Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Abstract

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0– 11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s–1, respectively. The catalytic efficiency (Kcat / Km) was 238 s–1 mM–1.

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Characterisation of thermostable trypsin and determination of trypsin
isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)
Sasimanas Unajaka,b, Piyachat Meesawata, Atchara Paemaneec, Nontawith Areechond,
Arunee Engkagula,b, Uthaiwan Kovitvadhib,e, Satit Kovitvadhib,f, Krisna Rungruangsak-Torrissenb,g,
Kiattawee Choowongkomona,b,h,⇑
aDepartment of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
bBiochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
cNational Science and Technology Development Agency, Pathum Thani 12120, Thailand
dDepartment of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand
eDepartment of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
fFaculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Bangkok 10600, Thailand
gInstitute of Marine Research, Ecosystem Processes Research Group, Matre Aquaculture Research Station, N-5984 Matredal, Norway
hCenter for Advanced Studies in Tropical Natural Resources (CASTNAR), National Research University–Kasetsart University (NRU-KU), Kasetsart University, Bangkok 10900, Thailand
a r t i c l ei n f o
Article history:
Received 20 July 2011
Received in revised form 29 February 2012
Accepted 19 March 2012
Available online 28 March 2012
Keywords:
Trypsin isozymes
Thermostable enzyme
Enzyme kinetics
Enzyme purification
Nile tilapia
a b s t r a c t
Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step
purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied
to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least
6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high
purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approxi-
mately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–
11.0 and an optimal temperature of approximately 55–60 ?C. The relative activity of the purified enzyme
was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or
alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (Km) and catalytic constant
(Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s?1, respectively. The catalytic efficiency
(Kcat/Km) was 238 s?1mM?1.
? 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Nile tilapia (Oreochromis niloticus L.) is an important economic
fish species in Thailand. The processing of fish generates a large
amount of waste. Different applications, in using the by-products
from this by product, have been developed to overcome the pollu-
tion problems. The majority of fish waste consists of viscera, which
are a potential source of many digestive enzymes, such as trypsin,
pepsin, chymotrypsin, collagenase and elastase. In the stomach,
the majority of proteolytic enzymes display high activity at pH
2.0–4.0, while the alkaline proteases in the intestine are highly ac-
tive over a pH range of 8.0–10.0 (Bezerra et al., 2005; El Hadj Ali,
Hmidet, Bougatef, Nasri, & Nasri, 2009; Supannapong et al.,
2008). Acidic proteases showed lower enzymatic activity than
did alkaline proteases when using the same substrate, such as
casein (Rungruangsak & Utne, 1981; Torrissen, 1984) or azocasein.
Trypsin is a serine protease, which is produced as an inactive
precursor. It has a function in the hydrolysis of target proteins at
the amino acids arginine and lysine. Many trypsins have been
isolated from the viscera of different fish species, such as tilapia
(Bezerra et al., 2005; El-Shemy & Levin, 1997; Wang et al., 2010),
smooth hound (Bougatef et al., 2010), skipjack tuna (Klomklao,
Kishimura, Nonami, & Benjakul, 2009), grey triggerfish (Jellouli
et al., 2009), Pacific cod and saffron cod (Fuchise et al., 2009),
and the Amazonian fish tambaqui (Marcuschi et al., 2010). Trypsin
has various industrial applications, especially in food industries,
due to its high stability and activity under harsh conditions, such
as in the presence of surfactants and oxidative agents. Although
the habitat temperature of cultivated fish seems to correlate with
0308-8146/$ - see front matter ? 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.03.074
Abbreviations: SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electro-
phoresis; Km, Michaelis–Menten constant; Kcat, catalytic constant; Kcat/Km, catalytic
efficiency; TAME, N-p-tosyl-L-arginine methyl ester hydrochloride; BAPNA, ben-
zoyl-DL-arginine-p-nitroanilide; BSA, bovine serum albumin; PMSF, phenylmethyl-
sulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl
sulphate; DTT, dithiothreitol; Vmax, maximum velocity.
⇑Corresponding author at: Department of Biochemistry, Faculty of Science,
Kasetsart University, Bangkok 10900, Thailand. Tel.: +66 2562 5555x2051; fax: +66
2561 4627.
E-mail address: fsciktc@ku.ac.th (K. Choowongkomon).
Food Chemistry 134 (2012) 1533–1541
Contents lists available at SciVerse ScienceDirect
Food Chemistry
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Page 2

a particular optimal temperature and heat stability (Kishimura,
Klomklao, Benjakul, & Chun, 2008), the optimal enzyme tempera-
ture and heat stability could be much higher than the habitat tem-
perature (Rungruangsak, 2007). However, trypsin from tropical fish
would be applicable for food industries.
Oneofthemostimportantprocessesforpredictingfishgrowthis
the efficiency of the digestive enzyme trypsin and the activity ratio
of trypsin to chymotrypsin, which influences the conversion of feed
to nutrients for utilisation (Rungruangsak, 2007; Rungruangsak,
Moss, Andresen, Berg, & Waagbø, 2006; Rungruangsak et al.,
2009; Sunde, Taranger, & Rungruangsak, 2001). Therefore, trypsin
is the key enzyme for food utilisation and growth. In the market,
most of selling tilapia has been captured at age 6th months. A lot
of waste products from fish viscera is discarded every day. The
implications in this study may increase the value of waste products
from the market. We used fish intestine at 6th months for purifica-
tion. Furthermore, characterisation of trypsin isoforms at each age
was also performed.
2. Materials and methods
2.1. Experimental fish
Eggs of Nile tilapia (O. niloticus L.) were incubated in a hatching
funnel. At 7 day post-hatching, the fish were sex-reversed to
males by feeding with shrimp feed (at least 45% protein) contain-
ing 17-a methyltestosterone, until one month old. After that, the
fish were reared in intensive net cages until 6 months old at
Khamphaengsaen Fisheries Research Station, Faculty of Fisheries,
Kasetsart University, Nakhon Pathom province. They were fed
with feeds containing mainly from plants with protein levels of
35%, 30% and 24–26%, during 2–3 months, for 4–5 months and
6–10 months, respectively. For sample collection, pooled samples
of intestine were collected at 6 months. The fish were kept on ice
while the intestines were dissected and briefly cleaned with
deionized water. Pooled samples of intestine were collected at
1 month, and at 3–7 months. The intestinal samples were imme-
diately placed in liquid nitrogen for transportation, and stored at
?80 ?C until used.
2.2. Preparation of crude enzyme extracts
The pooled samples of intestine were briefly thawed on ice.
Each pooled sample was homogenised at a ratio of 1:50 (w/v) in
50 mM Tris–HCl buffer, pH 8.0, containing 1 mM CaCl2, referred
to as starting buffer (SB). The homogenates were centrifuged at
10,000g for 30 min at 4 ?C to remove the tissue debris. The super-
natants were kept at ?80 ?C until used; these were referred to as
the ‘‘crude enzyme extracts.’’
2.3. Polyacrylamide gel electrophoresis (PAGE)
Protein samples were prepared for sodium dodecyl sul-
phate–polyacrylamide gel electrophoresis (SDS–PAGE) by mix-
ing each crude enzyme extract at a ratio of 1:1 (v/v) with
the sample buffer (0.5 M Tris–HCl buffer, pH 6.8, containing
10% SDS, 30% glycerol, and 10% b-mercaptoethanol), and subse-
quently boiling for 10 min. The mixed samples were cooled on
ice. Then 15 ll were loaded onto a gel made of 4% stacking
and 12% separating gels, and subjected to electrophoresis using
a Mini-PROTEAN II Cell apparatus (Atto Co., Tokyo, Japan).
After electrophoresis, the composite gel was stained with
0.1% Coomassie Brilliant Blue R-250 in 40% ethanol and 10%
acetic acid for 30 min. The gel was then destained with 40%
ethanol and 10% acetic acid. The molecular weights of particular
proteins were estimated using protein standards (Fermentas,
USA) as markers.
2.4. Zymography of trypsin
Zymography of trypsin was performed on native-PAGE accord-
ing to the substrate gel electrophoresis method modified by
Garcia-Carren ˇo, Dimes and Haard (1993). Briefly, the native-PAGE
was performed using 12% separating gels in a manner similar to
described above, except that the samples were mixed with sample
buffer without the reducing agent b-mercaptoethanol, and were
not heated. After electrophoresis, the gels were submerged in
100 ml of 5 M N-p-tosyl-L-arginine methyl ester hydrochloride
(TAME) substrate buffer (in 50 mM Tris–HCl buffer, pH 8, contain-
ing 1 mM CaCl2) for 30 min at 37 ?C, with gentle agitation. A clear
zone on the white background of the gel indicated the presence of
protease activity of trypsin.
2.5. Purification of trypsin
The crude enzyme extract from the pooled intestines of 6-
month-old tilapia was subjected to ammonium sulphate, at 20–
60% saturation, to fractionate particular proteins. The precipitated
proteins in the 40–60% saturation were collected by centrifugation
at 10,000g for 30 min at 4 ?C. The pellet was dissolved in a minimal
volume of SB and dialysed against SB overnight, with gentle stirring
at 4 ?C. The protein dialysate was then applied onto a Sephadex G-
100 column(1 ? 140 cm, Fluka,Switzerland), pre-equilibratedwith
approximately two column volumes of SB. The column was eluted
with the same buffer at a flow rate of 0.1 mlmin?1, and fractions
of 1 ml were collected. Protein concentration was determined by
the Lowry method, and trypsin activity was measured using ben-
zoyl-DL-arginine-p-nitroanilide (BAPNA; Sigma). The fractions con-
taining trypsin activity were pooled, and then chromatographed
using a Q Sepharose column (0.8 ? 5 cm), pre-equilibrated with
SB at a flow rate of 0.5 ml min–1. The column was washed with SB
until the absorbance at 280 nm (A280) reached baseline, and was
then eluted with linear gradient using 0.0–1.0 M NaCl in SB. Frac-
tions of 1 ml were collected; those with trypsin activity against
BAPNA were pooled, stored at ?20 ?C, and used for further study.
2.6. Determinations of protein concentration and trypsin activity
Protein concentration was measured by the Lowry method,
using bovine serum albumin (BSA) as standard. Protease activity
of trypsin was assayed by a method modified from Rungruangsak
(2007), based on Erlanger, Kokowsky, and Cohen (1961), using ini-
tial enzyme reaction rate and optimal temperature of Nile tilapia
trypsin activity. The enzyme solution (10 ll) was mixed with
50 ll of preheated 1.25 mM BAPNA in 50 mM Tris–HCl buffer, pH
8.0; the initial enzyme reaction rate of 1 min at 50 ?C was mea-
sured at 410 nm. The production of p-nitroaniline was calculated
by monitoring the increment in absorbance at 410 nm, which
was the difference between the sample and the blank (DA410).
TrypsinactivityðunitÞ¼ðDA410min?1Þ?mlof totalvolume?1000
8800?mlof enzymesolution
Trypsin specific activity ðunit=mgÞ
Enzyme activity
Protein concentrationðmgml?1Þ
The molarextinction
8800 M?1cm?1. One unit (U) of enzyme activity corresponds to
1 nmol p-nitroaniline released per min at 37 ?C.
¼
coefficientof
p-nitroaniline is
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2.7. Liquid chromatography–electrospray ionisation–tandem mass
spectrometry analysis (LC–ESI–MS/MS)
First, in-gel digestion was performed using a method modified
from that of Terry, Umstot, and Desiderio (2004). Stained gels on
SDS–PAGE of the purified protein from the NaCl gradient Q Sephar-
ose fraction were cut into cubes (1 ? 1 ? 1 mm3). Excised gel plugs
were rehydrated by acetonitrile and dried for 5 min. The gels were
then immersed in 10 mM dithiothreitol solution for 1 h to break
the disulphide bonds. Iodoacetamide (100 mM) was added and
incubated for a further 1 h in the dark. The gels were shrunk again
by acetonitrile and dried for 5 min. Proteins were digested over-
night at 37 ?C using 20 ll of 25 ngll?1of pepsin (Bio Basic, Canada)
at a weight ratio of 1:20 (protein:pepsin). The peptide solution
(pepsin-digested proteins) was extracted twice with 50% acetoni-
trile containing 0.05% formic acid, and evaporated overnight at
40 ?C. Extracted peptides were resuspended in 0.1% formic acid
and analysed by LC–MS/MS.
Second, nanoscale LC separation of suspended peptides was per-
formed with a NanoAcquity UPLC?System (Waters Corp., Milford,
Fig. 1. Column chromatography by (a) Sephadex G-100, and (b) Q Sepharose, showing protein levels (A280) and trypsin activity (mol p-nitroaniline min?1ml?1) of purified
trypsin from tilapia intestine.
Table 1
Purification and kinetic parameters of purified trypsin from Nile tilapia. The activity assay was performed at 37 ?C for 60 s with 50 mM Tris-HCl buffer, pH 8.0, by using varied
concentration of specific substrate ranging from 0.1 to 1 mM.
Procedure Protein (mg)Trypsin activity (unit) Trypsin specific activity (unit/mg)Purity (fold) Yield (%)
Crude extract
Ammonium sulphate precipitation (20–60%)
Sephadex G100 column
Q sepharose column
9.81
3.86
1.67
0.38
44.4
25.8
12.6
9
4.53
6.68
7.54
23.7
1
1.47
1.66
5.34
100
39.4
17
3.77
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MA), equipped with a Symmetry C18, 5 lm, 180 lm ? 20 mm trap
columnandaBEH130C18,1.7 lm,100 lm ? 100 mmanalyticalre-
versed-phase column (Waters). Mobile phase A was 0.1% formic
acid in water, and mobile phase B was 2–80% formic acid in acetoni-
trile. The peptides were initially transferred with mobile phase A to
the trap column with a flow rate of 3 llmin?1for 3 min. Subse-
quently, the peptides were separated with a gradient of 2–40% mo-
bile phase B over 15 min at a flow rate of 1 llmin?1, followed by a
3 min rinse with 80% of mobile phase B. The column temperature
was maintained at 35 ?C. Lock mass was delivered from the auxil-
iary NanoAcquity pump with a constant flow rate of 500 nlmin?1
at a concentration of 200 fmolll?1of [Glu1] fibrinopeptide B to
thereferencesprayeroftheNanoLockSpraysourceofthemassspec-
trometer. The peptides were analysed by using a SYNAPT HDMS
mass spectrometer (Waters). For all measurements, the mass spec-
trometer was operated in the V-mode of analysis. All analyses were
processed using positive nanoelectrospray ion mode. Time-of-flight
analyser of the mass spectrometer was externally calibrated with
[Glu1] fibrinopeptide B from m z?1of 100–1600. The reference
sprayer was sampled with a frequency of 30 s. Spectral acquisition
time was 0.5 s. The quadrupole mass analyser was adjusted such
that ions from m z?1of 100–1600 were efficiently transmitted.
Finally, the resulting MS/MS data set was exported in Micro-
mass (⁄.pkl) and (⁄.mgf) formats for automated peptide identifica-
tion using Mascot software (Matrix Science, London, UK) (Perkins,
Pappin, Creasy, & Cottrell, 1999). The data were searched against
the NCBI database for protein identification. Database selection
was: taxonomy (chordate vertebrates and relatives); enzyme (pep-
sin A); variable modifications (carbamidomethylation of cysteine,
oxidation of methionine residues); mass values (monoisotopic);
protein mass (unrestricted); peptide mass tolerance (±2 Da); frag-
ment mass tolerance (±1.5 Da); peptide charge state (1+, 2+ and
3+); maximum missed cleavages (3 positions). ESI-Quad-TOF was
selected as an instrument. The report top was set at 50 for the
increments of possible proteins. Proteins were identified with an
individual mascot score corresponding to p < 0.05.
2.8. Multiple sequence alignment
Multiple sequence alignment for fish trypsin was performed by
using deduced amino acid sequences and the corresponding Nile
tilapia sequence from MS/MS data by CLUSTAL W 2.0. The align-
ment file was then subjected to BOXSHADE.
2.9. Effects of pH and temperature on the activity of trypsin
The optimal pH for enzymatic activity of trypsin was assayed
over the pH range of 3.0–12.0 (50 mM acetate buffer for pH 3.0–
6.0, 50 mM Tris–HCl buffer for pH 7.0–9.0, 50 mM glycine–NaOH
for pH 9.0–11.0, and 50 mM KCl–NaOH for pH 12.0), using an initial
enzyme reaction rate of 1 min with BAPNA, as described above, at
30 ?C. The effect of pH on enzyme stability was evaluated by mea-
suring the residual activity after incubation with the various pH
buffers for 30 min at 30 ?C (Silva et al., 2011).
The optimal of temperature for trypsin activity was determined
using the initial enzyme reaction rate of 1 min with BAPNA at dif-
ferent temperatures (20–100 ?C) in preheated 50 mM Tris–HCl buf-
fer, pH 9.0 (the observed pH optimum). For thermal stability, the
enzyme was incubated with preheated 50 mM Tris–HCl buffer,
pH 9.0, at different temperatures (20, 30, 40, 50, 60, 70, 80, 90
and 100 ?C) for 30 min (Silva et al., 2011) in a temperature-con-
trolled heat box. Thereafter, the treated samples were immediately
chilled on ice. The residual activity was assayed using an initial en-
zyme reaction rate of 1 min with BAPNA at pH 8.0, as described
above, at around the optimal temperature observed of 60 ?C.
2.10. Effects of inhibitors and activators on trypsin activity
The effects of various inhibitors were determined on trypsin
activity using phenylmethylsulfonyl fluoride (PMSF), ethylenedi-
aminetetraacetic acid (EDTA), SDS, dithiothreitol (DTT), b-mercap-
toethanol, Triton X-100, Tween 20, Tween 80, H2O2 (hydrogen
peroxide), alkylbenzene sulphonate, and alcohol ethoxylate (kindly
Fig. 2. Electrophoresis of purified trypsin from tilapia intestine by: (a) 12% SDS–PAGE at each purification step, i.e. crude extract (1), 40%–60% (NH4)2SO4fraction (2),
Sephadex G-100 fraction (3), and NaCl gradient Q Sepharose fraction (4), showing standard protein markers in kDa (M); (b) native-PAGE zymography of the purified trypsin
from N. tilapia. MS/MS fragmentation is presented in the MS/MS spectrum. Each ion fragment corresponding to the spectrum is explained using b- and y-ions; (c) alignment of
the amino acid sequences from MS/MS spectrum of the purified N. tilapia trypsin (NtTryp1 and NtTryp2) with amino acid sequence from other fish species. Identical amino
acid residues are shaded in black and similar amino acid residues are shaded in grey. The GenBank Acc nos: XaTryp, Xiphister atropurpureus (AAX85688) (Gawlicka & Horn,
2006): CvTryp, Cebidichthys violaceus (AAX83265) (Gawlicka & Horn, 2006): SsTryp, Salmo salar (NP_001117183.1) (Male, Lorens, Smalås, & Torrissen, 1995): DlTryp,
Dicentrarchus labrax (CAA07315) (Péres, Zambonino Infante, & Cahu, 1998): PmTryp, Paranotothenia magellanica (CAA57701) (Genicot, Rentier-Delrue, Edwards, Van Beeuman
& Gerday, 1996): XmTryp, Xiphister mucosus (AAX85687) (Gawlicka & Horn, 2006): ApTryp, Anoplarchus purpurescens (AAX85686) (Gawlicka & Horn, 2006).
1536
S. Unajak et al./Food Chemistry 134 (2012) 1533–1541

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provided by Yosapong Temsiripong, Sriracha Moda Co., Ltd.,
Thailand). The purified enzyme solution was incubated with each
inhibitor at a ratio of 1:1 (v/v) in 50 mM Tris–HCl buffer, pH 8.0,
for 1 h at 37 ?C before the remaining enzyme activity was esti-
mated using an initial enzyme reaction rate of 1 min with BAPNA
as a substrate, as described above. The activity of the enzyme, as-
sayed in the absence of inhibitors, was valued as 100%.
2.11. Kinetic studies of trypsin
The purified trypsin (1 mgml?1) was assayed with different fi-
nal concentrations of BAPNA, ranging from 0.1 to 1 mM in
50 mM Tris–HCl buffer, pH 8.0, at 37 ?C, using a 1 min initial en-
zyme reaction rate, as described above. The determinations were
repeated twice, and the respective kinetic parameters, including
Vmax and Km, were evaluated using a Hanes plot. The turnover
number (Kcat) was calculated from the following equation:
Kcat¼ Vmax=½E?
where [E] is the active enzyme concentration.
3. Results and discussion
3.1. Purification of trypsin
Trypsin from pooled intestines of 6 month-old tilapia was puri-
fied using a three-step procedure: by ammonium sulphate precipi-
tation, Sephadex G-100, and Q Sepharose chromatography. At all
purification steps, trypsin activities in each fraction were deter-
mined by using BAPNA as a specific substrate. A single predominant
Fig. 3. Studies of (a) pH profile and pH stability at 30 ?C, and (b) temperature profile and temperature stability at pH 9.0 of purified trypsin from the NaCl gradient Q
Sepharose fraction.
S. Unajak et al./Food Chemistry 134 (2012) 1533–1541
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peak of trypsin was observed after the Sephadex G-100 column
(Fig. 1a), whereas two dominant peaks of trypsin were observed
after the Q Sepharose column: one during washing the column,
and the other in the fractions from NaCl gradient elution (Fig. 1b).
The purified trypsins obtained during washing the Q Sepharose col-
umnwerenotstudied,sincethemajorityof trypsinisinboundfrac-
tions. However the purified trypsin from the NaCl gradient pooled
fraction was studied, and showed a specific activity of 24.2 unit
mgprotein?1withapurityof5.34-foldand3.77%recovery(Table1).
The SDS–PAGE at each purification step was illustrated, indicating a
highly purified trypsin obtained at the final step of purification
(Fig. 2a). The native-PAGE zymography analysis also showed a sin-
gle band, indicating the homogeneity of the purified trypsin and
confirming the single isoform of purified trypsin (Fig. 2b). The puri-
fied enzyme from the NaCl gradient Q Sepharose fraction revealed a
single band (Figs. 2a and b) with a molecular weight of approxi-
mately 22.4 kDa.
The molecular weights of trypsin were similar among different
fish species, such as Nile tilapia (Supannapong et al., 2008; Wang
et al., 2010), smooth hound (Bougatef et al., 2010), skipjack tuna
(Klomklao et al., 2009), grey triggerfish (Jellouli et al., 2009), Pacific
cod and saffron cod (Fuchise et al., 2009), and the Amazonian fish
tambaqui (Marcuschi et al., 2010).
3.2. Protein identification by tandem mass spectrometry
After digestion of the expected proteins from the NaCl gradient
Q Sepharose fraction by SDS–PAGE with pepsin, the peptide frag-
ments were separated by LC–MS/MS and analysed by mascot
searching with Chordata taxonomy (vertebrates and relatives).
The peptide sequences, L.IQLSRPATL.N and L.IQLNRPATL.N were
identified in three and one (respectively) purified trypsin samples.
Although searching did not present a high score, the results re-
vealed exactly the same sequence of peptides in the genus Oreochr-
omis. Multiple sequence alignment of two amino acid sequences
identified from tandem mass spectrometry (NtTryp1 and NtTryp2)
shared significant similarity to those of trypsins from other teleosti
(Fig. 2c). This indicated that the purified protein with tryptic activ-
ity in this study was authentic trypsin.
3.3. Effect of pH on the activity and stability of Tilapia trypsin
Purified tilapia trypsin was active at pH between 6.0 and 11.0.
The relative activity of the enzyme profoundly increased at pH
around 6–8, with the optimal pH of trypsin activity observed at
around pH 9.0; its relative activity sharply decreased at pH greater
than 11.0 (Fig. 3a). The purified enzyme also showed high stability
at the pH range of 6.0–11.0. The activity of the enzyme was lost at
pH 6 5.0 and pH 12.0. This optimum pH of trypsin activity, in a pH
range of 8.0–10.0, has been observed in many fish species, such as
Nile tilapia (El-Shemy & Levin, 1997; Supannapong et al., 2008;
Wang et al., 2010), striped sea bream (El Hadj Ali, Hmidet,
Zouari-Fakhfakh, Ben Khaled, & Nasri, 2010), and yellowfin tuna
(Klomklao et al., 2006).
3.4. Optimum temperature and thermal stability
The activity of the purified trypsin in tilapia intestine was high
at a temperature range of 50–80 ?C, with an optimum temperature
around 55–60 ?C (Fig. 3b). Enzymatic activity decreased at temper-
atures below 40 ?C and above 80 ?C. The enzymatic activity of tryp-
sin from other fish species, such as skipjack tuna, smooth hound,
striped sea bream, and tambaqui (Bougatef et al., 2010; El Hadj
Ali et al., 2009; Klomklao et al., 2009; Marcuschi et al., 2010), as
well as Atlantic salmon (Rungruangsak, 2007; Rungruangsak &
Male, 2000), revealed similar activity profiles, with trypsin highly
active at an approximate temperature range of 40–60 ?C.
In terms of thermal stability, the activity of the purified trypsin
was stable after incubating the enzyme for 30 min at a tempera-
ture 680 ?C (Fig. 3b). Trypsin activity was undetectable after incu-
bating the enzyme for 30 min at a temperature above 80 ?C
(Fig. 3b). The purified trypsin from tilapia in this study showed a
very high tolerance to high temperature. The remaining trypsin
activity (100% when assayed at 60 ?C) after incubation for 30 min
at 70–80 ?C was higher than that reported for Atlantic salmon tryp-
sin from crude enzyme extract (Rungruangsak, 2007), as well as
those of other fish trypsins and trypsin isolated from tilapia in pre-
vious works (El-Shemy, & Levin, 1997; Supannapong et al., 2008;
Wang et al., 2010).
3.5. Effects of inhibitors and activators on trypsin activity
The effects of inhibitors on the enzymatic activity of the purified
tilapia trypsin are summarised in Table 2. The inhibition levels de-
pended on the concentrations of the inhibitors. For example, in the
presence of an inhibitor concentration of 1–5 mM, trypsin activity
was inhibited >22% by PMSF (a potent serine protease inhibitor),
>46% by EDTA (metalloprotease inhibitor), >96% by DTT, and com-
pletely 100% inhibited by 0.5–1% SDS. The result of full inhibition
of trypsin activity by SDS was surprising (Table 2), because trypsin
was still active on native-PAGE containing 10% SDS (Fig. 2b). This
may be due to the interaction between the SDS in monomer form
(1% SDS) and the active site of trypsin, which prohibits the enzyme
activity; while the SDS in micellular form (10% SDS in polyacryl-
amide) interacts with trypsin without interfering with the enzyme
active site.
Apart from the enzymatic activity of this purified trypsin at
alkaline pH, the imperishable nature of the enzyme activity was
observed. Under the same conditions, the enzyme was not active
at temperatures greater than 90 ?C. This indicated that the pH
change has no effect on protein structure (similar to temperature
change). The application of trypsin over the alkaline pH range of
8.0–11.0 is very useful for hydrolysing protein stains such as blood
Table 2
Effect of proteinase inhibitors and activators on enzyme activity.
InhibitorsClassificationConcentration Relative trypsin
activity (%)
Control
PMSF
–
Serine
Proteinase
–
5 mM
100
77.9
1 mM
5 mM
73.8
32.7EDTA Metallo
Proteinase
1 mM
5 mM
53.9
3.33
DTTReducing
reagent
1 mM
5 mM
0
77.6
b-MercaptoethanolReducing
reagent
1 mM
1% (w/v)
0.5% (w/v)
5% (v/v)
5% (v/v)
5% (v/v)
3% (v/v)
1% (v/v)
1% (v/v)
82
0
0
71.3
69.4
58.9
63.5
77.2
257
SDSDetergent
Triton x-100
Tween 20
Tween 80
H2O2
Detergent
Detergent
Detergent
Wetting NLF
(Alkylbenzene
sulphonate)
Detergent
0.1% (v/v)
1% (v/v)
80.6
129Adunil CEL (Alcohol
ethoxylate)
Detergent
0.1% (v/v)47.3
1538
S. Unajak et al./Food Chemistry 134 (2012) 1533–1541

Page 7

and milk, in addition to laundry detergents (El Hadj Ali et al., 2009).
To determine the stability of this alkaline protease, the purified
trypsin was pre-incubated for 1 h at 37 ?C in the presence of a sur-
factant, alcohol ethoxylate of 0.1% (v/v). The activity of the trypsin
was reduced by approximately 52% (Table 2). Interestingly, the
activity of trypsin was dramatically increased at a higher concen-
tration (1% v/v) of alcohol ethoxylate, and in the presence of
the other industrially used detergent, alkylbenzene sulphonate
(Table 2). This observation indicated that a purified trypsin from
intestine of tilapia had a higher stability in some detergents than
had the trypsin from Lithognathus mormyrus (El Hadj Ali et al.,
2009). The results indicated that this trypsin could be an effective
protease enzyme for industrial applications.
3.6. Kinetic studies of trypsin
Kinetic constants of the purified tilapia trypsin were analysed
using a Hanes plot. The Michaelis–Menten constant (Km) and
Fig. 4. (a) Electrophoresis native-PAGE zymography of trypsin isozymes from crude enzyme extracts of tilapia intestine at different fish ages of 1 month (1), 3 months (3),
4 months (4), 5 months (5), 6 months (6), and 7 months (7). Standard protein markers (M) with molecular weight in kDa and (b) Specific activities of trypsin at different fish
ages are shown.
S. Unajak et al./Food Chemistry 134 (2012) 1533–1541
1539

Page 8

catalytic constant (Kcat) were 0.16 mM and 23.8 s?1, respectively,
with catalytic efficiency (Kcat/Km) of 238 s?1mM?1. The Kmof the
purified trypsin was similar to that of trypsin isolated from grey
triggerfish (Jellouli et al., 2009), and had a lower value than that
of a previously isolated fish trypsin (Bougatef et al., 2010; El Hadj
Ali et al., 2009; Klomklao et al., 2009), suggesting higher affinity
for substrate than those of purified fish trypsin. However, the cat-
alytic efficiency of tilapia trypsin and trypsin from skipjack tuna
(266–316 s?1mM?1) revealed greater values than those of other
fish trypsins (Klomklao et al., 2009).
3.7. Trypsin isoforms at different fish ages
The analyses of protease isoforms in fish intestine in the first to
seventh month of fish age, were done by comparing their activity
in each fish age, together with zymography. Zymograms, to deter-
mine specific trypsin activity, revealed at least seven isoforms of
active trypsins present in the digestive tracts of Nile tilapia (Fig
4a). The enzyme extracted from pyloric caeca and intestine of
Atlantic salmon revealed six trypsin-like isozymes (Torrissen,
1987), which is similar to the number of trypsin isoforms in this
study. Among them, isoform 2 was obtained at all fish ages with
similar band intensity. Similar to isoform 2, isoform 7 was also ob-
served at all fish ages but it activity only disappeared in the first
month of fish culture. Interestingly, some trypsin isoforms were
activated only at some specific fish ages. Isoform 1 was activated
only at the fourth month and isoform 6 only at the third month.
Isoform 4 was strongly active at the fifth month and its activity
was dramatically decreased in fish rearing to the sixth month.
However, in fish, several trypsin isoforms were visualised at differ-
ent fish ages (Chakrabarti, 2006; Rathore, 2005). This suggests that
a specific type of protease was produced at a specific fish age by
mean of fish ontogenesis.
The analyses of overall trypsin activities, at all fish ages, were
performed under optimum conditions. Maximum specific trypsin
activity was detected at the first and the seventh months of rearing
whereas the lowest activity was detected in the sixth month
(Fig. 4b). By comparing with band intensity in the zymogram, the
highest activities were detected in the first 5 months of culture
which correlated with overall band intensity at each fish age. The
lowest trypsin specific activity was detected at the age of 6th
months which may result from the lack of trypsin isoform 3 and
isoform 4. However, from the specific trypsin activity, isoform 2
may serve as a basal trypsin enzyme which functions at all fish
ages. On the other hand, the highest specific trypsin activity was
observed in fish at 7th months when compared with all fish ages
(Fig 4b). The result from the zymogram revealed the presence of
isoform 2 and isoform 3 with lack of isoform 5 in fish cultured
for 7 months. This evidence may indicate that the activity of tryp-
sin isoform 5 may be minor in trypsins which differed from iso-
form 2 and isoform 3. Like isoform 5, isoform 7 which is strongly
detected at the fifth month, is not involved in increasing overall
trypsin specific activity.
Period of sample collection is important for determining the
activation or secretion of trypsin. The enzyme synthesis is usually
triggered by food ingestion and trypsin secretion. Trypsin secretion
levels were different in fed and fasting yellowtail (Seriola quinqu-
eradiata) (Murashita, Kubota, Kofuji, Hosokawa, & Masumoto,
2005). However, in this study, the basal trypsin, isoform 2, showed
similar band intensities at all fish ages. This demonstrated that the
tryptic activities in the zymogram, among different fish ages, were
comparable. Moreover, during culture of Nile tilapia, protein con-
tent in fish feed diet varied. The protein composition in feed mainly
arises from plants at protein levels of 35%, 30% and 24–26% during
2–3 months, 4–5 months and 6–10 months. The type of specific
trypsin isoforms and specific trypsin activities, at 1st–3rd,
4th–5th, and 6th months were not correlated with protein formula
in feed (Fig. 4a and b). Thus, we cannot summarise the relationship
between protein composition in fish feed diet and trypsin isoforms,
or trypsin specific activity at different fish ages. Taken together,
different active tilapia proteases were observed at different fish
ages; this indicated specific functions of particular enzyme iso-
forms in fish growth, by mean of fish ontogenesis, as described
above (Abi-Ayad & Kestemont, 1994; Bassompierre et al., 1998;
Cahu et al., 1998; Lazo, Holt & Arnold, 2000; Torrissen, 1987).
Due to the purification chromatogram (Fig. 1b), approximately
two-thirds of the protein with trypsin activity was lost during puri-
fication steps – only a single isoform of trypsin was isolated. By
comparison with the trypsin isoform of 6th month fish, the protein
band with highest trypsin activity accounted for more than half of
all trypsin activity (Fig. 4b). Thus, we cannot define the authentic
trypsin isoform purified. However, we suppose that the majority
of trypsin isoform, which is found at all fish ages might serve as
a house-keeping trypsin which provides common protease func-
tion for all fish ages. Moreover, our purified trypsin may play some
special role during fish development that may be expressed at a
specific age or depends on the composition of feed.
4. Conclusion
At least six trypsin isozymes (trypsin phenotypes), were ob-
served in Nile tilapia intestine. Most of them were lost during puri-
fication processes. However, the trypsin isozyme that was purified
from Nile tilapia intestine in this study showed stability at higher
temperature than did other fish trypsins. The development of this
trypsin isozyme for industrial use may be applicable for processes
that require high temperature, together with the use of detergents.
The variations in patterns of trypsin isozymes observed at different
ages may affect their catalytic efficiency by mean of fish ontology;
this may enable us, in the future, to predict feed efficiency and fish
growth performance under different growth conditions.
Acknowledgements
This work was financially supported by: the Reverse Brain Drain
Project (RBD), National Science and Technology Development
Agency (NSTDA), Thailand, and the Faculty of Science, Kasetsart
University, Bangkok, Thailand. We also thank Mr. Yosapong
Temsiripong (Sriracha Moda Co., Ltd., Thailand) for providing the
industrialdetergents, alkylbenzene
ethoxylate, used for this study.
sulphonate andalcohol
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
03.074.
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