Trypsins from fish processing waste: characteristics and biotechnological applications – comprehensive review

Journal of Cleaner Production 57 (2013) 257e265

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Review

Trypsins from fish processing waste: characteristics and biotechnological applications e comprehensive review Ali Bougatef* Higher Institute of Biotechnology of Sfax, Route Sokra Km 4, BP. 1175, 3038 Sfax, Tunisia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 October 2012 Received in revised form 2 June 2013 Accepted 4 June 2013 Available online 19 June 2013

Today, there is an increasing demand for fish proteolytic enzymes in food industries. During processing, large quantities of waste are generated and discarded. These wastes, which represent an environmental problem to the fishing industry, constitute an important source of proteolytic enzymes and protein. The most important digestive enzymes from fish and aquatic invertebrate viscera are trypsins. These enzymes have a high activity over a wide range of pH and temperature conditions and exhibit high catalytic activity at relatively low concentration. These characteristics have made them suitable for different applications in many food processing operations. Considering the specific characteristics of these enzymes, fish processing by-products are currently used for trypsins extraction. This review describes the characteristics and various applications of fish trypsins in detergents, carotenoproteins extraction from shrimp waste, and protein hydrolysates production. Considering their biological significance and their increasing importance in biotechnology, a thorough understanding of fish trypsins functioning is needed. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Trypsin Fish processing waste Biotechnological applications Biochemical characteristics

1. Introduction Proteases constitute the most important group of industrial enzymes used in the world today, accounting for about 50% of the total industrial enzyme market (Turk, 2006). They have diverse applications in a wide variety of industries, such as the detergent, food, agrochemical and pharmaceutical industries (Gupta et al., 2002; Zukowski, 1992). Proteases are derived from animal, plant and microbial sources. Seafood by-products and waste constitute at present a serious environmental problem (Arvanitoyannis and Kassaveti, 2008); those by-products and waste require appropriate management, especially because they are highly perishable. Fish viscera constitute approximately 20% of the marine biomass and are a rich source of digestive proteinases. If not used, this biomass would be discarded either as waste or as low value by-products, which would generate additional waste disposal and environmental problems. The recovery of proteinases from fishery by-products would, therefore, be of great importance since it would not only alleviate the serious concerns related to the management of visceral waste but would also help produce novel low-cost proteinases for industrial application (Simpson and Haard, 1999).

* Tel.: þ216 74 674 354; fax: þ216 74 674 364. E-mail addresses: [email protected], [email protected]. 0959-6526/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jclepro.2013.06.005

Fishes are poikilothermic, so their survival in cold waters required adaptation of their enzyme activities to low temperatures of their habitats. Enzymes from cold adapted fish species thus often have higher enzymatic activities at low temperatures than their counterparts from warm-blooded animals (Asgeirsson et al., 1989; Kristjansson, 1991). High activity of fish enzymes at low temperatures may be interesting for several industrial applications of enzymes, such as in certain food processing operations that require low processing temperatures. Furthermore, relatively lower thermal stability, often observed with fish enzymes, may also be beneficial in such applications as the enzymes can be inactivated more readily, with less heat treatment, when desired in a given process (Simpson and Haard, 1987; Kishimura et al., 2008). In EC system for enzyme nomenclature, all proteases (peptide hydrolases) belong to subclass 3.4, which is further divided into 3.4.11-19, the exopeptidases and 3.4.21-24, the endopeptidases or proteinases (Garcia-Carreno and Haard, 1993). Endopeptidases cleave the polypeptide chain at particularly susceptible peptide bonds distributed along the chain, whereas exopeptidases hydrolyse one amino acid from N terminus (aminopeptidases) or from C terminus (carboxypeptidases). Exopeptidases, especially aminopeptidases, are ubiquitous, but less readily available as commercial products, since many of them are intracellular or membrane bound. The most important digestive proteases of fish viscera are acid stomach enzymes and alkaline intestine enzymes. The main alkaline enzymes in fish viscera are trypsin, chymotrypsin and elastase,

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all belonging to the serine-protease family (EC. 3.4.21). They are characterized by serine, histidine and aspartic residues at the active site. As a group, serine proteases are inhibited by di-isopropyl phosphofluoride (DFP), they are active at neutral and alkaline pH, and inactive or unstable in acid pH (Simpson, 2000; Fuchise et al., 2009). Trypsins are present in fish as isoenzymes and they all have essentially the same specificity. They cleave the peptide bond on the carboxyl side of arginine and lysine, and have endopeptidase activity and molecular weight ranging from 22 to 28 kDa. They hydrolyze synthetic substrates like benzoyl-L-arginine-p-nitroanilide and tosyl-L-arginine methyl ester and are susceptible to the serine-protease inhibitors phenyl-methyl-sulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI) and aprotinin. They are optimally active at pH 7.5e10 and 35e45  C. They are unstable at lower temperatures and extreme pH (Whitaker, 1994). Trypsin is one of the major digestive enzymes, and it belongs to the serine protease family of enzymes with Enzyme Commission number EC 3.4.21.4. Trypsins act by cleaving the ester and peptide bonds involving the carboxyl groups of arginine or lysine. Pancreatic trypsins (and trypsinogens) from terrestrial animals (e.g., mammals and birds) have been well characterized and their structures are elucidated (Sweet et al.,1974). Examples of mammalian trypsins that have been studied include those from sheep (Schyns et al., 1969), bovine (Zwilling et al., 1969), dog (Pinsky et al., 1985), human (Johnson and Travis, 1976), rat (Gendry and Launay, 1988), pig (Charles et al., 1963) and horse (Johnson al., 2002). Avian trypsins that have been described include those from turkey (Ryan, 1965), ostrich (Bodley et al., 1995) and chicken (Guyonnet et al., 1999). An interesting alternative to convert the fish processing wastes into more marketable and acceptable products is to isolate and purify trypsins which can be used in the food industry or in fish protein hydrolysate preparation. The purpose of this review is to provide an overview of trypsins from fish processing waste and to discuss their potential uses in biotechnological applications. 2. Marine proteases Enzymatic processes have been implemented in a broad range of industries in recent decades because they are specific, fast in action and often save raw materials, energy, chemicals and/or water compared to conventional processes (Jegannathan and Nielsen, 2012). Alkaline proteases are robust enzymes with considerable industrial potential in detergents, leather processing, silver recovery, medical purposes, food processing, feeds, and chemical industries, as well as waste treatment. These enzymes contribute to the development of high value-added applications or products by using enzyme-aided digestion. Proteases for industrial use are produced by growing bacteria and fungi in submerged or solid state fermentation. With submerged being the primary fermentation mode, the unit operations in enzyme production involve fermentation followed by cell disruption and filtration. The crude enzyme is further purified by precipitation followed by centrifugation and vacuum drying or lyophilization, collectively known as “downstream processing” (Soetaert and Vandamme, 2010). Proteases constitute at present the dominant group of marine enzymes with a commercial expression. Gastric, intestinal and hepatopancreas proteinases have been scrutinized more thoroughly (Beynon and Bond, 2001); pepsin from polar cod is one of the most extracted gastric proteases, followed by collagenase, elastase, trypsin and chymotrypsin, as well as non proteolytic enzymes, such as transglutaminase, lipases and chitinolytic enzymes to lower extents (Rasmussen and Morrissey, 2007). Trypsin is the first discovered and probably the best characterized enzyme. It has been known for more than 130 years that

pancreatic juice is able to digest proteins (Graf and Szilágyi, 2003). This protein was among the first proteolytic enzymes isolated in pure form in enough amounts for exact chemical and enzymological studies. The structure of trypsin molecule and the mechanism of its action have been studied in considerable detail. Therefore, it provides an important and widely used model system for kinetic as well as physical and biophysical studies. 3. Characteristics of fish trypsins Over the past few decades, trypsin and trypsin-like enzymes have been isolated and identified in a wide array of cold water as well as warm water fish species. Trypsin and trypsin-like proteolytic enzymes have been purified and characterized in several fish species including stomachless bone fish Carassius auratus gibelio (Bloch) (Jany, 1976), sardine (Murakami and Noda, 1981), capelin (Hjelmeland and Raa, 1982), Greenland cod (Simpson et al., 1989), cunner (Simpson et al., 1989), Atlantic cod (Amiza et al., 1997; Asgeirsson et al., 1989; Han, 1993; Simpson et al., 1989), chum salmon (Uchida et al., 1984a, 1984b), Atlantic salmon (Male et al., 1995; Schroder et al., 1998), coho salmon (Haard et al., 1996), anchovy (Martinez et al., 1988), Atlantic white croaker (Pavlisko et al., 1997b), carp (Cyprinus carpio) (Cao et al., 2000), arabesque greenling (Pleuroprammus azonus) (Kishimura et al., 2006a) (Table 1). 3.1. Effect of pH and temperature on trypsins activities and stabilities Trypsins from marine animals resemble mammalian trypsins with respect to their molecular size (22e30 kDa), amino acid composition and sensitivity to inhibitors. Their pH optima for the hydrolysis of various substrates were from 7.5 to 11.0, while their temperature optima for hydrolysis of those substrates ranged from 35 to 65  C (De Vecchi and Coppes, 1996). In addition, fish trypsin is more sensitive to inactivation by heat, low pH, and autolysis than that of mesophilic analogues (Asgeirsson et al., 1989; Simpson and Haard, 1987). These enzymatic properties of fish trypsin are interesting for several industrial applications, such as in certain food processing operations that require low processing temperatures. Indeed, Atlantic cod trypsin has already been used in industrial applications (Bjarnason and Benediktsson, 2001; Bjarnason, 2000) and medical applications (Bjarnason, 2000). 3.1.1. pH Trypsins from marine animals tend to be more stable at alkaline pH, but are unstable at acidic pH. On the other hand, mammalian trypsins are most stable at acidic pH (Simpson, 2000). Ktari et al. (2012) purified a trypsin from zebra blenny (Salaria basilisca) viscera, which was active between pH 8.0 and 11.0, with an optimum around pH 9.5. This trypsin was highly stable over a wide pH range, maintaining 100% of its original activity between pH 7.0 and 12.0 (Table 2). Trypsin from tongol tuna spleen showed the high stability in the pH range of 6.0e11.0, but the inactivation was more pronounced at pH values below 6.0 (Klomklao et al., 2006a). Klomklao et al. (2007a) also reported that skipjack tuna spleen trypsins were stable in the pH ranging from 6.0 to 11.0 but were unstable at pH below 5.0. Elhadj-Ali et al. (2009) studied the effect of pH on the activity of striped seabream (Lithognathus mormyrus) trypsin and found that the purified enzyme was highly active between pH 7.0 and 12.0, with an optimum between pH 9.0 and 10.5. Striped seabream trypsin is highly stable over a wide pH range, maintaining more than 90% of its original activity between pH 5.0 and 12.0. Similar results were reported by Jellouli et al. (2009) for trypsin

A. Bougatef / Journal of Cleaner Production 57 (2013) 257e265

259

Table 1 Biochemical properties of different fish trypsins. Fish

MW (kDa)

Temperature ( C)

pH

Substrate

References

Barbel (Barbus callensis) Zebra blenny (Salaria basilisca) Pirarucu (Arapaima gigas) Sardinelle (Sardinella aurita)

24 27 28 28.8 28.8 28.8 24.0 23.0 22.0 28.4 24.0 23.0 24.0 24.0 23.9 23.2 23 24 25 28.0 24.0 24.0 24.0 24.0 24.0 24.0 23.8 28.0 24.0 24.0 24.0 24.0 25.0 38.5 24.0

55 60 65 55 50 55 60 55 60 45 60 60 50 50 50 40 50 45 60 55 60 50 65 55 65 60 55 60 60 50 60 60 50 60 65

10.0 9.5 9.0 9.0 9.0 9.0 8.5 9.0 9.0 9.0 8.5 8.5 8.5 8.0 9.0 10.5 10.0 8.5 8.0 9.5 8.0 8.0 8.5 8.5 8.5 8.0 8.0e11.0 8.0 8.0 8.0 8.0 8.0 8.0 9.5 8.5

BAPNAa BAPNA BAPNA BAPNA

Sila et al. (2012) Ktari et al. (2012) Freitas-Júnior (2012) Ben Khaled et al. (2011)

TAMEb BAPNA Casein BAPNA TAME BAPNA TAME TAME z-FR-MCAc BAPNA BAPNA TAME Casein BAPNA TAME TAME TAME TAME

Klomklao et al. (2010) Barkia et al. (2010) Wang et al. (2010) Espósito et al. (2010) Kanno et al. (2010) Khantaphant and Benjakul (2010) Bougatef et al. (2010) Kishimura et al. (2010) Marcuschi et al. (2010) Jellouli et al. (2009) Elhadj-Ali et al. (2009) Klomklao et al. (2009) Bougatef et al. (2007) Klomklao et al. (2007c) Kishimura et al. (2007) Kishimura et al. (2007) Klomklao et al. (2006a) Klomklao et al. (2006b)

TAME BAPNA BAPNA TAME TAME TAME

Kishimura et al. (2006b) Hau and Benjakul (2006) Kurtovic et al. (2006) Kishimura et al. (2006c) Kishimura et al. (2006c) Kishimura et al. (2005)

BAPNA Azocasein BAPNA

Castillo-Yanez et al. (2005) Bezerra et al., 2000 Pavlisko et al. (1997a)

Pacific saury (Cololabis saira) Bogue (Boops boops) Hybrid tilapia (O. niloticus  O. aureus) Lane snapper (Lutjanus synagris) Masu salmon (Oncorhynchus masou) Brownstripe red snapper (Lutjanus vitta) Smooth hound (Mustelus mustelus) Threadfin hakeling (Laemonema longipes) Amazonian fish tambaqui (Colossoma macropomum) Grey triggerfish (Balistes capriscus) Striped seabream (Lithognathus mormyrus) Pectoral rattail (Coryphaenoides pectoralis) Sardine (Sardine pilchardus) Bluefish (Pomatomus saltatrix) Jacopever (Sebastes schlegelii) Elkhorn sculpin (Alcichthys alcicornis) Tongol tuna (Thunnus tonggol) Yellowfin tuna (Thunnus albacores) Spotted mackerel (Scomber australasicus) Bigeye snaper (Priacanthus macracanthus) Chinook salmon (Oncorhynchus tshawytscha) Yellowtail (Seriola quinqueradiata) Brown hakeling (Physiculus japonicus) Japanese anchovy (Engraulis japonica) Monterey sardine (Sardinops sagax caerulea) Tambaqui (Colossoma macropomum) Palometa (Parona signata) a b c

N-a-benzoyl-DL-arginine-p-nitroanilide. N-a-tosyl-L-arginine methyl ester. carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin.

isolated from grey triggerfish intestines. Bougatef et al. (2007, 2010) found optimum pH activities for sardine (Sardina pilchardus) and smooth hound (Mustelus mustelus) trypsins to be 8.0 and 8.5, respectively. The stability of trypsins at a particular pH might be related to the net charge of the enzyme at that pH (Castillo-Yanez et al., 2005). Trypsin might undergo the denaturation under acidic conditions, where the conformational change took place and enzyme could not bind to the substrate properly (Klomklao et al., 2006b). 3.1.2. Temperature Temperature has a significant effect on the activity and stability of trypsins. Freitas-Júnior et al. (2012) purified a trypsin extracted

from giant Amazonian fish pirarucu (Arapaima gigas) viscera which showed an optimal temperature of 65  C. The purified trypsin was stable at temperature range 25e55  C for 30 min, losing only about 10% of its activity at 60  C (Table 2). Bezerra et al. (2001) reported an optimal temperature of 65  C for trypsin extracted from tambaqui (Colossoma macropomum) and observed that the enzyme was stable at 55  C up to 30 min. In the same context, Bougatef et al. (2007) purified a trypsin from sardine viscera which retained more than 80% of its initial activity after 4 h of incubation at 50  C. Additionally, two trypsins, TR-S and TR-P, were purified from the viscera of true sardine (Sardinops melanostictus) and pyloric ceca of arabesque greenling (Pleuroprammus azonus). Optimum temperatures of TR-S and TR-P were 60  C and 50  C, respectively

Table 2 Thermal and pH stability of different fish trypsins. Fish

Residual activity (%) 

Barbel Zebra blenny Sardinelle Pacific saury Bogue Lane snapper Brownstripe red snapper Smooth hound Grey triggerfish Striped seabream Pectoral rattail

References 

40 C (1 h)

50 C (1 h)

pH stability

95 100 70 100 (30 min) 80 100 (15 min) 100 (30 min) 94 100 100 100

88.6 65 41 e 60 100 (15 min) 100 (30 min) 73 78 14.45 90

5.0e12.0 7.0e12.0 7.0e10.0 e 6.0e11.0 7.0e11.0 7.0e10.0 7.0e10.0 6.0e12.0 5.0e12.0 6.0e11.0

Sila et al. (2012) Ktari et al. (2012) Ben Khaled et al. (2011) Klomklao et al. (2010) Barkia et al. (2010) Espósito et al. (2010) Khantaphant and Benjakul (2010) Bougatef et al. (2010) Jellouli et al. (2009) Elhadj-Ali et al. (2009) Klomklao et al. (2009)

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A. Bougatef / Journal of Cleaner Production 57 (2013) 257e265

(Kishimura et al., 2006a). Similar optimum temperatures were detected for trypsins extracted from M. mustelus (Bougatef et al., 2010), Boops boops (Barkia et al., 2010), S. basilisca (Ktari et al., 2012) and Sardinella aurita (Ben Khaled et al., 2008) (Table 2). High activity of trypsin at low temperatures may be interesting for many biotechnological and food processing applications (Haard, 1992). Two anionic trypsins (trypsin A and trypsin B) purified from the hepatopancreas of carp had optimal activity at 40 and 45  C, respectively (Cao et al., 2000). Jellouli et al. (2009) purified a trypsin acting at low and moderate temperatures with an optimum around 40  C and retained more than 80% of its maximum activity at 20  C. The optimum temperature for Balistes capriscus trypsin was similar to those of trypsins from cold-water fish, which had optimal temperatures in the range of 40e45  C (Lu et al., 2008; Simpson, 2000). 3.2. Effect of metal ions on trypsins activities Some enzymes require an additional chemical component (cofactor), such as inorganic ions, to be active. Silva et al. (2011) studied the effect of some metal ions on enzyme activity and found higher trypsin activity than the control (100%) when incubated in the presence of Kþ (34%), Liþ (46%) and Ca2þ (83%). FreitasJúnior et al. (2012) investigated the effect of metal ions at a concentration of 1 mM. At this concentration, the ions Kþ, Mg2þand Ba2þ did not promote any significant effect on enzyme activity. The ion Ca2þ has been reported in the literature as a trypsin activator in several organisms, especially mammals. However, pirarucu trypsin was slightly inhibited in the presence of low concentrations of this ion (1 mM). This same effect has been observed for trypsins from other tropical fish, such as Nile tilapia (Oreochromis niloticus) (Bezerra et al., 2005) and spotted goatfish (Pseudupeneus maculatus) (Souza et al., 2007). These findings point to a possible difference in the structure of the primary calcium binding site between mammalian pancreatic trypsin and the trypsin from these fish (Bezerra et al., 2005). A recent study, based on the use of fluorescent protease substrates and commercial inhibitors has indicated that fish trypsins may differ in structure and catalytic mechanism, when compared to mammalian enzymes (Marcuschi et al., 2010). Previous studies have shown that trypsin-like enzymes from other tropical fish also showed sensitivity to metallic ions (Bezerra et al., 2001, 2005; Bougatef et al., 2007; Souza et al., 2007), especially Cd2þ, Al3þ, Zn2þ, Cu2þ, Pb2þ and Hg2þ. It is known that Cd2þ, Co2þ and Hg2þ act on sulphhydryl residues in proteins. Bezerra et al. (2005) reported that the strong inhibition promoted by these metallic ions demonstrates the relevance of sulfhydryl residues in the catalytic action of this protease. The mechanism of the inhibitory action of certain ions is not clear. Metal ions may influence binding of substrate with enzyme by various mechanisms. For example, binding of metal ions with enzyme or substrate, or both, can lead to the formation of active complexes easily degraded to final products. Probably Agþ and Hg2þ ions affect electric charge of the molecules of reagents, which

can increase the ‘‘contact phase’’ between substrate and enzyme. Similar results were reported by Bougatef et al. (2010); Jellouli et al. (2009) and Ktari et al. (2012) who studied the inhibitory effect of certain metal ions on trypsins activities (Table 3). 3.3. Ca2þ dependent activities of trypsins Fish trypsin was stabilized by calcium ions similarly to porcine pancreatic trypsin (Kishimura et al., 2006a). Two calcium-binding sites are present in the bovine trypsinogen. The primary site, with a higher affinity for calcium ions, is common in trypsinogen and trypsin, and the secondary site is only found in the zymogen. The occupancy of the primary calcium-binding site stabilizes the protein toward thermal denaturation or autolysis (Kishimura et al., 2006a). The improvement in protease activity against thermal inactivation in the presence of Ca2þ may be explained by the strengthening of interactions inside protein molecules and by the binding of calcium ions to autolysis sites. In fact, many studies showed that serine-proteases contain binding site with a higher affinity for calcium ions, which play an important role in stabilizing the enzyme against thermal denaturation and autodegradation (Kishimura et al., 2007). The increased rate of proteolysis of proteases at elevated temperatures is one of the factors responsible for the rapid thermal inactivation of enzymes. Several works reported the role of calcium in enzyme stabilisation at high temperatures. The presence of calcium ions activates trypsinogen to trypsin and increases the thermal stability of the enzyme. This stabilizing effect is accomplished by a conformational change in the trypsin molecule, resulting in a more compact structure (Kim et al., 1994; Klomklao et al., 2004). Stabilization against thermal inactivation by calcium was also reported for the trypsins from true sardine (Kishimura et al., 2006a), bigeye snapper (Hau and Benjakul, 2006), pectoral rattail (Klomklao et al., 2009), eel (Yoshinaka et al., 1985) and rainbow trout (Kristjansson, 1991). Nevertheless, calcium ions did not show the enhancing effect on stability of trypsins from M. mustelus (Bougatef et al., 2010), B. boops (Barkia et al., 2010), S. basilisca (Ktari et al., 2012), S. aurita (Ben Khaled et al., 2008), sardine (Murakami and Noda, 1981), capelin (Hjelmeland and Raa, 1982) and Nile tilapia (Bezerra et al., 2005). These findings suggest a difference in the structure of the primary calcium binding site among different marine fish trypsins. 3.4. Kinetics parameters of trypsins Higher physiological efficiencies of fish trypsins contributed mainly by higher substrate-binding capacities (lower Km) than bovine trypsin may in fact be explained by the differences in their substrate-binding region. Electrostatic field has been proposed to increase the binding affinity by guiding the positively charged residues of the substrates efficiently into the specificity pocket (Gorfe et al., 2000). However, the importance of physiological efficiency

Table 3 Effect of various metal ions on trypsins activities. Relative activity (%)

Grey triggerfish Barbel Sardinelle Bogue Striped seabream Zebra blenny Silver mojarra Lane snaper

Ca2þ

Ba2þ

Zn2þ

Cu2þ

Mg2þ

Mn2þ

Hg2þ

Kþ

Naþ

References

100 100 103 100 114 114 183 115

92.6 95 48 99 60 70 108 72

97 68 63 82 117 0 28 56

100 75 e 86 110 0 69 13

105 98 65 76 100 86 e e

100 85.8 75 95 100 114 101 e

42.7 12.45 e 28 e 0 46 11

100 100 98 100 100 100 134 90.5

100 100 100 100 100 100 e e

Jellouli et al. (2009) Sila et al. (2012) Ben Khaled et al. (2008) Barkia et al. (2010) Elhadj-Ali et al. (2009) Ktari et al. (2012) Silva et al. (2011) Espósito et al. (2010)

A. Bougatef / Journal of Cleaner Production 57 (2013) 257e265

261

Table 4 Kinetic constants of different trypsins. Fish

Km

Kcat

Kcat/Km

Substrate

References

Barbel Zebra blenny Sardinelle Pacific saury Bogue Brownstripe red snapper Smooth hound Grey triggerfish Striped seabream

0.6 0.6 1.67 0.17 0.13 0.507 0.387 0.068 0.29

1.38 1.32 3.87 200 1.56 4.71 2.67 2.76 1.36

2.3 2.3 2.31 117.47 12 9.27 6.89 40.58 4.68

BAPNAa BAPNA BAPNA TAMEb BAPNA BAPNA TAME BAPNA BAPNA

Sila et al. (2012) Ktari et al. (2012) Ben Khaled et al. (2011) Klomklao et al. (2010) Barkia et al. (2010) Khantaphant and Benjakul (2010) Bougatef et al. (2010) Jellouli et al. (2009) Elhadj-Ali et al. (2009)

a b

N-a-benzoyl-DL-arginine-p-nitroanilide. N-a-tosyl-L-arginine methyl ester.

(kcat/Km) for a digestive enzyme, which functions in vivo at relatively high substrate concentration with zero order reaction kinetics, is unclear. It is assumed that an enzyme adapted to working at low temperatures needs a more flexible structure, which allows conformational changes to occur upon substrate binding despite the low thermal energy of the surroundings (Low and Somero, 1974). Such structural flexibility could be brought about by low intramolecular interaction (Hochachka and Somero, 1984) or reduced hydrophobic interaction (Smalås et al., 1994). However, Smalås et al. (1994) did not find any overall differences in intramolecular interaction or dynamic properties between salmon trypsin and its mammalian equivalent. Thus the molecular and biochemical bases of higher catalytic properties of cold-adapted fish trypsin appear to be more complex and await further elucidation. Michaelis constant (Km) indicates the affinity of the enzyme to the substrate, Kcat indicates molecular catalytic constant and kcat/ Km indicates its catalytic efficiency. A higher molecular activity (Kcat) denotes a greater amount of substrate molecules that are converted into product by a single enzyme. Table 4 summarizes kinetic constants of different fish trypsins. 3.5. Trypsins N-terminal sequences alignment N-terminal amino acid sequences are useful as tools to identify the type of enzymes and may be useful for designing primers for cDNA cloning of enzyme (Cao et al., 2000). Table 5 showed N-terminal amino acid sequences of different fish trypsins. Generally, fish trypsins had a charged Glu residue at position 6, where Thr is most common in mammalian pancreatic trypsins. Moreover, the Table 5 Alignment of the N-terminal amino acid sequence of different fish trypsins. Fish

N-terminal sequence

Reference

True sardine Atlantic salmon Jacopever (TR-J) Bluefish Smooth hound Arabesque geenling (TR-P) Common carp Tongol tuna Yellowfin tuna Skipjack tuna Mandarin fish Saffron cod Pacific cod Walleye pollock Zebra blenny Grey triggerfish Sardine Japanese anchovy Barbel Antarctic fish

IVGGYECKAYSQ IVGGYECKAYSG IVGGYECKPYSQ IVGGYECKPKSA IVGGYECKPHSQ IVGGYECTPHTQ IVGGYEXTPHSQ IVGGYECQAHSQ IVGGYECQAHSQ IVGGYECQAHSQ IVGGYECEAH– IVGGYECPRHSQ IVGGYECTRHSQ IVGGYECTKHSQ IVGGRECTEPSQ IVGGYECTPNST IVGGYECQPYSQ IVGGYECQKYSQ IVVGYECTPYSQ IVGGKECSPYSQ

Kishimura et al. (2006a) Outzen et al. (1996) Kishimura et al. (2007) Klomklao et al. (2007c) Bougatef et al. (2010) Kishimura et al. (2006a) Cao et al. (2000) Klomklao et al. (2006a) Klomklao et al. (2006b) Klomklao et al. (2006c) Lu et al. (2008) Fuchise et al. (2009) Fuchise et al. (2009) Kishimura et al. (2008) Ktari et al. (2012) Jellouli et al. (2009) Bougatef et al. (2007) Kishimura et al. (2005) Sila et al. (2012) Genicot et al. (1996)

sequence of all trypsins started with IVGG after limited proteolysis of inactive trypsinogen into the active trypsin form (Kanno et al., 2010, 2011a; 2011b). The first seven NH2-terminal amino acid residues (IVGGYEC) are conserved in the trypsin of all vertebrates. However, in mammals, glutamic acid (E) in position 6 is replaced by a threonine (T) (Roach et al., 1997; Huerou et al., 1990). The conservation of the NH2-terminal amino acid residues (isoleucine) is very important to trypsin activity, since it forms a salt bridge with the amino acid Asp-179 that promotes a molecular rearrangement, enabling the active conformation of the oxyanion hole in the trypsin (Hedstrom, 2002). Another important feature of protein structure is the disulfide bonds. There may be up to six bonds in the trypsin of vertebrates, one of which occurs between Cys-7 and Cys-142 (Roach et al., 1997). 3.6. Effect of bleaches and surfactants on trypsins stabilities The addition of proteases to detergents considerably increases the cleaning effect by removing protein stains and the consumption of surface active substances, thereby decreasing the pollution load (Rao et al., 2009; Moreira et al., 2002). L. mormyrus alkaline trypsin is stable in the presence of the nonionic surfactants like Tween 20 and Tween 80 and activity was increased in the presence of Triton X-100. However, L. mormyrus trypsin was less stable against the strong anionic surfactant (SDS) (Elhadj-Ali et al., 2009). L. mormyrus trypsin activity was little influenced by oxidizing agent, and retained about 72% and 40% of its activity after incubation for 1 h at 30  C in the presence of 0.2% and 1% sodium perborate, respectively (Elhadj-Ali et al., 2009). In the same context, Esposito et al. (2009), studied the trypsin activity from the lane snapper in the presence of nonionic (Tween 20 and Tween 80) and ionic (sodium choleate) surfactants. Alkaline trypsins from other fishes, like grey triggerfish and tambaqui were also stable to nonionic surfactants (Jellouli et al., 2009; Khantaphant and Benjakul, 2010) (Table 6). After 60 min of incubation with Tween 20 and sodium choleate, tambaqui trypsin retained 94.5% and 99.1% of its initial activity. Only sodium dodecyl sulphate (SDS) was capable of strongly inhibiting the enzyme after 60 min. Ionic surfactants, such as SDS, can bind to globular proteins, like trypsin, and form small aggregates around its polypeptide backbone, thus denaturing the protein native structure. Therefore, most enzymes from other fish are also not very stable in the presence of SDS and other similar surfactants (Ben Khaled et al., 2008; Jellouli et al., 2009; Ktari et al., 2012) (Table 6). 4. Biotechnological applications of fish trypsins 4.1. Detergent additive Alkaline proteases added to laundry detergents play a catalytic role in the hydrolysis of protein stains such as blood, milk, etc. An ideal detergent enzyme should be stable and active in the detergent

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Table 6 Effect of bleaches and surfactants on trypsins activities.

SDS (w/v) Triton X-100 5% (v/v) Tween 80 5% (v/v) Sodium perborate (w/v) H2O2 (v/v) Reference

Zebra blenny

Striped seabream

Grey triggerfish

Lane snapper

Sardinelle

68 (1%) 31 (5%) 100 86 91 (0.1%) 42 (1%) 75 (1%) 49 (5%) Ktari et al. (2012)

66 (1%) 23.5 (5%) 125.8 100 72.5 (0.1%) 40 (1%) e

28.8 23.8 125 115 96.4 86.8 79.8 39 Jellouli et al. (2009)

7.3

21

e 107.8 107.8

100 100 59 38.9 50

Elhadj-Ali et al. (2009)

solution for a long period of time. It should also maintain adequate temperature stability in order to be effective throughout a wide range of washing temperatures (Banik and Prakash, 2004). In order to be effective during washing, a good detergent protease must be compatible and stable with all commonly used detergent compounds such as surfactants, oxidizing agents and other additives, which might be present in the formulation (Kumar and Takagi, 1999; Gupta et al., 1999). Alkaline proteases, mainly trypsin and subtilisin, are the most important group of industrial enzymes, with applications in the leather, food and pharmaceutical industries as well as bioremediation processes (Anwar and Saleemuddin, 1998; Gupta et al., 2002). However, their major application (about 60% of all protease sold) is in the detergent industry. Biological detergents are commonly used in domestic laundry soaps because the enzymes provide the additional benefit of low temperature washes with improved cleaning performance. Studies of trypsins stabilities in commercial detergents have shown various differences. Ktari et al. (2012) show that S. basilisca trypsin is extremely stable in the presence of Dixan, Nadhif and Ariel after 1 h incubation at 40  C (Table 7). L. mormyrus trypsin retained more than 72% of its initial activity after 1 h incubation at 30  C in the presence of Axion, New Det, Dixan, Ariel and Nadhif, respectively. After 1 h at 40  C, lane snapper trypsin retained more than 60% of its activity in the presence of Surf detergent and retained about 50% of its activity in the presence of Ala and Bem-tevi detergents after 30 min. The Omo detergent inhibited the activity of the enzyme, with about 70% loss of activity after 30 min of incubation at 40  C (Table 7). Trypsins from fish waste processing demonstrate good potential for application in laundry detergents. Moreover, the economy in production would make this enzyme suitable for low-cost operations in the industry.

75 70 Espósito et al. (2010)

Ben Khaled et al. (2008)

carotenoids from shrimp waste using organic solvents (Sachindra et al., 2006) and vegetable oils (Sachindra and Mahendrakar, 2005). In crustaceans, carotenoids occur as carotenoproteins, which are stable complexes of carotenoids bound to a high-density lipoprotein (Shahidi et al., 1998). Studies have been carried out on the recovery of carotenoids from shrimp waste in the form of carotenoprotein by different techniques. Proteolytic enzymes were used to disrupt the proteinecarotenoid bond, thus, increasing carotenoid recovery (Chen and Meyers, 1982; Simpson and Haard, 1985; Cano-Lopez et al., 1987; Sila et al., 2012). Carotenoprotein has been isolated and characterized from several shellfish species, e.g., lobster (Homarus americanus) (Ya et al., 1991), shrimp (Pandulus borealis) (Simpson and Haard, 1985; Cano-Lopez et al., 1987), fairy shrimp (Streptocephlausdichotomus) (Velu et al., 2003), brown shrimp (Metapenaeus monoceros) (Chakrabarti, 2002), crayfish (Procambarus clarkia) (Cremades et al., 2003) and starfish (Linckia laevigata) (Clark et al., 1990). Simpson et al. (1992) have shown the effect of trypsin from bovine pancreas and Atlantic cod offal (crude cod enzyme and semi purified extracts) on the recovery of carotenoproteins from lobster waste. However, commercial trypsin afforded a higher yield of pigment than cod enzyme preparations. In the same context, Sila et al. (2012) reported that the addition of barbel trypsin to the shrimp shell homogenate was effective in improving the recovery of carotenoproteins. The results indicated that higher protein and carotenoid content recovery yields were obtained in shrimp shell homogenates treated with barbel trypsin. Similar results in isolation of carotenoproteins from crustacean processing discards by using fish trypsins were reported (Klomklao et al., 2007b; Chakrabarti, 2002; Cano-Lopez et al., 1987). 4.3. Production of protein hydrolysates

4.2. Carotenoproteins extraction from shrimp waste Shrimp waste has been explored as a source of carotenoid, protein, and chitin. Attempts have been made to recover

Enzymatic hydrolysis of proteins allows preparation of bioactive peptides and these can be obtained by in vitro hydrolysis of protein sources using appropriate proteolytic enzymes. The physico-

Table 7 Effect of various metal ions on trypsins activities. Commercial detergents

Zebra blennya

Striped seabreama

Grey triggerfishb

Lane snapperb

Dixan (Henkel, Spain) Nadhif Henkel-Alki, Tunisia) Ariel (Procter & Gamble, Switzerland) Axion (Colgate-Palmolive, France) New Det (Sodet, Tunisia) Ala (Procter & Gamble, Switzerland) Bem-te-vi (Alimonda) Surf (UniLever) Acão (UniLever) Reference

100 100 100 86 85 e e e e Ktari et al. (2012)

77 69 73.4 87.5 84 e e e e Elhadj-Ali et al. (2009)

90 38 100 100 80 e e e e Jellouli et al. (2009)

e e e e e 30 30 65 0 Espósito et al. (2010)

a b

trypsin was incubated 1 h at 30  C in the presence of solid detergents. trypsin was incubated 1 h at 40  C in the presence of solid detergents.

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chemical conditions of the reaction media, such as temperature and pH of the protein solution, must then be adjusted in order to optimize the activity of the enzyme used. Proteolytic enzymes from microbes, plants and animals can be used for the hydrolysis process of marine proteins to develop bioactive peptides (Simpson et al., 1998). Enzymes are biochemical catalysts vital for living beings, because they accelerate chemical reactions between organic constituents within the cells that otherwise would take an extremely long time to complete. Enzymes catalyse only one specific reaction and function by forming a complex with the substrate whose transformation they catalyse. Li et al. (2010) reported a comparative study between commercial enzymes (Alcalase and Delvo-Pro) and trypsin-like serine protease purified from the spleen of yellowfin tuna by membrane filtration. They showed that the purified trypsin-like could provide an equal degree of hydrolysis of casein comparing to Alcalase. In the same context, Klomklao et al. (2006c) investigated the hydrolysis of various sardine muscle proteins by a trypsin-like proteinase from skipjack tuna spleen. Bougatef et al. (2009) used a trypsin like-protease with other digestive proteases from smooth hound (M. mustelus) viscera for the generation of protein hydrolysates with antioxidative activity.

5. Recovery and purification of trypsins from fish waste The initial recovery steps of fish trypsins are: (i) extraction which includes preparation of crude material (entire viscera or separation of stomach and intestines), homogenization using a buffer to extract crude enzyme from the prepared material and centrifugation to separate the crude fish proteases and (ii) precipitation or fractionation to collect proteases. Precipitation is the most commonly used method for the isolation and recovery of proteins from crude biological mixtures (Bell et al., 1983). Chromatographic techniques have been used for partitioning trypsin mixtures depending on their affinity to either mobile or stationary phases. Once a crude trypsin extract is recovered, it must be purified using one of several chromatographic methods that can be classified as: gel filtration, ion exchange, hydrophobic interaction or affinity. It should be noted that these chromatographic techniques are commonly coupled together so that most purification schemes will begin with gel filtration, followed by either ion exchange or affinity chromatography or both, depending on the application. Many purification protocols can be used to purify trypsins from fish waste. Marcuschi et al. (2010) reported the purification of trypsin from the pyloric caecum of tambaqui (C. macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex-G-75 and p-aminobenzamidineeagarose affinity chromatography with a purification yield of 30.0%. In the same context, Silva et al. (2011) purified a trypsin from the viscera of the silver mojarra (Diapterus rhombeus) in a three step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex-G-75), with final specific activity 86fold higher than that of the enzyme extract and yield of 22.1%. A protocol based only on gel filtration separation (Sephacryl S-200 and Sephadex G-50) for trypsin purification from the pyloric ceca of walleye pollock (Theragra chalcogramma) was described by Kishimura et al. (2008). Ktari et al. (2012) reported the purification of an alkaline trypsin from the viscera of zebra blenny (S. basilisca) by ammonium sulphate (40e80% saturation) precipitation, Sephadex G-100, Mono Q-Sepharose and ultrafiltration with a yield of 12%. Interestingly, Bougatef et al. (2007) reported a one step trypsin purification from sardine (Sardina pilchardus) using gel filtration G-100.

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6. Opportunities and challenges for the future development of enzymes recovery technology According to the food and agriculture organization of the United Nations (FAO, 2010), more than 145.1 million ton of fish are actually caught or farmed annually worldwide. In 2010, the quantity of global releases was estimated at 24 million ton, i.e., about 16.54% of the total catches. Commercial fish production and seafood processing generate large amounts of fish waste, which create burdensome disposal problems and environmental concerns. Therefore, there is a great potential in marine bioprocess industry to convert and utilize more of these by-products as valuable products. The term by-products is however not clearly defined to distinguish from waste and in many instances it is identified as leftovers that are not ordinary saleable, but which can be recycled after treatment. To start up businesses based on the utilization of by-products, it is necessary to have stable and sustainable supply of raw material (Thorkelsson et al., 2009). By-products are highly perishable, and it is necessary to stabilise bulk fractions to produce products with a stable, reproducible quality. The use of marine by-products as sources of industrial enzymes is associated with some limitations such as seasonal availability, variations in the content and/or activity of enzymes due to nutritional status and the highly perishable nature of the raw material (Simpson et al., 1992). Although value-added utilization of these enzymes is desirable in many food applications, economic viability of the process and products must be realistically assessed. This is mainly because cost incurred in producing these enzymes by extraction from their natural sources is a limitation for their widespread use. More extensive research to identify the most specific and promising enzymes and to determine optimal conditions for their use is of utmost importance. In fact the results of such research would provide the incentive for commercial developments leading to large-scale production of enzymes at a much lower cost. 7. Conclusion Fish viscera have wide biotechnological potential as a source of trypsins. These enzymes may have the advantages for the applications in the food industry since their temperature and other characteristics differ from homologous proteinases from warmblooded animals. Therefore, digestive proteinases can be isolated as a value-added product from fish viscera and used as the processing aids in food industries to maximize the utilization of marine resources. Acknowledgements This work was funded by the “ Ministry of Higher Education, Scientific Research and Technology, Tunisia”. References Amiza, M.A., Galani, D., Owusu-Apenten, R.K., 1997. Cod (Gadus morhua) trypsin heat inactivation: a reaction kinetic study. J. Food Biochem. 21, 273e288. Anwar, A., Saleemuddin, M., 1998. Alkaline proteases. A review. Bioresour. Technol. 64, 175e183. Arvanitoyannis, I.S., Kassaveti, A., 2008. Fish industry waste: treatments, environmental impacts, current and potential uses. Int. J. Food Sci. Technol. 43, 726e 745. Asgeirsson, B., Fox, J.W., Bjarnason, J.B., 1989. Purification and characterization of trypsin from the poikilotherm Gadus morhua. Eur. J. Biochem. 180, 85e94. Banik, R.M., Prakash, M., 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res. 159, 135e140. Barkia, A., Bougatef, A., Nasri, R., Fetoui, E., Balti, R., Nasri, M., 2010. Trypsin from the viscera of bogue (Boops boops): Isolation and characterisation. Fish Physiol. Biochem. 36, 893e902.

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