6], carp (Cirrhinus mrigala) [7] and golden
grey mullet (Liza aurata) [8].
Albacore tuna (Thunnus alalunga) is one of the fish species used as
raw materials for production of canned tuna in Thailand [9]. During pro-
cessing, a large amount of viscera is generated as discards. Therefore,
the use of viscera as the source of proteinase should maximize the utili-
zation of this by-product from this species and reduce its dumping into
the environment to pollute it. Among all viscera, spleen has been re-
ported as an excellent source of trypsin [10].
Fish trypsins have been reported to be active in hydrolysis of pro-
teinaceous substrates. For instance, Cai et al. [11] reported that trypsin
from hepatopancreas of Japanese sea bass (Lateolabrax japonicus) was
active in hydrolyzing myofibrillar proteins of shrimp. Trypsin from he-
patopancreas of freshwater prawn (Macrobrachium rosenbergii) could
hydrolyse collagen from yellowtail (Seriola quinqueradiata) [12].
Poonsin et al. [9] also reported that trypsin from albacore tuna actively
hydrolysed proteins in Pacific white shrimp (Litopenaeus vannamei)
shells. To fully utilize the fishery resources, digestive proteinase

https://doi.org/10.1016/j.ijbiomac.2019.04.122
0141-8130/© 2019 Elsevier B.V. All rights reserved.
972 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979
(e.g., trypsin from albacore tuna spleen) could serve as an aid for a wide
range of industrial applications, e.g., in the food, detergent, leather and
pharmaceutical industries. However, the molecular and the biochemical
properties of trypsin in the spleen of albacore tuna still remain un-
known. Furthermore, although trypsin or trypsin-like serine proteinases
have been purified from different species of fish as mentioned above,
very little information regarding their digestive effect on food protein
has been reported. Thus, the aims of this study were to purify and char-
acterise trypsin from the spleen of albacore tuna and obtain the infor-
mation about their physicochemical properties and to investigate its
capacity to hydrolyse muscle proteins from threadfin bream
(Nemipterus virgatus).
2. Materials and methods
2.1. Chemicals
Q-Sepharose, Superdex 75 and Arginine Sepharose 4B were
purchased from GE Healthcare Bio-sciences AB (Uppsala, Sweden). t-
Butyloxycarbonyl-Val-Pro-Arg-4-methyl-coumaryl-7-amide (Boc-Val-
Pro-Arg-MCA) and other synthetic fluorogenic peptide substrates
(Boc-Phe-Ser-Arg-MCA, Boc-Leu-Lys-Arg-MCA, Boc-Gln-Arg-Arg-MCA,
Boc-Leu-Ser-Thr-Arg-MCA, Boc-Ile-Glu-Gly-Arg-MCA, Boc-Glu-Lys-
Lys-MCA, Suc-Leu-Leu-Val-Tyr-MCA, Suc-Ala-Ala-Pro-Phe-MCA, Z-
Phe-Arg-MCA, Z-Arg-Arg-MCA, Arg-MCA) were purchased from Pep-
tide Institute (Osaka, Japan). Soybean trypsin inhibitor, ethylenedi-
aminetetraacetic acid (EDTA), N p tosyl L lysine chloromethyl ketone
(TLCK), N tosyl L phenyl alanine chloromethyl ketone (TPCK), pepstatin
A, 1 (L trans epoxysuccinyl leucylamino) 4 guanidinobutane (E-64),
β mercaptoethanol (βME), bovine serum albumin and wide range mo-
lecular weight markers were procured from Sigma Chemical Co. (St.
Louis, MO, USA.). N,N,N′,N′ tetramethyl ethylenediamine (TEMED) and
Coomassie Brilliant Blue R-250 were purchased from Bio-Rad Laborato-
ries (Hercules, CA, USA). Pefabloc SC, Folin-Ciocalteu's phenol reagent,
tris (hydroxymethyl) aminomethane and sodium dodecyl sulfate
(SDS) were obtained from Merck (Darmstadt, Germany). Other
chemicals were all of analytical grade.
2.2. Preparation of fish sample and splenic extract
Albacore tuna internal organs were obtained from Tropical Canning
(Thailand) Public Co. Ltd., Hat Yai, Songkhla. Those samples kept in ice
with sample/ice ratio of 1:2 (w/w) were transported to the Department
of Food Science and Technology, Thaksin University, Phatthalung within
2 h. Only spleen was separated and collected. The defatted spleen pow-
der from albacore tuna was prepared according to the method of
Poonsin et al. [9] and used for trypsin extraction.
According to the method of Poonsin et al. [9], the trypsin extraction
was first prepared with a slight modification. Defatted spleen powder
was suspended in a “starting buffer” (SB) of 50 mM Tris-HCl containing
5 mM CaCl2 at a pH of 7.5 at a ratio of 1:10 (w/v) and stirred continu-
ously at 4 °C for 3 h. Then, using a Kubota Model 7780II centrifuge
(Tokyo, Japan), the suspension was centrifuged at 12,000g for 10 min
at 4 °C. Finally, for the remainder of the experiment, the supernatant
was referred to as “splenic extract.”
2.3. Purification of anionic trypsin from albacore tuna spleen
The splenic extract was purified using a series of chromatographies,
including ion-exchange column, gel filtration column and affinity col-
umn. All steps of purification were carried out (4 °C) in a walk-in cold
room. Fractions obtained from all steps of purification were subjected
to the determination of trypsin activity and protein content. Purify of
trypsin was examined using native-PAGE and SDS-PAGE.
2.3.1. Q-Sepharose anion exchange chromatography
The splenic extract was first applied to Q-Sepharose anion exchange
column (2.6 × 45 cm) pre-equilibrated with approximately two-bed
volumes of SB. Then, the column was washed with a flow rate of
0.5 mL/min using SB until A280 was b0.05. A linear gradient of
0.0–0.6 M NaCl in SB at a flow rate of 1.0 mL/min was used for elution.
Fractions of 5 mL/tube were collected and the fractions displaying tryp-
sin activity were pooled and concentrated by ultrafiltration with
Vivaspin turbo 15 (Sartorius stedim biotech, Goettingen, Germany).
2.3.2. Superdex 75 gel filtration chromatography
A Superdex 75 column (2.6 × 60 cm) was equilibrated with two-bed
volumes of 50 mM Tris-HCl containing 0.15 M NaCl at a pH of 7.0. Then,
the Q-Sepharose fractions concentrated using ultrafiltration were fil-
tered with a 0.2-μm filter and loaded to the column. The column was
eluted with the same buffer at a flow rate of 0.5 mL/min. Finally, frac-
tions were collected at a volume of 5 mL/tube and those with trypsin ac-
tivity were pooled, concentrated using ultrafiltration with Vivaspin
turbo 15 and further purified.
2.3.3. Arginine Sepharose 4B affinity chromatography
The pooled fractions with Boc-Val-Pro-Arg-MCA activity from
Superdex 75 column chromatography were chromatographed on an Ar-
ginine Sepharose 4B column (1 × 20 cm), which was equilibrated with
50 mM Tris-HCl at a pH of 8.8. Then, the same buffer at a flow rate of
0.2 mL/min was used for washing off the unbound protein in the col-
umn. When A280 was b0.05, the adsorbed protein was eluted using a lin-
ear gradient of KCl from 0 to 1.0 M at a flow rate of 0.2 mL/min. The
fractions of 2 mL were collected and the fractions with trypsin activity
were pooled and dialysed with SB for 12 h and used for further study.
2.4. Assay of trypsin activity
Trypsin activity was measured according to the method of Sriket
et al. [12]. An aliquot of enzyme solution was mixed with 100 μL of 50
μM fluorogenic substrate (Boc-Val-Pro-Arg-MCA) and 800 μL of buffer
(50 mM glycine-NaOH, pH 9.0, containing 5 mM CaCl2) in a total volume
of 1.0 mL, then incubated at 55 °C for 10 min. After addition of 1.5 mL of
the stopping solution (distilled water:methyl alcohol:n-butyl alcohol =
7:6:7, v/v), the liberated 7 amino 4 methylcoumarin (AMC) was mea-
sured using a fluorescence spectrophotometer (RF-1500, Shimadzu,
Kyoto, Japan) at an excitation wavelength of 380 nm and emission
wavelength of 470 nm. One unit of enzyme activity was defined as the
amount of the activity releasing 1 μmol of AMC per minute.
2.5. Measurement of protein concentration
Protein concentration was measured according to the method of
Lowry et al. [13] using bovine serum albumin as the standard. The pro-
tein content during purification steps was determined by monitoring
the absorbance at 280 nm.
2.6. Polyacrylamide gel electrophoresis
SDS-PAGE was performed according to the method of Laemmli [14].
Solutions of protein were mixed at 1:1 (v/v) ratio with sample buffer
(0.125 M Tris-HCl, pH 6.8, containing 4% (w/v) SDS and 20% (v/v) glyc-
erol) with or without 10% βME and boiled for 3 min. The samples at 10
μg protein concentration were loaded on the gel made of 4% stacking
and 12.5% separating gels. The electrophoresis was run at a constant
current of 15 mA per gel by a Mini-Protein II Cell apparatus (Atto Co.,
Tokyo, Japan). After the run, the gel was stained with 0.1% (w/v)
Coomassie Brilliant Blue R-250 in 50% (v/v) methanol and 10% (v/v)
acetic acid and destained in 7.5% (v/v) acetic acid and 50% (v/v)
methanol.
 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979
Native-PAGE was carried out similar to the procedure of SDS-PAGE,
except that the samples were not heated and were without addition of
SDS and reducing agents (βME) in both the samples and the gels.
2.7. Determination of N-terminal amino acid sequence
N-terminal amino acid sequence was performed according to the
method of Cai et al. [11]. For this, the purified anionic trypsin was ap-
plied to SDS-PAGE, and transferred to a polyvinylidene difluoride
(PVDF) membrane. After brief staining with Coomassie Brilliant Blue
R-250, the protein bands were excised, destained, and analysed by
PPSQ-33A protein sequencer (Shimadzu, Model PPSQ-33A. Kyoto,
Japan).
2.8. Biochemical properties
2.8.1. pH and temperature profiles
Activity of purified anionic trypsin was determined over the pH
range of 4.0–11.0. The pH 4.0–7.0 buffers were prepared using 50 mM
acetate buffer; the pH 7.0–9.0 buffers were prepared with 50 mM Tris-
HCl; and pH 9.0–11.0 buffers were prepared from 50 mM glycine-
NaOH. All reactions were carried out using 800 μL of buffer, 100 μL of
50 μM Boc-Val-Pro-Arg-MCA and 100 μL of purified anionic trypsin at
55 °C for 10 min. For the temperature profile study, trypsin activity
was assayed at various temperatures (20, 30, 40, 45, 50, 55, 60, 70 and
80 °C) for 10 min at pH 9.0 using Boc-Val-Pro-Arg-MCA as a substrate.
The relative trypsin activity (%) was calculated using the highest trypsin
activity obtained as 100%.
2.8.2. pH and thermal stabilities
The effects of pH and temperature on the stability of purified anionic
trypsin were evaluated by measuring the residual activity after incuba-
tion of the enzyme at various pHs and temperatures. For pH stability,
purified enzyme was mixed with different buffers mentioned above at
a ratio of 1:1 (v/v). The mixture was allowed to stand for 30 min at
room temperature (25-28 °C) prior to assay at optimal pH and temper-
ature, using Boc-Val-Pro-Arg-MCA as a substrate.
For the thermal stability, purified anionic trypsin was incubated at
different temperatures (20, 30, 40, 50, 60, 70, and 80 °C) for 20 min in
a temperature controlled water bath (Thermo Minder Mini-80, Taiyo,
Tokyo, Japan). Thereafter, the treated samples were rapidly cooled in
an ice bath. The residual trypsin activity was assayed as described
previously.
2.8.3. Effect of proteinase inhibitors
The effect of inhibitors on the activity of purified anionic trypsin was
determined according to the method of Khantaphant and Benjakul [1].
The enzyme solution was mixed with an equal volume of proteinase in-
hibitor solution to obtain the final concentration designated (1.0 mM
pepstatin A, 0.1 mM E-64, 1.0 g/L soybean trypsin inhibitor, 5.0 mM
TLCK, 5.0 mM TPCK, 5 mM Pefabloc SC and 10 mM EDTA). The mixture
was left at room temperature (26-28 °C) for 30 min. Thereafter, the re-
maining activity was measured and percent inhibition was calculated.
The control was conducted in the same manner except that distilled
water was used instead of inhibitors. The residual trypsin activity (%)
was calculated using the control as 100%.
2.8.4. Effect of NaCl
Effect of NaCl on the activity of purified anionic trypsin was investi-
gated. By assaying trypsin in the presence of NaCl at varying concentra-
tions (0, 2.5, 5, 10, 15, 20, 25 and 30% (w/v)). The residual activity was
measured at 55 °C and pH 9.0 for 10 min using Boc-Val-Pro-Arg-MCA
as a substrate. The relative trypsin activities (%) were calculated using
trypsin activity without NaCl as 100%.
973
2.8.5. Substrate specificity
The substrate specificity of purified anionic trypsin was investigated
using various fluorogenic MCA-substrates (Boc-Val-Pro-Arg-MCA, Boc-
Phe-Ser-Arg-MCA, Boc-Leu-Lys-Arg-MCA, Boc-Gln-Arg-Arg-MCA, Boc-
Leu-Ser-Thr-Arg-MCA, Boc-Ile-Glu-Gly-Arg-MCA, Boc-Glu-Lys-Lys-
MCA, Suc-Leu-Leu-Val-Tyr-MCA, Suc-Ala-Ala-Pro-Phe-MCA, Z-Phe-
Arg-MCA, Z-Arg-Arg-MCA, Arg-MCA). The activity of trypsin toward dif-
ferent substrates was measured as described above. The relative trypsin
activity (%) was calculated using the highest trypsin activity obtained as
100%.
2.8.6. Kinetic studies
Kinetic studies of purified anionic trypsin were investigated using
Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-MCA as a substrate with
different concentrations (0.1–1.0 μM). The final enzyme concentration
for the assay was 0.010 μg/mL. The kinetic parameters, apparent
Michaelis-Menten constant (Km), the maximum velocity (Vmax), and
the catalytic efficiencies (kcat/Km), were evaluated based on the
Lineweaver-Burk double-reciprocal plot [15]. Values of turnover num-
ber (kcat) were calculated from the following equation: kcat = Vmax /
[E], where [E] is the active enzyme concentration and Vmax is the maxi-
mal velocity.
2.9. Hydrolysis of threadfin bream actomyosin by purified albacore tuna
anionic trypsin
Threadfin bream (N. virgatus) samples were purchased from a local
market in Phatthalung, Thailand. The fish were stored in ice with a
fish/ice ratio of 1:2 (w/w) and transported to the laboratory at Thaksin
University, Phatthalung Campus, within 30 min of purchase. The fish
were filleted and the flesh was used for actomyosin extraction accord-
ing to the method of Klomklao et al. [4]. Purified anionic trypsin from al-
bacore tuna spleen (0.25 units) was added to the reaction mixture
containing 4 mg natural actomyosin (NAM) and 825 μL of 0.1 M
glycine-NaOH, pH 9.0. The hydrolysis was conducted by incubating
the mixture at 55 °C for 0, 5, 10, 20, 30 and 60 min. The control experi-
ment was performed in the same manner for 60 min, except distilled
water was added instead of enzyme. The reaction was terminated by
adding 5% (w/v) TCA solution. The mixture was centrifuged at 8000g
for 5 min at room temperature (25-28 °C) (Kubota, Model 3740,
Tokyo, Japan). TCA-soluble peptides in the supernatant were analysed
using the Lowry method [13].
To monitor the protein pattern of NAM hydrolysed by purified tryp-
sin, another lot of sample was added to the preheated solution contain-
ing 2% (w/v) SDS, 8 M urea, and 2% (v/v) βME (80 °C). The sample was
then incubated at 80 °C for 30 min to solubilize total proteins. All sam-
ples were subjected to SDS-PAGE using 10% separating and 4% stacking
gels.
2.10. Statistical analysis
A completely randomized design was used throughout this study.
The experiments were run in triplicate, except for the kinetic study,
which was carried out in duplicate. Data were presented as mean ±
standard deviation. Data were subjected to analysis of variance
(ANOVA) and comparison of means was carried out by Tukey's multiple
comparisons test [16]. Statistical analysis was performed using the Sta-
tistical Package for Social Science (SPSS 11.0 for Windows, SPSS Inc.,
Chicago, IL).
3. Results and discussion
3.1. Purification of anionic trypsin from albacore tuna spleen
Anionic trypsin from the spleen of albacore tuna was purified suc-
cessfully by a series of chromatographic separation as summarized in
974 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979

Table 1
Summary of purification of anionic trypsin from the spleen of albacore tuna.

Purification step Total activity (units)a Total protein (mg) Specific activity (units/mg protein) Purity (fold) Yield (%)

Crude enzyme 5565.18 3654.18 1.52 1.00 100.00

Q-sepharose 4095.36 54.54 75.09 49.40 73.59
Superdex 75 1171.14 7.87 148.81 97.90 21.04
Arginine sepharose 4B 575.07 1.2 477.51 313.54 10.33
a
The activities of the purified anionic trypsin were measured using Boc-Val-Pro-Arg-MCA at 55 °C for 10 min in 50 mM glycine-NaOH buffer (pH 9.0).

Table 1. The purification entailed separating the splenic extract using a Minor trypsin activity was found in unadsorbed fractions, while the
Q-Sepharose fast flow anion exchange column to produce two active major trypsin activity was found after eluting with a linear gradient
trypsin fractions as adsorbed and unadsorbed fractions. The results sug- of 0–0.6 M NaCl (Fig. 1A). The major trypsin fraction (anionic tryp-
gested that two groups of trypsin were present in the splenic extract. sin) was further purified using Superdex 75 column chromatography
to remove most of contaminating proteins, resulting in a substantial
increase in purification fold of 49.40 with a yield of 73.59%. The ion
exchange chromatography technique was previously used to remove
3.5 120
OD 280 Units/mL (A) the contaminating proteins and to separate different trypsin iso-
3 ( ) 100 forms [17]. Cao et al. [18] also purified two anion trypsins from the
Activity (units/mL) hepatopancreas of carp (Cyprinus carpio) by using a Q-Sepharose
2.5
80 anion exchanger column. Klomklao et al. [19] also isolated two iso-
2 forms of trypsin from the intestine of skipjack tuna (Katsuwonus
OD280

60 pelamis) using DEAE-cellulose.

1.5
The pooled active Q-Sepharose fractions were subsequently sepa-
40
1 rated on a Superdex 75 column. As shown in Fig. 1B, Superdex 75 col-
20 umn was highly effective in separating trypsin from other proteins
0.5
with higher molecular weight. A large amount of contaminated proteins
0 0 was removed, leading to an increase in purity by 97.90-fold. Sriket et al.
0 250 500 750 1000 1250 [12] also reported that the use of Superdex 75 for purification of trypsin
Fraction number (5 mL/tube) from the hepatopancreas of freshwater prawn (M. rosenbergii) led to
higher purity of trypsin.
0.7 25
OD 280 Units/mL (B) Next, the pooled Superdex 75 fractions with trypsin activity were
0.6 ( ) applied to Arginine Sepharose 4B column, and produced only one activ-
20
Activity (units/mL)

ity peak, suggesting the efficacy and specificity in binding trypsin of the
0.5
column (Fig. 1C). Pooled fractions from single activity peak obtained
0.4 15 after Arginine Sepharose 4B column showed high purity of about
OD280

313.54-fold with 10.33% yield.

0.3 10
0.2 3.2. Native versus SDS polyacrylamide gel electrophoresis
5
0.1 Purity of the anionic trypsin from albacore spleen was determined
0 0 using native gel electrophoresis. From native gel electrophoresis, only
0 10 20 30 40 50 60 70 80 90
Fraction number (5mL/tube) kDa
0.16 14 (A) 200 (B)
OD 280 Units/mL (C)
0.14 12 116
( ) 97
0.12
Activity (units/mL)

10 66
0.1
8
OD280

0.08
45
6
0.06
4
0.04
31 30 kDa
0.02 2

0 0
0 25 50 75 100 125 150 175 200 21.5
Fraction number (2mL/tube)

Fig. 1. Purification of trypsin from albacore tuna spleen. (A) Elution profile of trypsin on Q-
Sepharose anion exchange column. Elution was carried out with a linear gradient of
0–0.6 M NaCl in SB; (B) elution profile of trypsin on Superdex 75 gel filtration column;
(C) elution profile of trypsin on Arginine Sepharose 4B affinity column. Elution was
carried out with a linear gradient of 0–1.0 M KCl.
M 1 2
Fig. 2. Native-PAGE (A) and SDS-PAGE (B) of purified anionic trypsin from albacore tuna
spleen. Lanes 1 and 2: protein band of pooled Arginine Sepharose 4B fraction under
non-reducing and reducing, respectively. M: molecular weight marker.
 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979 975

a single protein band was obtained (Fig. 2A), confirming that anionic k
(A)
100
trypsin was purified to homogeneity. Also, the trypsin migrated as a sin-
gle band using SDS-PAGE under both reducing and nonreducing condi- j i
tions with a molecular weight of 30 kDa (Fig. 2B). The result indicated

Relative activity (%)

80
h
that anionic trypsin from albacore tuna spleen has no disulfide bond
in the interior of the protein molecule. Generally, trypsins from marine
60 g
animals have been reported to have molecular weights ranging from 20
to 30 kDa [1]. Examples include a 21.7 kDa trypsin from carp
f
(C. mrigala), a 23 kDa trypsin from golden grey mullet (L. aurata) [8], a 40
24 kDa trypsin from Tunisian barbel (B. callensis) [5], a 25 kDa trypsin e
from sardine (Sardine pilchardus) [20], a 28 kDa trypsin from pirarucu
20 d b
(Arapaima gigas) [21] and a 38.5 kDa trypsin from Tambaqui c
(Colossoma macropomum) [22]. The different molecular weights a a
among trypsins from different sources may be due to genetic variations 0
among species. 3 4 5 6 7 8 9 10 11
pH

3.3. N-terminal amino acid sequence of anionic trypsin from albacore tuna
spleen

The N-terminal amino acid sequence of purified anionic trypsin from i (B)
100 h
albacore tuna spleen was determined by the automated Edman method
g
after SDS-PAGE. The N-terminal 20 amino acids sequence of purified al-
bacore tuna trypsin was determined to be IVGGYECQAHSQPHQVSLNA 80

Relative activity (%)

f
(Fig. 3). This sequence showed uniformity, indicating that it was iso- e
lated in a pure form. When N-terminal amino acid sequences of purified d
albacore tuna anionic trypsin were compared with those of other fish 60
and mammals, it was found that the five sequences of purified albacore
tuna anionic trypsin (IVGGY) were identical to those of other fish and 40
mammal trypsins. Also, nearly all the trypsins shared the sequence c
(QVSLN) at position 15-19 (Fig. 3). Nevertheless, albacore tuna anionic
trypsin and other fish trypsins had a charged Glu residue at position 6, 20
b
a
whereas Thr is most common in mammalian pancreatic trypsins
(Fig. 3). Furthermore, albacore tuna anionic trypsin characteristically 0
conserved Cys residue at position 7 like the all other trypsins. From 10 20 30 40 50 60 70 80
the results, albacore tuna anionic trypsin showed high identities to
other animal trypsins. These results suggest the possibility that they Temperature (oC)
were genetically evolved from a common ancestor. Moreover, anionic
Fig. 4. pH (A) and temperature profiles (B) of purified anionic trypsin from albacore tuna
trypsin from the spleen of albacore tuna and other animal trypsin se- spleen. Bars represent the standard deviation (n = 3).
quences started with the conservative sequence, IVGG, which was a
part of the proteolytic cleavage site in inactive trypsinogen. Thus, the al- 3.4. Biochemical properties of purified anionic trypsin from albacore tuna
bacore tuna trypsin like its counterparts, is initially synthesized as an in- spleen
active proenzyme and then processed with proteolytic cleavage into an
active enzyme after translation. These data lend credence to the notion 3.4.1. pH and temperature profiles
that the anionic trypsin from albacore tuna spleen belong to the trypsin The pH activity profile of the purified albacore tuna spleen trypsin
family of enzymes. was investigated over a pH range of 4.0–11.0 as shown in Fig. 4A. The

Fish Albacore tuna IVGGYECQAH SQPH QVSLNA

Yellowfin tuna IVGGYECQAH SQP PQVS LNA
Japanese sea bass I V G G Y E C T PP YY S Q P H Q V S L N S
Jacopever I V G G Y E C K PP YYYSQ PH QVSLNS
Elkhorn sculpin I V G G Y E C T P HY S QAA H Q V S L N SS
Pacific cod IVGGYECTRH SQAHQVSLNS
Saffron cod IVG GYE CPR H SQ AH QVSLNS
Walleye pollock I V G G Y E C T K H S Q A H Q V S L N SS
True sardine I V G G Y E C K AY S Q P W Q V S L N S
Arabesque greenling I V G G Y E C T PP H T Q A H Q V S L N S
Mammal Rat Y Q V S L N SS
I V G G Y T C Q EE EN S V P Y
Dog I V G G YT C E E N S V PV Q V S LN A
Porcine I V G G Y T C A A N S V P Y Q V S L N SS
Bovine I V G G Y T C G AN T V P Y Q V S L N SS
NT

Fig. 3. Alignment of the N-terminal amino acid sequences of the purified anionic trypsin from spleen of albacore tuna with trypsins from other species including yellowfin tuna [23],
Japanese sea bass [11], jacopever, elkhorn sculpin [35], Pacific cod, saffron cod [3], walleye pollock [36], true sardine, arabesque greenling [37], rat [38], dog [39], porcine [40] and bovine
[41].
976 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979

trypsin exhibited a maximal activity at pH 9.0 (P b 0.05). The activity of c c c c (A)

100 c c
the enzyme was high in pH range of 8.0–9.5 (P b 0.05) but considerable
loss of activity was observed at highly acidic and alkaline pHs. No activ- b

Relative activity (%)

ity was found at pH 4.0 and 5.0. At very acidic and alkaline pHs, the 80
charge distribution and conformation of enzymes were changed and
enzymes could not bind to the substrate effectively, resulting in the
60 a
lower activity [23]. Generally, trypsins are more active at alkaline pH
with optimal pH ranging from 7.5 to 10.5. [24]. The optimal pH of puri-
fied albacore tuna spleen trypsin was similar with those of trypsins from 40
the viscera of pirarucu (A. gigas) [21] and hepatopancreas of Japanese
sea bass (L. japonicus) [11]. However, optimal pH can vary depending
20
upon factors such as temperature, substrate type and concentration,
ionic strength, and type of metal ions [23]. For instance, Martinez et al.
[25] reported that trypsin-like enzyme from the pyloric caeca of an- 0
chovy (Engraulis encrasicholus) had maximum activity at pH 8.0 for hy- 3 4 5 6 7 8 9 10 11
drolysis of BAPNA, but at 9.5 for the hydrolysis of casein as well as pH
myofibrillar proteins. The differences in optimal pH were attributed to
the accessibility of the substrate to the active site as affected by charge
on the substrate and on the active site at the particular pH of the me- d d d d (B)
dium [23]. Therefore, optimal pH of purified anionic trypsin from alba- 100
core tuna spleen may change when other substrates are used instead
c
of Boc-Val-Pro-Arg-MCA.

Relative activity (%)

80
The temperature-activity profile of purified anionic trypsin
is depicted in Fig. 4B. The optimal temperature of anionic trypsin
was 55 °C (P b 0.05) when Boc-Val-Pro-Arg-MCA was used as a sub- 60
strate. However, the activity markedly decreased at temperatures
above 60 °C (P b 0.05), mainly due to thermal denaturation of trypsin. 40 b
The same optimal temperature (55 °C) was reported for trypsin from in-
testine and pyloric caeca of silver mojarra (Diapterus rhombeus) [26],
viscera of Tunisian barbel (B. callensis) [5]. Nevertheless, anionic trypsin 20
from the spleen of albacore tuna had higher optimal temperature a
than those from cold-water fish, which had optimal temperatures 0
in the range of 40–45 °C such as Grey triggerfish (Balistes capriscus) 10 20 30 40 50 60 70 80
(40 °C) [2], lane snapper (Lutjanus synagris) (45 °C) [27] and Japanese
sea bass (L. japonicus) (40 °C) [11]. The differences in optimal tempera- Temperature (oC)
ture might be governed by habitat, environment and genetic factors
[12]. Fig. 5. pH (A) and thermal stabilities (B) of purified anionic trypsin from albacore tuna
spleen. Bars represent the standard deviation (n = 3).

3.4.2. pH and thermal stabilities

The effect of pH on the stability of anionic trypsin from the spleen of 3.4.3. Effects of various proteinase inhibitors on the activity of anionic tryp-
albacore tuna is shown in Fig. 5A. Albacore tuna trypsin was stable over sin from albacore tuna spleen
a broad pH range (pH 6–11) (P N 0.05). However, it was unstable below Proteinase inhibitors are very important tools for classifying the
pH 6 (P b 0.05). The stability of trypsins at particular pH may be due to types of proteinase [1]. Table 2 shows the effect of different proteinase
the net charge of the enzyme at that pH [28,29]. Trypsin generally be- inhibitors, such as chelating agents and specific group reagents on
longs to the alkaline protease group [24]. Thus, albacore tuna anionic the trypsin activity. The trypsin activity was strongly inhibited by
trypsin might undergo denaturation and/or conformational change compounds such as soybean trypsin inhibitor (99.80% inhibition),
under the acidic conditions to adversely impact the stability of the en- TLCK (96.64% inhibition) and Pefabloc SC (99.61% inhibition). Soybean
zyme [12]. Inactivation of enzyme activity at acidic pH was also reported trypsin inhibitor is a single polypeptide that forms a stable
for the anionic trypsins from various species including carp (C. carpio)
[18], smooth hound (Mustelus mustelus) [30] Japanese sea bass
(L. japonicus) [11] and Pacific saury (Cololabis saira) [31]. Table 2
The thermal stability of purified anionic trypsin subjected to heating Effects of various proteinase inhibitors on the activity of purified anionic trypsin from the
for 20 min at different temperatures (20–80 °C) is shown in Fig. 5B. The spleen of albacore tuna⁎.
result showed that purified trypsin was stable up to 50 °C (P N 0.05) and Inhibitors Concentration % Inhibition⁎⁎
slightly lost its stability at 60 °C (P b 0.05). However, the purified trypsin
Control No inhibition
was inactive at higher temperatures (P b 0.05). After heat treatment at
Pepstatin A 1 mM No inhibition
80 °C, the relative activity of the trypsin was about 3.8% of its initial ac- E-64 0.1 mM No inhibition
tivity. At high temperatures, the enzyme more likely underwent dena- Soybean trypsin inhibitor 1 g/L 99.80c ± 0.03
turation and lost its activity. Most fish trypsins are unstable at TLCK 5 mM 96.64b ± 0.13
TPCK 5 mM No inhibition
temperatures higher than 40–50 °C [31]. Sila et al. [5] reported that tryp-
Pefabloc SC 5 mM 99.61c ± 0.07
sin from the viscera of Tunisian barbel (B. callensis) was highly stable up EDTA 10 mM 11.56a ± 0.06
to 40 °C but the activity was rapidly lost at temperatures above 60 °C.
The different letters in the same column denote the significant differences (P b 0.05).
Trypsin from the digestive system of carp (C. mrigala) exhibited high ⁎ Activities of the purified anionic trypsin were analysed using Boc-Val-Pro-Arg-MCA as
thermal stability up to 50 °C for 1 h and this enzyme lost complete activ- a substrate for 10 min at pH 9.0 and 55 °C.
ity at 70 °C [7]. ⁎⁎ Values are mean ± standard deviation (n = 3).
 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979 977

stoichiometric enzymically inactive complex with trypsin, thereby re- Table 3

ducing the availability of trypsin [17]. TLCK deactivates only trypsin- Substrate specificities of purified anionic trypsin from the spleen of albacore tuna on
fluorescence synthetic substrates⁎.
like enzymes as a result of alkylation of the active center histidine. The
enzyme, in the presence of TLCK, is known to form a covalent bond Substrates (50 μM) Relative activity (%)⁎⁎
with histidine in the catalytic site and to block the substrate-binding Boc-Val-Pro-Arg-MCA 100.00g ± 1.37
portion of the active center of the molecule [32]. Pefabloc SC or Boc-Phe-Ser-Arg-MCA 71.09d ± 2.17
4 (2 Aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF) is an Boc-Leu-Lys-Arg-MCA 95.97f ± 1.22
Boc-Gln-Arg-Arg-MCA 90.37e ± 1.78
irreversible proteinase inhibitor with broad specificity for serine pro-
Boc-Leu-Ser-Thr-Arg-MCA 63.95c ± 1.88
teinases [12]. TPCK (a chymotrypsin specific inhibitor), E-64 (a cysteine Boc-Ile-Glu-Gly-Arg-MCA 65.08c ± 1.07
proteinase inhibitor) and pepstatin A (an aspartyl proteinase inhibitor) Boc-Glu-Lys-Lys-MCA 39.29b ± 0.58
did not show inhibitory effects toward the trypsin. The results from the Suc-Leu-Leu-Val-Tyr-MCA 0.00a
inhibition studies confirmed that the purified enzyme was a serine pro- Suc-Ala-Ala-Pro-Phe-MCA 0.00a
Z-Phe-Arg-MCA 0.00a
tease, most likely trypsin. Khangembam and Chakrabarti [7] reported Z-Arg-Arg-MCA 0.00a
that the activity of trypsin from the digestive system of carp Arg-MCA 0.00a
(C. mrigala) was effectively inhibited by soybean trypsin inhibitor and
The different letters in the same column denote the significant differences (P b 0.05).
TLCK. The enzymatic activities of cationic trypsin and anionic trypsin ⁎ Purified anionic trypsin activities were assay using various MCA substrates at
from the hepatopancreas of Japanese sea bass (L. japonicus) were pH 9.0 and 55 °C for 10 min.
also completely inhibited by Pefabloc SC [11]. However, the well- ⁎⁎ Values are mean ± standard deviation (n = 3).
known metalloproteinase inhibitor, EDTA, was less effective in
inhibiting trypsin activity (11.56% inhibition). In this regard, EDTA pos-
sibly chelated Ca2+, which is required for trypsin stability and activity 25% NaCl [33]. Therefore, anionic trypsin from albacore tuna spleen may
[17]. Removal of calcium ions might affect trypsin structure, resulting have a potential in accelerating protein hydrolysis under conditions of
in some losses in activity. From the results of the EDTA study, it is sug- high salinity such as in the production of fermented fish or shrimp paste.
gested that the enzyme is trypsin, which possibly required Ca ions for
stability and/or activity. 3.4.5. Substrate specificities of albacore tuna spleen trypsin
To characterise the substrate specificities of the enzyme, various
3.4.4. Effects of NaCl on the activity of anionic trypsin from albacore tuna fluorescent MCA-substrates were chosen to react with the purified alba-
spleen core tuna trypsin. As shown in Table 3, purified anionic trypsin hydro-
Fig. 6 shows the effect of varying concentrations of NaCl (0–30%) on lysed Boc-Val-Pro-Arg-MCA, Boc-Leu-Lys-Arg-MCA and Boc-Gln-Arg-
activities of the purified anionic trypsin. A progressive decrease in tryp- Arg-MCA more effectively than other specific synthetic substrates.
sin activity was observed with increasing NaCl concentration (P b 0.05). Other substrates containing arginine or lysine residues at P1 site were
The decrease in activity might be due to the denaturation of enzymes. also cleaved to some degree; however, the substrate containing lysine
The “salting out” effect was postulated to cause the enzyme denatur- residue was hydrolysed less than with arginine residue (Table 3). Chy-
ation. The water molecule is drawn from the trypsin molecule by salt, motrypsin substrates (Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-
leading to the aggregation of those enzymes [32]. This possibly led to Phe-MCA) and aminopeptidase substrate (Arg-MCA) were not hydro-
the loss in activity in the presence of high NaCl concentrations. How- lysed by purified anionic trypsin. No cleavage to Z-Phe-Arg-MCA, a sub-
ever, remaining activity of anionic trypsin from albacore tuna spleen strate for cathepsin L and Z-Arg-Arg-MCA, a substrate for cathepsin B,
was approximately 59% at high salt concentration (30%). Bougatef was identified. The substrate specificities of the enzyme were also sim-
et al. [30] found that activity of anionic trypsin from smooth hound ilar to trypsin from skeletal muscle of crucian carp (Carassius auratus)
(M. mustelus) intestine decreased with increasing NaCl concentration [34] and two trypsins from Japanese sea bass (L. japonicus) hepatopan-
and the activity at 30% NaCl was about 48% of the control. Trypsin creas [11]. All of these experimental results strongly indicated that puri-
from skipjack tuna (K. pelamis) was still active and able to degrade nat- fied enzyme from albacore tuna spleen was serine proteinase, most
ural actomyosin in the muscle of sardine (Sardinella gibbosa) containing likely trypsin.

3.4.6. Kinetic studies

Kinetic parameter for Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-
h (E) MCA hydrolysis by purified anionic trypsin from the spleen of albacore
100 g tuna were measured using Lineweaver-Burk plots. Km and kcat values
f were calculated as 0.23 ± 0.07 μM and 597.27 ± 15.61 s−1, respectively,
e for the hydrolysis of Boc-Val-Pro-Arg-MCA; and Km and kcat values for
Relative activity (%)

80 d
c the hydrolysis of Boc-Leu-Lys-Arg-MCA were determined as 0.53 ±
b a 0.06 μM and 42.81 ± 2.54 s−1, respectively. The Km value of Boc-Val-
60 Pro-Arg-MCA hydrolysis was lower than Boc-Leu-Lys-Arg-MCA hydro-
lysis. Km is often associated with the affinity of the enzyme for substrate.
This result suggested that albacore tuna anionic trypsin had higher af-
40
finity for Boc-Val-Pro-Arg-MCA than did Boc-Leu-Lys-Arg-MCA. When
comparing Km of purified albacore tuna anionic trypsin with anionic
20 trypsin from the hepatopancreas of Japanese sea bass (L. japonicus), it
was found that anionic trypsin from albacore tuna spleen had lower
Km values than anionic Japanese sea bass (L. japonicus) trypsin [11].
0
This indicated that anionic trypsin from the spleen of albacore tuna
0 5 10 15 20 25 30
had higher affinity to Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-MCA
NaCl concentration (%) than that from anionic trypsin from Japanese sea bass (L. japonicus)
hepatopancreas.
Fig. 6. Effect of NaCl concentration on activities of purified anionic trypsin from albacore For the kcat, anionic trypsin from albacore tuna spleen also exhibited
tuna spleen. Bars represent the standard deviation (n = 3). higher value on both Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-MCA
978 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979

than did trypsin from hepatopancreas of Japanese sea bass (L. japonicus)
[11] and freshwater prawn (M. rosenbergii) [12]. kcat indicates the max-
imum number of enzymatic reactions catalyzed per second [17]. Thus,
the higher kcat of albacore tuna anionic trypsin indicated a higher rate
of hydrolysis in comparison with trypsin from Japanese sea bass
(L. japonicus) and freshwater prawn (M. rosenbergii). The catalytic effi-
ciencies (kcat/Km), also known as substrate specificity, of the purified an-
ionic trypsin for hydrolysis of Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-
Arg-MCA were calculated to be 2.60 ± 0.07 s−1 mM−1 and 80.77 ±
4.80 s−1 μM−1, respectively. Purified anionic trypsin from the spleen
of albacore tuna also showed higher catalytic efficiencies than did tryp-
sin from Japanese sea bass (L. japonicus) and freshwater prawn
(M. rosenbergii) when Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-
MCA using as a substrate. The result suggested that anionic trypsin
from albacore tuna spleen would be more efficient in transforming
this particular substrate to product versus its counterpart from
Japanese sea bass (L japonicus).
3.5. Hydrolysis of threadfin bream natural actomyosin by albacore tuna
spleen trypsin
3.5.1. TCA-soluble peptide content in natural actomyosin hydrolysate
The hydrolysis of threadfin bream natural actomyosin (NAM) by pu-
rified albacore tuna spleen trypsin was also investigated. The
degradation of proteins in NAM of threadfin bream with and without
the enzyme addition at different incubation time expressed as TCA-
soluble peptide content is shown in Fig. 7A. The increase in TCA-
soluble peptide content became more pronounced with increasing in-
cubation time (P b 0.05). The highest content of TCA-soluble peptide
content was found in NAM treated with purified trypsin at 55 °C for
60 min. For the control (without purified trypsin), a slight degradation
of NAM was observed. It was noted that autolysis of sample occurred
to some extent during incubation at 55 °C. The result suggested that pu-
rified albacore tuna anionic trypsin had high proteolytic activity toward
NAM. This result was in agreement with the findings by Klomklao et al.
[4] who reported that trypsin from skipjack tuna (K. pelamis) was able
to hydrolyse NAM of sardine (S. gibbosa). The increase in TCA-soluble
peptide content in treated samples might be due to an increase in per-
meability of NAM structures, resulting in digestion and then the release
of peptides.
3.5.2. Protein pattern of hydrolysed natural actomyosin
Protein patterns of NAM extracted from threadfin bream incubated
with and without purified albacore tuna anionic trypsin at 55 °C for
0–60 min are shown in Fig. 7B. NAM contained actin and myosin
heavy chain (MHC) as major constituents. β-tropomyosin was found
as a minor component (Fig. 7B). Among all proteins, MHC was the
most susceptible to hydrolysis. MHC was effectively digested within

45
(A)
40 f
TCA-soluble peptide content
(nmol tyrosine/g sample)

35
e
30
d
25

20

15 c

10
b
5 a
a
0
0 5 10 20 30 60 C

kDa (B)

200 MHC
116
97
66
55
45 Actin
36 β-tropomyosin

29
24

M 0 5 10 20 30 60 C

Fig. 7. TCA-soluble peptide content (A) and protein patterns (B) of natural actomyosin incubated with purified anionic trypsin from albacore tuna spleen at 55 °C for different times. C:
control (incubated without trypsin addition for 60 min at 55 °C). Numbers denote the incubation time (min) at 55 °C. Bars represent the standard deviation (n = 3). Different letters
on the bars within the same gel indicate significant differences (P b 0.05).
 T. Poonsin et al. / International Journal of Biological Macromolecules 133 (2019) 971–979
5 min by purified trypsin, with concomitant occurrence of degradation
products having lower molecular weights (Fig. 7B). The degradation of
actin increased as the incubation time increased. Nevertheless, the deg-
radation rate was lower than that of MHC. For protein patterns of NAM
incubated without purified trypsin, the proteins were degraded slightly
by autolysis process as evidenced by the decrease in band intensities.
This result was consistent with the increase in TCA-soluble peptide con-
tent (Fig. 7A). The result reconfirmed that purified albacore tuna anionic
trypsin was able to degrade NAM. Cai et al. [11] reported that MHC and
actin were markedly degraded by trypsin from Japanese sea bass
(L. japonicus) hepatopancreas. From the result, it can be concluded
that purified trypsin from the spleen of albacore tuna hydrolysed myo-
fibrillar proteins extensively, particularly MHC which is the dominant
protein in fish muscle.
4. Conclusions
Anionic trypsin from albacore tuna spleen was purified and identi-
fied based on N-terminal amino acid sequencing, substrate specificity,
molecular weight, and inhibitor investigation. The enzyme also exhib-
ited high proteolytic activity toward threadfin bream muscle proteins.
Hence, albacore tuna spleen trypsin may be used as an enzyme source
for the preparation of products like fish sauce, based on its capacity to
hydrolyse fish muscle.
Acknowledgements
This work was supported by Thailand Graduate Institute of Science
and Technology (SCA-CO-2559-2285-TH). The Thailand Research Fund
and Thaksin University were also acknowledged.
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