Journals Homepage: www.sustenere.co/journals FISH PEPSIN: BASIC CHARACTERISTICS, EXTRACTION,
DETERMINATION AND BIOTECHNOLOGICAL
APPLICATIONS
ABSTRACT
Enzyme technology has been employed in various segments of the industry, food
production and consumer goods. Fish viscera are potential sources of proteins with
enzymatic properties appreciable in this market. Among them, pepsin (EC 3.4.23.x)
deserves special attention. The active enzyme has at least five isoforms which are
precursor groups of pepsins type: A, B, and F, gastricsin and chymosin. Their
zymogen (pepsinogen) is activated by autocatalysis at acidic pH in stomach.
Hemoglobin is their specific substrate. Features like optimal pH and temperature vary
according to species and even within the same species may be found different values.
The molecular weight also varies according to species and is generally between 25
and 35 kDa. The protocols for extraction and determination of pepsin fish follow a line
of standardization, varying concentrations of analysis. These data are basic steps to
detect the presence of the enzyme targeting a future purification process for its
effective biotechnological applications.
KEYWORDS: Digestive; Enzyme; Fish; Pepsin; Biotechnology.
Natural Resources, Aquidabã, v.4, n.1, Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014. ISSN 2237‐9290 SECTION: Articles TOPIC: Ictiologia DOI: 10.6008/SPC2237‐9290.2014.001.0001 Vagne de Melo Oliveira Universidade Federal de Pernambuco, Brasil http://lattes.cnpq.br/6160988158330428 [email protected] Ranilson de Souza Bezerra PEPSINA DE PEIXES: CARACTERÍSTICAS BÁSICAS,
EXTRAÇÃO, DETERMINAÇÃO E APLICAÇÕES
BIOTECNOLÓGICAS
RESUMO
Tecnologia enzimática vem sendo empregada em diversos segmentos da indústria,
da produção de alimentos e bens de consumo. Vísceras de peixes são potenciais
fontes de proteínas com propriedades enzimáticas apreciáveis neste mercado. Entre
eles, a pepsina (EC 3.4.23.x) merece atenção especial. A enzima ativa tem, pelo
menos, cinco isoformas, sendo elas precursoras do tipo pepsinas: A, B e F,
gastricsina e quimosina. O zimogênio (pepsinogênio) é ativado por autocatálise em
pH ácido do estômago que vem do ácido clorídrico. A hemoglobina é seu substrato
específico. Características como temperatura e pH ótimos variam de acordo com as
espécies e até mesmo dentro da mesma espécie podem ser encontrados valores
diferentes. O peso molecular varia também de acordo com a espécie e situa-se
geralmente, entre 25 e 35 kDa. Os protocolos de extração e de determinação de
pepsina de peixe seguem uma linha de normalização, com várias concentrações de
análise. Estes dados são passos básicos para detectar a presença da enzima
destinada a um futuro processo de purificação para aplicações biotecnológicas
eficazes.
PALAVRAS-CHAVES: Digestivo; Enzima; Peixe; Pepsina; Biotecnologia.
Universidade Federal de Pernambuco, Brasil http://lattes.cnpq.br/2205151409139871 [email protected] Caio Rodrigo Dias Assis Universidade Federal de Pernambuco, Brasil http://lattes.cnpq.br/0018678980235423 [email protected] Received: 17/09/2013 Approved: 29/09/2014 Reviewed anonymously in the process of blind peer. Referencing this: OLIVEIRA, V. M.; BEZERRA, R. S.; ASSIS, C. R. D.. Fish pepsin: basic characteristics, extraction, determination and biotechnological applications. Natural Resources, Aquidabã, v.4, n.1, p.6‐14, 2014. DOI: http://dx.doi.org/10.6008/SPC2237‐9290.2014.001.001 Natural Resources (ISSN 2237‐9290) © 2014 Sustenere Publishing Corporation; FMA. All rights reserved. Rua Dr. José Rollemberg Leite, 120, CEP 49050‐050, Aquidabã, Sergipe, Brasil WEB: www.sustenere.co/journals – Contact: [email protected] Fish pepsin: basic characteristics, extraction, determination and biotechnological applications INTRODUÇÃO
Enzymes are proteins specialized in the catalysis of organic reactions and efficient molecular
devices that determine the patterns of chemical transformations becoming essential for maintaining
life (ROEHM & KOOLMAN, 2005). Operate in a highly specific way with their substrates. The
enzymatic reaction occurs within the enzyme in a cavity called active site, where the substrate
interacts with the substituent groups of the amino acids which catalyze the transformation (NELSON
& COX, 2004). Pepsin (EC 3.4.23.x) is an acid protease, synthesized and secreted by gastric
membrane in its inactive state, pepsinogen (PG) which undergoes autocatalysis in the presence of
hydrochloric acid (HCl). The activation reaction is initiated by interruption of electrostatic interactions
between the terminal segment (NH2-terminal) and the active portion of the enzyme at acid pH values.
The zymogen is converted to the active form through conformational changes and bond cleavage,
leading to the opening of the active site by the removal of the prosegment from the active center of
the enzyme (RICHTER et al., 1998, SHAHIDI & KAMIL, 2001; KAGEYAMA, 2002; ROTTA, 2003;
KOOLMAN & ROEHM, 2005; DE LUCA et al., 2009; ZHOU et al., 2008; WU et al., 2009; ZHAO et
al., 2011).
It is an aspartyl protease, and as such depends on the aspartic acid residue present in its
active site for catalytic activity. It is included in the category of endopeptidases (only operates on
internal links of the chain) which cleave peptide bonds of proteins by their amino terminal side of
aromatic amino acid residues, such as tyrosine and tryptophan, breaking the long polypeptide chains
into smaller peptides and free amino acids in some (WHITAKER, 1994; DÍAZ-LÓPEZ et al., 1998;
RICHTER et al., 1998; RAO et al., 1998; SHAHIDI & KAMIL, 2001; ROTTA, 2003; KOOLMAN &
ROEHM, 2005; KLOMKLAO et al., 2007; DE LUCA et al., 2009; WU et al., 2009; ZHAO et al., 2011).
In comparison with pepsin, the pepsinogen contains additional 44 amino acids and is stable in weak
alkaline and neutral environments, but when exposed to HCl present in the gastric juice (pH 1.5-2.0),
the additional amino acids are removed proteolytically by autocatalysis to activate the pepsin
(RICHTER et al., 1998; ROTTA et al., 2003; KAGEYAMA, 2002; TANJI et al., 2007; WU et al., 2009;
ZHAO et al., 2011).
In mammals pepsinogens are classified into five groups, as described in table 1, differing
significantly in terms of their primary structures. These groups are precursors of pepsins type: A, B,
and F, gastricsin, and chymosin (KAGEYAMA, 2002). Fish synthesize two of the five types of pepsin:
pepsin I or A and pepsin II or C. Pepsin I and II have been described for the fish Epinephelus coioides
(CASTILLO-YAÑEZ et al., 2004; FENG et al., 2008; CAO et al., 2011). After autocatalysis of the
pepsinogen, the active pepsins present an approximate molecular weight of 35 kDa which varies
according to the species. Pepsin I or A has a range of isoelectric point from 6.5 to 7 while for the II
or C the point is the interval between 4.5 and 5. In general, the pI of mammalian pepsins are lower
than those of pepsins from fish, possibly because of the higher content of basic amino acids in fish
Natural Resources  v.4 ‐ n.1  Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014 P a g e | 7 OLIVEIRA, V. M.; BEZERRA, R. S.; ASSIS, C. R. D. pepsins (SHAHIDI & KAMIL, 2001; KAGEYAMA, 2002; CASTILLO-YAÑEZ et al., 2004). It was found
that pI of pepsin may vary even within the same species as in the Atlantic cod (Gadus morhua)
ranging from 4.1 to 6.9 (GILDBERG et al., 1990).
The three-dimensional structure of fish pepsin has been little explored. The first threedimensional structure has been proposed for pig (FRUTON, 2002). Cod pepsin resembles the amino
acid composition of porcine cathepsin D more than that of porcine pepsin (GILDBERG et al., 1990).
Cod pepsin was reported as a monomer with two similar domains bent forming a deep cleft. The
catalytic site of pepsin is formed at the junction of the domains and contain two aspartic acid
residues, Asp32 and Asp215 in each domain. In catalysis of pepsin, a water molecule acts between
the active carboxyls and help Asp215 (acting as a general base) and Asp32 (protonated) to become
negatively and positively charged, respectively, to break peptide bonds in proteins. A remarkable
property of the catalytic center is the adaptation to the action in a wide range of pH, from 1.0 to 7.0
(KARSEN et al., 1998; ANDREEVA & RUMSH, 2001; ZHAO et al., 2011).
Table 1: Classification of pepsinogens.
Group
Pepsinogen A
Pepsinogen B
Progastricsin
Prochymosin
Pepsinogen F
Active
precursors
Pepsin A
Pepsin B
EC
number
3.4.23.1
3.4.23.2
Other names
Pepsinogen I
-
Gastricsin
3.4.23.3
Pepsinogen C or II
Chymosin
3.4.23.4
Prochymosins A, B, and C (for bovine isozymogens)
Pepsin F
-
pregnancy-associated glycoprotein
Source: Kageyama (2002).
Factors Affecting the Activity (pH, Temperature, Activators and Inhibitors) Each type of fish has a distinct pepsin with an optimal pH, temperature and thermal stability
(El-BELTAGY et al., 2004; ZHAO et al., 2011). Pepsin presents higher activity in pH around 2, and
some fish species, may have a second optimum pH around 4. This finding could be explained by the
different optimal pH of pepsin types. Hemoglobin is efficiently hydrolyzed by type I pepsin between
pH 3 and 4, while for pepsin type II it occurs between pH 2 and 3. This endopeptidase is important
for carnivorous species to initiates the digestion of proteins, releasing some peptides and free amino
acids (GILDBERG et al., 1990; ROTTA, 2003). Pepsins from cold water fish and temperate climate
present maximum stability between pH 2 and 5, while the warm water species pepsins are stable
even at pH 7 (CASTILLO-YAÑEZ et al., 2004).
The amount of pepsinogen produced is strongly influenced by temperature, being directly
proportional thereto. The production of HCl is also proportional to the size of the meal. Stomach
distension appears to be the stimulus for early gastric secretion. Agastric fish do not produce HCl or
pepsinogen perform all the steps of digestion in alkaline medium (ROTTA, 2003). Fish from hot water
present stable activity between 40-50°C while the cold water fish pepsins are very susceptible to
higher temperatures (ZHAO et al., 2011).
Natural Resources  v.4 ‐ n.1  Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014 P a g e | 8 Fish pepsin: basic characteristics, extraction, determination and biotechnological applications The main inhibitors and activators of pepsin are shown in table 2. The enzymatic activity of
pepsin is almost completely inhibited by pepstatin A (aspartic proteinase competitive inhibitor that
binds strongly to residues of the active site of the enzyme) and partially inhibited by phenyl methyl
sulfonyl fluoride (PMSF, serinoproteinase inhibitor), L-3-carboxytrans-2, 3-epoxy-propionyl-L-leucyl4-guanidino-butylamide (E-64), cysteine, molybdate and EDTA (Ethylenediamine tetra acetic acid,
specific for metalloproteases) (DIAZ-LOPEZ et al., 1998; CASTILLO-YAÑEZ et al., 2004; TANJI et
al., 2007; BOUGATEF et al., 2008; KLOMKLAO et al., 2007; NALINANON et al., 2008; NALINANON
et al., 2010; WU et al., 2009; CAO et al., 2011; ZHAO et al., 2011). In contrast, the activity increases
in the presence of divalent cations such as CaCl2, MgCl2 and CoCl2 (El-BELTAGY et al., 2004;
KLOMKLAO et al., 2007). Sodium chloride has inhibitory effects (partial) or activator, depending on
the species studied and depending on the concentration used. Some authors have reported
increased activity by sodium chloride, such as El-Beltagy et al. (2004), while others attest enzyme
inhibition, as in the case of Gildberg et al. (1990). Pepsin activity is greatly dependent on the type of
substrate. Hemoglobin is the main substrate for the determination of pepsin activity (KLOMKLAO et
al., 2004; KLOMKLAO et al., 2007; KLOMKLAO, 2008; TANJI et al., 2009). Table 3 illustrates some
of the species models used for industrial applications, with their enzymatic characteristics for efficient
operation.
Table 2: Activators and inhibitors (full or partial) of pepsin.
Compound
Activators
Inhibitors (full)
Partial inhibition or no
effect
CaCl2
(calcium chloride)
MgCl2
(magnesium chloride)
CoCl2
(cobalt chloride)
NaCl*
(sodium chloride)
Pepstatin A
(aspartic proteinase inhibitor)
PMSF
(phenyl methyl sulfonyl fluoride, serinoproteinase inhibitor);
E-64
(L-3-carboxytrans-2, 3-epoxy-propionyl-L-leucin-4-guanidinobutylamide);
Cysteine;
SBTI
(soybean trypsin inhibitor)
EDTA
Reference
Gildberg et al. (1990);
El-Beltagy et al. (2004);
Klomklao et al. (2007);
Gildberg et al. (1990);
Díaz-López et al.
(1998);
Castillo-Yañez et al.
(2004);
Klomklao et al. (2007);
Tanji et al. (2007);
Bougatef et al. (2008);
Nalinanon et al. (2008);
Wu et al. (2009);
Nalinanon et al. (2010);
Cao et al. (2011);
Zhao et al. (2011)
Gildberg et al. (1990);
Xu et al. (1996);
Díaz-López et al.
(1998);
Castillo-Yañez et al.
(2004);
Klomklao et al. (2007);
Tanji et al. (2007);
Bougatef et al. (2008);
Natural Resources  v.4 ‐ n.1  Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014 P a g e | 9 OLIVEIRA, V. M.; BEZERRA, R. S.; ASSIS, C. R. D. (ethylenediaminetetraacetic acid), specific for
metalloproteases);
Adenosine tri phosphate (ATP),
Molybdate
SDS
(sodium dodecyl sulfate)
NaCl**
(sodium chloride)
Nalinanon et al. (2008);
Wu et al. (2009);
Nalinanon et al. (2010);
Cao et al. (2011);
Zhao et al. (2011).
Soucer: El-Beltagy et al., (2004); Gildberg et al., (1990).
Table 3: Enzymatic features of pepsin in different species.
Identified Species
Scientific
Name
°C
pH
pI
6.9
Molecular
Weight
( kDa)
35.5
Atlantic cod - I
Gadus morhua
37
3.5
Atlantic cod - II
Gadus morhua
37
Atlantic cod - b
Gadus morhua
Orange roughy - I
Gildberg et al. (1990)
3.5
4.0
34
Gildberg et al. (1990)
40
3.5
4.1
34
Gildberg et al. (1990)
Hoplostethus
atlanticus
37
2.5
4.3
33.5
Xu et al. (1996)
Orange roughy - II
Hoplostethus
atlanticus
37
3.5
4.40
34.5
Xu et al. (1996)
Monterey sardine
Sardinops sagax
caerulea
45
2.5
4.0
29
Castillo-Yañez et al.(2004)
Nile tilape
Oreochromis
niloticus
Thunnus alalunga
Coryphaenoides
pectoralis
Coryphaenoides
pectoralis
Sparus latus
Houttuy
Sparus latus
Houttuyn
35
2.5
-
31
El-Beltagy et al. (2004)
50
45
2
3
-
32.7
31
Nalinanon et al. (2010)
Klomklao et al. (2007)
45
3.5
-
35
Klomklao et al. (2007)
45
3
-
30
Zhou et al. (2007)
50
3.5
-
30
Zhou et al. (2007)
Smooth hound
Mustelus mustelus
40
2
-
35
Bougatef et al. (2008)
Albacore tuna
Skipjack tuna
50
50
2
2
-
-
Nalinanon et al. (2008)
Nalinanon et al. (2008)
Tongol tuna
Thunnus alalunga
Katsuwonus
pelamis
Thunnus tonggol
50
2
-
-
Nalinanon et al. (2008)
Eel
Anguilla anguilla
40
3.5
-
30
Wu et al. (2009)
Eel
Anguilla anguilla
40
2.5
-
30
Wu et al. (2009)
Eel
Anguilla anguilla
35
2.5
-
30
Wu et al. (2009)
Albacore tuna
Thunnus alalunga
50
2
-
39.9
Nalinanon et al. (2010)
Albacore tuna
Thunnus alalunga
50
2
-
32.,7
Nalinanon et al. (2010)
Albacore tuna
Rattail - I
Rattail - II
Sea bream - I
Sea bream - II
Reference
I – pepsin typo I; II – pepsin typo II; b – pepsin typo B; pI - isoelectric point; °C – temperature.
Natural Resources  v.4 ‐ n.1  Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014 P a g e | 10 Fish pepsin: basic characteristics, extraction, determination and biotechnological applications Preparation of crude extract
The methods for extracting enzymes from gastric cells are varied. All, however, involve at
least three basic steps: I - dissection and homogenized; II - solubilization in a suitable buffer; and III
- Separation by centrifugation to remove cell debris. Temperature and proper pH are essential during
the extraction process. The temperature of extraction should be between 0-4°C to prevent protein
denaturation and autolysis enzymes (DIAZ-LOPEZ et al., 1998; BOUGATEF et al., 2008;
NALINANON et al., 2008; NALINANON et al., 2010; ZHAO et al., 2011). Briefly: fish shall be
measured in terms of livestock (total length, length of head, tail length, and width). The sacrifice of
the fish may be by immersion in an ice bath or an appropriate anesthetic, according to recommended
protocol. After evaluating the biometric parameters, evisceration occurs. For stomach removal, an
L-shaped incision is made in the lateral muscles of the fish. The viscera is removed carefully to avoid
contamination and weighed. The samples are cut into pieces and homogenized with an extraction
buffer (glycine-HCl 0.01 mol/L pH 2.0) using a tissue homogenizer. The resultant preparations are
centrifuged at 10,000 x g for 10 min at 4°C. The supernatants are frozen at -20°C and used in
subsequent assays. Enzyme activity Proteolytic activity of enzyme can be determined using haemoglobin as a substrate according
to the method of Nalinanon et al. (2010). The figure 1 illustrates determination of enzymatic activity.
To initiate the reaction, 200 µL of enzyme solution were added into the assay mixture containing 200
µL of 2% haemoglobin, 200 µL of distilled water and 625 µL of reaction buffer. Appropriate dilution
was made to ensure that the amount of enzyme was not excessive for available substrate in the
assay system. The reaction was conducted at pH 2.0 and 50°C for 20 min. To terminate enzymatic
reaction, 200 µL of 50% (w/v) trichloroacetic acid (TCA) were added. Unhydrolysed protein substrate
was allowed to precipitate for 1 h at 4°C, followed by centrifuging at 15,000 x g for 10 min. The
oligopeptide content in the supernatant was measured at 280 nm. One unit of activity was defined
as the amount of product causing an increase of 1.0 in absorbance at 280 nm per min. A blank was
run in the same manner, except that the enzyme was added into the reaction mixture after the
addition of 50% (w/v) TCA. Protein concentration was determined by the method of Bradford (1976)
using bovine serum albumin as a standard, whilst during the course of enzyme purification by
measuring the absorbance at 280 nm. Natural Resources  v.4 ‐ n.1  Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014 P a g e | 11 OLIVEIRA, V. M.; BEZERRA, R. S.; ASSIS, C. R. D. Figure 1: Determination of enzyme activity Biotechnological applications
Biotechnologically purified pepsin is used in the extraction of collagen for pharmaceutical
industry and cosmetics; widely used in medical research for regulation of digestion, as an antiseptic
dental and treatment of some diseases, including dyspepsia, gastralgia, infant diarrhea and some
cancers. Swine pepsin is used for treating gastric ulcers and coagulation of the milk to form curd, an
important process in the dairy industry. The enzyme is also utilized in the production of fish silage,
in the use of byproducts of fish processing industry and in the animal feed industry for increase
protein digestibility. It is considered a promising protease with extensive industrial application of
efficient effect if effective techniques for their recovery and purification can be developed
(KAGEYAMA, 2002; KLOMKLAO et al., 2007; NALINANON et al., 2008; EISENMENGER & REYESDE-CORCUERA, 2009; ZHAO et al., 2011; ZENG et al., 2012).
CONCLUSION
Given the wide range of operating stability of pepsin and duplicity of parameters like
isoelectric point and optimal pH and temperature, it is possible to use this enzyme in several
biotechnological and industrial processes that pass through phases of different physicochemical
conditions.
Moreover, Fish processing waste may be proposed as source of pepsin, since they produce
large amounts of viscera and other discarded material in their processing activities. Such destination
of the disposed material avoids environmental impacts from the activities and is in accordance with
sustainability guidelines.
We presented here basic steps to detect the presence of the enzyme targeting a future
purification process for its effective biotechnological applications.
Natural Resources  v.4 ‐ n.1  Set, Out, Nov, Dez 2013, Jan, Fev, Mar, Abr, Mai, Jun, Jul, Ago 2014 P a g e | 12 Fish pepsin: basic characteristics, extraction, determination and biotechnological applications REFERENCES ANDREEVA, N. S.; RUMSH, L. D.. Analysis of crystal structures of aspartic proteinases: On the role of
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