Low molecular weight trypsin from hepatopancreas of freshwater prawn (Macrobrachium rosenbergii): Characteristics and biochemical properties

Article abstract of DOI:10.1016/j.foodchem.2012.02.173

Trypsin was purified to homogeneity from hepatopancreas of freshwater prawn (HFWP) (Macrobrachium rosenbergii) using a series of chromatographies including Q-Sepharose, Superdex 75 and MonoQ columns. HFWP trypsin was purified 525-fold with a yield of 10.6%. Based on native-PAGE, the purified trypsin showed a single band. Trypsin had a molecular weight of 17 kDa as estimated by SDS-PAGE. The optimal pH and temperature for Boc-Val-Pro-Arg-MCA hydrolysis were 8.0 and 55 °C, respectively. Trypsin was stable to heat treatment up to 40 °C, and over a pH range of 7.0-11.0. Trypsin activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and Pefabloc SC and was partially inhibited by ethylenediaminetetraacetic acid (EDTA). Apparent K_m value of trypsin was 0.24 μM and K _cat value was 607.56 s^-1 for Boc-Val-Pro-Arg-MCA. The N-terminal amino acid sequence of 20 residues of HFWP trypsin was IVGGDEAAPGEFPHQISMQV, which was highly homologous with those from other species of prawn. HFWP trypsin also showed high collagenolytic activity toward prawn, shrimp and fish collagens, suggesting its possible role in muscle softening of freshwater prawn during extended storage.

Full text of DOI:10.1016/j.foodchem.2012.02.173

Food Chemistry 134 (2012) 351–358
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Low molecular weight trypsin from hepatopancreas of freshwater prawn
(
Macrobrachium rosenbergii): Characteristics and biochemical properties
a
a,
⇑
b
c
c
Chodsana Sriket , Soottawat Benjakul , Wonnop Visessanguan , Kenji Hara , Asami Yoshida ,
Xiao Liang
a
d
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phaholyothin Rd., Klong 1, Klong Luang,
b
Pathumthani 12120, Thailand
c
Graduate School of Fisheries and Environment Studies, Nagasaki University, Bunkyo, Nagasaki 852-8521, Japan
Graduate School of Science and Technology, Nagasaki University, Bunkyo, Nagasaki 852-8521, Japan
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypsin was purified to homogeneity from hepatopancreas of freshwater prawn (HFWP) (Macrobrachium
rosenbergii) using a series of chromatographies including Q-Sepharose, Superdex 75 and MonoQ columns.
HFWP trypsin was purified 525-fold with a yield of 10.6%. Based on native-PAGE, the purified trypsin
showed a single band. Trypsin had a molecular weight of 17 kDa as estimated by SDS–PAGE. The optimal
pH and temperature for Boc-Val-Pro-Arg-MCA hydrolysis were 8.0 and 55 °C, respectively. Trypsin was sta-
ble to heat treatment up to 40 °C, and over a pH range of 7.0–11.0. Trypsin activity was strongly inhibited
by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and Pefabloc SC and was par-
Received 11 October 2011
Received in revised form 22 December 2011
Accepted 27 February 2012
Available online 6 March 2012
Keywords:
Purification
Trypsin
Collagenolytic activity
Hepatopancreas
Freshwater prawn
tially inhibited by ethylenediaminetetraacetic acid (EDTA). Apparent K
m
value of trypsin was 0.24
l
M and
À1
Kcat value was 607.56 s for Boc-Val-Pro-Arg-MCA. The N-terminal amino acid sequence of 20 residues of
HFWP trypsin was IVGGDEAAPGEFPHQISMQV, which was highly homologous with those from other spe-
cies of prawn. HFWP trypsin also showed high collagenolytic activity toward prawn, shrimp and fish col-
lagens, suggesting its possible role in muscle softening of freshwater prawn during extended storage.
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
Freshwater prawn (FWP) is widely consumed in Thailand due to
Bringas Alvarado, & Rouzaud Sandez, 2003). Hepatopancreas from
white shrimp, Penaeus vannamei (Ezquerra, Garcia-Carreño, & Haard,
1997) and Northern shrimp, Pandalus eous (Aoki, Ahsan, Matsuo,
Hagiwara, & Watabe, 2003) possessed collagenolytic/gelatinolytic
activities. Recently, serine protease, mainly trypsin-like protease
released from hepatopancreas, was reported to cause the softening
of FWP muscle during extended iced storage (Sriket, Benjakul,
Visessanguan, & Kishimura, 2011b). Therefore, better understanding
of trypsin from FWP, especially from the digestive organs, could pave
the way to prevent or lower the activity of those proteases, thereby
retarding the quality loss of FWP. Nevertheless, no information
regarding the characteristic and biochemical properties of purified
trypsin from hepatopancreas of freshwater prawn (HFWP) has been
reported. Therefore, this study aimed to purify and characterise
trypsin responsible for the muscle degradation from HFWP.
its delicacy. However, this species is not successfully marketable
due to the loss in textural acceptability. During postmortem han-
dling and storage, prawn meat becomes soft and mushy, probably
as a result of indigenous proteases. Softening of meat was devel-
oped within 4 days of iced storage and was most pronounced in
the first segment of abdomen (Sriket, Benjakul, & Visessanguan,
2
010). Sriket, Benjakul, and Visessanguan (2011a) reported that
proteases from FWP muscle had very low proteolytic activity and
no collagenolytic activity, whilst protease from hepatopancreas
showed high hydrolytic activity on natural actomyosin (NAM)
and pepsin soluble collagen (PSC) extracted from FWP muscle.
Shrimp hepatopancreas has been reported to have high proteo-
lytic activities (Oh, Kim, Kim, & Kim, 2000) and most digestive
enzymes in crustaceans were classified to be serine protease and
2
. Materials and methods
metalloprotease (Garcia-Carreño, Patricia Hernandez-Cortes,
Haard, 1994). Amongst proteolytic enzymes, serine collagenase has
an impact on the softening of muscle (Ezquerra Brauer, Salazar Leyva,
&
2.1. Chemicals
Q-Sepharose, Superdex 75 and MonoQ were obtained from
Amersham Pharmacia Biotech AB (Uppsala, Sweden). t-Butyloxy-
carbonyl-Val-Pro-Arg-4-methyl-coumaryl-7-amide (Boc-Val-Pro-
Arg-MCA) and other synthetic fluorogenic peptide substrates
⇑
Corresponding author. Tel.: +66 7428 6334; fax: +66 7455 8866.
E-mail address: soottawat.b@psu.ac.th (S. Benjakul).
0
308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2012.02.173

3
52
C. Sriket et al. / Food Chemistry 134 (2012) 351–358
(
MCA-substrates) were purchased from Peptide Institute (Osaka,
0.5 ml/min. The column was washed with SB until A280 was less
than 0.05 and then eluted with a linear gradient of 0.0–0.6 M NaCl
in SB at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected
and the fractions with trypsin activity were pooled, dialysed with
SB for 12 h and used for further study.
Japan). Pefabloc SC was obtained from Merck (Darmstadt, Ger-
many). Ethylenediaminetetraacetic acid (EDTA), pepstatin A, soy-
bean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone
(
(
TLCK), N-tosyl-L-phenyl-alanine chloromethyl ketone (TPCK), 1-
L-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane (E-64),
N-ethylmaleimide, b-mercaptoethanol (bME), bovine gelatin and
2.5. Trypsin activity assay
bovine serum albumin were procured from Sigma Chemical Co.
(
St. Louis, MO, USA.). Protein molecular weight markers were ob-
Trypsin activity was measured by the method of Yoshida et al.
(2009) using Boc-Val-Pro-Arg-MCA as a substrate. Appropriately
tained from Bio-Rad Laboratories (Hercules, CA, USA). All chemi-
cals used were of analytical grade.
diluted enzyme (100
buffer (pH 8.0). The reaction was immediately initiated by the
addition of 100 l of 50 M substrate and incubated at 55 °C for
0 min. To stop the reaction, 1.5 ml of the stopping agent (methyl
ll) was added to 800 ll of 50 mM Tris–HCl
2
.2. Sample preparation
l
l
1
Live FWP (M. rosenbergii) with a size of 30–35 prawn/kg were
alcohol:n-butyl alcohol:distilled water = 35:30:35, v/v/v) was
added. The fluorescence intensity of liberated 7-amino-4-methyl-
coumarin (AMC) was measured by a spectrofluorometer (Model
RF-1500, Shimadzu Co., Kyoto, Japan) at the excitation wavelength
of 380 nm and the emission wavelength of 450 nm. One unit of en-
zyme activity was defined as the amount of the enzyme, which re-
obtained from a farm in Phatthalung province, Thailand. The sam-
ples were ice-shocked with iced-water (0 °C). The hepatopancreas
was removed manually and immediately mixed with liquid nitro-
gen. The frozen sample was placed in a polyethylene bag, sealed
and stored at À40 °C until use.
leased 1
.6. pH and temperature profile
Trypsin activity was assayed over the pH range of 4.0–11.0
lmol AMC per min.
2
.3. Preparation of hepatopancreas extract
2
Hepatopancreas were homogenised with three volumes of ace-
tone at À20 °C for 3 min using an IKA homogeniser (Model T25,
Selangor, Malaysia) according to the method of Klomklao, Benjakul,
Kishimura, Osako, and Tanaka (2010). The homogenate was filtered
in vacuo through Whatman No. 4 filter paper. The residue obtained
was then homogenised in two volumes of acetone at À20 °C for
(50 mM acetate buffer for pHs 4.0–7.0; 50 mM Tris–HCl buffer
for pHs 7.0–9.0 and 50 mM glycine–NaOH for pHs 9.0–11.0) at
55 °C for 10 min. For the temperature profile study, the activity
was assayed at different temperatures (20, 30, 40, 50, 60, 70 and
80 °C) for 10 min at pH 8.0.
3
min, and then the residue was air-dried in a vacuum desiccator
until the sample was dry and free of acetone odour. Defatted hepa-
topancreas powder obtained was stored at À40 °C until use.
To prepare the hepatopancreas extract, the powder was sus-
2.7. pH and thermal stability
pended in 50 mM Tris–HCl, pH 7.5 containing 5 mM CaCl
to as ‘‘starting buffer; SB’’ at a ratio of 1:10 (w/v) and stirred con-
tinuously at 4 °C for 3 h. The suspension was centrifuged at
2
referred
The effect of pH on enzyme stability was evaluated by measur-
ing the residual activity after incubation at various pHs for 30 min
at 30 °C. Enzyme solution was mixed with different buffers men-
tioned above at a ratio of 1:1 (v/v). For thermal stability, the en-
zyme solution was mixed with 100 mM Tris–HCl, pH 8.0 at a
ratio of 1:1 (v/v) and incubated at different temperatures (20, 30,
1
2,000g for 10 min at 4 °C using a refrigerated centrifuge (Kubota,
Model 7780II, Tokyo, Japan) to remove the tissue debris, and the
supernatant obtained was referred to as ‘‘crude extract’’.
4
0, 50, 60, 70 and 80 °C) for 15 min in a temperature controlled
2
.4. Purification of trypsin from FWP hepatopancreas
water bath (Thermo Minder Mini-80, Taiyo, Tokyo, Japan). Thereaf-
ter, the treated samples were suddenly cooled in iced water. The
residual activity was assayed using Boc-Val-Pro-Arg-MCA as a sub-
strate at pH 8.0 and 55 °C for 10 min.
All purification processes were carried out at 4 °C in the labora-
tory of Faculty of Fisheries, Nagasaki University, Japan. Fractions
obtained from all purification steps were subjected to the mea-
surement of protein content and trypsin activity. Protein concen-
tration during purification steps was determined by monitoring
the absorbance of sample solution at 280 nm.
2.8. Effect of inhibitors
The effect of inhibitors on trypsin activity was determined
according to the method of Klomklao, Benjakul, Visessanguan,
Kishimura, and Simpson (2007). Enzyme solution was mixed with
an equal volume of protease inhibitor solution to obtain the final
concentration designated (0.1 mM E-64, 1.0 g/l soybean trypsin
inhibitor, 5 mM pefabloc SC, 5 mM TLCK, 5 mM TPCK, 1 mM pep-
statin A and 10 mM EDTA). The mixture was allowed to stand at
room temperature (26–28 °C) for 15 min. Thereafter, the remaining
activity was measured and percent inhibition was calculated.
After filtration through a 0.2-lm filter, the sample was then ap-
plied to a Q-Sepharose anion exchange column (3.0 Â 45 cm),
which was equilibrated with approximately two-bed volumes of
SB. The sample was loaded onto the column at a flow rate of
0
.5 ml/min. The column was washed with SB until A280 was less
than 0.05 and then eluted with a linear gradient of 0.0–0.6 M NaCl
in SB at a flow rate of 0.5 ml/min. Fractions of 5 ml were collected
and the fractions with trypsin activity were pooled and concen-
trated by ultrafiltration with YM-10 membrane (Millipore, Bed-
ford, MA, USA).
2.9. Determination of protein concentration
The concentrated Q-Sepharose fractions were filtered with a
0
.2-
l
m filter and further subjected to a Superdex 75 column
Protein concentration was determined by monitoring the absor-
bance of sample solution at 280 nm.
(
2.6 Â 60 cm) previously equilibrated with approximately two-
bed volumes of SB. The elution was performed with the same buf-
fer at a flow rate of 0.5 ml/min. Fractions of 6 ml were collected
and those with trypsin activity were pooled and further purified
by a MonoQ anion exchange column previously equilibrated with
SB. The sample was loaded onto the column at a flow rate of
2.10. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
SDS–PAGE was performed according to the method of Laemmli
(1970). Protein solutions were mixed at 1:1 (v/v) ratio with the

C. Sriket et al. / Food Chemistry 134 (2012) 351–358
353
SDS–PAGE sample buffer (0.125 M Tris–HCl, pH 6.8; 4% SDS; 20%
glycerol; 10% b-mercaptoethanol) and boiled for 3 min. The sam-
of Sriket, Benjakul, Visessanguan, and Kijroongrojana (2007). The
hydrolysis was conducted by incubating the mixture at 25 °C for
0, 5, 10 and 15 min. The control was performed in the same manner
for 15 min, except distilled water was added instead of trypsin. The
reaction was terminated by adding the preheated solution contain-
ing 2% SDS, 8 M urea and 2% bME (85 °C). The mixture was further
incubated at 85 °C for 60 min to solubilise total proteins. The solu-
tion was centrifuged at 5000g for 10 min at room temperature to re-
move the debris. The supernatant was then subjected to SDS–PAGE
using 7.5% separating gel and 4% stacking gel.
ples (10
5% separating gels and subjected to electrophoresis at a constant
current of 15 mA per gel using a Mini-Protean II Cell apparatus
Atto Co., Tokyo, Japan). After electrophoresis, the gels were
lg) were loaded onto the gel made of 4% stacking and
1
(
stained with 0.1% Coomassie brilliant blue R-250 in 50% methanol
and 7% acetic acid and destained with 7% acetic acid.
Native-PAGE was performed using 15% separating gels in a sim-
ilar manner, except that the sample was not heated and SDS and
reducing agent were left out.
2.16. Statistical analysis
2.11. Activity staining
The experiments were run in triplicate, except for the kinetic
Activity staining of purified trypsin towards bovine gelatin was
study, which was carried out in duplicate. Data were subjected to
analysis of variance (ANOVA) and mean comparison was carried
out using Duncan’s multiple range test (DMRT) (Steel & Torrie,
980). Statistical analyses were performed using the Statistical
Package for Social Science (SPSS 11.0 for Windows, SPSS Inc., Chi-
cago, IL, USA).
performed according to the method described by Lødemel and Ol-
sen (2003) with a slight modification. Purified trypsin was mixed
with sample buffer (0.125 M Tris–HCl, pH 6.8; 4% SDS; 20% glyc-
1
erol) at a ratio of 1:1 (v/v). The mixture containing 1 lg protein
was loaded onto the gel made of 4% stacking and 15% separating
gels containing 0.1% bovine gelatin. The electrophoresis was con-
ducted at 4 °C. After electrophoresis, the gel was washed twice in
3
. Results and discussion
2
.5% Triton X-100 for 15 min. The remaining Triton X-100 was re-
moved by washing the gel (three times) with 50 mM Tris–HCl buf-
fer pH 7.0. Gel was incubated in 50 mM Tris–HCl, pH 8.0 containing
3.1. Purification of HFWP trypsin
5
2 3
mM CaCl and 0.02% NaN at 37 °C for 6 h. The gel was rinsed
Purification steps of HFWP trypsin are summarised in Table 1.
using cold distilled water prior to staining and destaining, as de-
scribed above. Development of clear zones on a blue background
indicated gelatinolytic activity.
The purification of trypsin was started by separating the crude ex-
tract using a Q-Sepharose anion exchange column. After separa-
tion, the fractions with pronounced trypsin activity were pooled
and a purity of 14.3-fold was observed. A single activity peak
was obtained for the pooled Q-Sepharose fractions upon the subse-
quent chromatography on a Superdex 75 column (Fig. 1). The yield
of 23.8% and the purity of 27.7-fold were observed after this gel
filtration. The pooled fractions with trypsin activity were then sub-
jected to MonoQ anion exchange column. Ion-exchange chroma-
tography was used to remove contaminating proteins and to
separate different trypsin isoforms (Klomklao et al., 2006b). Pooled
fractions from single activity peak obtained after MonoQ anion ex-
change column showed high purity of 525-fold with 10.6% yield.
The results showed that Q-Sepharose anion exchange, Superdex
2.12. Determination of N-terminal amino acid sequence
Purified trypsin was subjected to SDS–PAGE and transferred to a
polyvinylidene difluoride (PVDF) membrane. The membrane was
briefly stained by Coomassie brilliant blue (CBB) and the protein
bands were excised. The N-terminal amino acid sequence of tryp-
sin was determined using a protein sequencer (Shimadzu, Model
PPSQ-33A, Kyoto, Japan).
2.13. Substrate specificity
7
5 and MonoQ anion exchange chromatographies effectively re-
To study the substrate specificity of the trypsin, various syn-
moved the contaminants from the crude extract. As a consequence,
the purified trypsin was obtained.
thetic fluorogenic MCA-substrates were used and the activity of
trypsin toward different substrates was determined as previously
described.
3.2. Purity and molecular weight
2.14. Kinetic studies
Based on native gel-electrophoresis, there was only a single pro-
tein band (Fig. 2a), indicating the high purity of trypsin. HFWP
trypsin also showed a single band on SDS–PAGE (Fig. 2b). Molecu-
lar weight of HFWP trypsin was estimated to be 17 kDa. This value
was in accordance with that determined by Superdex 75 column
The activity was assayed with different final concentrations of
Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-MCA. The final enzyme
concentration for the assay was 0.015 g/ml. The determinations
were repeated twice and the respective kinetic parameters,
including Vmax and K , were evaluated by plotting the data on a
Lineweaver–Burk double-reciprocal graph (Lineweaver & Burk,
934). Values of turnover number (Kcat) were calculated from the
l
(
data not shown). The result suggested that HFWP trypsin was
m
monomeric, however, the molecular weight of trypsin was quite
1
following equation: Vmax/[E] = Kcat, where [E] was the active enzyme
Table 1
concentration.
Purification of trypsin from hepatopancreas of freshwater prawn.
Purification
step
Total
activity
Total
protein
(mg)
Specific activity
(units/mg
protein)
Purity
(fold)
Yield
(%)
2.15. Hydrolysis of various collagens by purified trypsin
(
units)
Purified trypsin (100
mined by Boc-Val-Pro-Arg-MCA assay was added to the reaction
mixture containing 4 mg collagen and 800 l of 50 mM Tris–HCl,
pH 7.0, containing 5 mM CaCl . Pepsin soluble collagen (PSC) from
freshwater prawn, PSC from white shrimp and acid soluble collagen
ASC) from yellowtail meats were isolated according to the method
ll) with the activity of 0.01 unit deter-
Crude enzyme
Q Sepharose
Superdex 75
MonoQ
1418.59
1037.45
337.27
150.09
5827
301.9
50.76
1.19
0.24
3.44
6.64
126
1.0
100
14.3
27.7
525
73.1
23.8
10.6
l
2
Trypsin activity was assayed at pH 8 and 55 °C for 10 min using Boc-Val-Pro-Arg-
MCA as substrate.
(

3
54
C. Sriket et al. / Food Chemistry 134 (2012) 351–358
2
1
1
1
1
1
0
8
6
4
2
0
8
6
4
2
0
30
kDa
00
A 280
Unit/mL
(a)
(b)
(c)
(a)
2
(
----)
2
5
0
116
9
7
6
6
2
45
15
3
1
0
-0.6 M NaCl
1
5
0
0
2
1
1.5
17 kDa
4.4
6.5
0
50
100
150
200
250
300
350
Fraction No. (5 mL/tube)
1
M
1
1
2
1
0
.5
2
35
A 280
Unit/mL
Fig. 2. Protein pattern from native-PAGE (a), SDS–PAGE (b) and activity staining (c)
of purified trypsin from hepatopancreas of freshwater prawn. M, molecular weight
standard; lane 1, trypsin.
(b)
(
---)
30
25
20
15
10
5
0
molecular weight of trypsin-like enzymes from crustacea varies
widely. This present study therefore firstly reported the trypsin
with low molecular weight (17 kDa) in crustacean and fish. Never-
theless, the possibility that autolytic degradation causing such a
low molecular weight should not be excluded (Lu et al., 2008).
.5
1
.5
0
3
.3. pH and temperature profile
Purified HFWP trypsin had the increases in activity as the pH in-
0
10
20
30
40
50
60
70
creased and reached the maximum at pH 8.0 (P < 0.05) (Fig. 3a). The
optimal pH of purified HFWP trypsin was in accordance with those
of trypsins from other fish and shellfish (Castillo-Yanez, Pacheco-
Aguilar, Garcia-Carreno, & Navarrete-Del Toro, 2005; Hjelmeland
Fraction No. (6 mL/tube)
2
1
0
.5
2
180
160
A 280
Unit/mL
(
----)
(c)
&
Raa, 1982; Jiang, Moody, & Chen, 1991). Activity of trypsin
decreased when pHs were higher than the optimal pH. The lowered
activity at very acidic and alkaline pH was plausibly due to the
conformational changes of enzyme under harsh conditions
(Klomklao, Kishimura, Benjakul, Simpson, & Visessanguan, 2010).
The temperature profile of HFWP trypsin is depicted in Fig. 3b.
The optimal temperature of trypsin was 55 °C (P < 0.05) when
Boc-Val-Pro-Arg-MCA was used as a substrate. The optimal tem-
perature of purified HFWP trypsin was similar to that of trypsin
from other crustacean, which ranges from 50 to 60 °C (Jiang
et al., 1991; Maeda-Martinez, Obregon-Barboza, Navarrete-Del
Toro, Obregon-Barboza, & Garcia-Carreno, 2000). However, the
optimal temperature of purified HFWP trypsin was higher than
those of trypsins obtained from hepatopancreas from other fish
and shellfish such as Japanese sea bass (Lateolabrax japonicus)
1
1
1
40
20
00
.5
1
0
-0.6 M NaCl
80
60
40
20
0
.5
0
0
10
20
30
40
50
60
70
80
Fraction No. (0.5 mL/tube)
Fig. 1. Purification of trypsin from hepatopancras of freshwater prawn. (a) Elution
profile of trypsin on Q-Sepharose anion exchange column; (b) Superdex 75 gel
filtration column and (c) MonoQ anion exchange column. Fractions under the bars
were pooled.
(
40–45 °C) (Cai et al., 2011), carp (40–45 °C) (Cao et al., 2000)
and crab (45 °C) (Galgani & Nagayama, 1987). The differences in
optimal temperature might be governed by habitat, environment
and genetic factors (Klomklao, Benjakul, Visessanguan, Kishimura,
&
Simpson, 2006a). A sharp decrease in activity of HFWP trypsin at
different from those of mammalian, fish and shrimp trypsins. Tryp-
sins purified from black tiger prawn (27–29 kDa) (Lu, Liu, & Tsai,
temperatures above 60 °C was most likely due to thermal
denaturation.
1
990), white shrimp (Penaeus setiferus) (24 kDa) (Gates, 1969)
and crayfish (Procambarus clarkia) (23.8, 27.9, 24.8 and 31.4 kDa)
Kim, Meyers, & Godber, 1992) have been reported. The activity
3.4. pH and thermal stability
(
band was confirmed by substrate gel electrophoresis, in which a
clear band with the same molecular weight appeared on a blue
background (Fig. 2c). The result indicated that purified HFWP
trypsin was able to hydrolyse gelatin. Aoki et al. (2003) reported
that four gelatinolytic proteases were found in hepatopancreas of
Northern shrimp (Pandalus eous). Generally, trypsins have
molecular weights between 20 and 25 kDa (Klomklao, 2008). The
The pH stability of HFWP trypsin is shown in Fig. 3c. HFWP
trypsin was stable in pH values ranging from 7.0 to 11.0. The inac-
tivation was more pronounced at pH values below 7. A complete
loss in activity was observed after incubating trypsin at pH 4.
Osnes and Mohr (1985) reported that trypsins from krill, Euphausia
superba, were inactivated at pH 4.5, and fish trypsins are stable
only in alkaline pH (Simpson, 2000). The stability of trypsin at a

C. Sriket et al. / Food Chemistry 134 (2012) 351–358
355
(a)
(c)
(b)
(d)
Fig. 3. pH and temperature profiles (a and b) and pH and thermal stabilities (c and d) of purified trypsin from hepatopancreas of freshwater prawn.
particular pH might be related to the balance of charge of the en-
zyme (Castillo-Yanez et al., 2005). A similar pH effect on activity
has been reported for trypsin from several fish species (Cai et al.,
approximately 60% were retained. At high temperatures, the en-
zyme more likely underwent denaturation and lost its activity.
The results were in agreement with Kim et al. (1992) who reported
that trypsin activity of crayfish hepatopancreas was stable up to
40 °C and activity almost disappeared when heated at temperatures
higher than 60 °C. Trypsin from the hepatopancreas of Atlantic blue
crab was stable at temperatures ranging from 30 to 50 °C for
30 min, but the activity was rapidly lost at temperatures above
50 °C (Dendinger & O’Connor, 1990). Brun and Wojtowicz (1976)
reported that trypsin activity of rock crab hepatopancreas was re-
duced by 60% after incubation at temperatures >50 °C for 20 min.
Enzymes are inactivated at high temperature due to the partial
unfolding of their molecules. The mechanism for increasing thermal
stability of proteins appears to be due to a strengthening of hydro-
phobic interactions and disulphide bonds in the interior of the pro-
tein molecule (Kim, Meyers, Pyeun, & Godber, 1994). Numerous
disulphide linkages, as well as stronger hydrophobic interactions
in the interior of the protein contribute to protein thermal stability.
Disulphide bonds may stabilise a folded conformation of trypsin
(Klomklao et al., 2009). Fish and shellfish trypsins had a lower
2
&
011; Castillo-Yanez et al., 2005; Klomklao, Kishimura, Nonami,
Benjakul, 2009). Thus, HFWP trypsin might undergo denatur-
ation under the acidic conditions, where the conformational
change took place and enzyme could not bind to the substrate
properly. Inactivation at acidic pH is a common phenomenon for
anionic trypsins (Kim et al., 1992).
Thermal stability of HFWP trypsin is depicted in Fig. 3d. HFWP
trypsin was stable for 15 min when heated up to 40 °C (Fig. 3d).
The loss in activity was noticeable after incubation at temperature
above 40 °C for 15 min. When the enzyme was heated at suffi-
ciently high temperature, the configuration could be altered. As a
consequence, the lowered activity could be attained. In the pres-
ence of a substrate during the activity assay, the enzyme might
be stabilised to some extent. On the other hand, the enzyme was
directly subjected to heat treatment in the absence of a substrate
for the thermal stability study. No trypsin activities remained after
heat treatment at 80 °C. After heat treatment at 60 °C, activities of

3
56
C. Sriket et al. / Food Chemistry 134 (2012) 351–358
thermal stability than those from mammals, possibly caused by the
lower number of intramolecular disulphide bonds in fish and shell-
fish trypsins, compared with the mammal counterpart (Klomklao
et al., 2009; Lu et al., 1990). When the internal temperature during
cooking was not sufficiently high, trypsin still retained and caused
substantial softening of shrimp meat during storage or distribution.
HFWP trypsin more effectively than other specific synthetic sub-
strates. The result suggested that the substrates containing argi-
nine (Boc-Phe-Ser-Arg-MCA) or lysine (Boc-Glu-Lys-Lys-MCA and
Boc-Vla-Leu-Lys-MCA) residues at the P1 site were more preferable
for trypsin (Table 3). Nevertheless, the substrate containing argi-
nine residue was hydrolysed more than that with lysine residue
(
(
Table 3). A similar result was obtained for other fish trypsins
Cai et al., 2011), however, chymotrypsin substrates (Suc-Leu-
3
.5. Effect of inhibitors
Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA), cathepsin L sub-
strate (Z-Phe-Arg-MCA) and aminopeptidase substrate (Arg-MCA)
were not hydrolysed by HFWP trypsin. These results strongly indi-
cated that the purified HFWP was trypsin.
Since HFWP trypsin could cleave arginine and lysine residues,
this enzyme might be responsible for muscle softening of this spe-
cies during extended iced storage. FWP meat was found to have a
high content of arginine (34.91 mg/100 g) and lysine (27.11 mg/
mg) (Sriket et al., 2011c).
The effect of various inhibitors on the activity of HFWP trypsin
was determined as shown in Table 2. Trypsin was markedly inhib-
ited by trypsin inhibitors, such as soybean trypsin inhibitor (99.23%
inhibition), TLCK (97.94% inhibition) and pefabloc SC (99.35% inhi-
bition). No differences in % inhibition were found among those
three inhibitors (P > 0.05). Soybean trypsin inhibitor is a single
polypeptide that forms a stable stoichiometric enzymically inactive
complex with trypsin, thereby reducing the availability of trypsin.
TLCK is a competitive inhibitor with trypsin. When the enzyme
binds to TLCK at active site, the substrate cannot be hydrolysed.
Pefabloc SC or 4-(2-Aminoethyl)-benzenesulfonylfluoride hydro-
chloride (AEBSF) is an irreversible proteinase inhibitor with broad
specificity for serine proteinases. TPCK is a competitive inhibitor
of chymotrypsin. Inhibitors for cysteine and aspartic proteases
involving E-64 and pepstatin A did not show inhibitory effects to-
wards trypsin activity. No inhibition was also observed when TPCK,
a specific inhibitor for chymotrypsin, was used. The result confirms
that this purified enzyme was serine proteases, most likely trypsin.
Kim et al. (1992) reported that the activities of trypsins from hepa-
topancreas of crayfish were inhibited by soybean trypsin inhibitors
and TLCK. The enzymatic activities of two trypsins from the hepa-
topancreas of Japanese sea bass were also effectively inhibited by
pefabloc SC (Cai et al., 2011). EDTA, which chelates the metal ions
required for the enzyme, and partially lowered trypsin activity
Table 3
Substrate specificity of purified trypsin from hepatopancreas of freshwater prawn on
synthetic fluorogenic substrates.
Substrates (10
l
M)
Relative activity (%)
*
b
Boc-Val-Pro-Arg-MCA
Boc-Phe-Ser-Arg-MCA
Boc-Leu-Lys-Arg-MCA
Boc-Glu-Lys-Lys-MCA
Boc-Val-Leu-Lys-MCA
Arg-MCA
Z-Phe-Arg-MCA
Suc-Leu-Leu-Val-Tyr-MCA
Suc-Ala-Ala-Pro-Phe-MCA
100 ± 3.06
c
72.6 ± 4.45
120 ± 2.88a
d
38.1 ± 5.43
17.4 ± 3.55e
0
0
0
0
Trypsin activity was assayed at pH 8 and 55 °C for 10 min using various MCA-
substrates. Different superscripts indicate the significant differences (p < 0.05).
*
Means ± SD (n = 3).
(
9.71% inhibition). Removal of calcium ions might affect trypsin
structure, resulting in some losses in activity. The result suggests
that trypsin most likely required metal ions as cofactors for activity.
It has been reported that calcium ions are required for fish and
crustacean trypsins (Klomklao et al., 2009; Zwilling, Pfleiderer, Son-
neborn, Kraft, & Stucky, 1969). However, it was noted that soybean
trypsin inhibitor effectively inhibited trypsin from hepatopancreas
of FWP. Thus, the extracts from soybean or other legumes contain-
ing trypsin inhibitor could be used to inhibit the proteolytic activity
from hepatopancreas, which was more likely released to the flesh
and caused the softening of this species.
Table 4
Kinetic parameters of purified trypsin from hepatopancreas of freshwater prawn and
other fish trypsins.
Substrates
Trypsins
Km
M)
K
(S
cat
À1
K
(S
cat
À1
/Km
lM )
*
*
À1
(
l
)
Boc-Val-Pro-Arg-MCA Freshwater prawn
Sea bass trypsin A
0.24
0.58
0.59
0.56
0.39
0.82
607.56 2531.51
*
*
5.51
25.75
26.16
5.42
9.45
43.58
46.72
14.07
14.86
*
*
Sea bass trypsin B
Boc-Leu-Lys-Arg-MCA Freshwater prawn
Sea bass trypsin A
**
*
*
Sea bass trypsin B
12.19
3.6. Substrate specificity
*
*
K
m
and kcat values of all trypsins were determined at 37 °C under optimal pH.
*
Cai et al. (2011).
Among all fluorescent MCA-substrates tested, Boc-Val-Pro-Arg-
MCA and Boc-Leu-Lys-Arg-MCA were hydrolysed by purified
Table 2
Effect of various inhibitors on the activity of purified trypsin from hepatopancreas of
freshwater prawn.
Inhibitors
Concentration
% Inhibition
Control
Pestain A
E-64
SBTI
TLCK
Pefabloc SC
TPCK
EDTA
–
0
0
0
1 mM
0.1 mM
1 g/L
5 mM
5 mM
5 mM
10 mM
*
a
a
a
99.23 ± 0.02
97.94 ± 0.01
99.35 ± 0.03
0
9.71 ± 0.17b
Fig. 4. Comparison of N-terminal amino acid sequence of the purified trypsin from
hepatopancreas of freshwater prawn with other trypsins. Black tiger prawn (Lu
et al., 1990), sea bass (Cai et al., 2011), yellowfin tuna (Klomklao et al., 2006b),
skipjack tuna (Klomklao et al., 2009), arabesque greenling (Kishimura, Hayashi,
Enzyme solution was incubated with the same volume of inhibitor at 25 °C for
5 min and the residual activity was determined using Boc-Val-Pro-Arg-MCA as
1
substrate for 10 min at pH 8 and 55 °C. Different superscripts indicate the signifi-
cant differences (p < 0.05).
Means ± SD (n = 3).
Miyashita, & Nonami, 2006), dog (Pinsky, LaForge, & Scheele, 1985), porcine
(Hermodson, Ericsson, Neurath, & Walsh, 1973) and bovine (Walsh, 1970). Identical
amino acid residues were boxed.
*

C. Sriket et al. / Food Chemistry 134 (2012) 351–358
Freshwater prawn White shrimp
357
Yellowtail
(a)
(b)
kDa
00
kDa
00
β-chain
α-chain
2
2
1
16
1
16
9
7
9
7
6
6
66
45
4
5
M
C
5
10 15
C
5
10 15
M
C
5
10
15
Fig. 5. Protein patterns of pepsin soluble collagen from freshwater prawn and white shrimp (a) and acid soluble collagen from yellowtail (b) incubated with trypsin from
hepatopancreas of freshwater prawn at 25 °C for different times. M: molecular weight marker, C: collagen incubated without trypsin for 15 min. Numbers denote the
incubation time (min) at 25 °C.
3.7. Kinetic study
15 min are shown in (Fig. 5a). Marked degradation of the a-chain
was found after 5 min of hydrolysis, with concomitant occurrence
of degradation products having a lower molecular weight (Fig. 5a).
The protein pattern of ASC type I extracted from yellowtail muscle
incubated with purified HFWP trypsin is depicted in Fig. 5b. The
Kinetic data for purified HFWP trypsin are summarised in Ta-
ble 4. K
.24
m
and kcat for the hydrolysis of Boc-Val-Pro-Arg-MCA were
M and 607.5 s , respectively, and the corresponding values
À1
0
l
for the hydrolysis of Boc-Leu-Lys-Arg-MCA were 0.56
l
M and
degradations of b and
bation. The decrease in band intensity of b and
more pronounced with increasing incubation time.
a
-chains were observed after 5 min of incu-
À1
2
6.1 s , respectively. The catalytic efficiencies (kcat/K
m
) for the
a-chains became
hydrolysis of Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-MCA
À1
À1
were calculated to be 2531.5 and 46.72 s
When comparing K of purified HFWP trpysin with two tryp-
sins from Japanese sea bass hepatopancreas, it was found that
HFWP trypsin had lower K values than trypsins from Japanese
l
M
, respectively.
These results suggested that HFWP trypsin had high collageno-
lytic activity toward prawn and fish collagens. This result was in
agreement with Aoki et al. (2003) who found that proteases from
Northern shrimp (Pandalus eous) were able to hydrolyse collagen.
In general, collagens in fish and shellfish muscle contain lower
cross-links than those from warm-blooded vertebrates. Collagens
from fish and shellfish are more heat labile than land animal colla-
gen (Hernandez-Herrero, Duflos, Malle, & Bouquelet, 2003). There-
fore, trypsin released from FWP hepatopancreas may play a role in
postmortem degradation of prawn muscle during extended iced
storage.
m
m
sea bass (Table 4). The result indicated that HFWP trypsin had a
higher affinity for Boc-Val-Pro-Arg-MCA than did sea bass trypsins.
For the Kcat value, HFWP trypsin also showed higher Kcat value on
both Boc-Val-Pro-Arg-MCA and Boc-Leu-Lys-Arg-MCA than did
trypsins from hepatopancreas of Japanese sea bass. HFWP trypsin
also had higher catalytic efficiencies on both Boc-Val-Pro-Arg-
MCA and Boc-Leu-Lys-Arg-MCA than did trypsin from Japanese
sea bass. Thus, hepatopancreas trypsin might have higher hydro-
lytic activity on muscle proteins than did trypsins from hepatopan-
creas of Japanese sea bass.
4
. Conclusion
Low molecular weight trypsin from hepatopancreas of fresh
3
.8. N-terminal sequencing
water prawn showed high hydrolytic activity at pH 8.0 and 55 °C.
Based on substrate specificity, the inhibitor study and N-terminal
sequencing, the enzyme purified was found to belong to trypsin
family. Trypsin from FWP also showed high hydrolytic activity to-
ward prawn collagen. Therefore, trypsin released from hepatopan-
creas more than likely plays a role in muscle softening of this
species during extended iced storage.
The N-terminal amino acid sequence of HFWP trypsin was
aligned to compare with those of other trypsins as depicted in
Fig. 4. The N-terminal amino acid sequence of the first 20 amino
acids of purified trypsin was determined to be IVGGDEAAPGEFPH-
QISMQV (Fig. 4). When N-terminal amino acid sequences of puri-
fied trypsin were compared with those of other shrimp, fish and
three mammals, it was found that the sequence of the purified
HFWP trypsin displayed high homology to that of black tiger
prawn trypsin, especially the sequence from the first to fourth
Acknowledgements
This work was granted by Prince of Songkla University and the
Office of the Higher Education Commission, Ministry of Education,
Thailand. Mr. Chodsana Sriket was supported by a CHE PhD schol-
arship. The TRF Senior Research Scholar programme is also
acknowledged.
(
IVGG) and ninth to thirteenth (PGEFP) residues. However, HFWP
and all fish trypsins had a charged Glu residue at position 6,
whereas Thr is most common in mammalian pancreatic trypsin
(Fig. 4). Moreover, the sequences of HFWP trypsin and other tryp-
sins started with IVGG after the proteolytic cleavage of inactive
trypsinogen. The result confirmed that HFWP trypsin was most
likely a member of the trypsin family.
References
Aoki, H., Ahsan, N., Matsuo, K., Hagiwara, T., & Watabe, S. (2003). Purification and
characterization of collagenolytic proteases from the hepatopancreas of
Northern shrimp (Pandalus eous). Journal of Agricultural and Food Chemistry,
3.9. Collagen hydrolysis by purified HFWP trypsin
5
1(3), 777–783.
Protein patterns of PSC extracted from FWP and white shrimp
Brun, G. L., & Wojtowicz, M. B. (1976). A comparative study of the digestive
muscle incubated with purified HFWP trypsin at 25 °C for 0–
enzymes in the hepatopancreas of jonah crab (Cancer borealis) and rock crab

3
58
C. Sriket et al. / Food Chemistry 134 (2012) 351–358
Cancer irroratus). Comparative Biochemistry and Physiology Part B, 53(3), Purification and characterization. Comparative Biochemistry and Physiology Part
(
3
87–391.
B, 144(1), 47–56.
Cai, Q.-F., Jiang, Y.-K., Zhou, L.-G., Sun, L.-C., Liu, G.-M., Osatomi, K., et al. (2011).
Biochemical characterization of trypsins from the hepatopancreas of Japanese
sea bass (Lateolabrax japonicus). Comparative Biochemistry and Physiology Part B,
Klomklao, S., Kishimura, H., Benjakul, S., Simpson, B. K., & Visessanguan, W. (2010).
Cationic trypsin: A predominant proteinase in pacific saury (Cololabis saira)
pyloric ceca. Journal of Food Biochemistry, 34(5), 1105–1123.
159(3), 183–189.
Klomklao, S., Kishimura, H., Nonami, Y., & Benjakul, S. (2009). Biochemical
Cao, M. J., Osatomi, K., Suzuki, M., Hara, K., Tachibana, K., & Ishihara, T. (2000).
Purification and characterization of two anionic trypsins from the
hepatopancreas of carp. Fisheries Science, 66(6), 1172–1179.
Castillo-Yanez, F. J., Pacheco-Aguilar, R., Garcia-Carreno, F. L., & Navarrete-Del Toro,
M. d. l. A. (2005). Isolation and characterization of trypsin from pyloric caeca of
Monterey sardine Sardinops sagax caerulea. Comparative Biochemistry and
Physiology Part B, 140(1), 91–98.
Dendinger, J. E., & O’Connor, K. L. (1990). Purification and characterization of a
trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes
sapidus. Comparative Biochemistry and Physiology Part B, 95(3), 525–530.
Ezquerra Brauer, J. M., Salazar Leyva, J. A., Bringas Alvarado, L., & Rouzaud Sandez, O.
properties of two isoforms of trypsin purified from the intestine of skipjack
tuna (Katsuwonus pelamis). Food Chemistry, 115(1), 155–162.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature, 227(5259), 680–685.
Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociation
constants. Journal of the American Chemical Society, 56(3), 658–666.
Lødemel, J. B., & Olsen, R. L. (2003). Gelatinolytic activities in muscle of Atlantic cod
(Gadus morhua), spotted wolffish (Anarhichas minor) and Atlantic salmon (Salmo
salar). Journal of the Science of Food and Agriculture, 83(10), 1031–1036.
Lu, B. J., Zhou, L. G., Cai, Q. F., Hara, K., Maeda, A., Su, W. J., et al. (2008). Purification
and characterisation of trypsins from the pyloric caeca of mandarin fish
(Siniperca chuatsi). Food Chemistry, 110(2), 352–360.
(
2003). Effect of dietary protein on muscle collagen, collagenase and shear force
of farmed white shrimp (Litopenaeus vannamei). European Food Research and
Technology, 217(4), 277–280.
Ezquerra, J. M., Garcia-Carreño, F. L., & Haard, N. F. (1997). Effects of feed diets on
digestive proteases from the hepatopancreas of white shrimp (Penaeus
vannamei). Journal of Food Biochemistry, 21(5), 401–419.
Lu, P. J., Liu, H. C., & Tsai, I. H. (1990). The midgut trypsins of shrimp (Penaeus
monodon). High efficiency toward native protein substrates including collagens.
Biological Chemistry Hoppe-Seyler, 371(2), 851–860.
Maeda-Martinez, A. M., Obregon-Barboza, V., Navarrete-Del Toro, M. A., Obregon-
Barboza, H., & Garcia-Carreno, F. L. (2000). Trypsin-like enzymes from two
morphotypes of the ‘living fossil’ Triops (Crustacea: Branchiopoda: Notostraca).
Comparative Biochemistry and Physiology Part B, 126(3), 317–323.
Galgani, F., & Nagayama, F. (1987). Digestive proteinases in the Japanese spiny
lobster Panulirus japonicus. Comparative Biochemistry and Physiology Part B,
8
7(4), 889–893.
Garcia-Carreño, F. L., Patricia Hernandez-Cortes, M., & Haard, N. F. (1994). Enzymes
with peptidase and proteinase activity from the digestive systems of
freshwater and a marine decapod. Journal of Agricultural and Food Chemistry,
2(7), 1456–1461.
Oh, E. S., Kim, D. S., Kim, J. H., & Kim, H. R. (2000). Enzymatic properties of a protease
from the hepatopancreas of shrimp Penaeus orientalis. Journal of Food
Biochemistry, 24(3), 251–264.
Osnes, K. K., & Mohr, V. (1985). On the purification and characterization of three
anionic, serine-type peptide hydrolases from Antarctic krill, Euphausia Superba.
Comparative Biochemistry and Physiology Part B, 82(4), 607–619.
a
4
Gates, B. J. (1969). Isolation and comparative properties of shrimp trypsin.
Biochemistry, 8(11), 4483–4489.
Pinsky, S. D., LaForge, K. S.,
& Scheele, G. (1985). Differential regulation of
Hermodson, M. A., Ericsson, L. H., Neurath, H., & Walsh, K. A. (1973). Determination
of the amino acid sequence of porcine trypsin by sequenator analysis.
Biochemistry, 12(17), 3146–3153.
trypsinogen mRNA translation: full-length mRNA sequences encoding two
oppositely charged trypsinogen isoenzymes in the dog pancreas. Molecular and
Cellular Biology, 5(10), 2669–2676.
Hernandez-Herrero, M. M., Duflos, G., Malle, P., & Bouquelet, S. (2003). Collagenase
activity and protein hydrolysis as related to spoilage of iced cod (Gadus
morhua). Food Research International, 36(2), 141–147.
Simpson, B. K. (2000). Digestive proteinases from marine animals. In N. F. Haard & B.
K. Simpson (Eds.), Seafood enzymes: utilization and influence on postharvest
seafood quality (pp. 531–540). New York: Marcel Dekker.
Hjelmeland, K., & Raa, J. (1982). Characteristics of two trypsin type isozymes
isolated from the Arctic fish capelin (Mallotus villosus). Comparative Biochemistry
and Physiology Part B, 71(4), 557–562.
Sriket, C., Benjakul, S., & Visessanguan, W. (2010). Post-mortem changes of muscle
from fresh water prawn (Macrobrachium rosenbergii) as influenced by spawning
stages. LWT - Food Science and Technology, 43(4), 608–616.
Jiang, S.-T., Moody, M. W., & Chen, H.-C. (1991). Purification and characterization of
proteases from digestive tract of grass shrimp (Penaeus monodon). Journal of
Food Science, 56(2), 322–326.
Sriket, C., Benjakul, S., & Visessanguan, W. (2011a). Characterisation of proteolytic
enzymes from muscle and hepatopancreas of fresh water prawn
(Macrobrachium rosenbergii). Journal of the Science of Food and Agriculture,
91(1), 52–59.
Sriket, C., Benjakul, S., Visessanguan, W., & Kishimura, H. (2011b). Collagenolytic
serine protease in fresh water prawn (Macrobrachium rosenbergii):
Characteristics and its impact on muscle during iced storage. Food Chemistry,
124(1), 29–35.
Sriket, C., Benjakul, S., Visessanguan, W., Kishimura, H., Hara, K., & Yoshida, A.
(2011). Chemical and thermal properties of freshwater prawn (Macrobrachium
rosenbergii) meat. Journal of Aquatic Food Product Technology, Accepted
Manuscript.
Kim, H. R., Meyers, S. P., & Godber, J. S. (1992). Purification and characterization of
anionic trypsins from the hepatopancreas of crayfish, Procambarus clarkii.
Comparative Biochemistry and Physiology Part B, 103(2), 391–398.
Kim, H. R., Meyers, S. P., Pyeun, J. H., & Godber, J. S. (1994). Enzymatic properties of
anionic trypsins from the hepatopancreas of crayfish, Procambarus clarkii.
Comparative Biochemistry and Physiology Part B, 107(2), 197–203.
Kishimura, H., Hayashi, K., Miyashita, Y., & Nonami, Y. (2006). Characteristics of
trypsins from the viscera of true sardine (Sardinops melanostictus) and the
pyloric ceca of arabesque greenling (Pleuroprammus azonus). Food Chemistry,
9
7(1), 65–70.
Sriket, P., Benjakul, S., Visessanguan, W., & Kijroongrojana, K. (2007). Comparative
studies on chemical composition and thermal properties of black tiger shrimp
(Penaeus monodon) and white shrimp (Penaeus vannamei) meats. Food
Chemistry, 103(4), 1199–1207.
Steel, R. G. D., & Torrie, J. H. (1980). Principles and procedures of statistics: A
biometrical approach. New York: McGraw-Hill.
Walsh, K. A. (1970). [4] Trypsinogens and trypsins of various species. Methods in
Enzymology, 19, 41–63.
Yoshida, A., Bae, I., Sonoda, H., Masuo, R., Oda, S., Cao, M. J., et al. (2009).
Characterization of gelatinolytic enzymes in the skeletal muscle of red sea
bream Pagrus major. Fisheries Science, 75(5), 1317–1322.
Klomklao, S. (2008). Digestive proteinases from marine organisms and their
applications. Songklanakarin Journal of Science and Technology, 30(1), 37–46.
Klomklao, S., Benjakul, S., Kishimura, H., Osako, K., & Tanaka, M. (2010). Effect of
salts and polyethylene glycols on the partioning and recovery of trypsin from
hybrid catfish viscera in aqueous two-phase systems. Journal of Food
Biochemistry, 34(4), 730–747.
Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., & Simpson, B. K. (2006a).
Proteolytic degradation of sardine (Sardinella gibbosa) proteins by trypsin from
skipjack tuna (Katsuwonus pelamis) spleen. Food Chemistry, 98(1), 14–22.
Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., & Simpson, B. K. (2007).
Purification and characterisation of trypsins from the spleen of skipjack tuna
Zwilling, R., Pfleiderer, G., Sonneborn, H. H., Kraft, V., & Stucky, I. (1969). The
evolution of endopeptidases–V. Common and different traits of bovine and
crayfish trypsin. Comparative Biochemistry and Physiology Part B, 28(3),
1275–1287.
(Katsuwonus pelamis). Food Chemistry, 100(4), 1580–1589.
Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B. K., & Saeki,
H. (2006b). Trypsins from yellowfin tuna (Thunnus albacores) spleen: