Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization

Chemistry Central Journal

Helane MS Costa¹  Augusto CV Freitas Júnior¹  Ian PG Amaral¹  Izaura Y Hirata³  Patrícia MG Paiva²  Luiz B Carvalho¹  Vitor Oliveira³  Ranilson S Bezerra¹ 

[1] Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica (CCB) and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/n, Cidade Universitária, Recife, Pernambuco 50670-910, Brazil

[2] Laboratório de Glicoproteínas, Departamento de Bioquímica (CCB), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/n, Cidade Universitária, Recife, Pernambuco 50670-910, Brazil

[3] Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo 04044-020, Brazil

关键词: Waste recovery;    Thermostable trypsin;    N-terminal amino acid sequence;    Marine fish;    Fish trypsin;    Crevalle jack;    Caranx hippos;   

Others  :  787828
DOI  :  10.1186/1752-153X-7-166

 received in 2013-05-27, accepted in 2013-09-02,  发布年份 2013

【 摘 要 】

Background

Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.

Results

A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-_DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. K_m, k_cat, and k_cat/K_m values using BApNA as substrate were 0.689 mM, 6.9 s^-1, and 10 s^-1 mM^-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd²⁺, Al³⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Hg²⁺ at 1 mM, revealing high sensitivity of the enzyme to metal ions.

Conclusions

Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

【 授权许可】

   
2013 Costa et al.; licensee Chemistry Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Bezerra RS, Lins EJF, Alencar RB, Paiva PMG, Chaves MEC, Coelho LCBB, Carvalho LB Jr: Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus). Process Biochem 2005, 40:1829-1834.
• [2]Freitas-Júnior ACV, Costa HMS, Icimoto MY, Hirata IY, Marcondes M, Carvalho LB Jr, Oliveira V, Bezerra RS: Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin. Food Chem 2012, 133:1596-1602.
• [3]Silva JF, Espósito TS, Marcuschi M, Ribeiro K, Cavalli RO, Oliveira V, Bezerra RS: Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus). Food Chem 2011, 129:777-782.
• [4]Espósito TS, Marcuschi M, Amaral IPG, Carvalho LB Jr, Bezerra RS: Trypsin from the processing waste of the Lane Snapper (Lutjanus synagris) and its compatibility with oxidants, surfactants and commercial detergents. J Agric Food Chem 2010, 58:6433-6439.
• [5]Marcuschi M, Espósito TS, Machado MFM, Hirata IY, Machado MFM, Silva MV, Carvalho LB Jr, Oliveira V, Bezerra RS: Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum). Biochem Biophys Res Commun 2010, 396:667-673.
• [6]Souza AAG, Amaral IPG, Espírito Santo AR, Carvalho LB Jr, Bezerra RS: Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus). Food Chem 2007, 100:1429-1434.
• [7]Gupta R, Beg QK, Larenz P: Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 2002, 59:15-32.
• [8]Johnvesly B, Naik GR: Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem 2001, 37:139-144.
• [9]Castillo-Yáñes FJ, Pacheco-Aguiar R, García-Carreño FL, Navarrete-Del Toro MA: Isolation and characterization of trypsin from pyloric caeca of monterey sardine Sardinops sagax caerulea. Comp Biochem Physiol B 2005, 140:91-98.
• [10]Bougatef A, Souissi N, Fakhfakh N, Ellouz-Triki Y, Nasri M: Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus). Food Chem 2007, 102:343-350.
• [11]Kishimura H, Tokuda Y, Yabe M, Klomklao S, Benjakul S, Ando S: Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii) and elkhorn sculpin (Alcichthys alcicornis): isolation and characterization. Food Chem 2007, 100:1490-1495.
• [12]Klomklao S, Kishimura H, Nonami Y, Benjakul S: Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis). Food Chem 2009, 115:155-162.
• [13]Khantaphant S, Benjakul S: Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta). Food Chem 2010, 120:658-664.
• [14]Berry FH, Smith-Vaniz WF: Carangidae. In FAO species identification sheets for fishery purposes, Volume 1. Edited by Fischer W. Rome: West Atlantic (Fishing Area 31), FAO; 1978.
• [15]MPA: Boletim Estatístico da Pesca e Aquicultura - Brasil 2010. Brasília, Brasil: Ministério da Pesca e Aquicultura; 2012:129.
• [16]Alencar RB, Biondi MM, Paiva PMG, Vieira VLA, Carvalho LB Jr, Bezerra RS: Alkaline proteases from digestive tract of four tropical fishes. Braz J Food Technol 2003, 6:279-284.
• [17]Bezerra RS, Santos JF, Paiva PMG, Correia MTS, Coelho LCBB, Vieira VLA, Carvalho LB Jr: Partial purification and characterization of a thermostable trypsin from pyloric caeca of tambaqui (Colossoma macropomum). J Food Biochem 2001, 25:199-210.
• [18]Warburg O, Christian W: Isolierung und Kristallisation des Garunges Ferments enolasc. Biochem Z 1941, 3190:384-421.
• [19]Fuchise T, Kishimura H, Sekizaki H, Nonami Y, Kanno G, Klomklao S, Benjakul S, Chun B: Purification and characteristics of trypsins from cold-zone fish, Pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis). Food Chem 2009, 116:611-616.
• [20]Ktari N, Khaled HB, Nasri R, Jellouli K, Ghorbel S, Nasri M: Trypsin from zebra blenny (Salaria basilisca) viscera: Purification, characterization and potential application as a detergent additive. Food Chem 2012, 130:467-474.
• [21]Villalba-Villalba AG, Ramírez-Suárez JC, Valenzuela-Soto EM, Sánchez GG, Ruiz GC, Pacheco-Aguilar R: Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization. Food Chem 2013, 141:940-945.
• [22]Klomklao S, Benjakul S, Visessanguan W, Kishimura H, Simpson BK: Trypsin from the pyloric ceca of bluefish (Pomatomus saltatrix). Comp Biochem Physiol B 2007, 148:382-389.
• [23]Vojdani F: Methods of Testing Protein Functionality. Edited by Hall GM. New York: Chapman and Hall; 1996:11-60.
• [24]Jellouli K, Bougatef A, Daassi D, Balti R, Barkia A, Nasri M: New alkaline trypsin from the intestine of grey triggerfish (Balistes capriscus) with high activity at low temperature: purification and characterisation. Food Chem 2009, 116:644-650.
• [25]Maurer KH: Detergent proteases. Curr Opin Biotechnol 2004, 15:330-334.
• [26]Kishimura H, Klomklao S, Benjakul S, Chun B–S: Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma). Food Chem 2008, 106:194-199.
• [27]Haard NF: A review of proteolytic enzymes from marine organisms and their application in the food industry. J Aqua Food Prod Technol. 1992, 1:17-e35.
• [28]Genicot S, Rentier-Delrue F, Edwards D, Van Beeumen J, Gerday C: Trypsin and trypsinogen from an antarctic fish: molecular basis of cold adaptation. Biochim Biophys Acta 1996, 1298:45-57.
• [29]Moreira KA, Albuquerque BF, Teixeira MFS, Porto ALF, Lima Filho JL: Application of protease from Nocardiopsis sp. as a laundry detergent additive. World J Microbiol Biotechnol 2002, 18:307-312.
• [30]Barrett AJ: Classification of peptidases. New York: academic press; 1994:1-59. [Methods in Enzymology]
• [31]Pavlisko A, Rial A, De Vecchi S, Coppes Z: Properties of pepsin and trypsin isolated from the digestive tract of Parona signata “palometa”. J Food Biochem 1997, 21:289-308.
• [32]Mihalyi E: Application of proteolytic enzymes to protein structure studies. Boca Raton, FL: CRC Press; 1978.
• [33]Šafařík I, Ptáčková L, Koneracká M, Šafaříková M, Timko M, Kopčanský P: Determination of selected xenobiotics with ferrofluid-modified trypsin. Biotechnol Lett 2002, 24:355-358.
• [34]Aranishi F, Watanabe T, Osatomi K, Cao M, Hara K, Ishihara T: Purification and characterization of thermostable dipeptidase from carp intestine. J Mar Biotechnol 1998, 6:116-123.
• [35]Kishimura H, Hayashi K: Isolation and characteristics of trypsin from pyloric caeca of the starfish Asterina pectinifera. Comp Biochem Physiol B 2002, 132:485-490.
• [36]Gudmundsdóttir A, Gudmundsdóttir E, Óskarsson S, Bjarnason JB, Eakin AN, Craik CS: Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen. Eur J Biochem 1993, 217:1091-1097.
• [37]Kishimura H, Hayashi K, Miyashita Y, Nonami Y: Characteristics of trypsins from the viscera of true sardine (Sardinops melanostictus) and the pyloric ceca of arabesque greenling (Pleurogrammus azonus). Food Chem 2006, 97:65-70.
• [38]Hermodson MA, Ericsson LH, Neurath H, Walsh KA: Determination of the amino acid sequence of porcine trypsin by sequenator analysis. Biochemistry 1973, 12:3146-3153.
• [39]Walsh KA: Trypsinogens and trypsins of various species. Methods Enzymol 1970, 19:41-63.
• [40]Stroud RM, Kay LM, Dickerson RE: The structure of bovine trypsin: Electron density maps of the inhibited enzyme at 5 Å and 2.7 Å resolution. J Mol Biol 1974, 83:185-208.
• [41]Balti R, Barkia A, Bougatef A, Nasri M: Heat-stable trypsin from cuttlefish (Sepia officinalis) hepatopancreas: purification and characterization. Food Chem 2009, 113:146-154.
• [42]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.