Alkaline Proteases from the Digestive
Tract of Four Tropical Fishes
Proteases Alcalinas do Trato Digestório
de Quatro Espécies Tropicais Marinhas
AUTORES
AUTHORS
Rodrigo Barboza ALENCAR1,2
Michelle Miranda BIONDI2
Patrícia Maria Guedes PAIVA1
Vera Lúcia Almeida VIEIRA2
Luiz Bezerra CARVALHO JUNIOR1
Ranilson de Souza BEZERRA1,2
Laboratório de Enzimologia, Departamento de Bioquímica,
Universidade Federal de Pernambuco, Cidade Universitária,
50670-910, Recife, PE, Brasil.
Email: [email protected]
1 Laboratório de Imunopatologia Keizo Asami and
Laboratório de Enzimologia, Departamento de Bioquímica,
Universidade Federal de Pernambuco.
2 Laboratório de Fisiologia e Ecologia de Peixes,
Departamento de Pesca,
Universidade Federal Rural de Pernambuco.
SUMMARY
The occurrence of digestive alkaline proteases was investigated in four tropical fishes:
Crevalle Jack ( Caranx hippos), Spotted Goatfish (Pseudupeneus maculatus ); Parrotfish
(Sparisoma sp.) and Traira (Hoplias malabaricus). The highest enzymatic activities were found
in pyloric caeca of Crevalle Jack; Spotted Goatfish and Traira and in the intestine of Parrotfish.
The enzyme activities presented an optimum temperature of 55oC and remained unaltered
after 30min incubation at 45oC. This thermal property is similar to that reported for other
tropical fish enzymes. The optimum pH range for the enzymatic activity was established as
from 7.0 to 9.0, such a broad interval probably being due to the mixture of proteases. The
use of specific substrates and inhibitors showed strong evidences that trypsin, chymotrypsin
and leucine-aminopeptidase were present among the alkaline proteases of the digestive
tract (pyloric caeca and intestine) of these species. Large amounts of viscera are produced
by processing these fishes and are discarded. Therefore, these wastes could represent an
alternative source for obtaining these important proteases.
RESUMO
A ocorrência de proteases digestivas alcalinas foi investigada em quatro espécies de
peixes tropicais: xaréu (Caranx hippos), saramunete (Pseudupeneus maculatus), budião
(Sparisoma sp.) e traíra (Hoplias malabaricus). As maiores atividades enzimáticas foram
encontradas nos cecos pilóricos do xaréu, saramunete e traíra e no intestino do budião.
Apresentaram uma temperatura ótima de 55ºC e permaneceram inalteradas após incubação
por 30min a 45ºC. Esta última propriedade apresenta-se similar às descritas para outras
enzimas de peixes tropicais. A faixa de pH ótimo foi estabelecida entre 7,0 e 9,0 devido,
provavelmente, ao fato de tratar-se de uma mistura de proteases. A utilização de substratos
e inibidores específicos demonstraram fortes evidências da existência de tripsina, quimotripsina
e leucino-aminopeptidase entre as proteases alcalinas no ceco pilórico e intestino destas
espécies. O processamento desses peixes produz grandes quantidades de vísceras que são
descartadas. Portanto, esses subprodutos podem representar atrativas fontes alternativas
para obtenção dessas importantes enzimas proteolíticas.
PALAVRAS-CHAVE
KEY WORDS
Digestive protease; Traira (Hoplias malabaricus); Crevalle
Jack (Caranx hippos); Spotted Goatfish ( Pseudupeneus
maculatus); Parrotfish (Sparisoma sp.); Tropical fish.
Proteases Digestivas; Traira (Hoplias malabaricus); Xaréu
(Caranx hippos); Saramunete (Pseudupeneus maculatus);
Budião (Sparisoma sp.); Peixes tropicais.
Braz. J. Food Technol., v.6, n.2, p. 279-284, jul./dez., 2003
279
Recebido / Received: 18/07/2002. Aprovado / Approved: 21/07/2003.
ALENCAR, R. B. et al.
1. INTRODUCTION
Despite the large diversity of fish species in the aquatic
tropical zones, where the climatic conditions are propitious to
their development, only a few species have been studied from
the perspective of their the digestive proteases. There is little
information on proteases from tropical fishes and their
applications (DE VECCHI; COPPES, 1996). These enzymes play
an important role in the growth of these animals and their
diet formulation. They are also relevant in the development of
undesirable textural changes during storage and processing
(DIONYSIUS et al., 1994). Furthermore, such enzymes may
prove useful as processing tools in the food industry and are
used in almost half of all industrial enzymatic processes
(GUIZANI et al., 1991; HAARD, 1992; DE VECCHI; COPPES,
1996).
Crevalle Jack (Caranx hippos) is a marine fish found from
Nova Scotia through the whole of the Gulf of Mexico and the
Caribbean to off shore Uruguay. Its total length is usually about
100 cm, reaching sizes of 150cm. It feeds primarily on fish but
also on shrimp and other invertebrates (BERRY; SMITH-VANIZ,
1978).
Spotted Goatfish (Pseudupeneus maculatus) is also a
marine fish living in coastal waters throughout the same area,
including Bermuda and extending northward to New Jersey
and southward to Rio de Janeiro (Brazil). Its total length is
normally 22cm and the maximum is 30cm. It feeds mainly on
small bottom-dwelling invertebrates (VERGARA; RANDALL,
1978).
Parrotfish (Sparisoma sp.) is a coral reef fish, hence not
found in deep waters where such an environment does not
occur. Its total length is normally 35cm and the maximum is
59. It feeds on benthos plant life (RANDALL, 1978).
Traira (Hoplias malabaricus) is a predatory freshwater
fish living along the edges of streams, lakes and in flooded
forests. It is adapted to low oxygen conditions and occurs in
tropical waters over most of their range, but can also be found
in somewhat colder waters in Paraguay and Argentina
(COURTENAY; HENSLEY, 1979; TAPHORN, 1992). HENSLEY;
MOODY (1975) suggesting that temperature would not be a
limiting factor for this species in southern Florida. Traira is
considered mature when it reaches 15 to 25cm. Although it
can grow up to 50cm, the average adult length is usually about
30cm. It is mainly piscivorous, with the occasional ingestion
of crustacea (HENSLEY; MOODY, 1975; GÉRY, 1977).
These marine fishes have been caught (180ton/year)
by the company EMPAF Ltd. on the Pernambuco coast,
Northeast Brazil, and processed for exportation to European
Countries. On the other hand, traira is a relevant species in an
important group of fishes, the Characiformes, including the
major native fish species of Brazilian Aquaculture, an amount
reaching 13,080.5 ton having been reported as caught by Brazil
(IBAMA, 2000). As a result of processing this capture, a large
amount of residue has been produced as waste, which could
be used as an alternative source of proteases. These enzymes
show potential application in the food industr y and
biotechnology.
Braz. J. Food Technol., v.6, n.2, p. 279-284, jul./dez., 2003
Alkaline Proteases from the Digestive
Tract of Four Tropical Fishes
In this paper we report on some of the properties of
digestive alkaline proteases from three marine tropical fishes:
Crevalle Jack (xaréu), Spotted Goatfish (saramunete) and
Parrotfish (budião). A fourth freshwater predatory tropical fish,
traira, was included in the investigation.
2. MATERIAL AND METHODS
2.1 Enzyme extraction
The company EMPAF Ltd. kindly donated specimens of
Crevalle Jack, Spotted Goatfish and Parrotfish. Specimens of
traira were caught from the fishpond facilities at the “Estação
de Aquicultura Continental Prof. Johei Koike” (Departamento
de Pesca, Universidade Federal Rural de Pernambuco, Brazil).
They were kept at 4oC and processed in the laboratory within
20min. The pyloric caeca, intestine and liver were collected
and immediately homogenized with 25 parts 0.9% (w/v) NaCl
at 4 o C using a tissue homogenizer. The homogenized
preparations were centrifuged at 10,000 x g for 10min at 10°C
to remove cell debris and nuclei. The supernatants obtained,
referred to as the crude extracts, were frozen at –20oC (not
exceeding a storage time of 48h) and then used for the protease
assays.
2.2 Enzyme assay
The protease activities were assayed using azocasein
as substrate and adapted (LEIGHTON et al., 1973) to small
volumes, using micro plates and the ELISA reader. In duplicate,
using micro centrifuge tubes, 50µl of 1% (w/v) azocasein
(Sigma), prepared in 0.2 M Tris-HCl pH 7.2, was incubated
with 30µl of crude extract for 60min at 25°C. 240µL of 10%
(w/v) trichloroacetic acid (TCA) were then added to stop the
reaction. After 15min at rest, the tubes were centrifuged for
5min at 8,000xg. 70µl of the supernatant were then added to
130µl of 1 M NaOH on an ELISA plate, and the absorbance of
the mixture measured in an ELISA reader (Bio-rad 550) at
450nm against a blank prepared in the same way, replacing
the crude extract with 0.9 % (w/v) NaCl. Previous experiments
showed that under the conditions described above, the first
60min of the reaction follow first order kinetics. One unit (U)
of enzyme activity was defined as the amount of enzyme able
to hydrolyze azocasein giving an increase of 0.001 units of
absorbance per minute.
2.3 Enzyme inhibition
The inhibition effects of Phenylmethyl-sulfonyl fluoride
(PMSF), ethylenediamine tetracetic acid (EDTA) and L-transepoxysuccinyl-leucylamide – (4-guanidino)-butane (E-64) were
assayed in duplicate using micro centrifuge tubes, preincubating 15µl of crude extract with 15µl of 1 mM inhibitor
solution prepared in dimethylsulfoxide (DMSO) at 25oC for
280
ALENCAR, R. B. et al.
15min. Azocasein was then added and its hydrolysis followed
as described in the Enzyme Assay. The 100% value was that
established for the enzyme assay without inhibitors.
2.4 Specific enzyme inhibition
Twenty µl of the samples of crude extract and 30µl of
0.2 M Tris-HCl pH 8.0 buffer were placed on an ELISA plate
with 25µl of the following inhibitors, purchased from Sigma,
prepared in DMSO: 1 mM benzamidine; 1mM tosyl lysine
chloromethyl ketone (TLCK); 500µg/mL egg white trypsin
inhibitor (EWTI) and 100µg/mL Cratylia mollis trypsin inhibitor
(CmTI) prepared in our laboratory according to PAIVA (1998),
and incubated at 37oC for 15min. Afterwards, the volumes
were adjusted to 170µL with 0.9 % (w/v) NaCl and residual
proteolytic activities determined at 37oC by incubating with
(30µL) 4mM Benzoyl-DL-Arginine-p-Nitroanilide (BAPNA;
Sigma), prepared in DMSO according to ERLANGER et al.
(1961). The release of p-nitroaniline was followed by the
increase in absorbance at 405nm in an ELISA reader (Bio-rad
550). The enzyme and substrate blanks were similarly assayed
without substrate and enzyme solution, respectively. The 100%
values were established without inhibitors. Similar experiments
were carried out to evaluate the chymotrypsin activity using
succinyl-DL-phenylalanine-p-nitroanilide (Suc-Phe-p-Nan) as
substrate and tosyl phenylalanine chloromethyl ketone (TPCK)
as inhibitor, and the leucine-aminopeptidase activity using
leucine-p-nitroanilide (Leu-p-Nan) as substrate and bestatin as
inhibitor. All specific substrate assays either in the presence or
absence of inhibitors, were performed in duplicate. One unit
(U) of specific enzyme activity was defined as the amount of
enzyme capable of hydrolyzing BAPNA, Suc-Phe-pNAN and
Leu-pNAN to produce a 0.001 change in absorbance per
minute.
Alkaline Proteases from the Digestive
Tract of Four Tropical Fishes
caeca, except for the Parrotfish, which does not have this system
of blind sacs posterior to the stomach. Considering that 1ml
of each crude extract contains about 40mg of tissue and yields
proteolytic activity of from 50 to 92U/ml, one can deduce that
2,000-3,680U are produced per gram. Therefore, these viscera
are economically attractive since tons are discarded during
processing of these fish. Most teleostei do not have a defined
pancreas gland (MARTINEZ; SERRA, 1989) and ZENDZIAN;
BARNARD (1967) suggested that pyloric caeca proteases are
pancreatic-like enzymes. Table 1 also shows the inhibition of
the activity of the serine proteases, metallo proteases and
cysteine proteases induced by their respective inhibitors,
namely, PMSF, EDTA and E-64. This study was only carried out
with the tissues containing the highest enzymatic activity.
Serine protease was accountable for the highest alkaline
proteolytic activity of the pyloric caeca and intestine followed
by cysteine protease. Negligible metallo protease and cysteine
protease activities were found in the Traira intestine. However,
this result indicates that several enzymes are responsible for
the alkaline proteolytic activity. Similar results were reported
for the pyloric caeca of another tropical freshwater fish,
Colossoma macropomum tambaqui (BEZERRA et al., 2000) and
of Oreochromis niloticus Nile tilapia (BEZERRA et al., 2001b).
TABLE 1. Proteolytic activities of the digestive tracts of four
tropical fishes (1% w/v azocasein as substrate) and their
inhibitions by PMSF, EDTA and E-64.
Species
Organ
Inhibition [%]
Enzymatic
Activity (U/mL) PMSF EDTA E-64
Crevalle Jack
(Caranx hippos)
P yloric caeca
92.2
Intestine
10.8
Liver
16.2
Spotted Goatfish
(Pseudupeneus
maculatus)
P yloric caeca
51.7
Intestine
15.6
Liver
nd*
2.5 Optimum temperature and pH
Parrotfish
(Sparisoma sp.)
Intestine
50.1
Liver
nd
The influences of temperature and pH on the proteolytic
activity against azocasein were studied as follows: the crude
extracts were assayed at temperatures ranging from 10 to 80oC
and pH 7.2 to 9.0 (0.2 M Tris-HCl buffer).
Traira (Hoplias
malabaricus)
P yloric caeca
76.8
Intestine
38.1
Liver
nd
2.6 Thermal enzyme stability
The thermal stability of the enzyme was established by
incubating the crude extracts for 30min at temperatures
ranging from 25oC to 75oC. After equilibration at 25oC, their
activities against azocasein were assayed as described above.
3. RESULTS AND DISCUSSION
The proteolytic activities (azocasein as substrate) of the
digestive tracts of the four types of fish under investigation are
shown in Table 1. The highest activity was present in the pyloric
Braz. J. Food Technol., v.6, n.2, p. 279-284, jul./dez., 2003
51.7
7.4
27.6
23.8
15.4 20.9
35.5 22.4 24.9
43.9
1.6
2.9
*nd – not detected
The influence of pH and temperature on the activity of
the crude extracts from the fish tissues containing the highest
enzymatic activity, are presented in Figures 1 and 2. More than
one optimum pH was found between 7.0 and 9.0 for each of
the four fishes. Similar behavior was observed for the crude
extract from the Tambaqui pyloric caeca (BEZERRA et al., 2000).
However, a peak at pH 9.5 was found for the partially purified
trypsin (BEZERRA et al., 2001a). Probably, the present optimum
pH pattern is due to the mixture of proteases in the crude
extracts, as will be further demonstrated. All the proteolytic
enzymes showed an optimum temperature at 55oC (Figure 2).
According to Figure 3 one can see that their activities remained
unaltered after being treated for 30min at 45oC. The heat
treatment at 55oC (optimum temperature) for 30min, reduced
the activities to about 20% for crevalle jack and spotted goatfish
281
ALENCAR, R. B. et al.
pyloric caeca; 30% for traira pyloric caeca and 50% for parrotfish
intestine.
Proteolytic activity [%]
100
100
Proteolytic activity [%]
80
Alkaline Proteases from the Digestive
Tract of Four Tropical Fishes
[A]
[A]
80
60
40
20
60
0
40
0
20
40
60
Temperature ºC
20
40
60
Temperature ºC
80
20
40
60
Temperature ºC
80
20
40
60
Temperature ºC
20
7,5
80
60
40
60
40
20
0
7,5
8,0
pH
8,5
0
9,0
100
Proteolytic activity [%]
[C]
80
60
40
20
0
7,0
100
8,0
pH
8,5
9,0
60
40
20
0
100
[D]
80
60
40
20
0
7,0
[C]
80
0
7,5
[B]
80
20
100
Proteolytic activity [%]
100
9,0
[B]
0
7,0
Proteolytic activity [%]
8,5
Proteolytic activity [%]
Proteolytic activity [%]
100
8,0
pH
Proteolytic activity [%]
0
7,0
7,5
8,0
pH
8,5
80
9,0
FIGURE 1. Effect of pH on the alkaline proteolytic activities
from four tropical teleosts: crevalle jack [A], spotted goatfish
[B], parrotfish [C] and traira [D]. The crude extracts were assayed
in duplicate (10) at the pH values indicated, using 0.2 M TrisHCl buffers.
Braz. J. Food Technol., v.6, n.2, p. 279-284, jul./dez., 2003
[D]
80
60
40
20
0
0
80
FIGURE 2. Effect of temperature on the alkaline proteolytic
activities from four tropical teleosts: crevalle jack [A], spotted
goatfish [B], parrotfish [C] and traira [D]. The crude extracts
were assayed in duplicate at the temperatures indicated using
azocasein as substrate.
282
ALENCAR, R. B. et al.
Residual activity [%]
100
80
60
40
20
Residual activity [%]
100
30
60
70
80
60
40
20
100
Residual activity [%]
40
50
Temperature ºC
[B]
0
20
30
40
50
Temperature ºC
30
40
50
Temperature ºC
60
70
[C]
80
60
40
20
0
20
100
Residual activity [%]
The use of specific substrates and inhibitors is an
important strategy in the identification of biological proteases.
[A]
0
20
60
70
[D]
Table 2 shows the proteolytic activities of the crude
fish extracts on specific substrates, and their respective
inhibition by specific inhibitors. Therefore, by using specific
substrates and inhibitors, the presence of trypsin (BAPNA and
TLCK, CmTI, EWTI and Benzamidine); chymotrypsin (Suc-Phep-Nan and TPCK) and leucine aminopeptidase (Leu-p-Nan and
Bestatin) were investigated in the crude fish extracts. These
results suggest the presence of all three proteases in the tissues.
Trypsin and leucine aminopeptidase are responsible for the
highest proteolytic activity except for the Parrotfish intestine,
where all the enzymes showed similar activity. The trypsin
inhibitors presented different percent inhibitions. It is important
to draw attention to the fact that the degree of inhibition
presented by the different inhibitors varied considerably from
fish tissue to fish tissue. For instance, crevalle jack trypsin activity
was weakly inhibited by benzamidine, and the parrotfish
enzyme by CmTI and TLCK. These molecules are potent
inhibitors of trypsin activity. On the other hand, TPCK, a classic
chymotrypsin inhibitor, presented no inhibitory effect on the
spotted goatfish chymotrypsin activity. Low inhibition effect
was also observed for bestatin on the parrotfish leucine
aminopeptidase activity. Probably, the enzyme structures vary
from one species to another, resulting in these variations. The
presence of trypsin, chymotrypsin and leucine aminopeptidase
were demonstrated in the crude extracts obtained from the
four tropical fish digestive tracts. Therefore, these enzymes
could be extracted from the large amounts of viscera produced
in the fishery industry as waste. These enzymes have attractive
properties concerning their industrial applications, such as in
the production of fish protein hydrolysates (MACKIE, 1982;
HAARD, 1992), fish sauce (GILDBERG, 2001) and shrimp waste
recovery (GILDBERG; STENBERG, 2001).
TABLE 2. Action of the proteases from the digestive tracts of
four tropical fishes on specific substrates and under the
influence of specific inhibitors.
Species / Substrate
80
Enzymatic
Activity U/mL
Inhibition [%]
CmTI EWTI TLCK
Ben* TPCK Bes**
Crevalle Jack Caranx hippos
60
40
20
0
20
Alkaline Proteases from the Digestive
Tract of Four Tropical Fishes
BAPNA
1427
Suc-Phe-p-Nan
205
Leu-p-Nan
807
53.0
70.9
73.6
7.9
42.0
42.0
Spotted Goatfish Pseudupeneus maculatus
30
40
50
Temperature ºC
60
70
FIGURE 3. Thermal stability of the alkaline proteases from
four tropical teleosts: crevalle jack [A], spotted goatfish [B],
parrotfish [C] and traira [D]. The thermal stability of the enzymes
was established by incubating the crude extracts for 30min at
the temperatures indicated. After equilibration at 25oC, their
activities were assayed and compared to those of the untreated
crude extract (100%).
Braz. J. Food Technol., v.6, n.2, p. 279-284, jul./dez., 2003
BAPNA
920
Suc-Phe-p-Nan
1 68
Leu-p-Nan
1154
48.6
79.0
93.6
88.5
0
55.3
Parrotfish Sparisoma sp.
BAPNA
455
Suc-Phe-p-Nan
471
Leu-p-Nan
557
8.6
90.2
26.6
67.6
64.5
17.1
Traira Hoplias malabaricus
BAPNA
Suc-Phe-p-Nan
1029
184
Leu-p-Nan
1223
* Benzamidine ** Bestatin
283
63.6
80.7
95.2
78.2
33.4
64.6
ALENCAR, R. B. et al.
ACKNOWLEDGEMENTS
The authors would like to thank Mr. Otaviano Tavares
da Costa and Albérico Espírito Santo for their technical
assistance. This study was supported by CNPq/CTPETRO (grant
number 463655/001) and Japan International Cooperation
Agency (JICA).
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