Studies on activity, distribution, and zymogram of protease, a-amylase, and lipase in the paddlefish Polyodon spathula

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1 Fish Physiol Biochem (2012) 38: DOI /s 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 Ó Springer Science+Business Media B.V Abstract A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, 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 enzymatic 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 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 chloromethyl ketone was approximately 2.6-fold that of H. Ji H. T. Sun D. M. Xiong College of Animal Science and Technology, Northwest A & F University, Yangling , China H. Ji (&) Ankang Fisheries Experimental and Demonstration Station, Northwest A & F University, Ankang , China jihong0405@hotmail.com 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

2 604 Fish Physiol Biochem (2012) 38: 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 physiology, especially pertaining to characteristics of digestive 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 (Furné et al. 2005). The characterization 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 information 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 dependence, 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 metabolic 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 maintained 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; Corrêa 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 (Aristichthys 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 physiology of this sturgeon species and avail knowledge of its nutritional requirement, based on biochemical assays and electrophoretic observation. A comparative 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 commercial feed and natural live food on digestive enzymes in paddlefish. Materials and methods The present research work comprises two experiments. In the first experiment, the enzymatic activities, 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 feedreared paddlefish and natural live food-reared paddlefish 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

3 Fish Physiol Biochem (2012) 38: (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 kg and natural live food-reared fish in cage (NG) kg. All fish were starved for approximately 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 centrifuged (3 18 K, Sigma Ò, Germany) at 4 C at 10,0009g for 30 min. The supernatant containing enzymes was stored at -70 C (Forma-86C, Thermo Ò, USA) prior to analysis. The soluble protein content in the enzyme extract was measured according to the procedure described by Lowry et al. (1951) by using bovine serum albumin (CALBIOCHEM Ò ) 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 ), 0.1 M NaAC HAC (ph ), 0.2 M Na 2 HPO 4 NaH 2 PO 4 (ph ), 0.1 M Tris HCl (ph 8.1), and 0.1 M Na 2 HPO 4 NaOH (ph ). The enzyme reaction mixtures consisted of 1% (m/v) casein in 10 mm Tris HCl buffer of ph 8.5 containing 0.02 M CaCl 2 (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 Na 2 CO 3 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 Ò, 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 (Umg -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 CaCl 2. The remainder of the procedure for both proteases was the same as that for determination of total proteolytic activity. a-amylase (E.C ) Activity of a-amylase was evaluated using 1% starch solution in 20 mm sodium phosphate buffer, containing 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 (Umg -1 Protein). Lipase activity (E.C ) Enzyme reaction mixtures for determination of lipase activity comprised 50 mm sodium phosphate buffer

4 606 Fish Physiol Biochem (2012) 38: (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 (Umg -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 Ò ) in ethanol for serine protease inhibition, 10 mm tosyl-lysine chloromethyl ketone (TLCK; Sigma Ò ) in 1 mm HCl for trypsin inhibition, and 10 mm tosyl-phenylalanine chloromethyl ketone (TPCK; Sigma Ò ) in ethanol for chymotrypsin inhibition were used as alkaline protease inhibitor with casein as substrate (Natalia et al. 2004). The percentage of inhibition was calculated as 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 sulfate, 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 CaCl 2 (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 experimental procedure was the same as that followed for acid protease. A volume of 10 ll of molecular mass markers (SDS PAGE Standards, Biolabs Ò, New England) were used for molecular mass determination. a-amylase zymogram The protocol of the a-amylase zymogram was modified based on the procedure defined by Alvarez- Enzyme activity of control enzyme activity in the presence of inhibitors 100 Enzyme activity of control 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 Díaz-López 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 González 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.

5 Fish Physiol Biochem (2012) 38: 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 Ò, Sinopharm Chemical Reagent Co., Ltd) solution containing 10 mm fast blue BB salt (OURCHEM Ò, 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 Results Enzymatic activity Figure 1 demonstrates the ph dependence of proteolytic activity in the digestive tract of paddlefish, 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 bighead carp, and hybrid sturgeon, respectively. The highest activity was recorded in the ph range of and for alkaline proteases at a ph of 7.0 in paddlefish. The ph values with the highest activity observed were , , and 11.0 in bighead carp and , 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); however, 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 secondmost 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 protease 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 detectable activity in the duodenum and intestine in both

6 608 Fish Physiol Biochem (2012) 38: 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 different letters 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 ± ± ± ± 4.05 Bighead carp 95.8 ± ± ± ± 3.74 Hybrid sturgeon 96.0 ± ± ± ± 1.49 PMSF phenylmethylsulfonyl fluoride, TLCK tosyl-lysine chloromethyl kotone, TPCK tosyl-phenylalanine chloromethyl kotone Activity (U) Acid protease Alkalina protease -amylase Lipase 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) 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 demonstrated 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 undertaken, and the results are presented in Fig. 5. The gel image indicates the presence of at least six

7 Fish Physiol Biochem (2012) 38: Alkaline proteases Activity (U) CG NG 50 0 Oesophagus Stomach Duodenum Intestine Activity (U) Alkaline proteases CG NG 50 0 Oesophagus Stomach Duodenum Intestine Activity (U) amylases CG NG Oesophagus Stomach Duodenum Intestine Activity (U) Lipase CG NG Oesophagus Stomach Duodenum Intestine 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) ( kda), five ( kda), and three ( kda) different acid proteases in paddlefish, bighead carp, and hybrid sturgeon, respectively. Three types of alkaline protease were identified in paddlefish and hybrid sturgeon with molecular weight 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 paddlefish; B bighead carp; H hybrid sturgeon. Markers showing are MPB-b-galactosidases (126 kda, MPB maltose-binding protein); 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)

8 610 Fish Physiol Biochem (2012) 38: from to kda and to 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 kda), three ( kda), and four ( 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 predators (Chakrabarti et al. 1995). Paddlefish and bighead carp are filter-feeders, and they have similar food habits (zooplanktivores); however, their digestive 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, elastaselike 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 and the ph 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 classification for protease was possible based on feeding behavior (Furné 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 functions, such as activation of other zymogens; several research studies with regard to enzymatic development in fish reported that trypsin was present from the embryonic stage onward (Muhlia-Almazán, 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 chymotrypsin-like activity. In this study, the higher proteolytic activity of paddlefish at ph 7.0, as compared with that at ph (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

9 Fish Physiol Biochem (2012) 38: by various inhibitors with regard to proteolytic activities (Table 1). The rate of inhibition of TPCK (the inhibitor of chymotrypsin, 59.5%) was approximately 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, irrespective of the food habit of the fish, adaptations of the digestive system of different species exhibit closer correlation with their diet than their microenvironment 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 (commercial 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 esophagus 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 considerable 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 omnivorous or herbivorous fish (Tengjaroenkul et al. 2000; Furné 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 kda for amylase) or similar bands (154.4 kda in paddlefish and kda in hybrid sturgeon for acid protease, kda in paddlefish and kda

10 612 Fish Physiol Biochem (2012) 38: 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 categorization 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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