Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus)

Process Biochemistry 40 (2005) 1829–1834 www.elsevier.com/locate/procbio

Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus) Ranilson S. Bezerra*, Eduardo J.F. Lins, Rodrigo B. Alencar, Patrı´cia M.G. Paiva, Maria E.C. Chaves, Luana C.B.B. Coelho, Luiz B. Carvalho Jr. Laborato´rio de Imunopatologia Keizo Asami and Laborato´rio de Enzimologia, Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Cidade Universita´ria, 50670-910 Recife, PE, Brazil Received 24 June 2003; accepted 19 June 2004

Abstract An alkaline protease was extracted from the viscera (intestine) of Nile tilapia, Oreochromis niloticus, the second most important fish in Brazilian aquaculture. This enzyme is usually discarded among the tons of waste produced by its processing. The enzyme was purified in three steps: heat treatment, ammonium sulphate fractionation and Sephadex G-75 gel filtration, presenting an yield and purification of 30% and 22fold, respectively, and showing a single band by SDS-PAGE (23.5 kDa). This enzyme showed Km for the hydrolysis of benzoyl-DL-argininep-nitroanilide (BAPNA) equal to 0.755  0.008 mM, an optimum temperature at 50 8C, was stable for 30 min at 50 8C, and optimum pH of 8.0. The protease was strongly inhibited by Al3+ and Cd2+, followed by Cu2+, Hg2+, Zn2+ and Co2+. Inhibition by PMSF and specific trypsin inhibitors provided additional evidences that this activity can be attributed to a trypsin-like enzyme. # 2004 Elsevier Ltd. All rights reserved. Keywords: Enzyme; Oreochromis niloticus; Proteinase; Tilapia; Tropical fish; Trypsin

1. Introduction Nile tilapia (Oreochromis niloticus) is the second most important exotic fish species in Brazilian aquaculture and 40,000 tons were produced in 2000 [1]. It is predominantly herbivorous and able to produce high quality protein for human consumption [2]. Feeding and digestive mechanisms of tilapine fish have already been described [3–6]. Studies on the digestive proteases of O. niloticus reported in the literature include the purification and properties of a stomach protease [7], the effect of salinity [8], the distribution of digestive enzymes along the intestinal tract [9]. Also, the purification and characterization of an intestine trypsin from a hybrid Tilapia nilotica/aurea [10] and the effect of

* Corresponding author.Tel.: 55-8134638453; fax: 55-8132718485. E-mail address: [email protected], [email protected] (R.S. Bezerra). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.066

diets on the digestive enzymes from O. mossambicus [11] have been studied. Fish proteases have been studied since 1940, although, very few of these are from freshwater species [12,13]. There is very little information on proteases from tropical freshwater fishes and their applications. Digestive proteases represent an important class of industrial enzymes. These proteins are present in fish viscera, a by-product of the fishery industries, usually discarded in large amounts. Thus, this waste is a potential source of proteolytic enzymes. On the other hand, the use of fish proteases in biotechnological applications requires their purification and characterization. However, these studies are time consuming and, to some extent, expensive [14]. The present paper describes the purification of a trypsinlike enzyme from Nile tilapia, Oreochromis niloticus (OniT) following a procedure developed in our laboratory [15]. Some physicochemical properties were also evaluated, such as effect of metal ions, specific inhibitors and kinetic parameters.

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2. Material and methods 2.1. Enzyme extraction Specimens of O. niloticus were captured from the fish pond facilities at the ‘‘Departamento de Pesca, Universidade Federal Rural de Pernambuco, Northeast, Brasil’’. Intestines were collected and homogenized 40 mg of tissue/mL (w/v) in 0.9% (w/v) NaCl by using a tissue homogenizer. The resulting preparation was centrifuged at 10,000  g for 10 min at 10 8C to remove cell debris and nuclei. The supernatant (crude extract) was frozen at 20 8C and used for further purification steps. 2.2. Non-specific enzyme assay In a microcentrifuge tube (quadruplicates) 1% (w/v) azocasein (50 mL; Sigma), prepared in 0.2 M Tris–HCl, pH 7.2 was incubated with crude extract (30 mL) for 60 min at 25 8C. Then, 240 mL of 10% (w/v) trichloroacetic acid (TCA) was added to stop the reaction. After 15 min, centrifugation was carried out at 8000  g for 5 min. The supernatant (70 mL) was added to 1 M NaOH (130 mL) in a 96-well microtiter plate and the absorbance of this mixture was measured in a microtiter plate reader (Bio-rad 550) at 450 nm against a blank similarly prepared except that 0.9% (w/v) NaCl replaced the crude extract sample. Previous experiment showed that for the first 60 min the reaction carried out under the conditions described above follows first order kinetics. One unit (U) of enzymatic activity was defined as the amount of enzyme capable of hydrolysing azocasein to produce a 0.001 change in absorbance per minute. 2.3. Measurement of protein The protein content was estimated by measuring sample absorbance at 280 and 260 nm by using the following equation: [protein] mg/mL = A280 nm  1.5 A260 nm  0.75 [16]. 2.4. Enzyme purification This enzyme was purified in three steps: heat treatment at 45 8C for 30 min, ammonium sulphate fractionation inside the range 30–80% saturation and Sephadex G-75 gel filtration, according to Bezerra et al. [15]. 2.5. Electrophoresis SDS-PAGE Polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli [17], using a 6% (w/v) stacking gel and a 12.5% (w/v) separating gel. The gels were stained for protein overnight in 0.01% (w/v) Coomassie Brilliant Blue. The background of the gel was destained by washing in 10% (v/v) acetic acid. The mole-

cular weight of the Nile tilapia protease band was estimated using the protein standards (Sigma) bovine albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36.0 kDa), carbonic anhydrase (29 kDa), trypsinogen (24.0 kDa) and a-lactalbumin (14.2 kDa). 2.6. Physical chemical properties The influences of temperature and pH on the proteolytic activity of the enzyme preparation were studied as follows: the purified extract was assayed (quadruplicates) as described above at temperatures ranging from 10 to 60 8C and pH values from 7.2 to 10.0 (Tris–HCl buffer). The thermal stability of the enzyme was determined by assaying (quadruplicates) its activity (25 8C) after pre-incubation for 30 min at temperatures ranging from 30 to 60 8C. 2.7. Effect of metal ions The effect of various metal ions was determinated using benzoyl-DL-arginine-p-nitroanilide (BAPNA; Sigma) as substrate. Samples of the purified extract (20 mL) and Tris–HCl pH 8.0 buffer (30 mL) were added in a 96-well microtiter plate with the following metal ions solutions (25 mL at either 8 mM or 80 mM): AlCl3, BaCl2, CaCl2, CdSO4, CoCl2, CuSO4, HgCl2, KCl, LiCl, MgCl2, MgSO4, MnCl2, and ZnSO4. The volumes were adjusted to 170 mL with 0.9% (w/v) NaCl and residual proteolytic activities were determined at 25 8C (quadruplicates) by incubating with 4 mM BAPNA (30 mL), prepared in dimethylsulphoxide (DMSO) according to Erlanger et al. [18]. The release of p-nitroaniline was followed by the increase in absorbance at 405 nm in a microtiter plate reader (Bio-Rad 550). Controls were performed without enzyme and substrate solution. The 100% values were established without metal ions. 2.8. Enzyme inhibition Samples of the purified extract (20 mL) and Tris–HCl pH 8.0 buffer (30 mL) were added in a 96-well microtiter plate with the following inhibitors (25 mL), purchased from Sigma, prepared in DMSO: 8 mM ethylenediamine tetracetic acid; 8 mM b-mercaptoethanol; 8 mM phenylmethylsulfonylfluoride (PMSF); 8 mM benzamidine; 1 mM tosyl lysine chloromethyl ketone (TLCK); 8 mM tosyl phenylalanine chloromethyl ketone (TPCK); 320 mg Cratylia mollis trypsin inhibitor prepared in our laboratory according to Paiva [19] and incubated at 37 8C for 15 min. After the incubation period, the volumes were adjusted to 170 mL with 0.9% (w/v) NaCl and residual proteolytic activities were determined at 37 8C (quadruplicates) as described above. The release of p-nitroaniline was followed by the increase in absorbance at 405 nm using a microtiter plate reader. The enzyme and substrate blank were similarly assayed without enzyme and substrate solution, respectively.

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The 100% values were established using DMSO without inhibitors.

2.9. Kinetics parameters BAPNA prepared in DMSO was used as substrate (final concentration from 0.1 to 1 mM), in a total volume of 170 mL, at pH 8 (0.2 M Tris–HCl) in a 96-well microtitre plate. The reaction was started by addition of 30 mL of purified enzyme solution (420 mg protein/mL). It is important to register that previous experiment showed that the DMSO showed no effect on tilapia’s tryptic activity. The increase in absorbance at 405 nm was followed using a microtitre plate reader. The reaction rates obtained were fitted to Michaelis-Menten kinetics using Enzyme Fitter Software. Each reaction was performed in triplicate. The blanks were prepared similarly to the samples but, without enzyme or substrate solution.

3. Results The purification of an alkaline protease from Nile tilapia intestine in three steps is summarized in Table 1. In the first step (heat treatment of the intestine crude extract), a negligible change occurred in the purification but its inclusion enhanced the next step. From the three fractions obtained in the ammonium sulphate precipitation, F2 (30–80% saturation) showed a higher specific activity (22.98 U/mg of protein) than F1 (0–30% saturation; 8.32 U/mg of protein). No activity was detected in FS (final supernatant); however, it contained the highest amount of protein (about 70% of the total protein). Sephadex G75 chromatography of F2 fraction (third step) resulted a preparation (Fig. 1; second peak) with purification and yield of 22-fold and 30.0%, respectively, which showed only one polypeptide band under SDS-PAGE (Fig. 2). An apparent molecular weight of 23.5 kDa was calculated to this polypeptide by using standards on gel electrophoresis. This partial purified enzyme showed an optimum temperature of 50 8C (Fig. 3A), and was stable at this temperature for 30 min (Fig. 3B). High enzymtic activity was

Fig. 1. Elution pattern of the ammonium sulphate fraction (30–80% saturation) on sephadex G-75 filtration chromatography. A sample (5 mL containing 8 mg of protein) was applied on the top of a column measuring 1.2 cm  26 cm, eluted at a flow rate of 20 mL/h with 0.9% (w/v) NaCl and collected as 2 mL fractions. In each fraction protein and activity were, respectively, estimated at 280 and 450 nm (soluble coloured products released from azocasein).

detected in the pH range from 7.0 to 10; however, the optimum pH was 8.0 (Fig. 3C). The effect of metal ions on the activity of OniT was investigated (Table 2). This enzymtic activity was inhibited by almost all metal ions used. The notable ones were Al3+ and Cd2+, followed by Cu2+, Hg2+, Zn2+ and Co2+. The effects of Ba2+, Ca2+, K+, Li+, Mg2+, and Mn2+ were noticeable but not extreme. The effect of seven different inhibitors on the alkaline protease from Nile tilapia intestine is showed in Table 3. It was inhibited (approximately 55%) by PMSF, a potent serine protease inhibitor. TLCK and benzamidine, both synthetic trypsin inhibitors, showed strong inhibition effect (100 and 87.5%, respectively). TPCK, a synthetic chymotrypsin inhibitor, did not display any inhibitory effect. C. mollis trypsin inhibitor also inhibited (approximately 67%) the enzyme preparation. Furthermore, the enzymatic activity was decreased (38.5%) by b-mercaptoethanol. EDTA was capable of increasing this proteolytic activity by approximately 30%. The rates of BAPNA hydrolysis obeyed Michaelis-Menten kinetics over the concentration of substrate examined.

Table 1 Purification in three steps of trypsin-like enzyme from Nile tilapia intestine Samples

Total protein (mg)

Crude extract 365.48 Step 1: heat treatment Heated crude extract 354.00 Step 2: ammonium sulphate precipitation 1.68 F1, (NH4)2SO4 (0–30%) F2, (NH4)2SO4 (30–80%) 18.68 FS, (NH4)2SO4 (final) 252.78 Step 3: gel filtration chromatography Sephadex G75 5.06

Total activity (U)

Specific activity (U/mg of P)

Yield (%)

Purification

1239

3.39

100.0

1

1283

3.63

103.6

1.07

14 429 0

8.32 22.98 0

1.1 34.7 0

2.45 6.78 0

372

73.51

30.0

21.68

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Fig. 2. SDS polyacrylamide gel electrophoresis of intestine purified trypsin from Nile tilapia. It was carried out according to Laemmli [17], using a 6% (w/v) stacking gel and a 12.5% (w/v) separating gel. The gels were stained in 0.01% (w/v) Coomassie Brilliant Blue and destained by washing in 10% (v/v) acetic acid. The molecular weight was estimated using the protein standards (Sigma) bovine albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36.0 kDa), carbonic anhydrase (29 kDa), trypsinogen (24.0 kDa) and a-lactalbumin (14.2 kDa).

The Km and Vm values of OniT on BAPNA were 0.772  0.009 mM and 3.1  0.2 mU, respectively.

4. Discussion The purification of trypsin-like enzymes from fishes deserves considerable attention, since proteases constitute the most important class of industrial enzymes that are present in viscera, an important by-product of fishery industries [12,13,20]. It is also important to notice that the viscera account approximately 30% of the fish total weight. The total Brazilian tilapia’s production was about 40,000 tons in 2000 [1]. Therefore, almost 12,000 tons of Nile tilapia viscera are discarded in Brazil and they could be used as relevant biomolecule source. Bezerra et al. [15] reported a method for purification of a trypsin-like enzyme from tambaqui (Colossoma macropomum), based on the thermostability of this enzyme [21] and composed of three steps: heat treatment, ammonium sulphate fractionation and Sephadex gel filtration. The pretreatment enhanced the ammonium sulphate fractionation. Similar procedure followed for the crude extract from the tambaqui pyloric caeca was efficient to isolate a trypsin-like enzyme from O. niloticus intestine (Figs. 1 and 2). This enzyme had a molecular weight value of 23.5 kDa. It is within the fish trypsin range of 22.5–31.4 kDa [22]. Heat treatment has been proved to be an important strategy in fish protease purification, because it denatures and removes distinct heat-labile proteins in the crude extract [15]. More-

Fig. 3. The effect of temperature (A), thermal stability (B) and pH (C) on O. Niloticus intestine trypsin. The purified enzyme collected from the Sephadex G-75 filtration was incubated with azocasein (quadruplicates) at the indicated temperatures and pH for 60 min and after stopping the reaction with trichloroacetic the soluble coloured products were measured at 450 nm. The thermal stability was determined by assaying (quadruplicates) its activity (25 8C) after pre-incubation for 30 min at the indicated temperatures. The values (mean  S.D.) were expressed as percents of the highest one.

over, it is responsible for a significant breakdown (proteolytic action) of other undesired thermostable proteins, which turn them in more hydrophilic peptides. A trypsin from other important freshwater fish to tropical Aquaculture, common carp, Cyprinus carpio, was purified by using: DEAE Cellulose (anionic fraction) and affinity chromatography on a PABA-Sepharose column [23]. A trypsin from hybrid tilapia was also purified by affinity chromatography on soybean trypsin inhibitor bound to 4% beaded agarose column [10].

R.S. Bezerra et al. / Process Biochemistry 40 (2005) 1829–1834 Table 2 Effect of various metal ions on activity of trypsin-like enzyme from Nile tilapia intestine Ion (mM)

1

10

Controla Al3+ Cd2+ Cu2+ Zn2+ Hg2+ Co2+ Mn2+ Ca2+ Ba2+ K+ Mg2+ Li+

100.0 39.9 43.2 37.1 38.4 73.4 68.3 65.4 67.2 71.0 76.4 74.0 105.5

100.0 0 3.0 43.9 30.5 38.0 55.9 69.1 76.7 67.4 71.1 89.8 74.2

a

Proteolytic activity without any of these ion solutions.

OniT (50 8C) demonstrated an optimum temperature similar to mullet Mugil cephalus (50 8C), tambaqui C. macropomum (60 8C) and hybrid Tilapia nilotica/aurea (40 8C); however, it was observed that the Nile tilapia and tambaqui enzymes (50 and 55 8C) presented more thermal stability than mullet that lost about 30% of its activity at 50 8C [10,15,20]. These proteinases have optimum temperature slightly higher than those commonly reported for trypsins from other fish species (45 8C) [13]. These optimum temperatures can be explained to the fact that Nile tilapia, tambaqui and mullet live in warm waters, while most of the species reported live in cold waters. Hidalgo et al. [24] have studied the influence of temperature on the proteolytic activity of several freshwater fish species. They have found a decrease in this enzymtic action at environmental temperatures (25 and 15 8C to trout) in relation to 37 8C. OniT presented high proteolytic activity in the pH range from 7.0 to 10.0, with an estimated maximum at pH 8.0, similar to that reported for mullet [20]. Also, trypsin from Table 3 Effect of inhibitors on the activitya of trypsin-like enzyme from Nile tilapia intestine Inhibitor

Concentration

Inhibition [%]

PMSF TLCK TPCK EDTA CmTIb Benzamidine b-Mercaptoethanol

1 mM 1 mM 1 mM 1 mM 40 mg/mL 1 mM 1 mM

54.7 100.0 0 29.3 66.7 87.5 38.3

PMSF: phenylmethylsulfonylfluoride; TPCK: tosyl phenylalanine chloromethyl ketone; TLCK: tosyl lysine chloromethyl ketone; CMTI: C. mollis trypsin inhibitor; EDTA: ethylenediaminetetraacetic acid. a Trypsin was assayed for amidase activity at 25 8C as described in Section 2. A solution of 0.6 mM benzoyl-DL-arginine-p-nitroanilide (BAPNA) was used as substrate. Data shown in the table represent average values from quaduplicate determinations. b Inhibitor obtained from camaratu´ bean (Cratylia mollis) according to Paiva [19].

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pyloric caeca of tambaqui showed similar profile except that the optimum pH was equal at 9.5 [15]. El-Shemy and Levin [10] found an optimum pH of 9.0 for affinity-purified enzyme from hybrid tilapia T. mossambica/aurea. These values are common to fish trypsins [13]. This enzymatic property can be useful for specific technological applications, as for example in laundry detergents. The effect of metal ions on the activity of OniT shows that the majority metal ions inhibited the enzyme to various extents, and the effect was amplified with the increase of concentration to 10 mM (Table 2). It is known that Cd2+, Co2+ and Hg2+ act on sulphhydryl residues in proteins [25]. Inhibition caused by these metal ions suggests the relevance of sulfhydryl residues for the catalytic action of this protease. This is confirmed by b-mercaptoethanol inhibition (Table 3). The Ca2+ inhibition effect showed to be contradictory since this is a classical trypsin activator. This same effect has been observed for trypsins from tambaqui C. macropomum and spotted goatfish Pseudupeneus maculatus (unpublished data). Thermostable dipeptidase from common carp (Cyprinus carpio) intestine [25] and the trypsin from an aquatic invertebrate starfish (Asterina pectinifera) have been also reported as not activated by Ca2+ addition [26]. These findings suggest that a difference in the structure of the primary calcium-binding site may exist between mammalian pancreatic trypsin and Nile tilapia, tambaqui and spotted goatfish trypsin. Furthermore, the increase of OniT activity in presence of 1 mM EDTA showed an activation effect (Table 3). Probably, it should be due to its chelating action on metal presents in the assay mixture. The strong inhibitory effects of TLCK and benzamidine on the OniT indicates the involvement of a histidine residue at its active centre and provides an additional indication that the binding site exhibited resemblance to traditional mammalian trypsin (Table 3). Table 4 summarizes the Michaelis-Menten constant of the amidase activity from OniT (0.755 mM) compared to bovine and other tropical fish trypsin. It presents higher value than common carp (0.039 mM) and mullet (0.490 mM). However, it showed better affinity to BAPNA than bovine (0.939 mM) and hybrid tilapia trypsin. All these information emerged in this contribution indicate that OniT has characteristics compatible with other

Table 4 Michaelis-Menten constant (Km) of trypsin-like from Nile tilapia compared to other species trypsins Species

Km (mM)

References

Bovine Common carp Mullet Hybrid tilapia Nile tilapia

0.939 0.039a 0.490 2.5 0.755

[18] [27] [20] [10] This work

BAPNA as substrate at 25 8C. a Assayed at 30 8C.

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trypsin and could be isolated at low cost from the large amount of viscera produced as waste in tilapia industrial processing. Furthermore, this enzyme could be used to produce fish protein hydrolysates [12,28]; fish sauce [29]; shrimp waste recovery [30] and as a laundry detergent additive [31].

Acknowledgements The authors would like to thank Mr. Otaviano Tavares da Costa and Albe´ rico Espı´rito 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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