SciELO - Scientific Electronic Library Online
 
vol.12Comparación de la ganancia diaria de peso en dos grupos de cabritas alimentadas con leche entera o leche entera suplementada con biocolinaAmiloidosis sistémica AA: un problema potencial de salud pública índice de autoresíndice de materiabúsqueda de artículos 		Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

  • No hay artículos similaresSimilares en SciELO

Compartir

Abanico veterinario

versión On-line ISSN 2448-6132versión impresa ISSN 2007-428X

Abanico vet vol.12  Tepic ene./dic. 2022  Epub 31-Oct-2022

https://doi.org/10.21929/abavet2022.9 

Literature review

Crustacean chymotrypsin: estate of art

Carlos Castellanos-Ochoa 1  
http://orcid.org/0000-0002-8687-7123

Erika Torres-Ochoa*  1 
http://orcid.org/0000-0001-5252-7187

Juan Pacheco-Vega2 
http://orcid.org/0000-0001-9443-6849

Alejandro Cortés-Sánchez 3  
http://orcid.org/0000-0002-1254-8941

Daniel Espinosa-Chaurand**  3 
http://orcid.org/0000-0002-0587-5549

1Universidad Autónoma de Baja California Sur. Departamento Académico de Ingeniería en Pesquerías. Baja California Sur, México.

2Universidad Autónoma de Nayarit. Escuela Nacional de Ingeniería Pesquera. Nayarit, México.

3Consejo Nacional de Ciencia y Tecnología (CONACYT). Unidad Nayarit del Centro de Investigaciones Biológicas del Noroeste (UNCIBNOR+). Nayarit, México.

ABSTRACT

Chymotrypsin in crustaceans is an enzyme whose importance has not been fully recognized over time, despite being a fundamental component in the digestion of proteins in their food. Enzymes are basic catalytic components of cellular metabolism; of great diversity classified according to the function they exert (hydrolysis, oxidation-reduction, synthesis, isomerization, among others). Chymotrypsin belongs to hydrolyses, which catalyzes the breaking of peptide bonds adjacent to the carboxyl groups of the aromatic amino acids tryptophan, tyrosine and phenylalanine. Since the 1980s there have been few studies related to this enzyme in crustaceans, concentrating a third of these (13 studies) in the last five years. Therefore, the paper aims to present a review about the existing information on these enzymes in crustaceans from a general perspective, helping to identify areas of opportunity to expand the knowledge of their function and properties in these invertebrates, as well such as the ecological, ethological, management, feeding and nutrition implications of aquaculture.

Keywords: Enzymatic activity; digestive physiology; protease; crustaceans

RESUMEN

La quimotripsina en crustáceos es una enzima cuya importancia no ha sido del todo reconocida a lo largo del tiempo, a pesar de ser un componente fundamental en la digestión de las proteínas de sus alimentos. Las enzimas son componentes catalíticos básicos del metabolismo celular, de gran diversidad clasificadas acorde a la función que ejercen (hidrolisis, oxido-reducción, síntesis, isomerización, entre otros). La quimotripsina pertenece a las hidrolasas, que cataliza la ruptura de enlaces peptídicos adyacentes a los grupos carboxilo de los aminoácidos aromáticos triptófano, tirosina y fenilalanina. Desde la década de 1980 se han realizado pocos estudios relacionados con esta enzima en crustáceos, concentrando un tercio de estos (13 estudios) en los últimos cinco años. Por lo anterior, el presente artículo tiene como objetivo el presentar una revisión acerca de la información existente de estas enzimas en crustáceos desde una perspectiva general, contribuyendo a identificar las áreas de oportunidad para ampliar el conocimiento de su función y propiedades en estos invertebrados, así como las implicaciones ecológicas, etológicas, de manejo, alimentación y nutrición acuícola.

Palabras clave: actividad enzimática; hidrólisis; fisiología digestiva; proteasa; crustáceos

INTRODUCTION

Chymotrypsin is an enzyme that belongs to the hydrolases, which break covalent bonds by incorporating water between peptide bonds. It has a serine residue in the active site, this being the reason that classifies it as a serine endoprotease (Di Cera, 2009; Navarrete del Toro & García Carreño, 2019). This enzyme catalyzes the hydrolysis of peptide bonds adjacent to the carboxyl groups of the aromatic amino acids tryptophan, tyrosine and phenylalanine ((Zwilling & Neurath, 1981; Zhou et al., 2011), although it can also hydrolyze peptide bonds on the side of other large hydrophobic residues, such as methionine and leucine (Zwilling & Neurath, 1981; Zhou et al., 2011; Navarrete del Toro & García Carreño, 2019) to reduce the size of polypeptide chains and allow the action of exoproteases (McDonald, 1985; Barrett, 1994).

Studies on this biological catalyst have focused mainly on fish, mainly to seek alternatives for obtaining enzymes from waste materials generated by the fishing industry (Zhou et al., 2011). However, studies in crustaceans are limited when compared to those reported in fish and terrestrial organisms (Balti et al., 2012; Navarrete del Toro & García-Carreño, 2019), only approximately 36 investigations between 1980 to date that point to this enzyme and almost a third of them were conducted in the last five years (13 studies), this may be due to the fact that studies have been channeled and focused on trypsin, since this can represent up to 60% of the digestive proteolytic activity (Cruz-Suarez, 1996; Muhlia-Almazán et al., 2008).

Early research on the detection of this enzyme did not consider it relevant (Lee et al., 1984; Glass & Stark, 1994); however, this thinking has been changing as the presence of chymotrypsin has been found in several studies on crustaceans (Tsai et al., 1986; Tsai et al., 1991; Von Elert et al., 2004; Navarrete del Toro et al., 2015; Torres Ochoa, 2020) (Table 1).

Table 1 Studies in crustaceans dealing with the presence of chymotrypsin, period 1980-2020  

Species Common name Characterization Study
1. Artemesia longinaris Long-faced shrimp No Fernández-Gimenez et al., 2002.
2. Artemia salina Artemia Yes Serrano, 2015
3. Caridina cantonensis Bee shrimp, glass shrimp No Kattakdad et al., 2018
4. Daphnia magna Water flea, daphnia Yes VonElert et al., 2004
5. Homarus americanus American lobster No Brockerhoff et al., 1970
6. Lithodes santollla Patagonian spider crab No Bañuelos-Vargas et al., 2018
7. Macrobrachium amazonicum River shrimp, Amazonian shrimp No Da Silva et al., 2014
8. Macrobrachium australiense River shrimp, shrimp, prawn No Bonorino & Anderson, 2009
9. Macrobrachium carcinus Acamaya No Manriquez-Santos et al., 2018
10. Macrobrachium rosenbergii Malaysian shrimp No Tsai et al., 1986
11. Macrobrachium tenellum Shrimp, pigua No Espinosa-Chaurand et al., 2017; Montoya, 2018; Espinosa-Chaurand et al., 2019.
12. Metacarcinus edwardsii Marmola crab No Bañuelos-Vargas et al., 2018
13. Metapenaeus bennetae Shrimp No Bonorino & Anderson, 2009
14. Metapenaeus monoceros Spotted shrimp No Tsai et al., 1986
15. Penaeus californiensis Brown shrimp Yes Navarrete del Toro et al., 2015; Torres-Ochoa, 2020
16. Penaeus chinensis Meaty shrimp No Shiet al., 2008; Xue et al., 2013
17. Penaeus esculentus Brown tiger shrimp No Bonorino & Anderson, 2009
18. Penaeus indicus Indian shrimp Yes Omondi, 2005
19. Penaeus japonicus Kuruma shrimp No Tsai et al., 1986
20. Penaeus monodon Tiger shrimp Yes Tsai et al., 1986; Tsai et al., 1991; Jiang et al., 1991
21. Penaeus notialis Southern pink shrimp No Fernández et al., 1997
22. Penaeus paulensis Sao Paulo shrimp Yes Souza et al., 2009
23. Penaeus penicillatus Red tail shrimp No Tsai et al., 1991
24. Penaeus plebejus Shrimp No Bonorino & Anderson, 2009
25. Penaeus schmitti Southern white shrimp, white caribbean shrimp No Lemos et al., 2002
26. Penaeus stylirostris Blue shrimp Yes Navarrete del Toro et al., 2011
27. Penaeus subtilis Southern brown shrimp Yes Buarque et al., 2010
28. Penaeus vannamei White shrimp, Pacific white shrimp Yes Van Wormhoudt et al., 1992; Hernández-Cortes et al., 1997; Navarrete del Toro et al., 2011
29. Palaemon serratus Serrated shrimp Yes Trellu & Ceccaldi, 1980
30. Panulirus argus Common Caribbean lobster Yes Perera et al., 2008
31. Panulirus homarus Spiny lobster No Gora et al., 2018
32. Panulirus interruptus Red lobster Yes Bibo-Verdugo et al., 2015
33. Pleoticus muelleri Argentine shrimp No Fernández-Gimenez et al., 2001
34. Portunus pellagicus Blue crab No Bonorino & Anderson, 2009
35. Scylla paramamosain Mud crab No DuyKhoa et al., 2019
36. Scylla serrata Mangrove crab No Bonorino & Anderson, 2009; Serrano, 2015

Enzymatic characterization in crustaceans

In most of the research carried out in crustaceans, chymotrypsin is not specifically evaluated, but is part of a battery of digestive enzyme study protocols. Likewise, most of the characterizations have been made in decapod crustaceans such as the tiger shrimp Penaeus monodon, the Malaysian shrimp Macrobrachium rosenbergii, the white shrimp Penaeus vannamei, the common Caribbean lobster, Panulirus argus, the red lobster Panulirus interruptus and the brown shrimp Penaeus californiensis (Tsai et al., 1986; Tsai et al., 1991; Hernández-Cortes et al., 1997; Perera et al., 2008; Bibo-Verdugo et al., 2015; Navarrete del Toro et al., 2015; Torres-Ochoa, 2020); as they are exploited species or with potential to be exploited in aquaculture cultures (Espinosa-Chaurand et al., 2019; Torres-Ochoa et al., 2020).

In enzymatic characterization studies for the identification of chymotrypsin in crustaceans, parameters of optimum temperature, thermo stability, optimum pH, pH stability, isoelectric point and ion effect have been evaluated, finding that the ranges of activity can vary depending on the species between 30 to 60 °C in the optimum, between 0°C and 75 °C for its thermo stability, between 7 and 10 in the optimum pH and pH stability between 3 and 12 points (Table 2). In the particular case of isoelectric point analysis in chymotrypsin, studies mention the detection of its isoforms Navarrete del Toro et al., 2015), for example, Hernández-Cortes et al. (1997) identified a single isoelectric point in P. vannamei, as did Navarrete del Toro et al. (2015) in P. californiensis, which determines the presence of a single form of chymotrypsin; while, Tsai et al. (1991) report ed the presence of two isoelectric points in P. monodon with values of 3.0 and 3.2, indicating two isoforms. In most of the species studied, two isoforms of the enzyme have been found (Navarrete del Toro et al., 2011); with exceptions such as Panulirus interruptus, which presented five isoforms (Celis, 2003).

Table 2 Results of chymotrypsin characterization in different crustacean species, period 19802020  

Species Optimal pH Optimal temp pH stability Thermo- stability Study
1. Artemia salina 7.5 30°C 6.0-8.5 0-55°C Serrano, 2015
2. Daphnia magna 7 -- 3.0-12.0 -- VonElert et al., 2004
3. Penaeus californiensis 10 50°C 3.0-10.0 30-60°C Navarrete del Toro et al., 2015; Torres-Ochoa, 2020
4. Penaeus indicus 8 -- -- -- Omondi, 2005
5. Penaeus japonicus 7 -- 5.0-9.0 -- Tsai et al., 1986
6. Penaeus. monodon 7 40°C 4.0-10.0 25-70°C Tsai et al., 1986
7. Penaeus. paulensis 8 55°C --- 25-75°C Souza et al., 2009
8. Penaeus stilyrostris 7 60°C 4.0-11.0 10-70°C Navarrete del Toro et al., 2011
9. Penaeus subtilis 8 55°C --- 25-65°C Buarque et al., 2010
10. Penaeus vannamei 8 60°C 4.0-11.0 10-70°C Navarrete del Toro et al., 2011
11. Palaemon serratus --- 30°C --- 14-30°C Trellu & Ceccaldi, 1980
12. Panulirus argus 7.5 50°C 2.0-12.0 30-60°C Perera et al., 2008
13. Panulirus interruptus 8 55°C 3.0-12.0 25-65°C Bibo-Verdugo et al., 2015
14. Scylla serrata 8 30°C 6.5-8.5 0-45°C Serrano, 2015

Determination techniques

The techniques used for the determination of chymotrypsin-like activity in crustaceans are supported by different tools. It is possible to carry it out by using specific synthetic substrates, where techniques described by Hummel (1959), Erlanger & Edel (1964) and Del Mar et al. (1979) are used; on the other hand, enzymatic inhibitors can be used, such as those employed by Tsai et al. (1986), Vega-Villasante et al. (1995) and Navarrete del Toro et al. (2015).

It is possible that the simplest way to determine the presence of chymotrypsin in organisms is by using specific substrates for the enzyme, such as labenzoyl-tyrosine ethyl-ester (BTEE) proposed in the research of Hummel (1959); or 2-Nitro-4carboxyphenyl-N,N-diphenylcarbamate (NCDC), mentioned by Erlanger & Edel (1964); or Succinyl-Ala-Ala-Pro-Phe-P-nitroanilide (SAAPNA or SAAPFNA) which was proposed by Del Mar et al. (1979). The principle by which the above techniques work is the detection of changes in the interaction between the substrate and the enzyme, which promote the release of the dye molecule that can be read by colorimetry in spectrophotometry (Hummel, 1959; Erlanger & Edel, 1964; Del Mar et al., 1979).

With Erlanger & Edel (1964) method, it has been reported that no chymotrypsin-like activity was detected with P. vannamei and P. setiferus (Lee et al., 1984); while when using BTEE (Hummel, 1959) as substrate, contradictory results have been reported since in species such as H. gammarus no chymotrypsin enzymatic activity was found (Glass & Stark, 1994), but in P. bennetae, P. plebejus, M. austreliense and S. serrata activity was detected Bonorino & Anderson, 2009). SAAPNA use as a substrate (Del Mar et al., 1979) is one of the most widely used to date for its detection, since it is a very sensitive substrate to chymotrypsin activity (Tsai et al., 1986), showing its activity in all studies where it has been used, such as the investigations carried out in P. vannamei (Le Moullac et al., 1996; Hernández-Cortes et al., 1997), P. muelleri (FernándezGimenez et al., 2001), D. magna (Von Elert et al., 2004), P. interruptus (Celis-Guerrero et al., 2004), P. subtilis (Souza et al., 2009) and P. californiensis (Navarrete del Toro et al., 2015; Torres-Ochoa, 2020).

Just as it is desired to observe the activity of chymotrypsin, its inhibition is also studied, which is done at the same time as its characterization or presence through specific substrates, and which are intended to make evident the total or partial inhibition of enzymatic hydrolysis in the presence of these substances (García-Carreño, 1992). The most widely used enzymatic inhibitors for chymotrypsin are chymostatin (Tsai et al., 1986); tosyl-phenylalanine chloromethyl ketone (TPCK) (Tsai et al., 1986; Hernández Cortes et al., 1997; Omondi, 2005; Navarrete del Toro et al., 2015; Manriquez-Santos et al., 2018); carbobenzoxy-phenylalanine chloromethyl ketone (ZPCK) (Lemos et al., 2000); phenylmethylsulfonyl fluoride (PMSF) (Tsai et al., 1986; Hernández-Cortes et al., 1997; Omondi, 2005; Bibo-Verdugo et al., 2015; Manriquez-Santos et al., 2018); soybean-derived trypsin inhibitor (SBTI) (Tsai et al., 1986; Manriquez-Santos et al., 2018); and Z-L-allaniglycyl-L-phenylalanine-chloroketone (ZAGPCK) (Tsai et al., 1986; Chen et al., 1991).

Knowing the structure, function, physicochemical characteristics of activity thresholds, reaction substrates, as well as inhibition, can help us to adequately characterize this enzyme, which leads us to think about the formulation of functional foods for the species of interest, possible resources with which these organisms feed, or even its potential as a biomolecule in biotechnological processes for the treatment and use of different substrates. Thus, from basic physiological knowledge to industrial application, understanding this type of enzyme will provide an advance in its study and will denote avenues of research and application.

Function and action of chymotrypsin

Although chymotrypsin-type enzyme activity has been recorded since the 1980s with the studies of Galgani et al. (1984), it has always been considered as an enzyme with low catalytic activity in crustaceans (García-Carreño et al., 1994; Cruz-Suarez, 1996); its presence in these organisms has even been questioned, as in the research of Lee et al. (1984), where they specified that chymotrypsin-type enzyme activity was nonexistent in P. monodon juveniles, similar to what was reported in other crustacean species such as H. americanus (Brockerhoff et al., 1970), Lithodesa esquispinus and Paralithodescam tschaticus (Galgani & Nagayama, 1987), where the same conclusion is reached. Possibly because the activity of this enzyme was not detected in the first investigations, there was a decrease in interest in studies related to it (García-Carreño et al., 1994; Von Elert et al., 2004; Omondi, 2005), which caused a delay in the body of information on this enzyme, compared to trypsin (Muhlia-Almazán et al., 2008). Due to the above, an information gap has been generated on mechanisms of action of chemotrypsin in digestion processes in crustaceans (Bibo-Verdugo et al., 2015; Navarrete del Toro & García Carreño, 2019).

This lack of information becomes more evident when comparing the available information of chymotrypsin with respect to that of trypsin; which has been the most studied digestive enzyme in crustaceans and it is considered responsible for approximately 60% of the digestive proteolytic activity in these organisms (Vega Villasante et al., 1995; Albuquerque-Cavalcanti et al., 2001; Carrillo-Farnés et al., 2007; Muhlia- Almazan et al., 2008). The importance given to trypsin is mainly due to the fact that it was the first protease detected and characterized in crustaceans (Muhlia-Almazán et al., 2008; Sainz-Hernandez & Cordova-Murueta, 2009). For its detection, the techniques of Erlanger et al. (1961), which uses benzoyl-DL-arginine 4-nitroanilide chlorohydrate (BAPNA); and Hummel (1959), where tosyl-arginine-methylester hydrochloride (TAME) is used, are commonly used. Both methods were developed for the detection of trypsin in other organisms; however, their use has been ratified for the detection of chemotrypsin in crustaceans (Brockerhoff et al., 1970; Sainz et al., 2004; Von Elert et al., 2004; Omondi, 2005; Navarrete del Toro et al., 2011).

It was not until the development of other substrates, such as SAAPNA, that it was possible to detect the presence of the hydrolytic activity of chymotrypsin (García Carreño et al., 1994). Despite this, research on this enzyme in crustaceans continued to focus on the detection and analysis of trypsin, leaving aside the analysis of chymotrypsin, which was considered a complementary enzyme with low activity (Muhlia Almazán et al., 2008; Sainz-Hernández & Córdova-Murueta, 2009).

The probable reason why the enzyme does not hydrolyze the BTEE substrate is the one exposed by Tsai et al. (1986), where it is mentioned that although BTEE presents the amino acids on which chymotrypsin acts, it lacks other amino acids that interact in a secondary way with the enzyme so that it can carry out its function, this is also mentioned by Van Wormhoudt et al. (1992), when they report that crustacean chymotrypsin is more reactive to natural substrates than to synthetic ones, due to the length of the polypeptide chains alone.

This has led to consider trypsin as the protease responsible for the highest percentage of protein hydrolysis (Carrillo-Farnés et al., 2007; Muhlia-Almazán et al., 2008). However, subsequent studies consider that the enzyme with the highest hydrolysis capacity is chymotrypsin (Tsai et al., 1986; Buarque et al., 2010; Navarrete del Toro et al., 2011; Gora et al., 2018), and this enzyme has not been given greater importance. For this reason, information gaps are observed and new questions arise around the activity of this enzyme, because the lack of information forces researchers to use enzymatic activity models of other groups of organisms (Navarrete del Toro & GarcíaCarreño, 2019), thus establishing an area of opportunity to generate new and basic knowledge that affects the studies of the digestive physiology of this group of organisms so important as a source of food for humans.

Among the new aspects of study on chymotrypsin is its function within the collagenolytic activity (Navarrete del Toro et al., 2015), which involves review from classification to applications, and currently through more precise equipment, generated knowledge and adaptation of techniques make it feasible to reach specific results.

The collagenolytic function of chymotrypsin offers it a similarity with the enzyme brachyurin (Hernández-Cortes et al., 1997; Navarrete del Toro et al., 2015); in this sense Rudenskaya (2003) proposes an approach for the separation of chymotrypsin and brachyurin into two distinct groups of enzymes, since when analyzing the structure of the chain of amino acids that integrate brachyurins it has been observed that there are differences with chymotrypsin in the polypeptide chains, mainly in the amino acids in charge of anchoring the enzyme to the polypeptide chain to be hydrolyzed. Contrary to this, Navarrete del Toro & García-Carreño (2019) state that brachyurin classification is incorrect and should not be considered as a group of enzymes distinct from chymotrypsin, since in crustaceans the collagenolytic activity of chymotrypsin is a generality. Due to these different positions and the lack of more precise information in this regard, it is suggested that these groups should be called digestive enzymes with chymotrypsin-like activity (Navarrete del Toro & García-Carreño, 2019).

Chymotrypsin in crustaceans

Although there are few studies of chymotrypsin in economically important crustacean species, this is repeated in other groups of aquatic organisms such as fish (Lauff & Hofer, 1984; Rungruangsk-Torrissen et al., 2006; Castillo-Yañez et al., 2009; Hadj Ali et al., 2010). In this group, chymotrypsin is composed of a single polypeptide chain (Lauff & Hofer, 1984; Zhou et al., 2011), as in crustaceans (Hernández-Cortes et al., 1997; Navarrete del Toro et al., 2015), and studies have mainly focused on the comparison of chymotrypsin activity among organisms of this group, using standards of the enzyme derived from ruminants (Tsukada & Blow, 1985). According to these studies, it has been observed that chymotrypsin has been shown to have higher hydrolytic activity than its mammalian simile (Lauff & Hofer, 1984; Celis, 2003; Rungruangsk-Torrissen et al., 2006; Zhou et al., 2011; Navarrete del Toro et al., 2015)

As mentioned above, in commercially important crustaceans, research on the study of chymotrypsin, since 2000, has focused mainly on species such as P. chinensis (Shi et al., 2008), P. subtilis (Buarque et al., 2010), P. vannamei (Navarrete del Toro et al., 2011), as well as studies have also been carried out on this enzyme in species that present potential to be exploited in culture conditions, such as P. indicus (Omondi, 2005), P. californiensis (Navarrete del Toro et al., 2015; Torres-Ochoa, 2020), and M. tenellum (Espinosa-Chaurand et al., 2017); this because it is of great importance to have information on enzymatic dynamics and digestive physiology of organisms (Gamboa-Delgado et al., 2003; Simon, 2009) to influence and use their physiological potential within the formulation of balanced feed (Carrillo-Farnés et al., 2007; Simon, 2009; Buarque et al., 2010) and consequently increase the yield in commercial crops. This digestive physiological potential in crustaceans is evidenced by the results of chymotrypsin activity during protein digestion, since through its activity smaller polypeptides are released on which other enzymes act to obtain the different amino acids present (Tsai et al., 1991; Navarrete del Toro & García-Carreño, 2019).

Analyses of chymotrypsin activity between wild organisms and organisms in culture systems have been carried out, such as the one performed by Da Silva et al. (2014) in M. amazonicum, where they report that the enzymatic activity of chymotrypsin, and in general of proteases, is higher in cultured organisms; contrary to this, in P. californiensis, Torres-Ochoa (2020) observed that the activity of this enzyme was lower in cultured organisms than in wild organisms. These discrepancies could be due to two particular situations, the type of food or substrate and the time of consumption or time between feedings (sporadic starvation). It has been mentioned that chymotrypsin activity has been observed better from more specific substrates, such as SAAPNA, and in different magnitude according to the type of ingredient or diet evaluated, which in cultured organisms is supervised and guaranteed, promoting stability in the dynamics and digestive cycle of these organisms, contrary to what may occur with a wild organism that consumes whatever it has available, when it has it available and generally only one or two different ingredients, which would lead to the possible explanation where the feeding and food status of the wild samples in these studies is totally unknown.

In the starvation case, there is the possibility that wild organisms present a higher reserve of zymogens, which can only be activated in the presence of the substrate, so they may be prepared for the processing of these substrates, while fed organisms present lower values as they are constantly using enzymes for digestive processes (Gora et al., 2018; Torres-Ochoa, 2020). Gora et al. (2018) conducted a study with P. homarus, where they evaluated the differences in enzyme activity under starvation and dieting schemes, reporting that fed organisms presented lower enzyme activity than those in starvation. This is possible due to the fact that wild organisms may spend periods of starvation because they do not have food and when they find it, they must ingest it quickly to avoid competition with other animals. Therefore, it is necessary that they have a high reserve of zymogens, which when activated are able to quickly hydrolyze food to make the most of the available nutrients.

CONCLUSIONS

So far, chymotrypsin in crustaceans is considered as an enzyme with no or low activity within the digestive enzymatic dynamics in these species; however, its study is of utmost importance to understand the bioavailability of amino acids, especially tryptophan, tyrosine and phenylalanine. As well as to understand the mechanism of hydrolysis of large residues involving the presence of essential amino acids for these species such as methionine and leucine. Due to the advance of techniques for the assessment of enzymatic activity and the use of synthetic substrates such as SAAPNA, it has been possible on the one hand to detect the presence of this enzyme in crustaceans and, on the other hand, to understand in more detail the digestive physiology of these species. This will serve to define nutritional guidelines in which it is possible to optimize and efficiently take advantage of the ingredients in functional foods, establish feeding correlations and study the behavior in both wild and farmed organisms, and will allow exploring new biotechnological uses of this enzyme and its possible application in the hydrolysis of proteins.

ACKNOWLEDGMENTS

To Dr. Marco Antonio Cadena Roa, that provided support and guidance for the projects that resulted in this article. The support provided by the staff of the Seafood Workshop of UABCS Mexico, the members of the food laboratory of the Academic Unit Pichilingue of UABCS and the Laboratory of Ecophysiology of Aquatic Organisms of UNCIBNOR, for their support of this manuscript as part of Castellanos-Ochoa Carlos' bachelor's degree, is gratefully acknowledged.

REFERENCES

Albuquerque-Cavalcanti C, García-Carreño FL, Navarrete del Toro MA. 2001. Trypsin and trypsin inhibitors from penaeid shrimp. Journal of food biochemistry. 26: 233-251. ISSN 0145-8884. https://doi.org/10.1111/j.1745-4514.2002.tb00854.x [ Links ]

Balti R, Bougherra F, Bougateef A, Hayet BH, Nedjar-Arroume N, Dhulster P, Guillochon D, Nasri M. 2012. Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) with high activity in the hydrolysis of long chain peptide substrates: purification and biochemical characterization. Food Chemistry. 130:475-484. ISSN 0308-8146. https://doi.org/10.1016/j.foodchem.2011.07.019 [ Links ]

Bañuelos-Vargas I, Cárdenas-Chávez F, Paschke K, Román-Reyes JC, SalazarLeyva JA, Martínez-Montaño E. 2018. Partial biochemical characterization of digestive proteases presents in the gastric juices of two chilean crustaceans, Lithodes santolla (Molina, 1782) and Cancer edwardsii (Bell, 1835). Latin American Journal of Aquatic Research. 46:289-300. ISSN 0718-560X. https://doi.org/10.3856/vol46-issue2-fulltext-5 [ Links ]

Barrett A J. 1994.Classification of peptidases. Methods in Enzymology. 244:1-15. https://doi.org/10.1016/0076-6879(94)44003-4 [ Links ]

Bibo-Verdugo B, Rojo-Arreola L, Navarrete del Toro MA, García-Carreño F. 2015. A chymotrypsin from the digestive Tract of California Spiny Lobster, Panulirus interruptus: Purification and Biochemical characterization. Marine Biotechnology. 17:416-427. ISSN 1436-2236. https://doi.org/10.1007/s10126-015-9626-z [ Links ]

Bonorino MS, Anderson AJ. 2009. Digestive enzyme spectra un crustacean decapods (Paleomonidae, Portunidae and Penaeidae) feeding in the natural habitat. Aquaculture research. 40:282-291. ISSN 1365-2109. https://doi.org/10.1111/j.1365-2109.2008.02087.x [ Links ]

Brockerhoff H, Hoyle RJ, Hwang PC. 1970. Digestive enzymes of the American Lobster (Homarus americanus). Journal of the Fisheries Research Board of Canada. 27: 1357-1370. ISSN 0015-296X. https://doi.org/10.1129/f70-160 [ Links ]

Buarque DS, Castro PF, Santos FMS, Amaral IPG, Oliveira SM, Alves KB, Carvalho LB, Bezerra RS. 2010. Digestive proteinases and peptidases in the hepatopancreas of the southern brown shrimp (Farfantepenaeus subtilis) in two sub-adult stages. Aquaculture Nutrition. 16:359-369. ISSN 1365-2095. https://doi.org/10.1111/j.1365-2095.2009.00673.x [ Links ]

Carrillo-Farnés O, Forrellat A, Guerrero-Galván S., Vega-Villasante F. 2007. A review of digestive enzyme activity in penaeid shrimps. Crustaceana. 80:257-275. ISSN 0011-216X. https://doi.org/10.1163/156854007780162424 [ Links ]

Castillo-Yañez FJ, Pacheco-Aguilar R, Lugo-Sánchez ME, García-Sánchez G, Quintero-Reyes IE. 2009. Biochemical characterization of an isoform of chymotrypsin from the viscera of Monterey sardine (Sardinops sagaxcaerulea), and comparison with bovine chymotrypsin. Food Chemistry. 112:634-639. ISSN 0308-8146. https://doi.org/10.1016/j.foodchem.2008.06.023 [ Links ]

Celis LE . 2003. Caracterización de proteasas en el sistema digestivo de la langosta roja (Panulirus interruptus). Tesis de maestría, Centro de Investigaciones Biológicas de Noroeste. La Paz, BCS, México. Pp. 81. http://dspace.cibnor.mx:8080/bitstream/handle/123456789/465/celis_l.pdf;sequence=1 [ Links ]

Celis-Guerrero L, García-Carreño FL, Navarrete del Toro MA. 2004. Characterization of proteases in the digestive system of spiny lobster (Panulirus interruptus). Marine Biotechnology. 6:262-269. ISSN 1436-2236. https://doi.org/10.1007/s10126-003-0032-6 [ Links ]

Chen YL, Lu PJ, Tsai IH. 1991. Collagenolytic activity of crustacean midgut serine proteases: comparison with the bacterial and Mammalian enzymes. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 100:763-768. ISSN 0305-0491. https://doi.org/10.1016/0305-0491(91)90287-N [ Links ]

Cruz-Suarez LE. 1996. Digestión en camarón y su relación con formulación y fabricación de alimentos balanceados. Avances en nutrición acuícola. III: 207-276. http://eprints.uanl.mx/id/eprint/8350 [ Links ]

Da SILVA FM, Ribeiro K, Vasconcelos de Freitas AC, Bezerra de Carvalho L, Cotroni W, De Souza R. 2014. Digestive proteases from wild and farmed male morphotypes of the amazon river prawn (Macrobrachium amazonicum). Journal of Crustacean Biology. 34:189-198. ISN 0278-0372. https://doi.org/10.1163/1937240X-00002215 [ Links ]

Del Mar EG, Largman C, Brodrick JW, Geokas MC. 1979. A sensitive new substrate for chymotrypsin. Analytical Biochemistry. 99:316-320. ISSN 0003-2697. https://doi.org/10.1016/S0003-2697(79)80013-5 [ Links ]

Di Cera E. 2009. Serineproteases. IUBMB Life. 61: 510-515. ISSN 1521-6543. https://doi.org/10.1002/iub.186 [ Links ]

Duykhoatn, Mai NT, Linh NK, Mi LTY, Shaharom-Harrison F. 2019. Ontogenic development of digestive enzymes of Mud Crab (Scylla paramamosain) during larval stages. Thalassas: An International Journal of Marine Science.35:655-661. ISSN 26613239. https://doi.org/10.1007/s41208-019-00143-5 [ Links ]

Erlanger BF, Kokowsky N, Cohen W. 1961. the preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics. 95: 271-278. ISSN 0003-9861. https://doi.org/10.1016/0003-9861(61)90145-X [ Links ]

Erlanger BF, Edel F. 1964. The utilization of a specific chromogenic inactivator in an "All or none" assay for chymotrypsin. Biochemistry. 3:346-349. ISSN 0006-2960. https://doi.org/10.1021/bi00891a008 [ Links ]

Espinosa-Chaurand D, Vega-Villasante F, Carrillo-Farnés O, Nolasco-Soria H. 2017. Effect of circadian rhythm, photoperiod and molt cycle on digestive enzymatic activity of Macrobrachium tenellum juveniles. Aquaculture. 479:225-232. ISSN 00448486. https://doi.org/10.1016/j.aquaculture.2017.05.029 [ Links ]

Espinosa-Chaurand D, Carrillo-Farnés O, Vega-Villasante F, Nolasco-Soria H. 2019. Effect of protein level in diet and feeding schedule on the digestive enzymatic activity of Macrobrachium tenellum juveniles. Latin American Journal of Aquatic Research. 47:743-752. ISSN 0718-560X. https://doi.org/10.3856/vol47-issue5-fulltext-3 [ Links ]

Fernández-Gimenez AV, García-Carreño FL, Navarrete del Toro MA, Fenucci JL. 2001. Digestive proteinase of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): Partial Characterization and relationship with molting. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 130:331-338. ISSN 1096-4959. https://doi.org/10.1016/S1096-4959(01)00437-7 [ Links ]

Fernández I, Oliva M, Carrillo O, Van Wormhoudt A. 1997. Digestive enzyme activities of Penaeus notialis during reproduction and moulting cycle. Comparative Biochemistry and Physiology part A: Physiology. 118:1267-1271. ISSN 0300-9629. https://doi.org/10.1016/S0300-9629(97)86802-8 [ Links ]

Fernández-Gimenez AV, García-Carreño FL, Navarrete del Toro MA, Fenucci JL. 2002. Digestive proteinase of Artemesia longinaris (Decapoda, Penaeidae) and relationship with molting. Comparative Biochemistry and Physiology part B: Biochemistry and Molecular Biology. 132:593-598. ISSN 1096-4959. https://doi.org/10.1016/s10964959(02)00080-5 [ Links ]

Galgani F, Nagayama F. 1987. Digestive proteinases in five species of Lithodidae (Crustacea, Decapoda). Comparative Biochemistry and Physiology part B: Comparative Biochemistry. 87:103-107. ISSN 0305-0491. https://doi.org/10.1016/03050491(87)90476-7 [ Links ]

Galgani FG, Benyamin Y, Ceccaldi HJ. 1984. Identification of digestive proteinases of Penaeus kerathurus (Forskal): a comparison with Penaeus japonicus Bates. Comparative Biochemistry and Physiology part B: Comparative Biochemistry. 78:355-361. ISSN 0305-0491. https://doi.org/10.1016/0305-0491(84)90043-9 [ Links ]

Gamboa-Delgado J, Molina-Paveda C, Cahu C. 2003. Digestive enzyme activity and food ingesta in juvenile shrimp Litopenaeus vannamei (Boone, 1931) as a function of body weight. Aquaculture Research. 34: 1403-1411. ISSN 1365-2109. https://doi.org/10.1111/j.1365-2109.2003.00959.x [ Links ]

García-Carreño FL. 1992. Protease inhibition in theory and practice. Biotechnology Education. 2(4):150-153. ISSN 0717-3458. http://www.bashanfoundation.org/contributions/Garcia-F/carrenoinhibition.pdf [ Links ]

Garcia-Carreño FL, Hernández-Cortes MP, Haard NF. 1994. Enzymes with peptidase and proteinase Activity from the Digestive Systems of a Freshwater and a Marine Decapod. Journal of Agricultural and Food Chemistry. 42:1456-1461. ISSN 0308-8146. https://doi.org/10.1021/jf00043a013 [ Links ]

Glass HJ, Stark JR. 1994. Protein digestion in the european lobster, Humarus gammarus (L.) Comparative Biochemistry and Physiology part B: Comparative Biochemistry. 108:225-235. ISSN 0305-0491. https://doi.org/10.1016/0305-0491(94)90070-1 [ Links ]

Gora A, Jayasankar V, Rehman S, Kizhakudan JK, Laxmilatha P, Vijayagopal P. 2018. Biochemical responses of juvenile rock spiny lobster Panulirus humarus under different feeding regimes. Journal of Applied Animal Research. 46:1462-1468.ISSN 0974-1844. https://doi.org/10.1080/09712119.2018.1533475 [ Links ]

Hadj Ali NE, Hmidet N, Zouari-Fakhfakh N, Ben Khaled H, Nasri M. 2010. Alkaline Chymotrypsin from Striped Seabream (Lithognathus mormyrus) Viscera: Purification and Characterization. Journal of Agricultural and Food Chemistry. 58(17):9787-9792. ISSN 1520-5118. https://doi.org/10.1021/jf101667s [ Links ]

Hernández-Cortes P, Whitaker JR, García-Carreño FL. 1997. Purification and characterization of chymotrypsin from Penaeus vannamei (Crustacea: Decapoda). Journal of Food Biochemistry. 21:497-514. ISSN 01458884. https://doi.org/10.1111/j.1745-4514.1997.tb00202.x [ Links ]

Hummel BCW. 1959. A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin.Canadian Journal of Biochemistry and Physiology. 37:1393- 1399. ISSN 0576-5544. https://doi.org/10.1139/y59-157 [ Links ]

Jiang ST, Moody MW, Chen HC. 1991. Purification and Characterization of proteases from the Digestive tract of grass shrimp (Penaeus monodon). Journal of Food Science. 56:322-326. ISSN 1750-3841. https://doi.org/10.1111/j.1365-2621.1991.tb05271.x [ Links ]

Kattakdad S, Jintasataporn O, Worawattanamateekul W, Chumkam S. 2018. Successful nursing of Caridina cantonensis larvae with Ca-alginate microencapsulated diet in the first feeding. International Journal of Aquatic Science. 9(2):66-76. ISSN 20088019. http://www.journal-aquaticscience.com/article_70646_9abea4777e424db1dea7f86cfbe3ff05.pdf [ Links ]

Lauff M, Hofer R. 1984. Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture. 37:335-346. ISSN 0044-8486. https://doi.org/10.1016/0044-8486(84)90298-9 [ Links ]

Lee PG, Smith LL, Lawrence AL.1984. Digestive proteases of Penaeus vannamei Boone: relationship between enzyme activity, size and diet. Aquaculture. 42:225-239. ISSN 0044-8486. https://doi.org/10.1016/0044-8486(84)90103-0 [ Links ]

Lemos D, Ezquerra JM, Carcía-Carreño FL. 2000. Protein digestion in penaeid shrimp: digestive proteinases, proteinase inhibitors and feed digestibility. Aquaculture. 186:89-105. ISSN 0044-8486. https://doi.org/10.1016/S0044-8486(99)00371-3 [ Links ]

Lemos D, García-Carreño FL, Hernández P, Navarrete del Toro A. 2002. Ontogenic variation in digestive proteinase activity, RNA and DNA content of larval and postlarval white shrimp Litopenaeus schmitti. Aquaculture. 214:363-380. ISSN 0044-8486. https://doi.org/10.1016/S0044-8486(02)00253-3 [ Links ]

Lemoullac G, Klein B, Sellos D, Van Wormhoudt A. 1996. Adaptation of trypsin, chymotrypsin and α-amylase to casein level and protein source in Penaeus vannamei (Crustacea Decapoda). Journalof Experimental Marine Biology and Ecology. 208:107-125. ISSN 0022-0981. https://doi.org/10.1016/S0022-0981(96)02671-8 [ Links ]

Manriquez-Santos TJ,Alvarez-González CA, Peña M, Camarillo-Coop S, MartinezGarcía R, Vega-Villasante F. 2018. Partial characterization of digestive proteases in adults of big claw river shrimp Macrobrachium carcinus. Latin American Journal of Aquatic Research. 46:525-533. ISSN 0718-560X. https://doi.org/10.3856/vol46-issue3fulltext-5 [ Links ]

McDonald JJ. 1985. An overview of protease specificity and catalytic mechanisms: aspects related to nomenclature and classification. Histochemical Journal. 17:773-785. ISSN 0018-2214. https://doi.org/10.1007/BF01003313 [ Links ]

Montoya CE. 2018. Calidad de ingredientes proteínicos y actividad enzimática digestiva en Macrobrachium tenellum. Tesis de Doctora en Ciencias, Universidad de Guadalajara. Puerto Vallarta, Jalisco, México. Pp. 145. https://www.riudg.udg.mx/handle/20.500.12104/81122 [ Links ]

Muhlia-Almazán A, Sánchez-Paz A, García-Carreño FL. 2008. Invertebrate trypsins: a review. Journal of Comparative Physiology B. 178:655-672. ISSN 0174-1578. https://doi.org/10.1007/s00360-008-0263-y [ Links ]

Navarrete del Toro MA, García-Carreño FL, Cordova-Murueta JH. 2011. Comparison of digestive proteinases in three penaeids. Aquaculture. 317:99-106. ISSN 0044-8486. https://doi.org/10.1016/j.aquaculture.2011.03.035 [ Links ]

Navarrete del Toro MA, García-Carreño FL. 2019. The toolbox for protein digestion in decapod crustaceans: a review. Reviews in Aquaculture. 11:1005-1021. ISSN 1753-5131. https://doi.org/10.1111/raq.12276 [ Links ]

Navarrete del Toro MA, García-Carreño FL, Hernández-Cortés P, Molnar T, Graf L. 2015. Biochemical characterization of chymotrypsin from the midgut gland of yellowleg shrimp, Penaeus californiensis. Food Chemistry. 173:147-155. ISSN 0308-8146. https://doi.org/10.1016/j.foodchem.2014.09.160 [ Links ]

Omondi JM. 2005. Digestive endo-proteases from the midgut glands of the indian white shrimp, Penaeus indicus (Decapoda Penaeidae) from Kenya. Western Indian Ocean Journal of Marine Science. 4: 109-121. ISSN 2661-3239. https://doi.org/10.4314/wiojms.v4i1.28479 [ Links ]

Perera E, Moyano FJ, Díaz M, Perdomo-Morales R, Montero-Alejo V, Alonso E, Carrillo O, Galich GS. 2008. Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 150: 247-2554. ISSN 0305-0491. https://doi.org/10.1016/j.cbpb.2008.03.009 [ Links ]

Rudenskaya GN. 2003. Brachyurins, serine collagenolytic enzymes from crabs. Russian Journal of Bioorganic chemistry. 9:117-128. ISSN 0045-2068. https://doi.org/10.1023/A:1023248113184 [ Links ]

Rungruangsk-Torrissen K, Moss R, Andresen LH, Berg A, Waagbo R. 2006. Different expressions of trypsin and chymotrypsin in relation to growth in Atlantic Salmon (Salmo salar). Fish physiology and Biochemistry. 32:7-23. ISSN 1573-5168. https://doi.org/10.1007/s10695-005-0630-5 [ Links ]

Sainz JC, García-Carreño FL, Hernández-Cortes P. 2004. Penaeus vannamei isotrypsins: purification and characterization. Comparative Biochemistry and Physiology part B: Biochemistry and Molecular Biology. 138:155-162. ISSN 1096-4959. https://doi.org/10.1016/j.cbpc.2004.03.002 [ Links ]

Sainz-Hernandez JC, Cordova-Murueta JH. 2009. Activity of trypsin from Litopenaeus vannamei. Aquaculture. 290:190-195. ISSN 0044-8486. https://doi.org/10.1016/j.aquaculture.2009.02.034 [ Links ]

Serrano AE. 2015. Properties of chymotrypsin-like enzyme in the mudcrab Scylla serrata, Brine shrimp Artemia salina and rotifer Brachionus plicatilis. Der Pharma Chemica. 7:66-73. ISSN 0975-413X. https://www.derpharmachemica.com/pharmachemica/properties-of-chymotrypsinlike-enzyme-in-the-mudcrab-scylla-serrata-brineshrimp-artemia-salina-and-rotifer-brachionus-p.pdf [ Links ]

Shi X, Zhao X, Wang J. 2008. Molecular cloning and expression analysis of a chymotrypsin-like serine protease from the chinese shrimp, Fenneropenaeus chinensis. Fish and Shellfish Immunology. 25:589-597. ISSN 1050-4648. https://doi.org/10.1016/j.fsi.2013.03.360 [ Links ]

Simon CJ. 2009. Digestive enzyme response to natural and formulated diets in cultured juvenile spiny lobster, Jasus edwardsii. Aquaculture. 294: 271-281. ISSN 0044-8486https://doi.org/10.1016/j.aquaculture.2009.06.023 [ Links ]

Souza D, Fernandes P, Silva FM, Lemos D, Bezerra L, Bezerra RS. 2009. Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae). Aquaculture Research. 40: 861-870. ISSN 1365-2109. https://doi.org/10.1111/j.1365-2109.2009.02183.x [ Links ]

Torres-Ochoa E. 2020. Análisis del proceso digestivo de juveniles de Farfantepenaeus californiensis (Holmes, 1900) aclimatados a cultivo con tecnología de biofloc. Tesis de Doctorado en Ciencias, Universidad Autónoma de Baja California Sur. México. Pp. 149. https://www.researchgate.net/publication/355105308_Analisis_de_proceso_digestivo_de_juveniles_de_Farfantepenaeus_californiensis_Holmes_1900_aclimatados_a_condiciones_de_cultivo_con_tecnologia_de_biofloc [ Links ]

Trellu J, Ceccaldi HJ. 1980. Influence de la température sur quelques Activités Enqymatiques chez Palaemonserratus. Biochemical Systematics and Ecology. 8:171-179. ISSN 0305-1978. https://doi.org/10.1016/0305-1978(80)90010-1 [ Links ]

Tsai I, Chuang K,Chuan JL. 1986. Chymotrypsin in digestive tracts of crustacean decapods (shrimps). Comparative Biochemistry and Physiology part B: Comparative Biochemistry. 85: 235-239. ISSN 0305-0491. https://doi.org/10.1016/0305-0491(86)90248-8 [ Links ]

Tsai I, Lu P, Chuang JL. 1991. The midgut chymotrypsins of shrimps (Penaeus monodon, Penaeus japonicus and Penaeus penicillatus). Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1080:59-67. ISSN 0167-4838. https://doi.org/10.1016/0167-4838(91)90112-D [ Links ]

Tsukada H, Blow DM. 1985. Structure of α-chymotrypsin refined at 1.68A resolution. Journal of Molecular Biology. 184:703-711. ISSN 0167-4838. https://doi.org/10.1016/0022-2836(85)90314-6 [ Links ]

Van Wormhoudt A, Le Chevalier P, Sellos D. 1992. Purification, biochemical characterization and N-Terminal sequence of a serine-protease with chymotrypsic and collagenolytic activities in a tropical shrimp, Penaeus vannamei (Crustacea, decapoda). Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 103:675-680. SSN 0305-0491. https://doi.org/10.1016/0305-0491(92)90389-9 [ Links ]

Vega-Villasante F, Nolasco H, Civera R. 1995. The digestive enzymes of the Pacific brown shrimp Penaeus californiensis II. Properties of protease activity in the whole digestive tract. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 112:123-129. ISSN 1096-4959. https://doi.org/10.1016/03050491(95)00039-B [ Links ]

Von Elert E, Agrawal MK, Gebauer C, Jaensch H, Bauer U, Zitt A. 2004. Protease activity in gut of Daphnia magna:Evidence for trypsin and chymotrypsin enzimes. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 137:287-296. ISSN 1096-4959. https://doi.org/10.1016/j.cbpc.2003.11.008 [ Links ]

Xue S, Yang W, Sun J. 2013. Role of chymotrypsin-like serine proteinase in white spot syndrome virus infection in Fenneropenaeus chinensis. Fish and Shellfish Immunology. 34:403-409. ISSN 1050-4648. https://doi.org/10.1016/j.fsi.2012.10.017 [ Links ]

Zhou L, Budge SM, Ghaly AE, Brooks MS, Deepika D. 2011. Extraction, Purification and Characterization of fish Chymotrypsin: a review. American Journal of Biochemistry and Biotechnology. 7: 104-123. ISSN 1553-3468. https://doi.org/10.3844/ajbbsp.2011.104.125 [ Links ]

Zwilling R, Neurath H. 1981. Invertebrate proteases. Methods in enzymology. 80: 633-364. ISSN 0076-6879. https://doi.org/10.1016/S0076-6879(81)80050-X [ Links ]

Code: e2021-69.

Received: October 01, 2021; Accepted: March 05, 2022

Creative Commons License Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons

 
Tezontle 171, El Pedregal de San Juan, Tepic, Nayarit, MX, 63164, (52-311)1221626