Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the...

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: [email protected]

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


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


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)


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




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)


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


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


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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