Calcium dependent, alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

Article abstract of DOI:10.1016/j.procbio.2012.07.002

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa. The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^-1. The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.

Full text of DOI:10.1016/j.procbio.2012.07.002

Process Biochemistry 47 (2012) 1957–1964
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
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Calcium dependent, alkaline detergent-stable trypsin from the viscera of Goby
(Zosterisessor ophiocephalus): Purification and characterization
Rim Nasri, Assaâd Sila, Naourez Ktari, Imen Lassoued, Ali Bougatef, Maha Karra-Chaâbouni, Moncef Nasri∗
Laboratoire de Génie Enzymatique et de Microbiologie – Ecole Nationale d’Ingénieurs de Sfax, B.P. 1137-3038 Sfax, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2012
Received in revised form 29 June 2012
Accepted 1 July 2012
Available online 7 July 2012
An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was puri-
fied to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin
appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE.
The enzyme had an estimated molecular weight of 23.2 kDa.
The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH
7.0 and 11.0. The optimum temperature for enzyme activity was 60 ◦C, and the activity and stability
of trypsin was highly dependent on the presence of calcium ion. At 60 ◦C, Ca2+ (5 mM) stimulated the
Keywords:
Alkaline trypsin
protease activity by 220%. The trypsin kinetic constants, Km and kcat, were 0.312 mM and 2.03 s−1
.
Biochemical characterization
Goby
Zosterissessor ophiocephalus
Calcium-dependent
Detergent-stable
The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the
enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
pH 2.0 and 4.0, while alkaline digestive proteases, such as trypsin,
are most active between pH 8.0 and 10.0, and inactive or unstable at
Proteases constitute the most important group of industrial
enzymes and are widely used in different industrial processes
such as detergent, food, leather and pharmaceutical industries
[1]. Proteases with high activity and stability in high alkaline
range are interesting for biotechnological applications. Their major
application is in detergent industry because the pH of laun-
dry detergents is generally in the range of 9.0–12.0, accounting
for about 35% of the total enzymes sales [2]. Alkaline pro-
teases are particularly important because they are both stable
and active at high pH solutions and in the presence of deter-
gents [3]. Most of the commercial alkaline proteases were isolated
from Bacillus species [4]. The industrial demand of proteolytic
enzymes, with appropriate specificity and stability to pH, tem-
perature and surfactants, continues to stimulate the search for
new enzymes sources. Fish viscera, one of the most important by-
products of fishing industry, is a rich source of digestive enzymes
[5].
acidic pH. Trypsin is a member of a large family of serine proteinases
which specifically cleave proteins and peptides at the carboxyl
group of arginyl and lysyl residues and play major roles in biolog-
ical processes, including digestion and activation of zymogens of
chymotrypsin and other enzymes, including itself [6]. Trypsin (EC
3.4.21.4) is an important pancreatic serine protease synthetized as
a proenzyme in the pancreatic acinar cells and secreted into the
intestine of mammals.
Trypsin and trypsin-like proteolytic enzymes have been isolated
and characterized from the viscera of some marine invertebrates
and a wide range of cold water and warm water fish, including
the digestive gland (hepatopancreas) of cuttlefish (Sepia offic-
inalis) [7], the pyloric ceca of Chinook salmon (Oncorhynchus
tshawytscha) [8], and the entire viscera of smooth hound (Mustelus
mustelus) [9], Grey triggerfish (Balistes capriscus) [10], Amazo-
nian fish tambaqui (Colossoma macropomum) [11] silver mojarra
(Diapterus rhombeus) [12], and zebra blenny (Salaria basilisca)
[13].
The most important proteolytic enzymes from fish and aquatic
invertebrate’s viscera are the aspartic protease pepsin, and the
serine proteases, trypsin, chymotrypsin, collagenase and elastase.
Acidic proteases from fish stomachs display high activity between
The goby (Zosterisessor ophiocephalus) is common in Mediter-
ranean Sea, Black Sea, and Sea of Azovthe. It reaches a maximum
length of 25 cm and it is a carnivorous; juveniles feed on small crus-
taceans, polychaets and molluscs. It is relatively important in the
fish-catches of Tunisia, and is utilised for human consumption. In
Tunisia, goby (Z. ophiocephalus) catches were about 130 tonnes in
2004 [14].
∗
Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595.
E-mail addresses: moncef.nasri@enis.rnu.tn, mon nasri@yahoo.fr (M. Nasri).
1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2012.07.002

1958
R. Nasri et al. / Process Biochemistry 47 (2012) 1957–1964
7.5% acetic acid. The molecular weight of the trypsin was estimated using a low-
In a previous study, we reported the characterization of goby
molecular weight calibration kit as markers, consisting of: bovine serum albumin
(66,000 Da); ovalbumin (45,000 Da); glyceraldehyde-3-dehydrogenase (36,000 Da);
bovine trypsinogen (24,000 Da); soybean trypsin inhibitor (20,100 Da) and bovine
␣-lactoalbumin (14,200 Da).
Native-PAGE was performed according to the procedure of Laemmli [17], except
that the sample was not heated and SDS and reducing agent were left out. Protease
activity staining was performed on native-PAGE according to the method of Garcia-
Carreno et al. [18] with a slight modification [15].
crude alkaline enzyme extract, which contains several proteolytic
enzymes [15]. The crude enzyme extract showed an optimum activ-
ity at 50 ◦C and pH 8.0, using casein as a substrate.
In the present study, we describe the purification of an alka-
line detergent stable trypsin from goby (Z. ophiocephalus), and we
provide basic information about its main biochemical and kinetic
characteristics.
2.6. Determination of the N-terminal amino acid sequence of Z. ophiocephalus
trypsin
2. Materials and methods
2.1. Reagents
The purified enzyme, from Sephadex G-100 gel filtration, was applied to SDS-
PAGE. After brief staining with Coomassie Brilliant Blue R-250, the PVDF band
corresponding to the trypsin was excised and the N-terminal amino acid sequence
was determined by the Edman degradation method on an automated ABI Procise
494 protein sequencer (Applied Biosystems, Foster City, CA).
Casein sodium salt from bovine milk, ethylenediaminetetraacetic acid (EDTA),
phenylmethylsulphonyl fluoride (PMSF), dithio-bis-nitrobenzoic acid (DTNB), N˛-
benzoyl-dl-arginine-p-nitroanilide (BAPNA), benzamidine, glycine, bovine serum
albumin and protein markers for molecular weights 14,000–66,000 Da were pur-
chased from Sigma Chemical Co. (St. Louis, MO, USA). Soybean trypsin inhibitor
(SBTI) was obtained from Fluka Biochemica (USA). Sodium dodecyl sulphate
(SDS), acrylamide, ammonium persulphate, N,N,Nꢀ,Nꢀ-tetramethyl ethylenediamine
(TEMED) and Coomassie Brilliant Blue R-250 were from Bio-Rad Laboratories
(Mexico City, Mexico). Sephadex G-100 was from Amersham Pharmacia Biotech
(Uppsala, Sweden). Tris (hydroxymethyl) aminomethane was procured from Pan-
reac Quimica SA (Barcelona, Spain). Polyvinylidene difluoride (PVDF) membrane
was purchased from Applied Biosystems (Roissy, France). All other reagents were
of analytical grade.
2.7. Protein determination
Protein concentration was determined by the method of Bradford [19], using
bovine serum albumin as a standard. The concentration of protein during the purifi-
cation studies was determined by measuring the absorbance at 280 nm.
2.8. Biochemical properties
2.8.1. Effect of pH on activity and stability
Activity of the purified trypsin was assayed at different pH values for 10 min at
60 ◦C using BAPNA as a substrate. To check the pH stability, enzyme was preincu-
bated at various pHs (pH 7.0–12.0) for 60 min at 25 ◦C and the remaining activities
were measured under the standard assay condition. The following buffer systems
were used: 100 mM sodium acetate buffer, pH 6.0; 100 mM phosphate buffer, pH
7.0; 100 mM Tris–HCl buffer, pH 8.0; 100 mM glycine–NaOH buffer, pH 9.0–11.0;
100 mM Na2HPO4, pH 12.0.
2.2. Goby viscera
Goby (Z. ophiocephalus) was purchased from the fish market of Sfax City, Tunisia.
The samples were packed in polyethylene bags, placed in ice with the sample per
ice ratio of approximately 1:3 (weight per volume) and transported to the research
laboratory within 30 min. Viscera was separated, rinsed three times with cold dis-
tilled water to remove salts and contaminants, and then used immediately for the
extraction of digestive enzymes.
2.8.2. Effect of temperature on activity and stability
To investigate the effect of temperature, trypsin activity was determined by
incubating the reaction mixture at different temperatures ranging from 30 to 80 ◦C,
using BAPNA as a substrate for 10 min at pH 9.0. For thermal stability, the puri-
fied enzyme was incubated at different temperatures for 60 min, and then residual
activities were assayed at pH 9.0 and 60 ◦C for 10 min. Thermal inactivation was
also examined by incubating the purified trypsin at 50 ◦C for 4.5 h in the absence
or presence of 2 mM CaCl2 or 2 mM EDTA. Aliquots were withdrawn at desired
time intervals to test the remaining activity at standard conditions. The non-heated
enzyme was considered to be the control (100% activity).
2.3. Enzyme purification
Purification was carried out at 4 ◦C. The crude enzyme extract from 160 g of
viscera, prepared as previously described [15], was first subjected to ammonium
sulphate fractionation. Ammonium sulphate fractions of 0–20%, 20–60% and 60–80%
(w/v) were collected by centrifugation at 10,000 × g, and the resulting precipitates
obtained in each fraction were suspended in a minimal volume of buffer A (10 mM
Tris–HCl, pH 8.0). The precipitates were dialysed for 24 h at 4 ◦C against repeated
changes of the same buffer.
The 20–60% (w/v) ammonium sulphate fraction was subjected to gel filtration
on a Sephadex G-100 column (2.5 cm × 80 cm) which had been equilibrated with
buffer B (25 mM glycine–NaOH pH 9.0 containing 0.5 per mille Triton X-100). Frac-
tions of 4 ml were collected at a flow rate of 30 ml/h with the same buffer. Protein
content (Absorbance at 280 nm) and protease activity (using casein) and trypsin
activity (using BAPNA) were determined. The fractions with high trypsin activity
were pooled.
2.8.3. Effects of enzyme inhibitors, metal ions and denaturing reagents
The effects of enzyme inhibitors on trypsin activity were studied using PMSF,
SBTI, benzamidine, DTNB, Pepstatin A and EDTA. The purified enzyme was prein-
cubated with each inhibitor for 30 min at 25 ◦C, and then the remaining enzyme
activity was tested using BAPNA as a substrate. The activity of the enzyme assayed
in the absence of inhibitors was taken as 100%.
The effects of various metal ions (5 mM) on trypsin activity were investigated
by adding the monovalent (Na+ or K+) or divalent (Ca2+, Mn2+, Zn2+, Cu2+, Ba2+, Mg2+
or Hg2+) metal ions to the reaction mixture. The effect of CaCl2 concentration on
trypsin activity was also studied. The activity of the enzyme in the absence of metal
ions was taken as control.
The effects of some surfactants (Triton X-100, Tween 80 and SDS) and oxi-
dizing agents (sodium perborate and H2O2) on enzyme stability were studied by
pre-incubating the purified trypsin for 1 h at 30 ◦C. The residual activity was mea-
sured at pH 9.0 and 60 ◦C. The enzyme activity of the control (without any detergent)
was taken as 100%.
2.4. Trypsin activity assay
Enzyme activity was measured according to the method of Benjakul et al. [16],
using BAPNA as substrate specific for trypsin. An aliquot of the enzyme solution
(200 ␮l), with an appropriate dilution, was added to the pre-incubated reaction
mixture containing 1000 ␮l of 0.5 mM BAPNA in 0.1 M glycine–NaOH buffer, pH
9.0, and 200 ␮l of distilled water. The mixture was incubated for 10 min at 60 ◦C.
The enzymatic reaction was terminated by adding 200 ␮l of 30% (v/v) acetic acid,
and then centrifuged at 8000 × g for 3 min at room temperature. Trypsin amidase
activity was measured by the absorbance at 410 nm due to p-nitroaniline released.
One unit (U) of trypsin activity was defined as the amount that released 1 ␮mol
of p-nitroaniline per minute under the established conditions using a molecular
2.8.4. Kinetic studies
The activityof the purifiedtrypsin was evaluatedat 25 ◦C with differentfinal con-
centrations of BAPNA, ranging from 0 to 2000 ␮M. The final enzyme concentration of
the assay was 0.025 mg protein per ml. The determinations were repeated twice and
coefficient of 8800 M−1 cm−1
.
the respective kinetic parameters, including the apparent Michaelis–Menton (Km
)
2.5. Polyacrylamide gel electrophoresis and detection of protease activity by
zymography
and the maximum velocity (Vmax) were calculated from Lineweaver–Burk plots [20].
The value of the turnover number (kcat) was calculated from the following equation:
kcat = Vmax per [E], where [E] is the enzyme concentration.
SDS-PAGE was carried out for the control of the purity and determination of
molecular weight of the purified enzyme as described by Laemmli [17], using a 5%
(w/v) stacking and a 15% (w/v) separating gels. Samples were prepared by mixing
the purified enzyme at 1:5 (v/v) ratio with the SDS-PAGE sample buffer (10 mM
Tris–HCl (pH 8.0), 2.5% SDS, 10% glycerol, 5% ␤-mercaptoethanol and 0.002% bro-
mophenol blue). The samples were heated at 100 ◦C for 5 min before loading in
the gel. After electrophoresis, the gel was stained with 0.25% Coomassie Brilliant
Blue R-250 in 45% ethanol, 10% acetic acid and destained with 5% ethanol and
2.9. Detergent compatibility
The compatibility of the purified alkaline trypsin with commercial solid laundry
detergents was studied using Dixan (Henkel, Spain), Nadhif (Henkel-Alki, Tunisia),
Ariel (Procter and Gamble, Suisse), New Det (Sodet, Tunisia) and Axion (Colgate-
Palmolive, France). Commercial solid detergents were diluted in tap water to give a

R. Nasri et al. / Process Biochemistry 47 (2012) 1957–1964
final concentration of 7 mg per ml to simulate washing conditions. The compatibil-
1959
ity of the purified trypsin with commercial liquid detergents was also investigated
using Carrefour (Carrefour, France), Lav+ (Best LAV, Tunisia) and Tex’til (Belgique).
The liquid detergents were diluted 100-fold in tap water (1%) to simulate washing
conditions. The endogenous proteases contained in these detergents were inacti-
vated by heating the diluted detergents for 1 h at 65 ◦C prior to the addition of the
enzyme. The purified alkaline trypsin was incubated with different detergents for 1 h
at 30 and 40 ◦C, and then the remaining activities were determined under the stan-
dard assay conditions. The enzyme activity of a control, without detergent, incubated
under the similar conditions, was taken as 100%.
2.10. Statistical analysis
Statistical analyses were performed with statgraphics Ver. 5.1, professional edi-
tion (Manugistics Group, Rockville, MD) using ANOVA analysis. Differences were
considered significant at p < 0.05.
3. Results and discussion
3.1. Purification of Z. ophiocephalus trypsin
Trypsin from the viscera of Z. ophiocephalus was extracted and
purified by the two-step procedure described in Section 2. In
the first step, the crude enzyme extract was fractionated with
ammonium sulphate. The precipitate formed at 20–60% (w/v) sat-
uration showed higher specific activity (0.07 U/mg protein) than
precipitates formed at 0–20% (0.007 U/mg protein) and 60–80%
(0.017 U/mg protein). No activity was detected in the final super-
natant. The 20–60% fraction, with the greatest specific activity for
trypsin, was then subjected to Sephadex G-100 gel filtration. This
procedure yielded three peaks of protease activity using casein as
a substrate, and a single peak of trypsin activity using BAPNA as a
substrate (results not shown).
The specific activity in the initial enzyme extract was 0.05 U/mg
protein. After the final purification step, the trypsin was purified
16-fold, with a recovery of 20% and a specific activity of 0.8 U/mg
protein, using BAPNA as a substrate (Table 1).
Purified trypsin from goby migrated as a single band in
SDS-PAGE (Fig. 1a) and native PAGE (Fig. 1b), indicating the homo-
geneity of the enzyme and confirming that the purified trypsin
had only one isoform. The molecular weight of trypsin was esti-
mated to be 23.2 kDa based on SDS-PAGE (Fig. 1a), corresponding
to that determined by gel filtration. Most of fish trypsins have
been reported to have molecular weights in the range of 23–28 kDa
[21]. The molecular weight of Z. ophiocephalus trypsin was similar
to those from other fish species, such as walleye pollock (Ther-
agra chalcogramma) [22], true sardine (Sardinops melanostictus)
and arabesque greenling (Pleuroprammus azonus) [23], Amazonian
fish tambaqui (C. macropomum) [11], grey triggerfish (B. capriscus)
[10], and cuttlefish (S. officinalis) [7], and lower than those of
trypsins from zebra blenny (S. basilisca) [13], and silver mojarra
(D. rhombeus) [12].
Fig. 1. (a) SDS-PAGE of the purified trypsin from Z. ophiocephalus. Lane 1: standard
proteins marker of different molecular weights; lane 2: crude enzyme extract; lane
3: purified trypsin. (b) Native-PAGE (lane 1) and zymogram (lane 2) of the purified
trypsin from Z. ophiocephalus.
Z. ophiocephalus trypsin was identical to that of Japanese anchovy
(Engraulis japonica) [24]. The sequence of Z. ophiocephalus trypsin
showed high homology with those from smooth hound [9], grey
triggerfish [10] and arabesque greenling [23]. There is only one
amino acid residue in the 9-terminal sequence that differs in these
sequences. However, there are two amino acid residues in the 9-
terminal sequence that differ with trypsins from Amazonian fish
tambaqui [11], silver mojarra [12], walleye pollok [22], true sar-
dine (TR-S) [23], antarctic fish (Paranotothenia magellanica) [25],
mandarin fish (Siniperca chuasti) [26] and common Carp (Cyprinus
carpio) (trypsin A) [27]. The sequence of Z. ophiocephalus trypsin
showed also high homology with rat [28], dog [29], bovine [30],
porcine [31] and human [32] trypsins; they differ by three amino
acids at positions 6, 8 and 9.
Casein-zymogram staining, a very sensitive and rapid assay
method that detects nanograms of protein, revealed a unique clear
zone of proteolytic activity against the blue background of the gel,
indicating the purity of the trypsin (Fig. 1b).
3.2. N-terminal amino acid sequence of Z. ophiocephalus trypsin
The N-terminal 9 amino acids of the Z. ophiocephalus trysin were
determined to be IVGGYECQP. The N-terminal amino acid sequence
of Z. ophiocephalus trypsin showed uniformity, indicating that it
was isolated in a pure form and, if other isoforms are present, their
amounts must be small.
The N-terminal amino acid sequence of Z. ophiocephalus trypsin
was aligned with the sequences of other animal trypsins (Fig. 2).
The alignment indicates that the first four NH2-terminal amino
acid residues (IVGG) are conserved in all trypsins. The sequence of
3.3. Enzyme characterization
3.3.1. Effects of enzyme inhibitors on trypsin activity
Proteases can be classified by their sensitivity to various
inhibitors. To confirm the nature of the purified protease, activi-
ties were measured in the presence of different enzyme inhibitors
(Table 2). Trypsin from Z. ophiocephalus was strongly inhib-
ited by the well-known trypsin inhibitor investigated, namely,
SBTI (1 mg/ml) and by PMSF and benzamidine, serine protease

1960
R. Nasri et al. / Process Biochemistry 47 (2012) 1957–1964
Table 1
Summary of the purification of trypsin from Z. ophiocephalus viscera.
Purification steps
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Recovery (%)
Purity (fold)
Crude extract
Ammonium sulphate precipitation (20–60%)
Sephadex G-100
45
30
8.9
850
430
11.36
0.05
0.07
0.8
100
0.66
20
1
–
16
All operations were carried out at 4 ◦C.
inhibitors. On the other hand, the metalloprotease inhibitor (EDTA,
5 mM) inhibited the enzyme by 59%. The relatively high activity of
the trypsin in the presence of EDTA is very useful for application
as detergent additive because chelating agents are components of
most detergents. Chelating agents function as water softeners and
also assist in the removal of stain. These agents specifically chelate
metal ions making them unavailable in the detergent solution.
In addition, Pepstatin A, an aspartic protease inhibitor and DTNB,
specific for thiol proteases, did not show any inhibitory effects
against the purified trypsin. The inhibition results indicate that the
purified enzyme is a serine-protease based on inhibition by PMSF
and authentic trypsin based on its catalytic specificity for BAPNA
and inhibition by SBTI.
S. officinalis, which showed maximum activity at pH 8.0 [7] and
that of trypsin from D. rhombeus which exhibited higher enzyme
activity at pH 8.5 [12]. However, the optimum pH was lower than
that of B. capriscus trypsin which exhibited maximum activity at
pH 10.5 and showed about 91% activity at pH 11.0 [10].
The pH stability profile of trypsin from Z. ophiocephalus is shown
in Fig. 3b. The enzyme is highly stable in a broad pH range, and more
than 95% activity was retained even after incubation for 1 h at 25 ◦C
at pH between 7.0 and 11.0. However, it was unstable at pH 12.0.
The pH stability of Z. ophiocephalus trypsin is higher than monterey
sardine trypsin, which was stable in the pH range from 7.0 to 8.0
[33] and D. rhombeus trypsin, which maintained over 85% of its
optimum activity between pH 8.5 and 11.0 [12]. These results sug-
gest that the viscera of goby would be a potential source of trypsin
for certain food processing operations that require high alkaline
conditions.
3.3.2. Effect of pH on activity and stability of Z. ophiocephalus
trypsin
The pH activity profile of the purified Z. ophiocephalus trypsin
was investigated at 25 ◦C between pH 6.0 and pH 12.0. As can
be seen in Fig. 3a, the optimum pH value of the purified enzyme
was 9.0. The relative activities at pH 10.0 and 11.0 were about
91% and 50%, respectively, of that at pH 9.0. Therefore, Z. ophio-
cephalus trypsin could be a potential candidate for addition in
commercial laundry detergents, because the pH in laundry deter-
gent is generally in the range of 9.0–11.0. The optimum pH activity
of Z. ophiocephalus trypsin was higher than that of trypsin from
3.3.3. Effect of temperature on the activity and stability of Z.
ophiocephalus trypsin
Temperature profile of trypsin from Z. ophiocephalus is depicted
in Fig. 3c. The enzyme was active at temperatures from 30 to 80 ◦C
with an optimum around 60 ◦C. The relative activity at 50 ◦C was
about 85% of that at 60 ◦C. However, enzyme activity decreased
rapidly at higher temperatures. The sharp decrease in hydrolysis
of BAPNA might be attributed to irreversible protein denaturation
Table 2
Effect of various enzyme inhibitors, metal ions and additives on the activity of the purified trypsin from Z. ophiocephalus viscera.
Ions, inhibitors, surfactant, oxidizing agent, detergent
Concentration
Target proteases
Relative activity (%)
None
EDTA
DTNB
Pepstatin A
PMSF
–
5 mM
5 mM
0.1 mM
1 Mm
5 mM
5 mM
1 mg/ml
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
0.1% (w/v)
0.5%
–
100
41
99
100
58
0
1.15
0
102
98
112
110
10
Metallo-proteases
Cysteine proteases
Some aspartic proteases
Serine proteases
–
Benzamidine
SBTI
Serine proteases
Trypsine-like serine proteases
Na+
K+
Mg2+
Mn2+
Zn2+
Cu2+
0
0
220
95
52
Hg2+
Ca2+
Ba2+
SDS
40
1%
20
Tween 80
1% (v/v)
5%
1% (v/v)
5%
0.2% (w/v)
1%
1% (v/v)
5%
100
100
100
96
89.5
51.7
66.8
40.1
Triton X-100
Sodium perborate
H2O2
Purified enzyme was pre-incubated with various enzyme inhibitors for 30 min at 25 ◦C and the remaining activity was determined at pH 9.0 and 60 ◦C. Enzyme activity
measured in the absence of any inhibitor was taken as 100%.
The effect of metal ions on the activity of the purified trypsin was determined by incubating the enzyme in the presence of various metal ions for 10 min at 60 ◦C and pH
9.0. The enzymes were pre-incubated with surfactants, oxidizing agents for 1 h at 30 ◦C and the remaining protease activities were measured under standard conditions. The
activity is expressed as a percentage of the activity level in the absence of additives.

R. Nasri et al. / Process Biochemistry 47 (2012) 1957–1964
1961
due to the partial unfolding of the enzyme molecule. The optimal
temperature for Z. ophiocephalus protease was similar to trypsin
from S. basilisca [13], higher than that of trypsin from B. capriscus
[10], which had an optimum temperature at 40 ◦C and lower than
that of cuttlefish trypsin which had an optimum temperature at
70 ◦C [7].
The thermal stability profile showed that the purified trypsin
was highly stable at 30 and 40 ◦C for 1 h incubation, retaining 100%
and 96% of its activity, respectively, but was inactivated at temper-
atures over 40 ◦C (Fig. 3d). The enzyme retained 52% and 13% of
its initial activity after 60 min incubation at 50 and 60 ◦C, respec-
tively. The thermal stability profile is similar to trypsins from grey
triggerfish [10] and zebra blenny [13].
3.3.4. Effect of calcium on trypsin thermostability
The increased rate of proteolysis of proteases at elevated tem-
peratures is one of the factors responsible for the rapid thermal
inactivation of enzymes. Several works reported that calcium ion
plays a major role in enzyme stabilization at high temperatures. To
determine whether Ca2+ enhanced thermal stability of trypsin dur-
ing thermal inactivation, the stability was examined by incubating
the purified trypsin for 4 h 30 min at 50 ◦C in the absence and pres-
ence of 2 mM CaCl2 or 2 mM EDTA. The thermal stability profiles
reported in Fig. 4a showed a continuous decrease in trypsin activity
in the absence of CaCl2 or in the presence of EDTA with increasing
incubation time. The enzyme retained 50% and 34% of its activity
after 60 min incubation in the absence of CaCl2 or in the presence
of EDTA, respectively. However, the stability of the enzyme was
Fig. 2. Alignment of the N-terminal amino acid sequence of the purified trypsin
from Z. ophiocephalus with the sequences of other trypsins. Amino acid residues
identical to Z. ophiocephalus trypsin are shaded: walleye pollok (T. chalcogramma)
[22]; arabesque greenling (P. azonus) and true sardine (TR-S) (S. melanostictus) [23];
antarctic fish (Paranotothenia magellanica) [25]; Japanese anchovy (E. japonica) [24];
grey triggerfish (B. capriscus) [10]; cuttlefish (S. officinalis) [7]; smooth hound (S.
aurita) [9]; zebra blenny (S. basilisca) [13]; mandarin fish (Siniperca chuasti) [26];
common Carp (C. carpio) (trypsin A) [27]; silver mojarra (D. rhombeus) [12]; Tam-
baqui (C. macropomum) [11], rat [28], dog [29], bovine [30], porcine [31] and human
[32].
Fig. 3. pH (a) and temperature profiles (c), and pH (b) and thermal (d) stability of the purified trypsin. The pH profile was studied at 60 ◦C at various pHs. For temperature
profile, the activity of trypsin was determined at pH 9.0 using BAPNA as a substrate. For pH and thermal stability, the remaining activities were assayed at pH 9.0 and 60 ◦
using BAPNA as a substrate. The activity of the enzyme before incubation was taken as 100%. Buffer solutions used for pH activity and stability are presented in Section 2.
C

1962
R. Nasri et al. / Process Biochemistry 47 (2012) 1957–1964
promote the formation of active trypsin from trypsinogen and sta-
bilize trypsin against autolysis. Similarly, trypsins from other fish
species were activated by calcium ion [13,27]. On the contrary,
trypsin from pyloric ceca of the starfish Asterina pectinifera was
neither activated nor stabilized by calcium ion [34].
Protease activity was not affected by Ba2+, NaCl and KCl at a
concentration of 5 mM. The trypsin activity was reduced by Zn2+ to
10%. Cu2+ and Hg2+ completely inhibited trypsin activity.
Since CaCl2 enhances trypsin activity, the effect of its concen-
tration on enzyme activity was also studied. As shown in Fig. 4b,
maximum activity was obtained with 5 mM CaCl2 and there was
220% increase in the activity compared to that realized without
CaCl2. Beyond 5 mM Ca2+, trypsin activity decreased. These results
indicated that the enzyme require Ca2+ for its optimal activity. The
calcium requirement for trypsin activity is highly specific since
other metal ions (such as Mn2+, Mg2+, and Ba2+) are not able to
enhance, or slightly stimulated the enzyme activity.
3.3.6. Effects of oxidizing agents and surfactants on protease
stability
The suitability of Z. ophiocephalus trypsin as a detergent addi-
tive was determined by testing its stability against oxidants and
surfactants. As shown in Table 2, the alkaline trypsin is stable in
the presence of the non-ionic surfactants, like Tween 80 and Triton
X-100. Interestingly Z. ophiocephalus trypsin was little influenced
by oxidizing agent, and retained about 89% and 52% of its activ-
ity after incubation for 1 h at 40 ◦C in the presence of 0.2% and
1% sodium perborate, respectively. In the presence of 1% and 5%
hydrogen peroxide the enzyme retained about 67% and 40% of its
initial activity, respectively. The relative stability of the enzyme in
the presence of oxidizing agents is a very important characteristic
for its eventual use in detergent formulations. The stability of Z.
ophiocephalus trypsin against sodium perborate was higher than
S. basilisca trypsin which retained about 40% of its initial activ-
ity after incubation for 1 h at 30 ◦C in the presence of 1% sodium
perborate [13]. The Z. ophiocephalus trypsin was, however, unsta-
ble against the strong anionic surfactant (SDS) and retained 40%
and 20% activity in the presence of 0.5% and 1% SDS, respectively.
Fig. 4. (a) Effect of calcium ion (2 mM) and EDTA (2 mM) on the stability of the
purified tryspsin from Z. ophiocephalus. The stability was tested by incubating the
purified enzyme at 50 ◦C in the absence or presence of CaCl2 or EDTA and resid-
ual enzyme activity was determined from 0 to 4 h 30 min at 30-min intervals. The
non-heated enzyme was considered as control (100% activity). (b) Effect of CaCl2
concentrations on the trypsin activity. The activity of the enzyme without CaCl2
was considered as 100%.
3.4. Stability of the alkaline trypsin with commercial solid and
liquid detergents
considerably enhanced by Ca2+. After incubation for 150 min the
enzyme retained more than 85% of its activity, while no activity
was detected in the absence of CaCl2. The half-lives of the purified
trypsin at 50 ◦C were determined to be of the order of 1 h and >10 h,
in the absence and presence of 2 mM CaCl2, respectively.
The improvement in protease stability against thermal inactiva-
tion in the presence of Ca2+ may be explained by the strengthening
of interactions inside protein molecules and by the binding of
calcium ion to autolysis sites. In fact, many studies showed that
serine-proteases contain binding site with a higher affinity for
calcium ions, which play an important role in stabilizing the
enzyme against thermal denaturation and autodegradation [34].
These results suggest that the purified trypsin possesses a pri-
mary calcium-binding site like mammalian and other fish species
trypsins.
In order to confirm the potential of the purified Z. ophiocephalus
trypsin as a detergent additive, we examined its compatibility and
stability towards some commercial liquid and solid detergents after
incubation for 1 h at 30 and 40 ◦C.
The data presented in Fig. 5a shows that the alkaline trypsin
showed excellent stability and compatibility in all liquid detergents
tested. The enzyme retained 100% activity in the presence of Car-
refour and Lav+, and about 91, 87 and 94% in Tex’til, Ariel and Persil,
respectively, after 1 h incubation at 30 ◦C. The enzyme showed also
relative stability and compatibility at 40 ◦C.
The data presented in Fig. 5b shows that the enzyme is also
highly stable in the presence of solid detergents, retaining 90, 100,
60, 93 and 100% of its initial activity after 1 h incubation at 30 ◦C in
the presence of Nadhif, Ariel, Axion, New-Det and Dixan, respec-
tively. The enzyme was, however, less stable at 40 ◦C than 30 ◦C.
The enzyme was found to be affected by axion, retaining only 43%
of its initial activity after 1 h incubation at 40 ◦C.
Since the proteolytic activity varied with each laundry detergent
tested, the obtained results clearly indicated that the performance
of enzymes in detergents depends on number of factors, including
the detergents compounds. Esposito et al. [35] reported also the sta-
bility of tambaqui proteases in the presence of several commercial
detergents.
3.3.5. Effects of metal ions
The effects of some metal ions on the trypsin activity were stud-
ied (Table 2). Addition of CaCl2 to the assay medium enhanced the
protease activity to 220%. However calcium at 5 mM increased the
activity of the crude protease extract from goby to only 110% [15].
Further, Mg2+ and Mn2+ exhibited a slight stimulatory effect on
the enzyme. On the contrary, Mn2+ decreased the protease activ-
ity of the crude extract by 52.5% [15]. It is known that calcium ion

R. Nasri et al. / Process Biochemistry 47 (2012) 1957–1964
1963
Table 3
Kinetic constants of Z. opheocephalus trypsin and other trypsins.
Trypsins
Km (mM)
kcat (s−1
)
kcat/Km (s−1 mM−1
)
References
Goby
(Z. ophiocephalus)
Cuttlefish
0.312
2.03
6.51
This study
[7]
0.064
0.068
0.312
0.051
0.266
0.6
2.32
2.76
1.06
2.12
0.93
1.38
36.25
40.6
3.4
(S. officinalis)
Grey triggerfish
(B. capriscus)
Bigeye snapper
(P. macracanthus)
Montery sardine
(S. sagax cearula)
Silver mojarra
(D. rhombeus)
Zebra blenny
(S. basilisca)
[10]
[36]
41.0
3.48
2.32
[33]
[12]
[13]
3.5. Kinetic properties
4. Conclusion
Kinetic constants Km and kcat of the purified Z. ophiocephalus
trypsin were determined using Lineweaver–Burk plots. The Km
and kcat of the purified enzyme, using BAPNA, were 0.312 mM
and 2.03 s−1, respectively, and were close to those reported for
trypsins from bigeye snapper (Pricanthus macracanthus) [36] and
silver mojarra [12]. The catalytic efficiency (kcat/Km) of Z. ophio-
cephalus trypsin, 6.51 s−1 mM−1, was close to trypsins from silver
mojarra [12] and bigeye snapper [36] (Table 3).
In this report, an alkaline calcium dependent trypsin from
Z. ophiocephalus was purified and identified based on molecular
weight, inhibitor sensibility, substrate specificity and N-terminal
amino acids sequencing. The enzyme showed an optimum temper-
ature at 60 ◦C and optimum pH of 9.0. The enzyme was highly stable
at a pH range of 7.0–11.0 and at temperatures below 40 ◦C. Further,
the activity of the enzyme was highly enhanced by the addition of
calcium ion in the reaction mixture. On the other hand, the enzyme
possesses some other special characteristics, stability against the
non-ionic surfactants and oxidizing agents, and compatibility with
some commercial liquid and solid detergents.
Considering these properties, Z. ophiocephalus trypsin may find
application in certain industrial applications that require alkaline
conditions, such detergent industry. Further research is needed
to determine properties of Z. ophiocephalus trypsin as a possible
biotechnological tool in the fish processing and food industries.
Acknowledgement
This work was funded by the Ministry of Higher Education and
Scientific Research, Tunisia.
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