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1 DOI /s Partial characterisation of digestive proteases of the Mayan 4 cichlid Cichlasoma urophthalmus 5 C. A. Cuenca-Soria C. A. Álvarez-González J. L. Ortiz-Galindo 6 H. Nolasco-Soria D. Tovar-Ramírez R. Guerrero-Zárate A. Castillo-Domínguez 7 M. A. Perera-García R. Hernández-Gómez E. Gisbert 8 Received: 9 April 2013 / Accepted: 3 October Ó Springer Science+Business Media Dordrecht AQ1 Abstract The characterisation of digestive proteases 11 in native freshwater fish such as the Mayan cichlid 12 Cichlasoma urophthalmus provides scientific ele- 13 ments that may be used to design balanced feed that 14 matches with the digestive capacity of the fish. The 15 purpose of this study was to characterise the digestive 16 proteases, including the effect of the ph and the 17 temperature on enzyme activity and stability, as well 18 as the effect of inhibitors using multienzymatic 19 extracts of the stomach and intestine of C. urophthal- 20 mus juveniles. Results showed that the optimum 21 activities of the acid and alkaline proteases occurred 22 at ph values of 3 and 9, respectively, whereas their 23 optimum temperatures were 55 and 65 C, A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 C. A. Cuenca-Soria J. L. Ortiz-Galindo Centro Interdisciplinario de Ciencias Marinas, Av. Instituto Politécnico Nacional s/n, Col. Playa Palo de Santa Rita, Apdo Postal 592, La Paz, BCS, Mexico C. A. Cuenca-Soria A. Castillo-Domínguez M. A. Perera-García R. Hernández-Gómez División Académica Multidisciplinaria de los Ríos, Universidad Juárez Autónoma de Tabasco, Carretera Tenosique-Estapilla km 1, Tenosique, Tabasco, Mexico A16 A17 A18 A19 A20 A21 A22 A23 respectively. The acid proteases were most stable at ph values of 2 3 and at temperatures of C, whereas the alkaline proteases were most stable at ph values of 6 9 and at C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory. H. Nolasco-Soria D. Tovar-Ramírez Centro de Investigaciones Biológicas del Noroeste, S.C. Mar Bermejo No. 195, Col. Playa Palo de Santa Rita, Apdo Postal 128, La Paz, BCS, Mexico E. Gisbert IRTA, Centre de Saint Carles de la Ràpita (IRTA-SCR), Ctra. Poble Nou, km 6, Saint Carles de la Ràpita, Tarragona, Spain A11 C. A. Álvarez-González (&) R. Guerrero-Zárate A12 Laboratorio de Acuicultura Tropical, DACBIOL-UJAT, A13 Carretera Villahermosa-Cárdenas km 0.5, A Villahermosa, Tabasco, Mexico A15

2 38 Keywords Cichlasoma urophthalmus 39 Characterisation Digestive proteases Inhibitors ph Temperature Introduction 44 To date, many studies that were designed to determine 45 the effects of experimental diets on the growth and 46 survival of fish have only considered growth perfor- 47 mance parameters and biotechnical aspects, but with- 48 out including aspects related to the physiology of the 49 organism. Regarding this issue, the understanding of 50 how digestive enzymes work makes possible to 51 explain the hydrolysis of the diverse nutrients in the 52 diet (Glass et al. 1989; Kolkovski 2001). Studies on 53 nutritional physiology and digestive secretions in fish 54 have been proposed to solve nutritional problems 55 linked with diet formulation and ingredient selection, 56 such as the composition of artificial diets in relation to 57 the digestion capacity and the nutritional requirements 58 of a several fish species (Furné et al. 2005). Studies on 59 the digestive enzymes of marine fish included those on 60 European seabass Dicentrarchus labrax, red seabream 61 Pagrus major, Monterey sardine Sardinops sagax 62 caerulea, Pacific bluefin tuna Thunnus orientalis, 63 spotted sand bass Paralabrax maculatofasciatus and 64 some Mediterrean teleosts (Lindner et al. 1995; Iijima 65 et al. 1998; Castillo-Yáñez et al. 2005; Matus de la 66 Parra et al. 2007; Álvarez-González et al. 2008; 67 Caruso et al. 2009, respectively). Studies on the 68 digestive physiology of freshwater fish are becoming 69 increasingly relevant; for example, Natalia et al. 70 (2003) characterised the digestive enzymes of the 71 ornamental carnivore Scleropages formosus and found 72 that, despite its carnivorous habits, it may assimilate 73 inert artificial feed. In addition, Lundstedt et al. (2004) 74 worked with dietary levels of % of crude 75 protein and of % of starch to determine their 76 relationship with the enzymatic activity of the fresh- 77 water catfish Pseudoplatystoma corruscans, and found 78 the best levels to be % protein and % 79 starch, based on the enzymatic response. Furné et al. 80 (2005) carried out a comparative study to evaluate the 81 activity of the proteases, amylases and lipases in the 82 sturgeon Acipenser naccarii and the freshwater trout 83 Oncorhynchus mykiss, and indicated that the first 84 presented characteristics of an omnivore, with a high amylase activity and a lower protease and lipase activity. Debnath et al. (2007) studied the effect of diet composition on the activity of the digestive enzymes in the rohu carp Labeo rohita and found that a diet with 30 % crude protein responded best to the enzymatic machinery of the fish, obtaining the lowest hepatosomatic index and one of the greatest yields in weight. Zhong-yi et al. (2008) isolated cathepsin D from the intestine of the herbivorous carp Ctenopharyngodon idellus in order to carry out inhibition essays and found the optimum values of ph and temperature. Perales- García (2006) characterised the proteases of the Bay snook Petenia splendida and found them similar to those of other species and enzymatic machinery typical of a strictly carnivorous fish. Finally, Guerrero-Zárate (2010) studied the digestive enzymes of the tropical gar Atractosteus tropicus, evaluated the activity of the lipases, recorded the resistance of acid proteases in alkaline media and of alkaline proteases in acid media and observed a joint enzymatic activity of acid and alkaline proteases in the intestine. On a different note, López-Ramírez et al. (2010) studied the development of the digestive enzymes of the Mayan cichlid Cichlasoma urophthalmus through 60 days of the larval period. The authors concluded that the species is capable of digesting a wide spectrum of food sources of plant and animal origin, including artificial feed, during this period. There is at present no relevant information on the characterisation of the enzymatic machinery of C. urophthalmus juveniles, for which reason this study focused on characterising operational parameters and the effect of inhibitors of the digestive enzymes present in multienzymatic extracts of the stomach and the intestine. Materials and methods Larval culture Cichlasoma urophthalmus larvae were obtained from broodstock located in the Laboratorio de Acuicultura Tropical of the División Académica de Ciencias Biológicas (DACBIOL) of the Universidad Juárez Autónoma de Tabasco (UJAT), Tabasco, Mexico. Larvae were kept in three 100-l tanks for 30 days post-fertilisation until juvenile stage. Water quality parameters were recorded daily: temperature with a thermometer (Brannan USA), dissolved oxygen with an oxymeter (YSI 55, AQ

3 130 California, USA) and ph with a ph meter (Denver 131 Instrument UB-10, Denver, Colorado, USA), and the 132 values were maintained at 28.7 ± 0.6 C, 5.7 ± mg l -1 and 7.3±0.1, respectively. The C. uroph- 134 thalmus larvae were fed non-enriched Artemia nauplii 135 ad libitum (INVE Aquaculture Nutrition, Belgium) three 136 times a day up to day 15 after hatching (dah). From then 137 on, they were fed balanced trout feed (45 % protein, % lipids, Nelson & Sons Inc., USA). After larvicul- 139 ture, juveniles (5.7±3.4 g wet weight, n=50) were 140 placed in 1.7-m 3 circular plastic tanks with an open 141 circulation system for additional 30 days until 142 processing. 143 Multienzymatic extracts preparation 144 The juveniles of the Mayan cichlid C. urophthalmus 145 were killed with an MS-222 anaesthetic (tricaine 146 methanesulfonate, Argent Laboratories, Redmond, 147 WA) and dissected while cold in order to isolate the 148 stomachs and intestines from the gut cavity. The wet 149 weight of the stomachs and intestines of each spec- 150 imen was recorded using an analytical scale (Denver 151 Instrument APX-200, resolution 0.1 mg). The tissues 152 were homogenised as a pool (stomachs and intestines) 153 in an electrical grinder (Ultra Turrax Ò Ika T18 Basic) 154 (tissue/distilled water, w/v of ratio 1:5). The mixtures 155 were centrifuged at 14,000 rpm (20,8179g) and 4 C 156 for 30 min in a centrifuge (Eppendorf 5810-R). The 157 supernatants were collected and stored at-20 C for 158 future analysis. 159 Biochemical analyses 160 The concentration of soluble protein in stomach and 161 intestine extracts of C. urophthalmus was determined 162 following Bradford (1976) and used bovine albumin as 163 a standard. The activity of the acid proteases in the 164 stomach extract was recorded according to Anson 165 (1938) with slight modifications. In brief, the reaction 166 mixture consisted of 1 ml of haemoglobin (in 1 % 167 glycine-100 mmol l -1 HCl buffer, ph 2) and 20 llof 168 stomach multienzymatic extract of C. urophthalmus. 169 It was incubated at 25 C for 30 min. The reaction was 170 stopped with 500 ll of 20 % trichloroacetic acid 171 (TCA) and was left to stand at 4 C for 15 min. The 172 samples were then centrifuged at 10,000 rpm under 173 the same standing conditions. The supernatants were 174 collected and diluted with distilled water (1:10) to take a reading of the absorbance at 280 nm in a spectrophotometer (Jenway 6405 UV/Visible) using quartz cells (1 cm 2 ). One unit of activity was defined as the amount of enzyme needed to catalyse the liberation of an equivalent of 1 lg of tyrosine per minute. The activity of the alkaline proteases in the intestine multienzymatic extract was recorded according to the method of Kunitz (1947), modified by Walter (1984), using 1 ml of Hammarsten casein (ICN Biomedicals No , Aurora, OH, USA) in 1 % Tris- 100 mmol l -1 HCl buffer, ph 9 as substrate, and 5 ll of intestine multienzymatic extract of C. urophthalmus. The mixture was incubated at 25 C for 30 min. The reaction was stopped with 500 llof20 % TCA. The samples were centrifuged and the absorbance read as were those of the stomach proteases, in order to obtain the amount of equivalents of tyrosine liberated by the proteolytic reaction. All assays were carried out by triplicate. Trypsin activity was evaluated following the technique of Erlanger et al. (1961). The BAPNA (N-abenzoyl-DL-arginine 4-nitroanilide) used as substrate was previously diluted in 200 ll of dimethylsulfoxide (DMSO) and taken to 3.5 mmol l -1 in a buffer (Tris HCl 50 mm, CaCl 2 20 mm, ph 8). The reaction took place with the addition of 10 ll of intestine multienzymatic extract to 990 ll of substrate, incubated at 25 C for 30 min. The reaction was stopped with 250 ll of 30 % acetic acid. The absorbance was recorded at 410 nm. The enzymatic activity was defined as 1 lmol of p-nitroanilide liberated per minute, using an 8.8 ml lmol -1 cm -1 molar extinction coefficient (Dimes and Haard 1994). Chymotrypsin activity was determined following Asgeirsson and Bjarnason (1991). The BTEE (Nbenzoyl-L-tyrosine ethylester) was previously diluted in DMSO and taken to a final concentration of 5 mmol l -1 in a buffer (Tris HCl 44.4 mmol l -1, ph 7.8, 25 C). The reaction was started by adding 623 ll of buffer to the quartz cell (1 cm 2 ) to stabilise the spectrophotometer at cero. An amount of 70 ll of substrate was added and a reading was taken at 256 nm every 20 s for 2 min. This was followed by adding 10 ll of intestine multienzymatic extract and again recording the absorbance every 20 s for 2 min. The delta value of the absorbance between the catalysed reaction and the substrate reaction was recorded to calculate the activity, considering a ml lmol -1 cm -1 molar extinction coefficient

4 224 A unit of chymotrypsin was defined as the amount of 225 enzyme required to liberate 1 lmol of tyrosine per 226 minute at 25 C. 227 Leucine aminopeptidase activity was determined 228 following the method proposed by Maraux et al. 229 (1973). The substrate used was 1 mm leucine-p- 230 nitroanilide diluted in sodium phosphate buffer 231 (50 mm, ph 7.2). To 980 ll of substrate was added ll of intestine multienzymatic extract. The mixture 233 was incubated at 25 C for 30 min and the reaction was 234 stopped with 250 ml of 30 % acetic acid. The absor- 235 bance was read at 410 nm and the activity was defined 236 as the amount of enzyme required to liberate 1 lmol of 237 p-nitroanilide per minute. An 8.2 ml lmol -1 cm molar extinction coefficient was considered. 239 Carboxypeptidase A activity was recorded following 240 the method of Folk and Schirmer (1963). The substrate 241 for the assay was 1 mm hippuryl-l-phenylalanine in 242 Tris HCl (25 mmol l -1 NaCl 500 mmol l -1,pH7.5). 243 An amount of 600 ll of buffer was added to the quartz 244 cell (1 cm 2 ). The spectrophotometer was stabilised at 245 zero, 60 ll of substrate was added, and the absorbance 246 was recorded at 254 nm every 20 s for 2 min at 25 C. 247 This was followed by adding 5 ll of intestine multien- 248 zymatic extract and again recording the absorbance 249 every 20 s for 2 min. Calculations were carried out in 250 the same way as was done for chymotrypsin. The 251 enzyme unit was defined as the liberation of 1 lmol l of hippuric acid per minute. A 0.36 ml lmol -1 cm molar extinction coefficient was used. 254 In order to determine the effect of the ph and the 255 temperature on protease activity, the previous proce- 256AQ3 dure was followed: ph values of 2, 4, 6, 8 and 10 (acid 257 proteases) and of 4, 6, 8, 10 and 12 (alkaline proteases) 258 were evaluated using a universal buffer solution 259 (Stauffer1989). Temperatures of C were used 260 for both types of proteases. For the protease stability 261 assays, the samples were pre-incubated at 25 C for 0, , 60 and 90 min for each of the indicated ph values 263 (stability at different values of ph). The stability at 264 different temperatures was recorded by pre-incubating 265 the multienzymatic extract of C for 0, 30, and 90 min. 267 Protease inhibition (biochemical techniques) 268 The enzymatic characterisation of the different prote- 269 ases in C. urophthalmus juveniles followed the meth- 270 odology described by Dunn (1989). Pepstatin A (1 mmol l -1 ) was used as an inhibitor to characterise the acid protease activity. 20 ll of concentrated inhibitor solution was pre-incubated with 20 ll of multienzymatic extract for 1 h at 25 C. The residual activity was recorded as the difference between a control activity (without inhibitor) as 100 % and the activity of the extract after 1 h in the presence of the inhibitors. In the case of the inhibition assays of the alkaline proteases, the inhibitors used were 100 mmol l -1 phenylmethylsulfonyl fluoride (PMSF), 10 mmol l -1 tosyl-lysine-chloromethyl ketone (TLCK), 250 mmol l -1 trypsin soybean inhibitor (SBT1), 10 mmol l -1 tosyl-phenylalanine-chloromethyl ketone (TPCK), 10 mmol l -1 phenanthroline (Phen), 250 mmol l -1 ovalbumin (Ovo) and 10 mmol l -1 ethylenediaminetetraacetic acid (EDTA). 5 ll of intestine multienzymatic extract was pre-incubated at 25 C for 1 h with 5 ll of the inhibitors used in the essay. The residual activity was determined as mentioned previously, and it was expressed as the percentage of the activity with respect to a control without inhibitors (100 % activity) for both cases. Each assay was carried out by triplicate. Protease inhibition (electrophoretic techniques) casein solution (2 % in a Tris HCl 100 mmol l -1 Protease characterisation was complemented by elec- 294 trophoresis and the use of specific inhibitors. The 295 number of enzymatic activity bands and the types of 296 proteases in the multienzymatic extracts of C. uroph- 297 thalmus were quantified. The multienzymatic extracts 298 were pre-incubated for 1 h with the previously 299 described inhibitors, from which a 20-ll sample was 300 taken to carry out electrophoresis. In the case of the 301 acid proteases, electrophoresis was run under native 302 conditions (PAGE), according to Davis (1964), in % acrylamide continuous gels (80 V for 15 min). 304 The gels were submerged in a 100 mmol l -1 HCl 305 solution to obtain a ph of 2 to detect the acid 306 proteolytic activity, after which they were placed in a 307 haemoglobin solution (in 0.25 % 100 mmol l -1 gly- 308 cine HCl buffer, ph 2, at 4 C). In the case of the 309 alkaline proteases, electrophoresis was run under 310 denaturalising conditions (SDS-PAGE), according to 311 Laemmli (1970) and adapted by García-Carreño et al. 312 (1993), with a SDS 0.1 % buffer, Tris (25 mmol l -1 ) 313 and glycine (192 mmol l -1, ph 8.3, 100 volts, min). The gels were submerged in a Hammerstein buffer, ph 9, for 1 h at 4 C) to detect the alkaline 317

5 Table 1 Characterisation of digestive enzymes in C. urophthalmus juveniles 318 protease activity. The dyeing of the gels followed 319 Weber and Osborn (1969). The relative electromobil- 320 ity (Rf) was calculated for all zymograms (Igbokwe 321 and Downe 1978), and the molecular weight (MW) of 322 each band in the SDS zymograms (alkaline protease) 323 was calculated by a linearly adjusted model between 324 the Rf and the decimal logarithm of MW proteins 325 using Quality One version (Hercules, CA) 326 software program. 327 Results 328 Biochemical analyses 329 Enzyme activity levels in C. urophthalmus juveniles 330 are shown in Table 1. The activity values for acid 331 proteases were 1.8±0.5 U mg protein -1 and for the 332 alkaline proteases were 3.1±0.5 U mg protein The endopeptidases such as trypsin and chymotrypsin 334 presented activities of 0.52 ± 0.02 and 0.8 ± U mg protein -1, respectively, whereas the exopeptid- 336 ases such as aminopeptidase and carboxypeptidase A 337 presented activity levels of ± and ±0.1 U mg protein -1, respectively. 339 Effect of ph and pre-incubation on protease 340 activity and stability 341 Protease activity considering the ph and its stability 342 considering the ph and the time of pre-incubation are 343 shown in Fig. 1. The acid protease activity presented 344 differences depending on the ph considered, with an 345 optimum value at a ph of 3 and a drastic decrease in 346 activity as the ph increased (Fig. 1a). At ph 2, acid 347 protease presented the highest residual activity 348 (around 80 %) after 60 min of incubation. Also the 349 residual activity increased as the time of pre- Uml -1 U g tissue -1 U mg protein -1 Acid proteases 2.7± ± ±0.5 Alkaline proteases 10.6 ± ± ± 0.5 Trypsin 1.9 ± ± ± 0.02 Chymotrypsin 3.1 ± ± ± 0.3 Aminopeptidase leucin ± ± ± Carboxypeptidase A 1.3 ± ± ± 0.1 incubation increased at ph values 4 and 6, whereas the acid protease stability decreased at ph values 8 and 10 (Fig. 1b). The alkaline protease activity was also affected by the ph with an optimum activity at a ph of 9 (Fig. 1c). The residual activity of the alkaline proteases presented a high resistance at different ph values. It increased to more than 650 % in relation to the initial activity (100 %), especially at ph value of 4 and 6 after 30 min of pre-incubation. As the time of pre-incubation increased, however, the residual activity decreased around 450 % after 60 and 90 min of pre-incubation (Fig. 1d). The effects of temperature on protease activity may be seen in Fig. 2. The greatest activity was recorded at 55 C (Fig. 2a). The stability of the acid proteases was very high at all the pre-incubation times at 35 and 45 C, whereas the residual activity decreased rapidly at greater temperatures, especially when pre-incubating the extracts at 65 C (Fig. 2b). The temperature had a positive effect on the activity of alkaline proteases, with the optimum activity at 55 C (Fig. 2c). The stability of the alkaline proteases with regard to temperature changes showed a high resistance, especially at 45 C, reaching the greatest value after 90 min, followed by a temperature of 35 C and then 55 C. The lowest value was recorded when preincubating the multienzymatic extract at 65 C (Fig. 2d). Protease inhibition (biochemical techniques) The effects of the different inhibitors on the activity of the acid and alkaline proteases of C. urophthalmus are shown in Table 2. Inhibition of the alkaline proteases was high when the specific inhibitors TPCK, TLCK and EDTA and the general inhibitor ovalbumin (greater than 80 %) were used, while degrees of inhibition of 60 and 43.8 %, respectively, were AQ

6 Fig. 1 Protease characterisation. a Optimum ph for acid protease activity, b residual activity to evaluate the stability of acid proteases, considering the ph and the time of incubation, c optimum ph for alkaline protease activity, d residual activity to evaluate the stability of alkaline proteases, considering the ph and the time of incubation Fig. 2 Protease characterisation. a Optimum temperature for acid protease activity, b residual activity to evaluate the stability of acid proteases, considering the temperature and the time of incubation, c optimum temperature for alkaline protease activity, d residual activity to evaluate the stability of alkaline proteases, considering the temperature and the time of incubation 386 observed when the general inhibitors PMSF and SBT1 387 were used. The inhibition generated by pepstatin A 388 (pepsin A type inhibitor) in the stomach multienzy- 389 matic extract decreased activity by 96 %. Protease inhibition (electrophoretic techniques) Figure 3a shows that the stomach enzymatic extract of C. urophthalmus (Control lane) presented one single

7 Table 2 Effect of inhibitors on protease activity in C. urophthalmus juveniles Inhibitor Inhibitor concentration (mmol l -1 ) Residual activity (%) PMSF ± 8.3 SBT ± 6.4 Ovo ± 1.6 TPCK ± 1.5 TLCK ± 2.0 Phen ± 5.5 EDTA ± 2.2 Pepst A 1 4.0±1.1 PMSF = phenylmethylsulfonyl fluoride, SBT1 = trypsin soybean inhibitor, Ovo = ovalbumin, TPCK = tosyl-phenylala nine-methyl ketone, TLCK = tosyl-lysine-methyl ketone, Phen = phenanthroline, EDTA = ethylenediaminetetraacetic acid, Pepst A=pepstatin A 393 isoform that was completely inhibited when using 394 pepstatin A (CI lane). When comparing with pig 395 pepsin (P lane) and porcine pepsin inhibited with 396 pepstatin A (PI lane), the band disappeared. In Fig. 3b, 397 it is possible to see the protease bands from the effect 398 of the inhibitors in the intestine multienzymatic extract 399 of C. urophthalmus, with six bands that correspond to 400 six isoforms with alkaline proteolytic activities (Control lane) of 16.1, 21.5, 43.1, 72.1, 82.2 and 92.9 kda. The six bands were inhibited in the presence of ovalbumin (Ovo lane). All the bands were inhibited when the inhibitors SBT1 (SBT1 lane) and PMSF (PMSF lane) were used, except for those with the greatest molecular weights (82.2 and 92.9, and 92.9 kda, respectively). No band was inhibited with phenanthroline (Phen lane) except for the band with the lowest molecular weight (16.1 kda), while most of the bands were inhibited with EDTA (EDTA lane) except for the bands that corresponded to the most extreme molecular weights (16.1 and 92.9 kda). Most of the bands were not inhibited when the specific inhibitors TPCK (TPCK lane) and TLCK (TLCK lane) were used, except for the band with the lowest molecular weight (16.1 kda). Discussion The enzymatic activity of the intestinal proteases recorded in this study (3.1±0.5 U mg protein -1 )in C. urophthalmus was greater than the activity derived from stomach proteases (1.8±0.5 U mg protein -1 ). These results have also been observed in other omnivorous fish species such as tilapia Oreochromis niloticus (Klahan et al. 2009), Pagellus bogaraveo (Caruso et al. 2009) and the planktivorous Fig. 3 Zymogram of acid (a) and alkaline (b) proteases with the action of the respective inhibitors on the isoforms of the multienzymatic stomach and intestine extracts of C. urophthalmus. CI: control with pepstatin A inhibitor, P: pig pepsin, PI: pig pepsin with pepstatin A inhibitor, PMSF: phenylmethylsulfonyl fluoride, SBT1: trypsin soybean inhibitor TPCK: Tosylphenylalanine-methyl ketone, TLCK: Tosyl-lysine-methyl ketone, Phen: Phenanthroline, Ovo: Ovalbumin, EDTA: ethylenediaminetetraacetic acid, M: molecular weight marker (kda): bovine serum albumin (66 kda), egg albumin (43 kda), carbonic anhydrase (29 kda), trypsinogen (24 kda), trypsin soybean inhibitor (20 kda)

8 426 Hypophthalmichthys molitrix (Kumar et al. 2007). 427 These results suggested that the intestinal proteases in 428 omnivorous and planktivorous fish might play a more 429 important role than the stomach proteases, whereas in 430 carnivorous fish such as the catfish Pseudoplatystoma 431 corrucans and Senegal sole Solea senegalensis, the 432 alkaline proteolytic activity in the intestine is more 433 limited than the proteolytic activity in the stomach 434 (Lundstedt et al. 2003; Sáenz de Rodrigáñez et al ). 436 At the same time, the acid proteases in the stomach 437 multienzymatic extracts of C. urophthalmus presented 438 a maximum activity at a ph of 3, whereas the alkaline 439 proteases in the intestine multienzymatic extracts 440 presented a maximum activity at a ph of 9. Jun-sheng 441 et al. (2006) recorded optimum ph values for acid and 442 alkaline proteases in juveniles of the hybrid O. 443 niloticus 9 Oreochromis aureus of 2 and 10.5, 444 respectively, which were not too different from those 445 recorded in the present study. However, it is also 446 possible to find similarities in carnivorous fish such as 447 salmonids in which the acid proteases worked opti- 448 mally in a ph range of 1 2 (Munilla-Moran and Stark ), and in the carnivorous fish S. formosus in which 450 the alkaline proteases worked in an optimum ph range 451 of 9 10 (Natalia et al. 2003). This result suggested that 452 the optimum activity of the proteases at a specific ph 453 is not related to the feeding habits of the species. 454AQ5 On the other hand, acid proteases remained stable 455 until ph value of 6 and present an intermediate activity min after pre-incubation, although this decreased 457 markedly at pre-incubation temperatures of 60 and C. This suggested that their proteolytic capacity, 459 though optimum at acid ph values, was relatively 460 stable even at a value of ph 6, making it possible for 461 the acid proteases to act as potential activators of 462 zymogens of the alkaline proteases secreted by the 463 pancreas into the intestine (Nolasco-Soria, personal 464 communication). Digestion with alkaline proteases 465 takes place with the digestion of acid proteases 466 (Chakrabarti et al. 1995; Hi et al. 2012). This has 467 been observed in the strictly carnivorous tropical gar 468 A. tropicus (Guerrero-Zárate 2010) and the tuna 469 Tunnus thynnus (Essed et al. 2002). The results 470 obtained thus showed that C. urophthalmus presented 471 a certain tendency to carnivory and its alkaline 472 proteases presented an ample stability throughout the 473 range of tested ph values. It has been showed in the 474 Mediterranean fish P. bogaraveo, which is considered as omnivorous species, although it is a preferable consumer of crustaceans, molluscs, worms and small fish (Caruso et al. 2009). This indicated that these proteases have a marked plasticity that might be even greater than that of carnivorous fish. The stability of the acid proteases at ph values outside their optimum range might be related to the fact that C. urophthalmus is a species for which a functional digestive zonation has not been well defined. In the higher vertebrates, the activity of the digestive enzymes is restricted to different regions of the intestine, showing a clear functional zonation, as occurs in most fish (Lundstedt et al. 2003). However, proteinases with optimum ph values of 5 and 8.5 have been found in the gastric juice of fish (Kuzmina 1991), whereas alkaline proteases have been also found in the stomach of species such as Sparus aurata (Deguara et al. 2003). Regarding the effects of temperature on enzyme activity, Jun-sheng et al. (2006) determined the optimum temperature for the acid and alkaline proteases of the hybrid O. niloticus9o. aureus and found that optimum conditions were at 55 C for both proteases. These results were somewhat similar to the values recorded for the acid (65 C) and alkaline (55 C) proteases of C. urophthalmus. Although it is a carnivore species, the activity of the acid and alkaline proteases in A. tropicus presented an optimum temperature of 65 % (Guerrero-Zárate 2010); thus, the optimum temperatures recorded for C. urophthalmus felt within the range of values recorded for other species. Regarding the stability of the alkaline proteases considering the temperature, the stability of the acid proteases might be seen through the small effect recorded at an incubation temperature of 45 C and the small loss of protease activity (even after 90 min of pre-incubation). From 55 C onwards, they started to present a 50 % loss of activity that became more marked at 65 C. In contrast, the stability of the alkaline proteases was affected little or nothing by the pre-incubation temperature, as well as by the temperature at which they presented proteolytic activity with the exception of 65 C as in the previous case. The relatively high plasticity of the digestive proteases in C. urophthalmus might be closely related to its wide spectrum of habitats. The Mayan cichlid C. urophthalmus is a eurytopic species, and consequently, it might be found in freshwater and brackish karstic water bodies on the Yucatán peninsula. The probable explanation of this wide ecological tolerance might AQ

9 524 thus be linked to the great plasticity of its physiolog- 525 ical characteristics and feeding habits (Chávez-Sán- 526 chez et al. 2000). 527 According to the present results, trypsin activity 528 (0.52±0.02 U mg protein -1 ) was slightly below 529 that of chymotrypsin (0.8±0.3 U mg protein -1 )in 530 C. urophthalmus. This has also been recorded for 531 species with similar feeding habits such as L. rohita, 532 Catla catla and H. molitrix in which chymotrypsin 533 activity is greater than trypsin activity (Kumar et al ). In this context, Jonas et al. (1983) stated that 535 chymotrypsin activity tends to predominate in omniv- 536 orous fish. 537 The results obtained by means of biochemical 538 techniques regarding enzyme inhibition with pepstatin 539 A confirmed the absence of bands that corresponded to 540 pepsin in the PAGE gel, like the pepsin inhibitor in the 541 stomach of C. urophthalmus, and pointed to an 542 enzymatic tendency that was characteristic of carniv- 543 orous fish. This has been observed for omnivores and 544 carnivores (Chiu and Pan 2002). Regarding the 545 alkaline proteases, the inhibition with ovalbumin and 546 PMSF resulted in an almost total decrease in activity 547 ([80 and 60 %, respectively) and proved the impor- 548 tance of the serine proteases in the alkaline digestion 549 of fish. Jonas et al. (1983) mentioned an important 550 inhibition of serine-protease activity in carnivorous 551 and omnivorous fish in the presence of PMSF, which 552 was confirmed by the almost total absence of bands in 553 the SDS-PAGE gels, specifically in lane 1 (PMSF) 554 where only one band remains (92.9 kda isoform), 555 which might correspond to a aminopeptidase-like 556 enzyme, in contrast with the ovalbumin that inhibited 557 all the bands, which agreed with Wu et al. (2008) who 558 reported that leucine aminopeptidase had a molecular 559 weight of approximately 96 kda in P. major. The 560 inhibition reactions also showed that chymotrypsin 561 presented almost double the inhibition (with TPCK) 562 that was recorded for trypsin (with TLCK) in the 563 intestine multienzymatic extract of C. urophthalmus. 564 This provided an additional element when suggesting 565 that chymotrypsin might be more important than 566 trypsin in omnivorous fish such as C. urophthalmus. 567 Recent studies of juveniles have recorded an increase 568 in the importance of chymotrypsin with respect to 569 trypsin in well-developed larvae. At the same time, 570 this enzyme might triplicate trypsin s activity (Lazo 571 et al. 2007). Additionally, the presence of metal 572 proteases (that depend on divalent metallic ions for their activity) was made evident through the inhibition of their activity by at least 50 % (with phenanthroline), and even greater with EDTA (10 % of the initial enzymatic activity), which indicated the importance of this type of proteases in the alkaline digestion of C. urophthalmus. The use of these last two inhibitors in other freshwater species such as A. tropicus had proved the presence of metal proteases in the gut, as Guerrero-Zárate (2010) concluded. Regarding the electrophoretic techniques that were carried out with the SDS-PAGE gel, the presence of bands associated with most of the six isoforms present in the intestine multienzymatic extract (lanes 5 and 6) demonstrated the specificity of the inhibitors TPCK and TLCK. However, these inhibitors of chymotrypsin and trypsin respectively did not turn out to be specific as it was not possible to tell each endoprotease apart due to the fact that both the TPCK and the TLCK inhibited the last band that corresponded to the isoform with the lowest molecular weight (16.1 kda), although it was possible to establish a range of molecular weights of kda for both endoproteases. Castillo- Yáñez et al. (2004) reported a molecular weight of 25 kda for trypsin in the Monterey sardine Sardinops sagax caerulea, whereas Heu et al. (1995) recorded weights of 25.6 and 26.1 kda for trypsin and chymotrypsin in the anchovy Engraulis japonica, and Chong et al. (2002) reported kda for chymotrypsin in the discus Symphysodon aequifasciata. The absence of several bands in lane 9 (EDTA), except for those corresponding to the extreme values (92.9 and 16.1 kda, respectively), confirmed the results obtained by the biochemical techniques that provided evidence of the presence of metal proteases in the intestine multienzymatic extract of C. urophthalmus. However, the presence of the band associated with the 16.1 kda isoform in lane 9 (EDTA) and its absence in lanes TPCK and TLCK might indicate that neither trypsin nor chymotrypsin required metallic ions to become active. However, the presence of most of the bands of the six isoforms observed in lane 7 (Phen) proved the high specificity of the inhibitor 1, 10 phenanthroline to the activity of the metal proteases, specifically trypsin and chymotrypsin. The differences in the degree of inhibition of these endoproteases generated by the presence of the 1, 10 phenanthroline and EDTA might be due to different degrees of sensitivity (of one of the inhibitors) to the effect of the SDS present in the gel, and suggested that this issue

10 622 requires further study. The degree of inhibition of 623 trypsin and chymotrypsin generated by the 1, phenanthroline and EDTA might also vary from 625 species to species, as Heu et al. (1995) stated. 626 The low levels of exopeptidases (carboxypeptidase 627 A and leucine aminopeptidase) detected in C. uroph- 628 thalmus might indicate that most of the alkaline 629 digestion of proteins was carried out jointly by the 630 endopeptidases and the metal proteases, which have 631 been shown to play an important role in the intestine of 632 the discus S. formosus (Natalia et al. 2003). The role of 633 the exopeptidases in C. urophthalmus might be limited 634 to degrading small short peptide chains to liberate 635 terminal amino acids (López-Ramírez et al. 2010), 636 once pepsin, the endoproteases and the metal proteases 637 had carried out most of their digestive proteolytic 638 activity in C. urophthalmus. 639 The results of this study confirmed that C. uroph- 640 thalmus is an omnivorous species capable of degrad- 641 ing substrates that are characteristic of carnivores, as it 642 showed an important activity that was specific to 643 alkaline proteases, together with an acid protease 644 activity. 645 Acknowledgments Authors thank the Consejo Nacional de 646 Ciencia y Tecnología from Mexico for providing grant for 647 postgraduate studies, Carlos Alfonso Frías Quintana 648 (Laboratorio de Acuicultura Tropical, DACBIOL-UJAT, 649 Tabasco, Mexico) for providing advice, as well as the project 650 Identificación de ingredientes en alimentos balanceados y su 651 digestibilidad en el cultivo experimental de peces nativos en 652 Tabasco financed by the Fondos Mixtos CONACyT, Mexico, 653 for supporting the development of the study. 654 References 655 Álvarez-González CA, Moyano-López FJ, Civera-Cerecedo R, 656 Carrasco-Chávez V, Ortiz-Galindo JL, Dumas S (2008) 657 Development of digestive enzyme activity in larvae of 658 spotted sand bass Paralabrax maculatofasciatus, bio- 659 chemical analysis. Fish Physiol Biochem 34: Anson ML (1938) The estimation of pepsin, trypsin, papain and 661 cathepsin with hemoglobin. J Gen Physiol 22: Asgeirsson B, Bjarnason JB (1991) Structural and kinetic 663 properties of chymotrypsin from atlantic cod (Gadus 664 morhua): comparison with bovine chymotrypsin. Comp 665 Biochem Physiol 99B(2): Bradford MM (1976) A rapid and sensitive method for the 667 quantization of microgram quantities of protein utilizing 668 the principle of protein dye binding. Anal Biochem 72: Caruso G, Denaro MG, Genovese L (2009) Digestive enzymes 671 in some teleost species if interest for mediterranean aqua- 672 culture. Open Fish Sci J 2:74 86 Castillo-Yáñez FJ, Pacheco-Aguilar R, García-Carreño FL, Navarrete-Del Toro MA (2004) Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea. Comp Biochem Physiol 140(B):91 98 Chavez-Lomelí MO, Mattheeuws MH, Vega P (1989) Biología de los peces del Río San Pedro en vista de determinar su potencial para la piscicultura. Xalapa, Veracruz, México. 222 pp Chávez-Sánchez C, Martínez-Palacios CA, Martínez-Pérez G, Ross LG (2000) Phosphorus and calcium requirements in the diet of the American cichlid Cichlasoma urophthalmus. Aquac Nutr 6:1 9 Chong ASC, Hashim R, Chow-Yang L, Ali AB (2002) Partial characterization and activities of proteases from the digestive tract of discus fish (Symphysodon aequifasciata). Aquaculture 203: Davis BJ (1964) Disc electrophoresis-ii. Method and application to human serum proteins. Ann NY Acad Sci 121: Debnath D, Pal AK, Sahu NP, Yengkokpam S, Baruah K, Choudhury D, Venkateshwarlu G (2007) Digestive enzymes and metabolic profile of Labeo rohita fingerlings fed diets with different crude protein levels. Comp Biochem Physiol B 146: Deguara S, Jauncey K, Agius C (2003) Enzyme activities and ph variations in the digestive tract of gilthead sea bream. J Fish Biol 62: Dimes LE, Haard N (1994) Estimation of protein digestibility: development of an in vitro method for estimating protein digestibility in salmonids. Comp Biochem Physiol 108(A): Dunn BM (1989) Determination of protease mechanism. In: Beynon RJ, Bond JS (eds) Proteolytic enzymes: a practical approach. I.R.L Press, Oxford, pp Erlanger B, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95: Essed Z, Fernández I, Alarcón FJ, Moyano FJ (2002) Caracterización de la actividad proteasa digestiva de atún rojo Thunnus thynnus (Linnaeus, 1758). Boletín del Instituto Español de Oceanografía 18(1 4): Folk J, Schirmer E (1963) The porcine pancreatic carboxypeptidase A system: three forms of the active enzyme. J Biol Chem 238:38 84 Furné M, Hidalgo MC, López A, García-Gallego M, Morales AE, Domezain A, Domezainé J, Sanz A (2005) Digestive enzyme activities in Adriatic sturgeon Acipenser naccarii and rainbow trout Oncorhynchus mykiss: a comparative study. Aquaculture 250: García-Carreño FL, Dimes LE, Haard NF (1993) Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal Biochem 214:65 69 Glass HJ, McDonald NL, Moran RM, Stark JR (1989) Digestion of protein in different marine species. Comp Biochem Physiol 94(B): Guerrero-Zárate R (2010) Evaluación de la capacidad digestiva del pejelagarto (Atractosteus tropicus). Tesis de maestría, Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias Biológicas AQ

11 734 Heu MS, Kim HR, Pyeun JH (1995) Comparison of trypsin and 735 chymotrypsin from the viscera of anchovy, Engraulis 736 japonica. Comp Biochem Physiol II2B(3): Igbokwe EC, Downe AER (1978) Electrophoretic and histo- 738 chemical comparison of three strains of Aedes aegypti. 739 Comp Biochem Physiol 60B: Iijima N, Tanaka S, Ota Y (1998) Purification and character- 741 ization of bile salt-activated lipase from the hepatopancreas 742 of red sea bream, Pagrus major. Fish Physiol Biochem : Jonas E, Ragyanssszki M, Olah J, Boross L (1983) Proteolytic 745 digestive enzymes of carnivorous (Silurus glanis L.), her- 746 bivorous (Hypophtlamichthys molitrix Val.) and omnivo- 747 rous (Cyprinus carpio) fishes. Aquaculture 30: Jun-sheng L, Jian-lin L, Ting-ting W (2006) Ontogeny of pro- 749 tease, amylase and lipase in the alimentary tract of hybrid 750 juvenile tilapia (Oreochromis niloticus X Oreochromis 751 aureus). Fish Physiol Biochem 32: Klahan R, Areechon N, Yoonpundh R, Engkagul A (2009) 753 Characterization and activity of digestive enzymes in dif- 754 ferent sizes of Nile tilapia (Oreochromis niloticus L.). 755 Kasetsart J Nat Sci 43: Kolkovski S (2001) Digestive enzymes in fish larvae and 757 juveniles, implications and applications to formulated 758 diets. Aquaculture 200: Kumar S, García-Carreño FL, Chakrabarti R, Toro MAN, 760 Córdova-Murueta JH (2007) Digestive proteases of three 761 carps Catla catla, Labeo rohita and Hypophthalmichthys 762 molitrix: partial characterization and protein hydrolysis 763 efficiency. Aquac Nutr 13: Kunitz M (1947) Crystalline soybean trypsin inhibitor II. Gen- 765 eral properties. J Gen Physiol 30: Kuzmina VV (1991) Evolutionary features of the digestive- 767 transport function in fish, vol 27. Plenum Publishing Cor- 768 poration, pp Translated from Zhurnal Evolyut- 769 sionnoi Biokhimii i Fiziologii 770 Laemmli UK (1970) Cleavage of structural proteins during the 771 assembly of the head of bacteriophage T4. Nature : Lazo JP, Mendoza R, Holt GJ, Aguilera C, Arnold CR (2007) 774 Characterization of digestive enzymes during larval 775 development of red drum (Sciaenops ocellatus). Aqua- 776 culture 265: Lindner P, Eshell A, Kolkovski S, Tandler A, Harpaz S (1995) 778 Proteolysis by juvenile sea bass (Dicentrarchus labrax) 779 gastrointestinal enzymes as a method for the evaluation of 780 feed proteins. Fish Physiol Biochem 14(5): López-Ramírez G, Cuenca-Soria CA, Álvarez-González CA, 782 Tovar-Ramírez D, Ortiz-Galindo JL, Perales-García N, 783 Márquez-Couturier G, Arias-Rodríguez L, Indy JR, Contreras-Sánchez WM, Gisbert E, Moyano FJ (2010) Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus. Fish Physiol Biochem 37(1): Lundstedt LM, Melo JFB, Moraes G (2004) Digestive enzymes and metabolic profile of Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition. Comp Biochem Physiol 137(B): Maraux S, Louvard D, Baratti J (1973) The aminopeptidase from hog-intestinal brush border. Biochim Biophys Acta 321: Matus de la Parra A, Rosas A, Lazo JP, Viana MT (2007) Partial characterization of the digestive enzymes of Pacific bluefin tuna Thunnus orientalis under culture conditions. Fish Physiol Biochem 33: Munilla-Moran R, Stark JR (1990) Metabolism in marine flatfish VI. Effect of nutritional state of digestion in turbot, Scophtalmus maximus (L.). Comp Biochem Physiol 95B: Natalia Y, Hashim R, Ali A, Chong A (2003) Characterization of digestive enzymes in a carnivorous ornamental fish, the Asian bony tongue Scleropages formosus (Osteoglossidae). Aquaculture 233(1 4): Perales-García N (2006) Ontogenia enzimática de la mojarra tenguayaca Petenia splendida. Tesis de maestría. Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias Biológicas Sáenz de Rodrigáñez M, Alarcón FJ, Martínez MI, Ruiz F, Díaz M, Moyano FJ (2005) Characterisation of digestive proteases in the Senegal sole Solea senegalensis Kaup, Boletín-Instituto Español de Oceanografía 21(1 4): Stauffer C (1989) Enzyme assays for food scientists. Van Nostand Reinhold/AVI, Nueva York Walter HE (1984) Proteinases: methods with hemoglobin, casein and azocoll as substrates. In: Bergmeyern HJ (ed) Methods of enzymatic analysis, vol 5. Verlag Chemie, Weinham, pp Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J Biol Chem 244: Wu GP, Cao MJ, Chen Y, Liu BX, Su WJ (2008) Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution. J Agric Food Chem 56(20): Zhong-yi L, Zhang W, Jian Z (2008) An acidic protease from the grass carp intestine (Ctenopharyngodon idellus). Comp Biochem Physiol 149(B):

12 Journal : Article : 9876 the language of science Author Query Form Please ensure you fill out your response to the queries raised below and return this form along with your corrections Dear Author During the process of typesetting your article, the following queries have arisen. Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proof/online grid or in the Author s response area provided below Query Details Required AQ1 AQ2 AQ3 AQ4 AQ5 AQ6 AQ7 Please check and confirm the author names and initials are correct. Also, kindly confirm the details in the metadata are correct. Perales-García (2009) has been changed to Perales-García (2006) so that this citation matches the list. Please check that the phrase 2, 4, 6, 8 y 10 (acid proteases) and of 4, 6, 8, 10 y 12 (alkaline proteases) has been changed to 2, 4, 6, 8 and 10 (acid proteases) and of 4, 6, 8, 10 and 12 (alkaline proteases). The labels in figure (2) is not readable. Please provide a new figure with legible labels in Vector EPS or tiff / jpeg format with 600 dpi resolution. Essed et al. (2010) has been changed to Essed et al. (2002) so that this citation matches the list. References Lundstedt et al. (2003), Chakrabarti et al. (1995), Hi et al. (2012), Chiu and Pan (2002) are cited in text but not provided in the reference list. Please provide references in the list or delete these citations. Reference Chavez-Lomelí et al. (1989) is given in list but not cited in text. Please cite in text or delete from list. Author s Response

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