RESEARCH PAPER
Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification
and characterization
1
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto Nacional de Investigación y Desarrollo Pesquero (INIDEP),
Mar del Plata, Argentina
2
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ingeniería Universidad Nacional de Mar del Plata,
Mar del Plata, Argentina
Submission date: 2016-07-29
Final revision date: 2017-02-06
Acceptance date: 2017-02-09
Publication date: 2017-07-18
BioTechnologia 2017;98(2):103-112
KEYWORDS
TOPICS
ABSTRACT
Marine by-products contain valuable protein fractions. To ensure a profitable utilization of wastes from the fish industry, the knowledge about their quality and composition is necessary. Fish digestive proteolytic enzymes from cold-adapted ectothermic organisms have found applications in several industries because their temperature requirements and other characteristics differ from those of homologous proteases from warm-blooded animals. Herein, we describe detection, partial purification, and characterization of proteolytic enzymes from the viscera and heads of Engraulis anchoita. Enzymatic activities of the by-products were assayed using azocaseín as a substrate. To characterize the alkali protein fraction, the activity against inhibitors and their molecular weights were studied. The crude protein extract exhibited maximal activity at pH 8.0 and 60EC. Results relative to the substrate-specific Nabenzoyl-DL-arginine-p-nitroanilide and the 25 kDa molecular weight indicated that the recovered protease was trypsin. The activity showed an increment in presence of SDS and a slight decrease when it was incubated with EDTA. Increasing the concentration of NaCl up to 5% did not significantly decrease the protein’s activity. The results obtained suggest that by-products of anchovy industry could be used in the detergents industry.
REFERENCES (60)
1.
Anwar A., Saleemudin M. (1998) Alkaline proteases – a review. Biores. Technol. 6: 175-183.
2.
Balti R., Barkia A., Bougatef A., Nasri M. (2009) Heat-stable trypsin from cuttlefish (Sepia officinalis ) hepatopancreas: purification and characterization. Food Chem. 113: 146-154.
3.
Burkert J.M.F., Maugeri F., Rodrigues M.I. (2004) Optimization of extracellular lipase production by Geotrichum sp. using factorial design. Biores. Technol. 91: 77-84.
4.
Banerje U.C., Sani R.K., Azmi W., Soni R. (1999) Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process. Biochem. 35: 213-219.
5.
Blanco M., Simpson B.K., Perez-Martín R.I., Sotelo G.C. (2014) Isolation and partial and characterization of trypsin from pancreas of small-spotted catshark (Scyliorhinus canicula). J. Food Biochem. 38: 196-206.
6.
Bougatef A., Souissi N., Fakhfakh N., Ellouz-Triki Y., Nasri M. (2007) Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus). Food Chem. 102: 343-350.
7.
Cao M.J., Osatomi K., Suzuki M., Hara K,. Tachibana K., Ishihara T. (2000) Purification and characterization of two anionic trypsins from the hepatopancreas of carp. Fish Sci. 66: 1172-1179.
8.
Castillo-Yańez F.J., Pacheco-Aguilar R., García-Carreńo F.L., Navarrete del Toro M. (2005) Isolation and characterization of trypsin from pyloric caeca of Monterrey sardine (Sardinops sagax caerulea). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 140: 91-98.
9.
Castro Ceseńa A.B., Sánchez-Saavedra M.P., Márquez-Rocha F.J. (2012) Characterization and partial purification of proteolytic enzymes from sardine by-products to obtain concentrated hydrolysates. Food Chem. 135: 583-589.
10.
Cohen T., Gertler A., Birk Y. (1981) Pancreatic proteolytic enzymes from carp (Cyprinus carpio). Purification and physical properties of trypsin, chymotrypsin, elastase and carboxypeptidase B. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 69: 639-646.
11.
Copeland R.A. (2000) Enzymes: a practical introduction to structure, mechanism and data analysis. Ed. Wiley-VCH, New York, NY.
12.
Czerner M., Yeannes M.I. (2010) Brining kinetics of different cuts of anchovy (Engraulis anchoita). Int. J. Food Sci. Technol. 45: 2001-2007.
13.
Diaz-Lopez M., Garcia-Carreńo F.L. (2000) Applications of fish and shellfish enzymes in food and feed products. Food Sci. 97: 571-618.
14.
Ferraro V., Cruz I.B., Jorge R.F., Malcata F.X., Pintado M.E., Castro P.M.L. (2010) Valorisation of natural extracts from marine source focused on marine by-products: a review. Food Res. Int. 43: 2221-2233.
15.
Ferraro V., Carvalho A.P., Piccirillo C., Santos M.M., Castro P.M.L., Pintado M.E. (2013) Extraction of high added value biological compounds from sardine, sardine-type fish and mackerel canning residues. Mater Sci. Eng. C. 33: 3111-3120.
16.
Gbogouri G.A., Linder M., Fanni J., Parmentier M. (2004) Influence of hydrolysis degree on the functional properties of salmon by-product hydrolysates. J. Food Sci. 69: 615-622.
17.
Gupta R., Beg Q.K., Lorenz P. (2002) Bacteria alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59: 15-32.
18.
Haard N.F. (1992) A review of proteolytic enzymes from marine organism and their application in the food industry. J. Aquat. Food Prod. Technol. 1: 17-35.
19.
Hau P.V., Benjakul S. (2006) Purification and characterization of trypsin from pyloric caeca of bigeye snapper (Priacanthus macracanthus). J. Food Biochem. 30: 478-495.
20.
Heu M.S., Pyeun J.H., Kim H.R., Godber J.S. (1991) Purification and characterization of alkaline proteinases from the viscera of anchovy Engraulis japonica. J. Food Biochem. 15: 51-66.
21.
Heu M.S., Kim H.R., Pyeun J.H. (1995) Comparison of trypsin and chymotrypsin from the viscera of anchovy (Engraulis japonica). Comp. Biochem. Physiol. Part B. 112: 557-567.
22.
Ishida M., Sugiyama N., Sato M., Nagayama F. (1995) Two kinds of neutral serine proteinases in salted muscle of anchovy, Engraulis japonica. Biosci. Biotechnol. Biochem. 59: 1107-1112.
23.
Jeong Y., Wei C.I., Preston J.F., Marshall M.R. (2000) Purification and characterization of proteases from hepatopancreas of crawfish (Procambarus clarkii). J. Food Biochem. 24: 311-332.
24.
Jeloulli K., Bougatef A., Daassi D., Balti R., Nasri M. (2009) New alkaline trypsin from the intestine of grey triggerWsh (Balistes capriscus) with high activity at low temperature: purification and characterisation. Food Chem. 116: 644-650.
25.
Joo H.S., Park G.C., Kim K.M., Paik S.R., Chang C.S. (2001) Novel alkaline protease from the polychaeta, Periserrula leucophyrna: purification and characterization. Process Biochem. 36: 893-900.
26.
Kanner J., Rosethal I. (1992) An assessment of lipid oxidation in foods. Pure Appl. Chem. 64: 1959-1964.
27.
Ketnawa S., Benjakul S., Ling T.C., Martínez-Alvarez O., Rawdkuen S. (2013) Enhanced recovery of alkaline protease from fish viscera by phase partitioning and its application. Chem. Cent. J. 7: 79-88.
28.
Khaled H.B., Bougatef A., Balti R., Triki-Ellouz Y., Souissi N., Nasri M. (2008) Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera. J. Sci. Food Agric. 88: 2654-2662.
29.
Kishimura H., Hayashi K., Miyashita Y., Nonami Y. (2006) Characteristics of trypsins from the viscera of true sardine (Sardinops melanostictus) and the pyloric caeca of arabesque greenling (Pleuroprammus azonus). Food Chem. 97: 65-70.
30.
Klomklao S., Benjakul S., Visessanguan W. (2004) Comparative studies on proteolytic activity of spleen extracts from three tuna species commonly used in Thailand. J. Food. Biochem. 28: 355-372.
31.
Klomklao S., Benjakul S., Visessanguan W., Simpson B.K., Kishimura H., Saeki H. (2006) Trypsins from yellowfin tuna (Thunnus albacores) spleen: Purification and characterization. Comp. Biochem. Physiol. B. 144: 47-56.
32.
Klomklao S., Benjakul S., Visessanguan W., Kishimura H., Simpson B.K. (2007) Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis). Food Chem. 100: 1580-1589.
33.
Klomklao S., Kishimura H., Benjakul S. (2008) Endogenous proteinases in true sardine (Sardinops melanostictus). Food Chem. 107: 213-220.
34.
Klomklao S., Songklanakarin J. (2008) Digestive proteinases from marine organisms and their applications. J. Sci. Technol. 30: 37-46.
35.
Kurtovic I., Marshall S.N., Simpson B.K. (2006) Isolation and characterization of a trypsin fraction from the pyloric caeca of chinook salmon (Oncorhynchus tshawytscha). Comp. Biochem. Physiol. B. 143: 432-440.
36.
Laemmli U. (1970) Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nat. London 277: 680-685.
37.
Lamas D.L., Yeannes M.I., Massa A.E. (2015) Partial purification of proteolytic enzymes and characterization of trypsin from Merluccius hubbsi by-products. Int. J. Food Nut. Sci. 4: 121-130.
38.
Lineweaver H., Burk D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 665-666.
39.
Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 256-275.
40.
Matsushita S., Kobayashi M., Nitta Y. (1970) Inactivation of enzymes by linoleic acid hydroperoxides and linoleic acid. Agric. Biol. Chem. 34: 817-824.
41.
Martinez A., Gildberg A. (1988) Autolytic degradation of belly tissue in anchovy (Engraulis encrasicholus). Intern. J. Food Sci. Technol. 23: 185-194.
42.
Martinez A., Olsen R.L., Serra J.L. (1988) Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 91: 677-684.
43.
Macouzet M., Simpson B.K., Lee B.H. (1999) Cloning of fish enzymes and other fish protein genes. Crit. Rev. Biotechnol. 19: 179-196.
44.
Macouzet M., Simpson B.K., Lee B.H. (2005) Expression of a cold-adapted fish trypsin in Pichia pastoris. Fems Yeast Res. 5: 851-857.
45.
Michail M., Vasiliadou M., Zotos A. (2006) Partial purification and comparison of precipitation techniques of proteolytic enzymes from trout (Salmo gairdnerii) heads. Food Chem. 97: 50-55.
46.
Olivas-Burrola H., Ezquera-Brauer M.J., Rouzaud-Sandez O., Pacheco-Aguilar R. (2001) Protease activity and partial characterization of the trypsin-like enzyme in the digestive tract of the tropical sierra scomberomorus cocncolor. J. Aquat. Food Product Technol. 10: 51-63.
47.
Pastous Madureira L.S. et al. (2009) Current and potential alternative food uses of the Argentine anchoita (Engraulis anchoita) in Argentina, Uruguay and Brazil. [in:] Fish as feed inputs for aquaculture: practices, sustainability and implications. Eds. Hasan M.R., Halwart M. FAO Fisheries and Aquaculture Technical Paper. No. 518. Rome, FAO, p. 269-287.
48.
Rao Y.K., Bahadur P., Ghosh S. (1989) Stability andkinetic behaviour of some enzymes in surfactant environment. Indian J. Biochem. Biophys. 26: 390-39.
49.
Rawdkuen S., Vanabun A., Benjakul S. (2012) Recovery of proteases from the viscera of farmed giant catfish (Pangasianodon gigas) by three-phase partitioning. Process. Biochem. 47: 2566-2569.
50.
Regeinstein J.M., Regeinstein C.E. (1991) Introduction to Fish Technology. Van Nostrand Reinshold, New York.
51.
Salazar-Leyva J.A., Lizardi-Mendoza J., Ramírez-Suarez J.C., García-Sanchez G., Ezquerra-Brauer J.M., Valenzuela-Soto E.M., Carvallo-Ruiz M.G., Lugo-Sanchez M.E., Pacheco- Aguilar R. (2014) Utilization of chitin and chitosan based aterials for protease inmobilization: stabilization effects and applications. Rev. Mex. Ing. Qca. 13: 129-150.
52.
Shahidi F. (2007) Maximising the value of marine by-products. Woodhead publishing in food science, technology and nutrition. Cambridge, England.
53.
Shahidi F., Kamil J.Y.V.A. (2001) Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci. Technol. 12: 435-464.
54.
Simpson B.K. (2000) Digestive proteases from marine animals. [in:] Seafood enzymes. Ed. Haard N.F., Simpson B.K. New York, NY: Marcel Dekker: 191-213.
55.
Simpson B.K., Haard N.F. (1987) Trypsin and trypsin-like enzyme from the stomachless cunner. Catalytic and other physical characteristics. J. Agric. Food Chem. 35: 652- 656.
56.
Siringan P., Raksakulthai N., Yongsawatdigul J. (2006) Autolytic activity and biochemical characteristics of endogenous proteinases in Indian anchovy (Stolephorus indicus). Food Chem. 98: 678-684.
57.
Siringan P., Raksakulthai N., Yongsawatdigul J. (2007) Partial puriWcation and characterization of trypsin-like proteinases in Indian anchovy (Stolephorus spp.). Food Chem. 01: 82-89.
58.
Sweadner K.J. (1991) Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulphate, facilitating proteolytic fingerprinting. Anal. Biochem. 194: 130-135.
59.
Trincone A. (2011) Marine biocatalysts: enzymatic features and applications. Mar. Drugs 9: 478-499.
60.
Zhang C., Kim S.K. (2012) Application of marine microbial enzymes in the food and pharmaceutical industries. Adv. Food Nutr. Res. 64: 423-435.
| eISSN: | 2353-9461 |
| ISSN: | 0860-7796 |
We process personal data collected when visiting the website. The function of obtaining information about users and their behavior is carried out by voluntarily entered information in forms and saving cookies in end devices. Data, including cookies, are used to provide services, improve the user experience and to analyze the traffic in accordance with the Privacy policy. Data are also collected and processed by Google Analytics tool (more).
You can change cookies settings in your browser. Restricted use of cookies in the browser configuration may affect some functionalities of the website.
You can change cookies settings in your browser. Restricted use of cookies in the browser configuration may affect some functionalities of the website.
