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Studies on activity, distribution, and zymogram of protease,a-amylase, and lipase in the paddlefish Polyodon spathula
H. Ji • H. T. Sun • D. M. Xiong
Received: 20 January 2011 / Accepted: 26 July 2011 / Published online: 6 September 2011
� Springer Science+Business Media B.V. 2011
Abstract A series of biochemical determination and
electrophoretic observations have been conducted to
analyze the activities and characteristics of prote-
ase, a-amylase, and lipase of paddlefish Polyodon
spathula. The results obtained have been compared
with those of bighead carp (Aristichthys nobilis) and
hybrid sturgeon (Huso dauricus $ 9 Acipenser
schrenki Brandt #), in order to increase available
knowledge of the physiological characteristics of this
sturgeon species and to gain information with regard to
its nutrition. Further, a comparative study of enzy-
matic activity, distribution, and characterization
between commercial feed-reared paddlefish (CG)
and natural live food-reared (NG) paddlefish was
conducted. Results showed that higher proteolytic
activity was observed in the pH range 2.5–3.0 and at a
pH of 7.0 for paddlefish. Levels of acid protease
activity of paddlefish were similar to that of hybrid
sturgeon, and significantly higher than that of bighead
carp. The inhibition assay of paddlefish showed that
the rate of inhibition of tosyl-phenylalanine chloro-
methyl ketone was approximately 2.6-fold that of
tosyl-lysine chloromethyl ketone. There was no
significant difference observed for acid protease
activity between PG and CG groups, whereas the
activity of alkaline protease, a-amylase, and lipase in
the PG group were significantly lower than those in the
CG group. The substrate sodium dodecyl sulfate
polyacrylamide gel electrophoresis analysis further
showed that there were certain types of enzymes,
especially a-amylase, with similar molecular mass in
the paddlefish and hybrid sturgeon. It can be inferred
that acid digestion was main mechanism for protein
hydrolysis in paddlefish, as reported for other fishes
with a stomach. This indicates that the paddlefish
requires higher alkaline protease, a-amylase, and
lipase activity to digest natural live food.
Keywords Polyodon spathula � Protease �a-Amylase � Lipase � Substrate SDS–PAGE
Introduction
Paddlefish belongs to the family Polyodontidae; this
is a species of the largest freshwater fish found in the
river basins of North America. Highly valued for
their grayish black roe (eggs)—which is processed
into caviar—and their boneless, firm, white meat
(Phillip and George 2006), paddlefish have been
successfully introduced and cultured in countries
such as the People’s Republic of China, Russia. On
the other hand, research on paddlefish appears to be
H. Ji � H. T. Sun � D. M. Xiong
College of Animal Science and Technology,
Northwest A & F University, Yangling 712100, China
H. Ji (&)
Ankang Fisheries Experimental and Demonstration
Station, Northwest A & F University,
Ankang 725000, China
e-mail: jihong0405@hotmail.com
123
Fish Physiol Biochem (2012) 38:603–613
DOI 10.1007/s10695-011-9541-9
scarce across the world. For example, in the field of
nutritional physiology, although the development of
feeding organs (Liu et al. 1998) and feeding behavior
(Russell et al. 1999) have been studied, few reports
are available with regard to the nutritional physiol-
ogy, especially pertaining to characteristics of diges-
tive enzymes, in this high-value species.
In fish, digestive enzymes play the principal role in
the hydrolysis of protein, carbohydrate, and lipid to
form small absorbable units. The hydrolysate formed
is transported into tissues by the circulatory system
and transformed into energy or material for growth
and reproduction (Furne et al. 2005). The character-
ization and quantification of protease, amylase, and
lipase activities may contribute to better understand
the digestive physiology of the paddlefish, improve
feeding regimes, and develop formulated diet for the
farming of this species. Therefore, learning informa-
tion about protease, amylase, and lipase from this
species is needed.
There are several differences among digestive
enzymes in fish species with different nutritional
habits due to their varied diets and digestive structure.
Hidalgo et al. (1999), researching digestive enzymes
in fish with different nutritional habits, reported that
trout and carp demonstrated the highest digestive
proteolytic activity. With regard to amylase activity,
the omnivorous species demonstrated higher activity
than the carnivores. A commercial diet, which has
been reported to improve weaning efficiency and to
reduce the expensive period during live food depen-
dence, would be of great benefit in finfish culture
(Watanabe and Kiron 1994). There were differences
in growth and survival rates between fish that were
fed on live and compound diets. These differences
were attributed to the nutritional value of the feed
and/or food digestion, nutrient absorption, and met-
abolic factors (Segner and Rosch 1992). Ribeiro et al.
(2002) reported that activities of amylase, trypsin,
alkaline phosphatase, and leucine–alanine peptidase
showed differences between the Solea senegalensis
following a larvae diet and Artemia that was main-
tained on a compound diet. There are other factors
that influence the characteristics of digestive enzymes
in fish; this includes the composition of diet (Cahu
and Zambonino Infante 1995; Kolkovski et al. 1997;
Ribeiro et al. 2002; Lundstedt et al. 2004; Perrin et al.
2004; Correa et al. 2007; Debnath et al. 2007;
Santigosa et al. 2008; Chatzifotis et al. 2008; Cedric
2009), age (Kuz’mina 1996) and the environment
(Zhi et al. 2009).
Although it belongs to the sturgeon family,
characteristics of the feeding organ, the gill raker,
for instance, distinguish the paddlefish from other
sturgeons that are zoobenthivores; however, its
stomach differentiates it from bighead carp (Aris-
tichthys nobilis), a zooplanktivores, and stomach-less
cyprinid fish species. The work presented in this
article analyzed activities and characteristics of
protease, a-amylase, and lipase in paddlefish and
compared the results with that of bighead carp and
hybrid sturgeon (Huso dauricus $ 9 Acipenser
schrenki Brandt #). This study aimed to increase
the available information with regard to the physiol-
ogy of this sturgeon species and avail knowledge of
its nutritional requirement, based on biochemical
assays and electrophoretic observation. A compara-
tive study of enzymatic activity, distribution, and
characterization between commercial feed-reared
paddlefish and natural live food-reared paddlefish
was also conducted to discuss the effect of commer-
cial feed and natural live food on digestive enzymes
in paddlefish.
Materials and methods
The present research work comprises two experi-
ments. In the first experiment, the enzymatic activ-
ities, effect of pH on total proteolytic activity, and
zymogram of paddlefish were compared with those of
bighead carp and hybrid sturgeon. In the second
experiment, a comparative study on enzymatic
activities and distribution between commercial feed-
reared paddlefish and natural live food-reared pad-
dlefish was conducted.
Experimental fish species
In the first experiment, a total of six paddlefish
(weight: 0.52 ± 0.05 kg) were obtained from the
Ankang Fisheries Experimental and Demonstration
Station (AFEDS) of the Northwest Agriculture and
Forestry University, and the same number of bighead
carp (weight: 1.25 ± 0.28 kg) and hybrid sturgeon
(weight: 0.86 ± 0.09 kg) were purchased from the
local market. For second experiment, fish were
hatched at the same time and cultured in a pond
604 Fish Physiol Biochem (2012) 38:603–613
123
(with commercial feed) and in a cage (with natural
live food) for at least 4 months. A total of 16 fish
were sampled, eight each from pond and cage,
respectively. The body weight of the commercial
feed-reared fish in pond (CG) was in the range of
0.61–0.80 kg and natural live food-reared fish in cage
(NG) 0.46–0.58 kg. All fish were starved for approx-
imately 24 h prior to sampling.
Preparation of crude enzyme extract
Fish were dissected and the complete digestive tracts
(from esophagus to anus) were removed and as
sample for the first stage of the experiment. The
esophagus, stomach, duodenum, and intestine with
spiral valve were separated for fish from CG and NG.
After washing with cold deionized water to remove as
much mucus as possible, tissues were homogenized
in cold sodium phosphate buffer (0.1 M, at pH 7.0,
and 4�C) by a ratio of 1:9 (m/v) (Chong et al. 2002;
Liu et al. 2008). The homogenate was then centri-
fuged (3–18 K, Sigma�, Germany) at 4�C at
10,0009g for 30 min. The supernatant containing
enzymes was stored at -70�C (Forma-86C,
Thermo�, USA) prior to analysis. The soluble protein
content in the enzyme extract was measured accord-
ing to the procedure described by Lowry et al. (1951)
by using bovine serum albumin (CALBIOCHEM�)
as the standard reference.
Enzyme assay
Effect of pH on total protease activity
The effect of pH on the total proteolytic activity of
crude enzymatic solutions in the whole digestive tract
was studied following casein digestion assay, but
with a slight modification (Chong et al. 2002; Liu
et al. 2008). The assay was conducted using a wide
range of pH values: 0.1 M KCl–HCl (pH 1.5), 0.2 M
glycine–HCl (pH 2.0–3.0), 0.1 M NaAC–HAC (pH
4.0–5.5), 0.2 M Na2HPO4–NaH2PO4 (pH 6.5–7.4),
0.1 M Tris–HCl (pH 8.1), and 0.1 M Na2HPO4–
NaOH (pH 8.5–12.0). The enzyme reaction mixtures
consisted of 1% (m/v) casein in 10 mM Tris–HCl
buffer of pH 8.5 containing 0.02 M CaCl2 (0.5 ml);
the selected buffer (1.0 ml) and enzyme sample
(0.2 ml) were incubated for 30 min at 37�C. The
reaction was terminated by the addition of 2.0 ml of
10% (m/v) trichloroacetic acid. After holding for
10 min, samples were centrifuged at 3,0009g for
3 min. The supernatant obtained was mixed with
2.5 ml of 0.4 M Na2CO3 and 0.1 ml Follin’s reagent.
The mixture was incubated for 20 min at 37�C and
then cooled in an ice bath. The absorbance of the
reaction mixture was recorded at 680 nm (1240,
SHIMADZU�, Japan) to measure the amount of
tyrosine produced. All samples were assayed in
triplicate, and the tyrosine was used as the standard
reference. One unit of specific activity was defined as
the amount of enzyme required to produce 1 lg of
tyrosine per minute per milligram of soluble protein
of enzyme solution at 37�C (U mg-1 Protein).
Acid protease and alkaline protease
The acid protease activity was determined with 2%
hemoglobin solution in 0.04 M HCl as the substrate.
The mixture was incubated for 30 min at pH 2.0 and
37�C. The alkaline protease activity was determined
using 1% casein solution as the substrate at a pH 8.5
and 37�C in 50 mM Tris–HCl buffer containing
0.02 mM CaCl2. The remainder of the procedure for
both proteases was the same as that for determination
of total proteolytic activity.
a-Amylase (E.C. 3.2.1.1)
Activity of a-amylase was evaluated using 1% starch
solution in 20 mM sodium phosphate buffer, contain-
ing 6.0 mM NaCl as substrate, at a pH of 6.9 (Natalia
et al. 2004). A quantity of 0.5 ml of substrate solution
was added to 0.2 ml of enzyme preparation, and the
mixture was incubated at 25�C for 5 min precisely.
This was followed by the addition of 1.0 ml of
dinitrosalicylic acid, and the mixture was incubated in
a boiling water bath for 5 min. The absorbance value
was recorded at 540 nm. The amount of maltose
released from this assay was determined from the
standard curve. One unit of specific activity was
defined as the amount of enzyme needed to produce
1 lmol maltose per minute per milligram of soluble
protein in enzyme solution at 25�C (U mg-1 Protein).
Lipase activity (E.C. 3.1.1.3)
Enzyme reaction mixtures for determination of lipase
activity comprised 50 mM sodium phosphate buffer
Fish Physiol Biochem (2012) 38:603–613 605
123
(2.0 ml) at a pH of 9.0, olive oil (0.5 ml), and the
enzyme sample (0.1 ml). The mixture was incubated
at 37�C for 10 min; the reaction was terminated by
the addition of 4.0 ml of methylbenzene. The sample
was centrifuged, and 4.0 ml of supernatant was
mixed with 1.0 ml of 5% copper acetate reagent
(m/v; pH 6.1). The absorbance of the supernatant was
recorded at 710 nm to measure the amount of fatty
acid produced, and oleic acid was used as the
standard reference (Jiang et al. 2007). One of unit
of specific activity was defined as the amount of
enzyme needed to produce 1 lmol fatty acid per
minute per milligram of soluble protein in enzyme
solution at 37�C (U mg-1 Protein).
Classification of proteases by inhibitory studies
A quantity of 20 lM Pepstatin A was used as the acid
protease inhibitor with hemoglobin as substrate, in a
procedure described by Bezerra et al. (2000). Further,
10 mM phenylmethylsulfonyl fluoride (PMSF;
Sigma�) in ethanol for serine protease inhibition,
10 mM tosyl-lysine chloromethyl ketone (TLCK;
Sigma�) in 1 mM HCl for trypsin inhibition, and
10 mM tosyl-phenylalanine chloromethyl ketone
(TPCK; Sigma�) in ethanol for chymotrypsin inhibi-
tion were used as alkaline protease inhibitor with
casein as substrate (Natalia et al. 2004). The
percentage of inhibition was calculated as
Classification of enzymes by SDS–PAGE
Protease zymogram
Substrate SDS–PAGE was applied to characterize the
protease present in the crude enzyme, in a procedure
described by Liu et al. (2008) and Dıaz-Lopez et al.
(1998), but with a slight modification. The crude
enzyme extract was mixed with sample buffer (1 M
Tris–HCl at a pH of 6.8, glycerol, SDS, and
bromophenol blue) at a ratio (v/v) of 4:1. A quantity
of 20 ll of the sample buffer mixture was loaded into
SDS–PAGE gels with a thickness of 1.0 mm. The gel
consisted of 4% stacking gel and a 15% separating
gel. Electrophoresis was conducted at 80 V for
approximately 30 min at 4�C with an electrophoresis
buffer comprising Tris–glycine–sodium dodecyl sul-
fate, and then again at 110 V for 3.5 h.
After electrophoresis, for the acid protease, the
sample was soaked in 0.04 M HCl (pH 2.0, 4�C) for
enzymes to become active. Following soaking for
30 min, the gel was soaked for 30 min in 0.2%
hemoglobin in 0.04 M Gly–HCl buffer (pH 2.0, 4�C),
and then again for 120 min at 37�C. The gel was
washed in deionized water, fixed for 20 min in 10%
tricholoracetic acid (TCA) solution, stained with 0.1%
(m/v) Coomasie Blue for 120 min, and destained. For
the alkaline protease, the gel was soaked for 60 min in
2% of the casein solution in 0.1 M Tris–HCl buffer
containing 20 mM CaCl2 (pH 8.5, 4�C); the gel was
then removed and placed in a water bath at 37�C for an
additional 120 min. The remainder of the experimen-
tal procedure was the same as that followed for acid
protease. A volume of 10 ll of molecular mass
markers (SDS–PAGE Standards, Biolabs�, New
England) were used for molecular mass determination.
a-Amylase zymogram
The protocol of the a-amylase zymogram was modi-
fied based on the procedure defined by Alvarez-
Gonzalez et al. (2010) and Tian et al. (2008). The
SDS–PAGE gels (4% of stacking gel and a 15%
separating gel) were stabilized for 30 min at
80 V, and this was increased to 110 V for 3 h.
After electrophoresis, the gel was soaked in 2%
starch solution at 25�C for 1 h and then soaked in
0.15 M sodium acetate trihydrate solution at 37�C
for 1 h. The gel was stained with a saturated
iodine/potassium iodide solution, and clear zones
became apparent after approximately 30 min of
staining.
Enzyme activity of control� enzyme activity in the presence of inhibitors
Enzyme activity of control� 100
606 Fish Physiol Biochem (2012) 38:603–613
123
Lipase zymogram
The procedure for electrophoresis of the lipase
zymogram was the same as that of a-amylase. The
gel was soaked and stained in 50 mM of a-naphtyl
caprylate/b-naphtyl acetate (SCRC�, Sinopharm
Chemical Reagent Co., Ltd) solution containing
10 mM fast blue BB salt (OURCHEM�, Sinopharm
Chemical Reagent Co., Ltd) at 37�C, and clear zones
were revealed after approximately 15 min of staining.
Data analysis
The results are expressed as mean ± SD. The
comparison of values obtained for enzyme activities
was carried out using analysis of variance (ANOVA)
and where applicable, and Tukey’s HSD. The level of
significance employed was 0.05.
Results
Enzymatic activity
Figure 1 demonstrates the pH dependence of proteo-
lytic activity in the digestive tract of paddlefish,
bighead carp, and hybrid sturgeon, respectively. The
highest activity was recorded in the pH range of
2.5–3.0 and for alkaline proteases at a pH of 7.0 in
paddlefish. The pH values with the highest activity
observed were 4.0–4.5, 8.0–8.5, and 11.0 in bighead
carp and 2.5–3.0, 8.0–8.5 in the hybrid sturgeon,
respectively.
The activity of acid protease (43.7 ± 0.08 U mg-1
Protein) and lipase (8.66 ± 0.77 U mg-1 Protein) of
paddlefish was significantly higher than those of
bighead carp and hybrid sturgeon (P \ 0.05); how-
ever, alkaline protease activity (1.90 ± 0.01 U mg-1
Protein) and a-amylase activity (1.47 ± 0.20 U mg-1
Protein) of paddlefish were lower (P \ 0.05) than
those of two species (Fig. 2).
The effects of different protease inhibitors on the
proteolytic ability of extracts of digestive tract from
paddlefish are shown in Table 1. The results indicate
that 20 lM Pepstatin A inhibited 89.6, 95.8, and
96.0% of acid protease activity in paddlefish, bighead
carp and hybrid sturgeon, respectively. The percentage
of inhibition of the alkaline protease was highest with
PMSF at 68.9 ± 0.42, 48.3 ± 8.56, and 35.7 ± 1.15%
in paddlefish, bighead carp, and hybrid sturgeon,
respectively; TPCK was second-most inhibitive, with
inhibition of 59.5 ± 4.05%, followed by TLCK with
inhibition of 23.1 ± 7.01% in paddlefish. However, in
bighead carp and hybrid sturgeon, TLCK was second-
most inhibitive, with inhibition of 28.9 ± 2.99 and
27.3 ± 1.38%, followed by TPCK, with inhibition of
27.5 ± 3.74 and 23.0 ± 1.49%, respectively.
There was no significant difference in acid prote-
ase activity between CG and NG; however, alkaline
protease, a-amylase, and lipase activities of PG were
significantly lower than those of NG (P \ 0.05;
Fig. 3). Figures 2 and 3 showed that alkaline protease
and amylase activity in paddlefish fed on commercial
feed (25.0 ± 1.11 U) was significantly lower than
that of bighead carp (132.0 ± 35.9 U), whereas
paddlefish filtering a natural diet in the NG group,
similar to that of bighead carp, had higher alkaline
protease and amylase activity (118 ± 6.18 U) than
fish in the CG group.
In general, positions in the digestive tract for
presence of enzymes activity were similar in both CG
and NG groups (Fig. 4). For example, acid protease
activity was detected in the esophagus, stomach, and
intestine, and alkaline protease demonstrated detect-
able activity in the duodenum and intestine in both
Fig. 1 Effect of incubation pH on the proteolytic activity of
extract from whole digestive tracts of paddlefish, bighead carp
and hybrid sturgeon. Results are mean ± SD from triplicate
assays
Fish Physiol Biochem (2012) 38:603–613 607
123
groups. The a-amylase showed activity across the
entire digestive tract, whereas lipase activity was only
detected in the stomach.
The activity of acid protease in the intestine of the
CG group was significantly higher than that in the NG
group (P \ 0.05); however, there was no difference
in enzyme activity of esophagus and stomach
between both groups. The alkaline protease activity
of CG was significantly lower than that of NG in both
duodenum and intestine (P \ 0.05). In addition, the
a-amylase activity of CG was significantly lower than
that of NG in the esophageal and duodenal sections
(P \ 0.05); however, there was no difference in the
activity in the stomach and intestine. Lipase demon-
strated higher activity in the stomach of the NG group
than that of the CG group (P \ 0.05).
Zymogram
Further characterization of digestive enzymes using
substrate SDS–PAGE electrophoresis was under-
taken, and the results are presented in Fig. 5. The
gel image indicates the presence of at least six
Fig. 2 Acid proteinase, alkaline proteinase, a-amylase, and
lipase activities recorded from whole digestive tracts of
paddlefish, bighead carp, and hybrid sturgeon. Results are
mean ± SD from triplicate assays. The same letters indicate
statistically no significant differences (P [ 0.05) and differentletters indicate statistically significant differences (P \ 0.05)
Table 1 Inhibition of proteolytic activities of the acidic and alkaline proteases by various inhibitors
Fish species Inhibition (%)
For acid protease For alkaline protease
Pepstain A PMSF TLCK TPCK
Paddlefish 89.6 ± 6.62 68.9 ± 0.42 23.1 ± 7.01 59.5 ± 4.05
Bighead carp 95.8 ± 2.91 48.3 ± 8.56 28.9 ± 2.99 27.5 ± 3.74
Hybrid sturgeon 96.0 ± 1.70 35.7 ± 1.15 27.3 ± 1.38 23.0 ± 1.49
PMSF phenylmethylsulfonyl fluoride, TLCK tosyl-lysine chloromethyl kotone, TPCK tosyl-phenylalanine chloromethyl kotone
Acid protease Alkalina protease -amylase Lipase
*
*
*
Act
ivity
(U
)
Fig. 3 Comparison of enzymatic activity from digestive tracts
of commercial feed-reared paddlefish (CG) and natural live
food-reared paddlefish (NG). Results are mean ± SD from
triplicate assays. *Signify statistically significant differences
(P \ 0.05)
608 Fish Physiol Biochem (2012) 38:603–613
123
(154.4–12.3 kDa), five (106.8–17.7 kDa), and three
(159.6–40.5 kDa) different acid proteases in paddle-
fish, bighead carp, and hybrid sturgeon, respectively.
Three types of alkaline protease were identified in
paddlefish and hybrid sturgeon with molecular weight
0
50
100
150
200
250
300
Act
ivit
y (U
)
CGNG
CGNG
CGNG
CGNG
Alkaline proteases*
*
0
50
100
150
200
250
300
Oesophagus Stomach Duodenum Intestine
Oesophagus Stomach Duodenum Intestine
Oesophagus Stomach Duodenum Intestine
Oesophagus Stomach Duodenum Intestine
Act
ivit
y (U
)
Alkaline proteases*
*
0
10
20
30
40
50
60
70
Act
ivit
y (U
)
- amylases
*
*
0
5
10
15
20
25
30
35
40
Act
ivit
y (U
)
* Lipase
Fig. 4 Distribution of enzymatic activity from different
sections of digestive tract between commercial feed-reared
paddlefish (CG) and natural live food-reared paddlefish (NG).
Results are mean ± SD from triplicate assays. *Signifies
statistically significant differences (P \ 0.05)
Fig. 5 Substrate SDS–PAGE electrophoresis gel showing
existence of different enzymes bands from digestive tract
extract (n = 3) of paddlefish, bighead carp and hybrid
sturgeon. Lands are described as follows: M marker; P paddle-
fish; B bighead carp; H hybrid sturgeon. Markers showing are
MPB-b-galactosidases (126 kDa, MPB maltose-binding pro-
tein); MPB-trancated-b-galactosidases (65 kDa); MBP-CBP
(45 kDa, MBP-CBP fusion of maltose-binding protein and
chitin-binding domain); CBD-MXE Intein-2CBD (35 kDa);
CBD-MXE Intein (25 kDa); CBD-E.Colipar (17 kDa)
Fish Physiol Biochem (2012) 38:603–613 609
123
from 139.5 to 101.0 kDa and 138.3 to 118.5 kDa;
similarly, there were two types of alkaline proteases
in bighead carp (129.6 and 99.3 kDa). The molecular
weight of a-amylases in paddlefish and hybrid
sturgeon was higher than that in bighead carp. The
types of a-amylase were two (156.3 and 129.9 kDa),
three (111.2–74.2 kDa), and four (156.3–116.2 kDa)
for paddlefish, bighead carp, and hybrid sturgeon,
respectively. There was only one type of lipase
detected in bighead carp (127.9 kDa) and hybrid
sturgeon (40.5 kDa); however, no band was observed
in gel electrophoresis for lipases in paddlefish.
Discussion
Protease
Similar to other vertebrates, fishes are conventionally
grouped as carnivores, omnivores, and herbivores on
the basis of their food habit. They may also be
grouped as filter-feeders, detritus-feeders or as pre-
dators (Chakrabarti et al. 1995). Paddlefish and
bighead carp are filter-feeders, and they have similar
food habits (zooplanktivores); however, their diges-
tive tracts are vastly different. The former, such as the
hybrid sturgeon analyzed in this study, has stomach
for digesting food, and the later is a stomach-less fish.
One of the major functions of the vertebrate
stomach is the initiation of protein digestion by the
action of pepsin and HCl. Most of the available
reports on pepsins in fish indicate an optimum
functional pH of 2.0 (Clark et al. 1985). In the
present study, higher proteolytic activity in the acid
pH ranging from 2.5 to 3.0 in paddlefish and hybrid
sturgeon indicated that pepsins play a role in protein
digestion. Low proteolytic activity was observed at
acidic pHs in the bighead carp (stomach-less species)
in this study, and similar results have been reported in
other stomach-less species, such as the common carp,
Cyprinus carpio L. (Hidalgo et al. 1999) and the grass
carp, Ctenopharyngodon idella Val. (Liu et al. 2008).
This decreased proteolytic activity could be attributed
to cellular proteases present in the homogenate
(Kuz’mina 1990). However, high acid protease
activity, similar to that detected in the stomach, was
present in the esophagus of paddlefish in CG and NG
groups (Fig. 3). Therefore, the hypothesis presented
by Kuz’mina (1990) does not shed light on the basis
of high acid proteases activity in the esophagus;
further study with regard to histology and physical
construction of this enzyme should be conducted for
clarity on this activity.
Higher proteolytic activity was observed at high
alkaline pHs (11) in bighead carp and hybrid sturgeon
(Fig. 1); this can probably be attributed to alkaline
proteases possessing carboxypeptidase-like, elastase-
like or collagenase-like activities, as has previously
been reported (Clark et al. 1985; Hidalgo et al. 1999).
The bighead carp demonstrated two peak-activity
regions between the pH range of 7.5–8.5 and the pH
11.0. This indicates the existence of two groups of
alkaline protease, and the findings are similar to that
reported for discus fish (Chong et al. 2002).
Previous research demonstrated that no classifica-
tion for protease was possible based on feeding
behavior (Furne et al. 2005). Hidalgo et al. (1999)
reported that rainbow trout and common carp had
high protease activity levels whereas certain other
carnivorous fish such as European eel and gilthead
seabream had lower activities. In the present work,
protease activity, especially acid protease activity,
was completely different between paddlefish and
bighead carp (Fig. 2), although both have the same
feeding behavior. Similar protease activities were
recorded in paddlefish and hybrid sturgeon because of
the similar structure of their digestive tracts.
Alkaline proteases include trypsin, chymotrypsin,
carboxypeptidase, elastase, and collagenase. Both
trypsin and chymotrypsin belong to the trypsin
superfamily, which are ubiquitous in animals; they
possess a catalytic triad that characterizes all serine
proteinases, consisting of His, Asp, and Ser amino
acid residues. In addition to its protein-digestion
capabilities, trypsin has several physiological func-
tions, such as activation of other zymogens; several
research studies with regard to enzymatic develop-
ment in fish reported that trypsin was present from
the embryonic stage onward (Muhlia-Almazan, et al.
2008). Some authors (Clark et al. 1985; Uys and
Hecht 1987) reported that the optimum pH for
trypsin-like activity was higher than that for chymo-
trypsin-like activity. In this study, the higher prote-
olytic activity of paddlefish at pH 7.0, as compared
with that at pH 8.0–8.5 (the optimum pH for trypsin),
suggests that chymotrypsin is the main component of
the trypsin superfamily in the paddlefish. This
hypothesis is supported by results of inhibitory action
610 Fish Physiol Biochem (2012) 38:603–613
123
by various inhibitors with regard to proteolytic
activities (Table 1). The rate of inhibition of TPCK
(the inhibitor of chymotrypsin, 59.5%) was approx-
imately 2.6-fold that of TLCK (the inhibitor of
trypsin, 23.1%).
Live food is very important to the development
and growth of larvae because live food affects
digestive enzymes and thereby assists the digestive
process. Kolkovski et al. (1997) proposed that live
food facilitated the larval digestive process via a
contribution of gastric hormones that could improve
gastric activation. In addition, live food could delay
the development and maturation of certain digestive
processes in larvae, such as the onset of pancreatic
secretory functions and enterocyte differentiation
(Kolkovski et al. 1997). In this study, paddlefish fed
with commercial feed had significantly lower alkaline
protease activity than that of fish fed on a filtered
natural diet (Fig. 3). The probable reasons for this
result need more in-depth study.
The acid protease activity was detected in the
esophagus and stomach, whereas alkaline protease
activity was observed in the intestine of paddlefish in
both CG and NG groups (Fig. 4). This indicates that
digestion with alkaline protease takes place following
acid protease digestion. A similar observation was
reported for other stomach fish in previous reports
(Chakrabarti et al. 1995).
a-Amylase
Amylase activity, in general, has been considered by
most authors to be more dependent on nutritional
habits rather than proteolytic activity. It is postulated
that herbivorous and omnivorous fish have higher
amylase activity than carnivorous fish. For example,
the Rainbow trout, a carnivorous fish, has low
amylase activity; in the European eel, amylase
activity is higher than that in trout (Hidalgo et al.
1999). Hidalgo et al. (1999) reported that, irrespec-
tive of the food habit of the fish, adaptations of the
digestive system of different species exhibit closer
correlation with their diet than their microenviron-
ment and taxonomic category. However, there are
some data contradicting this statement. Hidalgo et al.
(1999) demonstrated that the production of amylase
was neither food dependent nor of reflexive origin. It
is evident that the type of diet, apparently, has no
bearing on the amylase production in fish. Our results
demonstrate that amylase activity was different
between paddlefish fed with different diets (commer-
cial feed or natural live diet) (Figs. 2, 3).This
indicates that the type of diet influences the amylases
activity of paddlefish.
The observation of amylase activity in the esoph-
agus of paddlefish, in this study, is not surprising
(Fig. 4). Chakrabarti et al. (1995) demonstrated that
most of the omnivorous (with the exception of
C. catlu) and herbivorous fish exhibited a consider-
able amount of amylase activity in the esophagus. Of
the two carnivore fishes studied, the esophagus of
N. notopterus exhibited no amylase activity. It is
remarkable that amylase activity was detected in the
stomach of paddlefish in both CG and NG groups
(Fig. 4), whereas it was completely absent in the
stomach extract of Notopterus chitala (Ghosh 1985).
Lipase
Fish are hypothesized to consume a fat-rich food.
Thus, the occurrence of lipase in the digestive tract in
fish appears to be justified. The presence of lipases in
the liver is attributed to the adherence of fragments of
pancreatic tissue, whereas proponents of the opposite
viewpoint argue that the presence of lipase is not
necessarily the result of production by the pancreas
but is a property of hepatic tissue (Chakrabarti et al.
1995). In general, it is considered that the presence of
lipases in carnivorous fish is greater than in omniv-
orous or herbivorous fish (Tengjaroenkul et al. 2000;
Furne et al. 2005). In this study, lipase activities in
paddlefish were significantly higher than in bighead
carp, whereas there was no difference in lipase
activity between bighead carp and hybrid sturgeon
(Fig. 2). On the other hand, paddlefish fed with
commercial feed had greater amounts of lipases
detected than those filtering the natural diet (Fig. 3).
This implies that the type of diet might influence the
production of lipases.
Zymogram
The results of the zymogram on acid protease,
alkaline protease, and amylase indicated that there
were certain same (69.9 kDa for acid protease and
156.3 kDa for amylase) or similar bands (154.4 kDa
in paddlefish and 154.9 kDa in hybrid sturgeon for
acid protease, 139.5 kDa in paddlefish and 138.3 kDa
Fish Physiol Biochem (2012) 38:603–613 611
123
in hybrid sturgeon for alkaline protease) in paddlefish
and hybrid sturgeon, and the characterization of
zymogram was greatly different between paddlefish
and bighead carp (Fig. 5). We hypothesize that types
of proteases and amylase were relative to categori-
zation rather than food habits.
Conclusion
It can be inferred that acid digestion was main
mechanism for protein hydrolysis in paddlefish, as
reported for other fishes with a stomach. This
indicates that the paddlefish requires higher alkaline
protease, a-amylase, and lipase activity to digest
natural live food.
Acknowledgments We are grateful for financial support from
the construction project of Ankang Fisheries Experimental and
Demonstration Station of Northwest A & F University and the
agricultural research project of Science and Technology
Department of Shaanxi Province. Thanks Ph.D Xuebo Liu for
supporting for Substrate SDS–PAGE analysis.
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