Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

Digestive proteases of Morelet’s crocodile (Crocodylus moreletii)

in three life stages

Manuel Alejandro Castillo-Rodríguez1; https://orcid.org/0009-0008-9578-2821

Judith Andrea Rangel-Mendoza2; https://orcid.org/0000-0003-1189-9835

Emyr Saul Peña-Marín3; https://orcid.org/0000-0002-4736-9089

Carlos Alfonso Álvarez-González1; https://orcid.org/0000-0001-9240-0041

Marco Antonio López-Luna2; https://orcid.org/0000-0003-2259-544X

Claudia Ivette Maytorena-Verdugo4*; https://orcid.org/0000-0001-7417-858X

1 Laboratorio de Fisiología en Recursos Acuáticos (LAFIRA)-DACBIOL, Universidad Juárez Autónoma de Tabasco,

Carretera Villahermosa Cárdenas Km 0.5, 86139 Villahermosa, Tabasco, México; alejandro1708ava@gmail.com,

alvarez_alfonso@hotmail.com

2 Centro de Investigación para la Conservación de Especies Amenazadas (CICEA)-DACBIOL, Universidad Juárez

Autónoma de Tabasco, Carretera Villahermosa Cárdenas Km 0.5, 86139 Villahermosa, Tabasco, México;

judith.rangel@ujat.mx, marco.lopez@ujat.mx

3 Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California (UABC), km 107 carretera Tij/

Eda, 22860 Ensenada, Baja California, México, emyr.pea@uabc.edu.mx

4 Universidad Politécnica del Centro, Carretera Federal, Villahermosa-Teapa Km 22.5, Tumbulushal Centro, 86290

Villahermosa, Tabasco; clau.maytorena@gmail.com (*Correspondence)

Received 07-X-2023. Corrected 01-III-2024. Accepted 12-IV-2024.

ABSTRACT

Introduction: Morelet’s crocodile (Crocodylus moreletii) is a species distributed in the Mexican southeast and

threatened due to multiple pressures.

Objective: To characterize the digestive proteases in the acid phase (stomach) and alkaline phase (intestine) of

three life stages of C. moreletii in captivity (hatchling, juvenile, and adult).

Methods: Total alkaline and acid protease activities were quantified using casein and haemoglobin as substrates.

Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were quantified using synthetic substrates.

Protease profiles were analysed by SDS-PAGE and Native-PAGE.

Results: The specific activity of acid and alkaline proteases showed differences between the three stages, finding

the highest activity in the juveniles. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were

higher in hatchlings. There were differences in optimum pH and temperature of acid and alkaline proteases,

trypsin, and leucine aminopeptidase between the three stages, demonstrating the diversification of the enzymes

according to different stages, as well as the presence of specific isoforms in each stage of C. moreletii. The acid

phase zymogram showed four bands with pepsin-like acid activity in the hatchling and juvenile crocodile, while

in the adult only two of the four bands were detected. The alkaline zymogram showed that the hatchling had

the highest number of activity bands compared to the other stages, corresponding to the high specific activity

reported in the alkaline phase.

Conclusions: Digestive proteases of Morelet’s crocodile differ in their biochemical characteristics and the num-

ber of proteases between hatchling, juvenile, and adult. This could help in the future design of balanced diets as

well to the sustainable management and production of this species.

Key words: pepsin; trypsin; leucine aminopeptidase; hatchling; juvenile; adult.

https://doi.org/10.15517/rev.biol.trop..v72i1.56736

VERTEBRATE BIOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

INTRODUCTION

Digestion involves cooperation between

the gastrointestinal tract (GIT) and accessory

secretory organs, such as the pancreas and

liver. Food in the GIT stimulates the produc-

tion of digestive enzymes from the stomach

and pancreas, and bile from the liver. Diges-

tive enzymes, including proteases, lipases, and

amylase, are secreted by the pancreas into the

intestine for the final breakdown and absorp-

tion of nutrients (Chikwati et al., 2013). Pep-

sins are one of the main proteases found in

the stomach and are responsible for breaking

down proteins to peptides. These enzymes are

secreted in the form of pepsinogens, which are

activated by the acidic pH generated by hydro-

chloric acid poured into the stomach (Clarks et

al., 1985). Pepsin has a broad specificity. The

optimum pH of pepsins is around 2, which

maximises the digestion process because the

substrate (protein) is under an acid denatur-

ation (Rawlings and Salvesen, 2013). The two

primary alkaline digestive proteases responsible

for the initial protein hydrolysis in the intestine

are trypsin and chymotrypsin. The exocrine

pancreas synthesizes both proteases and stores

them as non-active zymogen forms (trypsino-

gen and chymotrypsinogen). In the intestinal

lumen, enterokinase activates trypsinogen by

cleaving a short peptide, converting it to tryp-

sin (Solovyev et al. 2023). The breakdown of

di- and tripeptides is facilitated by Leucine ami-

nopeptidase (LAP). It represents peptide diges-

tion and leucine release, as well as amino acid

absorption in the small intestine. The function

of this exopeptidase is poorly understood, aside

from its secretion by the mucosa of the small

intestine (Bhardwaj, 2013).

In crocodiles, the digestion process begins

when crocodiles swallow small preys, and larg-

er preys are masticated before deglutination,

though an excessively larger prey is reduced by

ripping off bits and limbs. Next, reduction is

performed in the stomach where the swallowed

food is exposed to the action of hydrochloric

acid and pepsin to digest protein and dissolve

bones. Then, digestion continues in the upper

small intestine under the action of bile and

RESUMEN

Proteasas digestivas del cocodrilo de pantano (Crocodylus moreletii) en tres etapas de vida

Introducción: El cocodrilo de pantano (Crocodylus moreletii) es una especie distribuida en el sureste mexicano y

amenazada por múltiples presiones.

Objetivo: Caracterizar las proteasas digestivas en fase ácida (estómago) y fase alcalina (intestino) en tres etapas

de vida de C. moreletii en cautiverio (cría, juvenil y adulto).

Métodos: Se cuantificaron las actividades de proteasas alcalinas y ácidas totales utilizando caseína y hemoglobina

como sustrato. Las actividades de tripsina, quimotripsina, leucina aminopeptidasa y elastasa se cuantificaron

utilizando sustratos sintéticos. Los perfiles de proteasas se analizaron mediante SDS-PAGE y PAGE nativa.

Resultados: La actividad específica de las proteasas ácidas y alcalinas mostró diferencias entre las tres tallas,

encontrándose la mayor actividad en el estadio juvenil. Las actividades de tripsina, quimotripsina, leucina amino-

peptidasa y elastasa fueron mayores en las crías. Hubo diferencias en el pH y temperatura óptimos de las proteasas

ácidas y alcalinas, tripsina y leucina aminopeptidasa entre las tres tallas, demostrando la diversificación de las

enzimas según las diferentes tallas, así como la presencia de isoformas específicas en cada talla de C. moreletii. El

zimograma en fase ácida mostró cuatro bandas con actividad similar a pepsina en la cría y juvenil, mientras que

en el adulto solo se detectaron dos de las cuatro bandas. El zimograma alcalino mostró que la cría tuvo el mayor

número de bandas de actividad en comparación con las otras tallas, correspondiente a la alta actividad específica

reportada en la fase alcalina.

Conclusiones: Las proteasas digestivas del cocodrilo de pantano presentaron características bioquímicas y en

número de proteasas diferentes entre cría, juvenil y adulto. Esto podría ayudar en el futuro diseño de dietas

balanceadas, así como al manejo y producción sustentable de esta especie.

Palabras clave: pepsina; tripsina; leucina aminopeptidasa; cría; juvenil; adulto.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

pancreatic secretions (Huchzermeyer, 2003).

Digestive enzymes in reptiles have not been

studied as much in other groups, for example,

mammals and fishes. Unlike digestive enzymes

from other groups, some reptiles’ digestive

enzymes are not in the form of zymogens

before ingestion, for example, in the water

snake (Natrix tessellata), acinar cells of the pan-

creas are loaded with zymogen granules after 24

h of ingestion (Zhalka & Bdolah, 1987).

Body size in the different life stages of a

species affects an organism’s energetic require-

ments and nutrient necessities (Radloff et al.,

2012). Studies point out that nutrition in the

first life stages of crocodiles is the most impor-

tant for their development and future use if

they want to reach a commercial size more

quickly (Huchzermeyer, 2003). Proteases play a

very important role in the survival and growth

of any living organism, since they hydrolyse

peptide bonds (Klomklao, 2008). In this sense,

there are few studies that address the digestive

physiology in crocodiles, including the func-

tionality and type of proteases in these species.

In general, the digestive physiology and

nutrition of crocodiles are poorly studied. In

the spectacled caiman (Caiman crocodilus),

when fed with 5 % of the body weight, complete

gastric emptying takes an average of 99 h at 30

°C, to an average of 315 h at 15 °C, increasing

the frequency and amplitude with temperature

or upon feeding (Diefenbach, 1974). For the

American alligator (Alligator mississipiensis), a

diet based only on gizzard for 9 to 15 months

reduced the levels of thiamine in blood and

muscle, and over 40 % of the crocodiles died,

probably because of the nutritional deficien-

cies in the diet (Ross & Honeyfield, 2008). In

another work, the American crocodile (Cro-

codylus acutus) showed that when feeding with

fish, hatchlings grew 1.35 times more (3.5

mm per day) than with a mixture of fish and

cow liver and lung, and a diet with fly larvae

(Pérez-Gómez et al., 2009). For crocodilians,

growth rates differ between life stages; during

the first months of life, crocodiles feed at least

6-7 times a week, and sub-adult crocodiles only

need to consume 8-10 % of their body weight

once a week (Whitaker & Andrews, 1998). In

the broad-snouted caiman (Caiman latirostris),

the growth and efficiency of food conver-

sion are higher at 33 °C compared to 29 °C.

Specimens maintained at 33 °C for 72 days were

longer (9.2 ± 1 cm) and heavier (79.6 ± 13.2 g)

than those maintained at 29 °C (6 ± 0.9 cm,

42.2 ± 8.5 g) (Parachú-Marcó et al. 2009).

In general, we know very little about croco-

diles compared to other species, and research

into their biology has been carried out for

biological studies and conservation efforts.

Crocodiles belong to the Crocodylidae fam-

ily and are classified as reptiles, together with

lizards, snakes, tuataras, tortoises, terrapins,

and turtles (Huchzermeyer, 2003). Morelet’s

crocodile (Crocodylus moreletii) inhabits main-

ly freshwater areas (marshes, swamps, ponds,

rivers, lagoons, and artificial water bodies in

the Atlantic lowlands of the Gulf of Mexico

(Mexico) and the Yucatán Peninsula (Mexico,

Belize, Guatemala) (Platt et al., 2010).

Platt et al. (2006) classified Morelet’s croco-

diles by hatchlings, small juveniles, large juve-

niles, subadults, and adults, and found that

natural preys include aquatic and terrestrial

insects, arachnids, aquatic gastropods, crus-

taceans, fish, amphibians, reptiles, birds, and

mammals, finding that smaller crocodiles fed

mostly on insects and arachnids, followed by

large juveniles which fed on aquatic gastro-

pods, crustaceans, fish, and non-fish verte-

brates. Adults feed on aquatic gastropods, fish,

and crustaceans, however, there are reports

of adult Morelet’s crocodiles consuming large

mammals (over 15 kg) by kleptoparasitism

(Platt et al., 2007).

In this work, we attempt to increase the

basic knowledge of the digestive process of

crocodiles by using Morelet’s crocodile (Croco-

dylus moreletii) as a model to describe digestive

proteases in three life stages. This information

could help in the future design of balanced diets

as well to the sustainable management and pro-

duction of this species.

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

MATERIALS AND METHODS

Sampling: Crocodylus moreletii (Family:

Crocodylidae, Order: Crocodylia) samples, one

sample per stage, came from crocodiles that

underwent unexpected death associated with

the cold season or attacks by organisms of

the same species (cannibalism), donated from

the Research Centre for the Conservation of

Threatened Species in the División Académica

de Ciencias Biológicas of the Universidad Juárez

Autónoma de Tabasco. The donated crocodiles

corresponded to three different stages: one

hatchling (H), one juvenile (J) and one adult

(A). All crocodiles were weighed and measured

(Total length) before making a longitudinal

cut in the abdomen from the beginning of the

larynx to the beginning of the cloaca, extract-

ing the complete digestive system (Fig. 1).

Stomachs and intestines of the three stages

of crocodile were separated. The intestine of

each crocodile was measured by length and the

relative intestine length (RIL) was determined

by using the formula RIL = Total Length of

the intestine / Total length of the crocodile

(Moreira et al. 2020). Biometrics are shown

in Table 1. The samples obtained were frozen

(-20 °C) until processing.

Digestive enzyme activities: Dissected tis-

sues (stomach and proximal small intestine)

were homogenised in a 1:10 (w/v) ratio with

distilled water using an Ultra Turrax disperser

(model IKA T18 Basic). The homogenised sam-

ples were centrifuged at 12 000 rpm for 15 min

at 4 °C, and subsequently, the supernatant was

distributed in 2 ml tubes and stored at -20 °C

until further analysis. The soluble protein con-

centration of the supernatant was determined

by the technique of Bradford (1976).

Table 1

Biometrics of hatchling, juvenile and adult of Morelet’s crocodiles (Crocodylus moreletii) used for digestive proteases

characterization. RIL: relative intestine length.

Crocodile Stage Total length (cm) Total weight (Kg) Intestine length (cm) RIL

Hatchling 33 0.14 34 1.03

Juvenile 94 2.3 100 1.06

Adult 184 28 182 0.99

Fig. 1. Digestive system of a Morelet’s crocodile (Crocodylus moreletii) hatchling. St: stomach; Int: intestine; Cl: sewer.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

The total acid protease activity of the

stomach extracts was quantified using 1 % hae-

moglobin as substrate in 100 mM glycine-HCl

buffer at pH 2.0, the reaction comprised 5 µl of

the sample and 300 µl of the substrate-buffer

mixture. After 15 min of incubation at 37 °C,

the reaction was stopped with 200 µl of 10 %

trichloroacetic acid (TCA), then, the samples

were centrifuged at 10 000 g for 15 min at 4 °C

and the absorbance of the tyrosine released

in the supernatants was measured at 280 nm

(Anson, 1938). Total alkaline protease activity

in the intestine was determined using the tech-

nique described by Walter (1984), using 1 %

casein in 100 mM Tris-HCl buffer + 10 mM

CaCl2 at pH 9.0. The reaction comprised 10 µl

of the sample and 300 µl of the substrate-buffer

mixture. After 30 min of incubation at 37 °C,

the reaction was stopped with 200 µl of 10 %

TCA, then, the samples were centrifuged at

10 000 g for 15 min at 4 °C and the absorbance

of the tyrosine released was measured at 280

nm. The product absorbance of both enzymes

was quantified in a Genesys 10S UV-Vis spec-

trophotometer (ThermoScientific, Waltham,

MA) and a molar extinction coefficient (ε) of

0.005 µM-1 cm-1 was used for calculations.

Chymotrypsin activity was quantified

using SAApNA as substrate (Cat. No. S7388,

Sigma-Aldrich, Saint Louis, MO). A 50 mM

stock solution of the substrate was prepared

with DMSO, then the working solution was

prepared at a concentration of 1.25 mM with

50 mM Tris-HCl, pH 8.0. Fifteen microliters

of each intestine extract were mixed with 135

µl of the buffered substrate to measure the

absorbance of the released nitroanilide at 410

nm in a kinetics of 30 min (García-Carreño et

al., 1994). Trypsin activity was quantified using

BAPNA as a substrate (Cat. No. B4875, Sigma-

Aldrich). A stock solution of 122.78 mM of the

substrate was prepared with DMSO and the

working solution was prepared by diluting the

stock solution to a concentration of 2 mM with

50 mM Tris-HCl, pH 8.0. Fifteen microliters of

each intestine extract were mixed with 135 µl of

the substrate to measure the absorbance of the

nitroanilide released at 410 nm in a kinetics of

15 min (García-Carreño et al., 1994).

Leucine aminopeptidase activity was quan-

tified using L-leucine-p-nitroanilide as sub-

strate (Cat. No. L9125, Sigma-Aldrich). A 250

mM stock solution of the substrate was pre-

pared with DMSO, and the working solution

was prepared at a concentration of 4 mM dilut-

ed with 50 mM sodium phosphate, pH 7.2. Fif-

teen microliters of each intestine extract were

mixed with 135 µl of the substrate and buffer

mixture to measure the absorbance of the

released nitroanilide at 410 nm over a 30 min

kinetics (Maroux et al., 1973). Elastase activity

was measured using the method of Ishida et al.

(1987) with some modifications.

In this work we quantified the activity

using N-Succinyl tri-L-alanine-4-nitroanilide

(SAAANA; Cat. No. S4760, Sigma-Aldrich)

as substrate in a kinetics instead a final point

measurement. A 50 mM stock solution of the

substrate was prepared with DMSO, and the

working solution was prepared at a concentra-

tion of 1.5 mM diluted with 100 mM Tris-HCl

buffer at pH 8. Fifteen microliters of each

intestine extract were mixed with 135 µl of

the substrate and buffer mixture to measure

the absorbance of the released nitroanilide at

410 nm over a 30 min kinetics. The product

absorbance was quantified in a xMark spec-

trophotometer (Bio-Rad, Hercules, CA), and a

molar extinction coefficient (ε) of 0.0088 µM-1

cm-1 was used for calculations. Total activ-

ity (Units/ml) = [ Δabs*reaction final volume

(ml)] / [MEC*time (min)*extract volume (ml)]

and Specific activity (Units / mg prot) = Total

activity / soluble protein (mg / ml), where

Δabs represent the increase in absorbance, and

MEC represents the molar extinction coeffi-

cient (García-Carreño et al., 1994).

Optimum pH and temperature: For acid

proteases, total alkaline proteases, trypsin, and

leucine aminopeptidase, the optimum tem-

perature was determined using the enzymatic

techniques described above, changing the tem-

perature to 10, 20, 30, 40, 50, 60, 70, and 80 °C.

Optimum pH was also determined for the

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

same enzymes mentioned above using previ-

ously described enzymatic techniques at 37 °C

but adjusted with the following buffers: 100

mM glycine-HCl for pH 1, 2, 3 and 4; 100 mM

sodium acetate for pH 5 and 6; 100 mM Tris-

HCl for pH 7, 8 and 9; 100 mM glycine-NaOH

for pH 10 and 11.

Electrophoretic analyses: Electrophoretic

analyses were performed in a Mini-PROTEAN

3 Cell electrophoretic chamber (Bio-Rad) using

vertical gel plates (8 × 10 × 0.1 cm). For the

analysis of acid proteases, the stomach extracts

of C. moreletii were separated by non-denatur-

ing native conditions (Native-PAGE) using a

continuous acrylamide gel (10 %) in 25 mM Tris

and 192 mM glycine buffer at pH 8.3. After the

electrophoresis, the gels were incubated in hae-

moglobin to reveal protease isoforms according

to the procedure of Díaz-López (1998). The gels

were soaked in 100 mM glycine-HCl buffer at

pH 2.0 to activate acid proteases. After 15 min,

the gels were immersed for 60 min at 4 °C in 2

% haemoglobin in 100 mM glycine-HCl buffer,

pH 2.0, and then placed for 60 min in the same

solution, but at 37 °C. After the incubation, the

gels were washed with distilled water and fixed

in a 12 % trichloroacetic acid (TCA) solution

for 15 min.

For the analysis of alkaline proteases, the

intestine extracts of C. moreletii were used.

The electrophoresis gel was prepared with a

4 % polyacrylamide (PAA) stacking gel and

a 10 % PAA resolving gel. Electrophoresis

was performed under denaturing conditions

(SDS-PAGE) with SDS in 0.1 % in 25 mM Tris

and 192 mM glycine buffer at pH 8.3, adapted

by García-Carreño et al. (1993). After alka-

line SDS-PAGE electrophoresis, the gels were

immersed for 60 min at 4 °C in a 2 % casein

solution in 0.1 M Tris-HCl buffer, pH 9. Then,

gels were incubated for 60 min in the same

solution at 37 °C, then washed and fixed with

TCA as described above. To detect enzyme

activity, after incubation with the substrates,

the gels were stained using a Coomassie R-250

brilliant blue solution at 0.1 %, while destain-

ing was carried out in a 35:10:55 solution

of methanol-acetic acid-water. Clear bands

revealed protease activity within a few min-

utes, although well-defined bands were only

obtained after a 2–4-hour staining period.

Electrophoresis with stomach extracts was

complemented with the use of the specific

inhibitor pepstatin A (PepA, 1 mM), incubating

for 1 hour at 37 °C. A set of molecular weight

markers (Broad range leader, Cat #161-0318,

Bio-Rad) was applied on each Native-PAGE

and SDS-PAGE (5 μl per well) and the molecu-

lar weight (MW) of each band was calculated

using a linearly fitted relationship between Rf

and log10 of the molecular weight markers,

using Quality One software version 4.6.5 (Bio-

Rad, Hercules, CA).

Statistical analysis: Even though this is

a descriptive study using an organism per

stage, all enzymatic assays were performed in

triplicate. Data of specific activity of the dif-

ferent proteases between the different stages,

as well as the characterisation of pH and tem-

perature between each stage were analysed for

normality and homoscedasticity, and in the

case of fulfilling these assumptions, a para-

metric one-way ANOVA analysis was used.

If the aforementioned assumptions were not

met, a non-parametric Kruskal-Wallis analysis

was performed. The tests showing differences

between the variables were analysed with a

Tukey’s posterior test. All analyses were per-

formed in the Sigma Plot 11.0 program with a

significance of P < 0.05.

RESULTS

Biometrics of hatchling, juvenile and adult

of Morelet’s crocodiles (Crocodylus moreletii)

used for digestive proteases characterisation

are shown in Table 1. The relation between

total length and intestine length, reported as

relative intestine length, was near to one in the

three stages.

The specific activity of acid proteases

showed significant differences between the

three stages, presenting greater activity in the

juvenile and less activity in the hatchling. Total

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

alkaline protease activity showed significant

differences between the three stages, with high-

er activity in the hatchling and lower activity

in the adult. Trypsin, chymotrypsin, elastase,

and leucine aminopeptidase activities showed

higher activity (P < 0.05) in hatchling and less

activity in juvenile and adult (Table 2).

In Fig. 2A, an optimum pH of 2 is observed

for acid proteases of the juvenile and adult

crocodiles, while, for hatchling, its optimum

activity was found at pH 2 and 3. It is worth

noting the decrease in activity at pH 4 and 5

for the adult stage, while hatchling and juvenile

stage showed activities near to 50 % of relative

activity. Fig. 2B shows the maximum of alkaline

protease activity between pH 8 to 11 for hatch-

ling. The juvenile presented its maximum peak

of activity at pH 9 also showing a peak of activ-

ity at pH 5, with 80 % of relative activity, while

the adult stage presented the maximum peak of

activity at pH 8, also showing a peak of activity

at pH 5, with 60 % of relative activity.

Fig. 2C shows that trypsin from hatchling

has a maximum activity at pH 10, showing a

Table 2

Specific activity (U/mg protein) of acid and alkaline proteases, trypsin, chymotrypsin, elastase, and leucine aminopeptidase

(mean ± SD) in hatchling, juvenile and adult of Morelet’s crocodiles (Crocodylus moreletii). *, **, *** indicate differences

between each enzyme activity and stage.

Specific activity (U/mg protein) Hatchling Juvenile Adult

Acid proteases 69.68 ± 6.19*** 5 819.7 ± 6.12*518.44 ± 18.35**

Alkaline proteases 43.04 ± 0.59*19.22 ± 4.14** 2.02 ± 0.31***

Trypsin 22.12 ± 0.8*0.7 ±0.3** 0.9 ± 0.4**

Chymotrypsin 1.1 ± 0.2*0.2 ± 0.2** 0.1 ± 0.1**

Elastase 0.9 ± 0.1*0.1 ± 0.1** 0.3 ± 0.1***

Leucine aminopeptidase 29.5 ± 0.8*15.1 ± 1.1** 13.9 ± 0.3**

Fig. 2. Effect of pH on the relative activity (%) of acid proteases (A), alkaline proteases (B), trypsin (C) and

leucine aminopeptidase (D) of digestive enzymes obtained from hatchling, juvenile and adult of Morelet’s crocodiles

(Crocodylus moreletii).

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

peak of activity at pH 7 and 8 with a relative

activity of approximately 60 %. Trypsin activity

in the juvenile has a maximum activity at pH 9,

showing a peak of activity at pH 6 with a rela-

tive activity of approximately 60 %, while the

adult stage presents its maximum peak of tryp-

sin activity at pH 9. Fig. 2D shows the activity of

leucine aminopeptidase with a maximum peak

in the three stages at pH 8, while in adult stage

showed a second peak at pH 6 with a relative

activity higher than 60 %.

In Fig. 3A, differences were observed in the

optimum temperature for acid protease activity

in the three stages, finding the maximum activ-

ity at 30, 40 and 50 °C for hatchling, juvenile

and adult, respectively. Fig. 3B shows differ-

ences in the optimum temperature for alkaline

proteases between the three stages, finding the

maximum activity at 50 and 60 °C for juvenile

and hatchling, respectively, where the juvenile

shows approximately 60 % of the total activ-

ity at 10 °C. The adult stage showed two peaks

of maximum activity (40 and 60 °C). Fig. 3C

shows two peaks of trypsin activity in hatchling

and adult at 30 and 50 °C, finding almost no

activity for the three stages at 40 °C, while the

juvenile showed maximum activity at 30 °C,

and as observed for alkaline proteases, trypsin

activity in the juvenile shows approximately

50 % of the total activity at 10 °C. Fig. 3D shows

the differences in the optimum temperature for

leucine aminopeptidase activity between the

three stages, showing maximum activity at 40

and 50 for juvenile and hatchling, respectively,

and between 50 and 60 °C for adult stage. As

observed in alkaline proteases and trypsin,

the relative activity of LAP in juveniles shows

approximately 75 % of the total activity at 10 °C.

The results of the electrophoresis (Native-

PAGE) are shown in Fig. 4, which shows four

bands with acid protease activity from the

hatchling and juvenile stomachs with calcu-

lated weights of 51.2, 39.4, 30.9 and 21.2 kDa,

while the adult only revealed two bands (39.4

and 30.9 kDa). The same figure shows that the

incubation with pepstatin A inhibited the four

bands in all the crocodile stages. In Fig. 5, the

results of SDS-PAGE revealed six bands with

Fig. 3. Effect of temperature on the relative activity (%) of acid proteases (A), alkaline proteases (B), trypsin (C) and leucine

aminopeptidase (D) of digestive enzymes obtained from baby, juvenile and adult of Morelet’s crocodiles (Crocodylus moreletii).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

stomach after feeding in other reptile species

such as white turtle (Dermatemys mawii) and

the red-eared turtle (Trachemys scripta elegans)

that show optimum acid protease activity at pH

2 and pH 2.5, respectively (Rangel-Mendoza et

al., 2018; Sun et al., 2007).

Pepsins reported in marine fish range

between 27 and 42 kDA (Simpson, 2000). In

the present study, the electrophoresis results

showed four bands with proteolytic activity in

the stomach (21.2, 30.9, 39.4 and 51.2 kDa),

which were inhibited with pepstatin A. The

four bands were found in extracts from hatch-

ling and juvenile, while in adult, only two bands

were revealed (30.9 and 39.4 kDa). In this

case, the presence of four pepsin isoforms can

generate digestive adaptations as found in the

hatchling, where the optimal pH of pepsin was

found at 2 and 3, while in juvenile and adult,

the optimum pH was 2. This diversification was

Fig. 5. SDS-PAGE electrophoresis of total alkaline proteases

obtained from the proximal intestine of hatchling, juvenile

and adult of Morelet’s crocodiles (Crocodylus moreletii) at

pH 9.

Fig. 4. Native-PAGE electrophoresis (Native-PAGE) of acid

proteases obtained from the hatchling, juvenile and adult

stomach of Morelet’s crocodile (Crocodylus moreletii) at

pH 2.

alkaline protease activity from the hatchling

proximal intestine with calculated weights of

51.2, 41.7, 33.1, 26.3, 22.2, 15.2 kDa. The juve-

nile stage showed no activity bands, while in

the adult, two bands were revealed (26.3 and

15.2 kDa).

DISCUSSION

Pepsins are very important endopeptidases

in carnivorous species, as they initiate protein

digestion, releasing peptides and some free

amino acids (Gilberg et al., 1990). In fasted

painted turtles (Chrysemys picta), the pH var-

ies between cardiac and pyloric stomach (1.8

and 2.1, respectively), and after feeding, the pH

increases, reporting average values from 2.2 to

4.0 (Fox & Musacchia, 1959). To date, there are

no reports of pepsin characterization in croco-

diles, this being the first report in C. more-

letii, where the optimum pepsin activity found

agrees with that reported in most fishes, show-

ing two or three forms of pepsin with optimum

activity between 2 and 4 (Klomklao, 2008).

Unfortunately, we were not able to measure the

pH of the sampled stomachs nor having more

organisms to sample, however, the optimum

pH found for pepsins agrees with the pH of the

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

also observed for optimum temperature, which

was different for the three stages.

Pancreatic digestive proteases play a central

role in digestion by hydrolysing proteins into

peptides and amino acids, which are essential

for proper growth, development, repair, energy,

and every type of body function (Whitcomb &

Lowe, 2007). Pancreatic digestive enzymes in

snakes increase after ingestion, for example, in

the water snake (Natrix tessellate), the activity

of chymotrypsin and elastase increased over

three-fold after 24 h of ingestion (4.1 ± 1.1 to

13.0 ± 3.4 U/mg for chymotrypsin, and 4.1 ±

2.5 to 13.8 ± 7.1 U/mg for elastase) and in the

sand viper (Vipera ammodytes), the activity

increased tenfold after 24 h of ingestion (1.1 to

12.7 U/mg for chymotrypsin, and 0 to 17.8 U/

mg for elastase) (Alcon & Bdolah, 1975). In our

work, the activity of chymotrypsin and elastase

was lower (Table 2), however, the activity of

trypsin and leucine aminopeptidase were in

similar values for the ones reported for snakes.

Chymotrypsin and elastase are enzymes with

a specific range of cleavage sites, so their low

activity could suggest that Morelett’s crocodile

need protein with a high content of lysine

and arginine corresponding to carnivorous diet

(Rawlings and Salvesen, 2013).

Coulson & Coulson (1986) reported that

temperature affects the rates of digestion,

amino acid absorption, and assimilation in

alligators (Alligator mississipiensis); at 25 °C,

digestion was slow and incomplete, at 28 °C,

digestion was about 50 %, and 31 °C, was the

optimum temperature for digestion since the

initial rate of the rise in total and essential

amino acids in plasma, and specifically, ala-

nine, was greater, since at 31 °C, the highest

concentration of alanine in plasma was found

after 40 min after ingestion, compared to 28 °C

(70 min) and 25 °C (100 min). Those results

could be related to the efficiency of digestive

proteases, in this work, the optimum tem-

perature for trypsin was found at 30 °C for

the three animals, even though, physiological

temperature does not determine the optimum

activity of an enzyme, for C. moreletii, its

natural habitat varies between 26 and 28 °C,

which opens new questions about the opti-

mum temperature for the species cultivation

(Casas-Andreu et al., 2011).

Total alkaline protease activity in C. more-

letii hatchling was the highest (43.04 ± 0.59 U/

mg) compared to the other stages. This high

activity is also reflected in pancreatic (tryp-

sin, chymotrypsin, and elastases) and cytosolic

(leucine aminopeptidase) proteases, denoting

a high diversity of proteases in hatchling. The

results of optimal pH of alkaline protease activ-

ity in C. moreletii hatchling show stability

between pH 8 and pH 11, suggesting that the

sum of the optimum pH of several types of pro-

teases, confer the ability to the hatchling to have

high activity in a wide pH range. This assump-

tion is corroborated by the optimum activity

obtained for trypsin and leucine aminopepti-

dase, which shows the highest activity at pH

10 and pH 8, respectively. Studies in reptiles

such as the white turtle (Dermatemys mawii),

show optimum alkaline protease activity at pH

9 (Rangel-Mendoza et al., 2018), while the red-

eared turtle (Trachemys scripta elegans) shows

optimum pH in pancreas, and foregut, midgut,

and hindgut of 8.0, 7.0, 8.0, and 8.5, respectively

(Sun et al., 2007). Tracy et al., (2015), reported

the digestive morphology, the activity of mem-

brane enzymes (aminopeptidases, maltase and

sucrase) and the absorption of nutrients in

Alligator mississippiensis and Crocodylus poro-

sus, where it was found that the villi of the

proximal intestine varied in the two species,

having A. misisipiensis the highest absorption

and aminopeptidase activity compared to C.

porosus. For C. moreletii, the activity of leucine

aminopeptidase was detected, having a higher

activity compared to other digestive proteases

for this species, denoting the importance of the

cytosolic hydrolysis of peptides for the digest-

ibility of proteins in the species.

In addition to the above, the results of

SDS-PAGE in C. moreletii hatchling showed six

bands with proteolytic activity with molecular

weights of 15.2, 22.2, 26.3, 33.1, 41.7, 51.2 kDA,

of which two (22.2 and 26.3 kDa) are within the

molecular weights reported for trypsins in fish

(Jesús-De la Cruz et al., 2018) and they show

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56736, enero-diciembre 2024 (Publicado Abr. 16, 2024)

the highest activity in the zymogram. No bands

were revealed in the juvenile, while two bands

(15.2 and 26.3 kDA) were revealed in the adult,

which are also present in the hatchling, where

one of these bands corresponds to the molecu-

lar weights reported for trypsins.

Some species have better catalytic efficien-

cy at low temperatures (Klomklao, 2008). In the

case of C. moreletii, it is denoted in the juvenile

stage that presents a peak of activity of alkaline

proteases, trypsin, and leucine aminopeptidase

at a temperature of 10 °C, an effect not found

in hatchling and adult. This result denotes

the importance of studying the diversification

of enzymes and/or isoforms that improve the

digestion capacity under extreme conditions,

especially in ectotherms.

The current investigation was carried out

using only the stomachs and intestines of a

single specimen of each stage, this is because

the species is regulated (NOM-059-SEMAR-

NAT-2010), which generates difficulty in the

process of obtaining the species’ biological

material. Working with only one organism of

each stage can influence the results, since these

will not only depend on age but also on the

nutritional status and possible digestive phe-

notypes in reptiles (Venesky et al., 2013). How-

ever, the results obtained lay the foundations

for understanding the digestive physiology of

the Morelet’s crocodile (C. moreletii), opening

opportunities for future research. In this sense,

it is recommended to carry out research related

to the activity and functionality of lipases and

carbohydrases, as well as to evaluate the in vitro

digestibility of different commercial flours and

oils, with the aim of selecting ingredients as the

basis for the formulation of specific diets for

the species.

CONCLUSION

There are differences in the specific activ-

ity of the Morelet’s crocodile between the three

stages, presenting a greater activity in acid pro-

teases in the juvenile unlike the other stages,

while the hatchling presented greater activity

for alkaline proteases, trypsin, chymotrypsin,

elastase, and leucine aminopeptidase compared

to the other stages, denoting greater protein

digestion capacity in the alkaline digestive

phase. There are differences in the optimum pH

and temperature for the activity of acid, alka-

line, trypsin, chymotrypsin, and leucine ami-

nopeptidase proteases between the three stages,

an effect that demonstrates the diversification

of the enzymes according to different ages, as

well as the presence of specific isoforms in each

age of C. moreletii. The acid phase zymogram

showed four bands with pepsin-like acid activ-

ity in the hatchling and juvenile crocodiles,

while in the adult, only two of the four bands

were present. The zymograms showed that the

hatchling crocodile presented the highest num-

ber of bands in the alkaline phase compared to

the other stages, which corresponds to the high

specific activity reported in the alkaline phase.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGEMENTS

The authors express their gratitude to the

Laboratory of Physiology of Aquatic Resources,

where all biochemical assays were performed,

and the Research Centre for the Conservation

of Threatened Species for donating the samples.

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