Extraction And Characterization Of Protease From The Viscera Of Skipjack Tuna Fish

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Extraction And Characterization Of Protease From The Viscera Of Skipjack Tuna Fish

  1. 1. EXTRACTION AND CHARACTERIZATION OF PROTEASE FROM THE VISCERA OF SKIPJACK TUNA FISH (Katsuwonus pelamis Linnaeus) ABSTRACT Extraction and characteristics of protease from the viscera of skipjack tuna fish were studied.Viscera of skipjack tuna fish (consist of intestines, stomach, pancreas, and liver) was extracted usingpotassium phosphate solution 20mM (pH 7.5) and precipitated by using cold acetone (ratio proteaseextract to cold acetone were 1:1 and 1:2) and ammonium sulfate (30%, 40%, 50%, and 60%, w/v).The enzyme showed the highest activity when precipitated by using acetone 1:2. The optimaltemperature and pH of the cold acetone (1:2) precipitated were 50 oC and 8 respectively. The enzymewas stable in 40oC for 3 hours incubation but less stable in 70oC. The enzyme retained more than 50%of its activity after heating 50 oC for 30 minutes. The enzyme activity was decreased to 11.29% whenincubated in 70oC for 30 minutes. The enzyme was more stable in pH 7 and less stable in pH 10. Theenzyme activity was 5.40, 0.29, and 0.15 units/mg protein in casein (0.65%, w/v), BSA (0.65%, w/v)and chicken feather powder (0.65%, w/v) substrate respectively. The presence of NaCl 4.6 mMconcentration increased the activity of enzyme (control activity was 100%) to 104.10% whereas thepresence of CaCl2 4.6 mM didn’t increase the activity of enzyme significantly. The presence of EDTA4.6 mM concentration decreased the activity to 84.43%.Keywords: Skipjack tuna, protease, extraction, characterizationINTRODUCTION Skipjack/cakalang fish (Katsuwonus pelamis Linnaeus) is one of the most species tuna caughtin Indonesia. Fish viscera are rich of many kinds of enzyme (Venugopal, 2006). Enzyme is commonlyused in food or chemical industry and also as food supplement to help food digestion. Protease is thehighest production among the other kinds of enzyme widely used. The objectives of this research wereto study the efficiency of protease precipitation by using acetone (ratio protease extract to acetone) orammonium sulfate and to characterize protease from viscera of skipjack tuna such as optimum pH,optimum temperature, pH stability, temperature stability, substrate specificity, and effect of EDTA,NaCl, and CaCl2 on protease activity.MATERIALS AND METHODSMaterials Skipjack tuna viscera were obtained from Muara Baru Harbor, North Jakarta, Indonesia,frozen at ±-20oC. Chemicals used were from Merck such as ethanol, cold acetone (pa), ammoniumsulfate, potassium phosphate (pa), casein, sodium hydroxide (pa), hydrochloric acid (pa),Trichloroacetic Acid (pa), Folin & Ciocalteu’s phenol reagent, Sodium carbonate anhydrous (pa), L-Tyrosin, Bovine Serum Albumin, ortho-phosphoric acid 85% (pa), Coomassive brilliant blue G-250,chicken feather, calcium chloride, sodium chloride, EDTA, citric acid, and boric acid; screen fabric 60mesh, and demin. water.METHODS1. Crude Protease Extraction (Yaneza et al. (2004), modified): Viscera were washed by using water and cold acetone 70%. Viscera were blended in cold potassium phosphate 20 mM solutions (pH 7.5) for 1 min, filtered by using screen fabric 60 mesh, refrigerated (±4oC) and centrifuged at 6000rpm for 20 min. Supernatant was precipitated over night at ±4oC by using cold acetone (ratio protease extract to cold acetone, 1:1 and 1:2) or by using ammonium sulfate (30, 40, 50, and 60%) and then centrifuged at 6000rpm for 30 min. Crude protease with the highest activity would be used for calculating purification and characterization.2. Optimum Temperature and pH (El-Beltagy et al. (2004), Bougatef et al. (2007), Lopez and Norman, 2007) modified): measuring the activity of enzyme extracts using casein (0.65%) as substrate at pH 7.5 and various temperatures (10, 30, 40, 50, and 70oC) for 10 min (for temperature optimizing); and at 50oC and various pH (3, 5, 7, 8 and 10) in buffer universal for 10 min (for pH optimizing). Percentage of enzyme activity was estimated at the highest activity detected. 1
  2. 2. 3. Temperature and pH Stability (El-Beltagy et al. (2004), modified): estimated by incubating enzyme extracts at various temperatures (40, 50, and 70oC) for 0, 30, 60, 90, and 180 min at pH 7.5 (for temperature stabilization analyses); and incubating enzyme extracts and various pH (7, 8, and 10) using buffer universal for 0, 30, 60, 90, and 180 min at 37 oC (for pH stabilization analyses). Residual activities were estimated at 37oC and pH 7.5 for 10 min of incubation. The 100% of the enzyme activity was the activity of enzyme without incubation.4. Substrate specificity (Guangrong et al. (2006) and Syed et al. (2008), modified): The hydrolysis activity toward a variety of protein including casein (0.65%, w/v), BSA (0.65%, w/v), and chicken feather powder (0.65%, w/v) were estimated. Protease activity was estimated by incubating enzyme extract in each substrate at 50oC (pH 8) for 10 min. The 100% of the enzyme activity was the activity of enzyme on casein substrate.5. Effect of EDTA, NaCl, dan CaCl2 (Bougatef et al. (2007), Balti et al. (2008), and Syed et al. (2008), modified): estimated and evaluated at 50oC, pH 8. 50mM of solution (0.6mL) was added to the mixture of enzyme extract and substrate for 10 min of incubation. The activity of enzyme was calculated and the 100% of the enzyme activity was the activity of enzyme without addition.6. Protein Evaluation (Bradford (1976): using Bovine Serum Albumin standard (1.25mg/ml).7. Assay of Protease Activity ( colorimetric methods) by using casein as substrate: Casein substrate 5ml (0.65%, w/v) was mixed with 1ml enzyme extract and incubated at 37 oC, pH 7.5 for 10 min. The same procedure was done for blank but enzyme extract addition was added after 10 min of incubation. The reaction was stopped by adding 5ml TCA 110mM solution and mixture was incubated at 37oC, pH 7.5 for 30 min. The mixture was centrifuged at 6000rpm for 10 min. Supernatant was mixed with 5ml Na2CO3 500mM solution and 1ml F-C solution (F-C solution have been thinned by using demin. water). The mixture was incubated for 30 min at 37 oC and centrifuged at 6000rpm for 10 min. The absorbance of supernatant at 660nm. One unit of activity was defined as the amount hydrolyzing casein to produce color equivalent to 1.0 μmol of tyrosine per minute at assayed temperature and pH.8. Statistical Analysis: A completely randomized design was used throughout this study and experiments were done in duplicate. Data were subjected to ANOVA and t-test for 2 sample comparison and further analyses of Tukey and Dunnet multiple range test. Statistical analysis was performed using the SPSS version 16.RESULTS AND DISCUSSIONCrude Protease Extraction 1.200 1.12e 1.021d Specific activity (unit/mg protein) 1.000 0.800 0.61c 0.600 0.52b 0.37a 0.37a 0.400 0.200 0.000 1:1 1:2 30 40 50 60 Ratio extract:cold acetone %Ammonium sulfate Different notation indicate value have significant difference at α=0.05 Figure 1. Crude protease activity after precipitation using acetone and ammonium sulfate The highest enzyme activity was showed at precipitation by using cold acetone (ratio proteaseextract to cold acetone, 1:2) and this cold acetone precipitation method was used to purify enzymeextract henceforth. Extracted enzyme could be in the inactive form (zymogen) but activation ofenzyme could happen cause of other enzyme consisted in intestine mucosa. This enzyme could 2
  3. 3. activate zymogen likes trypsinogen and trypsinogen that has been activated could activate the otherzymogen likes chymotrypsinogen. So, the presence of inactive form of enzyme in enzyme extractcould be minimized. Protein concentration was decreased but the enzyme specific activity was increased after theprecipitation (Table 1.). Generally, these data are in agreement with those reported by Balti et al. (2008)that studied about protease from Cuttlefish hepatopancreas, Bougatef et al. (2007) that studied aboutprotease from sardine viscera, and Yaneza et al. (2004) that studied about protease from Montereysardine viscera. Protein concentration might decrease caused by soluble protein that castaway withsupernatant after centrifugation. But, specific activity was increase caused by the increasing ofpurification level after precipitation using cold acetone. Table 1. Protein concentration and crude protease activity before and after precipitation Protein concentration Activity of crude protease Specific activity Purification Precipitation (mg/ml crude (unit/ml crude protease (unit/mg protein)* fold* protease extract)* extract)* Before 8.48 8.54 1.007 1.00 After 8.25 12.1 1.47 1.46* indicate value have significant difference at α= 0.05Crude Protease CharacterizationOptimum Temperature and pH 120.00 120.00 d 100.00 100.00 c d 92.38 c 100.00 100.00 Relative activity (%) 94.00 Relative activity (%) 80.00 80.00 83.91c 60.00 60.00 b 49.14 b 51.69 40.00 40.00 20.00 b 20.00 0.97 a 13.64 8.90 a 0.00 0.00 0 2 4 6 8 10 12 0 10 20 30 40 50 60 70 80 pH Temperature (oC) Different notation indicate value have significant difference at α=0.05Different notation indicate value have significant difference at α=0.05 Figure 2. Optimum temperature (left) and pH (right) of skipjack tuna viscera crude protease The partially purified protease had the highest activity at 50oC, pH 7.5 and it decreased withthe increasing of temperature (Figure 2, left). Enzyme activity was increased caused by the increasingof reaction constanta when the temperature was increased but the activity was decrease after optimumtemperature caused by denaturation of protein at high temperature. Optimum temperature of each kindof protease was varying according to its temperature endurance (Reed a, 1975). This protease hashigher optimum temperature than protease from Bolti fish viscera (35oC) (El-Beltagy et al., 2004),papaya and pineapple (30-40oC) (Aurand et al., 1987) but it has lower optimum temperature thanprotease from sardine viscera (Bougatef et al., 2007), skipjack tuna spleen (Klomklao a et al., 2007),and Colossoma macropomum viscera (Esposito et al., 2008) which have optimum temperature at 60 oC,and protease from cuttlefish hepatopancreas at 70oC (Balti et al., 2008). Optimum pH of partially purified skipjack tuna protease was 8 at 50 oC (shown in Figure 3).The protease activity was low in low pH value and shown higher activity in neutral and high pH value(7-10) so this kind of protease could be employed at neutral to alkalis condition. This might be causedby ionic group in active site that more active in neutral to alkalis condition. Similar result was reportedfor Colossoma macropomum protease (Esposito et al., 2008). Different results were reported forMonterey sardine viscera which has optimum pH 2.5 (Yaneza et al., 2004), bolti fish viscera which hasoptimum pH 2.5 (El-Beltagy et al., 2004), and hepatopancreas of Jumbo squid which has optimum pH4.5 (Lopez and Norman, 2007). Protease that was produced by papaya and pineapple were more activein acid to neutral pH condition. Generally, proteases application at 50 oC and pH 8 were used inproduction of protein hydrolysates from soybean (Suhartono, 1989), production of gelatin hydrolysatesfor low-calories beverages production (Suhartono, 1989), production of Fish Protein Hydrolysates(FPH) (Gomez et al., 2007), production of amino acid from fish waste (Gomez et al., 2007), andextraction of astaxanthin pigment from shrimp waste (Armenta and Isabel, 2008). Besides that, 3
  4. 4. protease also could be employed to repair the functional properties of protein (Gomez et al., 2007) and to overcome waste from food industry likes chicken feather or animal skin (Suhartono, 1989). Temperature and pH Stability Skipjack tuna protease was more stable at 40 oC and less stable at 70oC which is shown in Figure 4. Similar result was shown for bolti fish viscera (El-Beltagy et al., 2004). So, this enzyme was better employed at room temperature or temperature below 40 oC if it needs a longer process time in application. Protease from Colossoma macropomum viscera was reported had better temperature stability at optimum temperature (Esposito et al., 2008). Protease from cuttlefish hepatopancreas was reported stable at 50oC in 1 hour incubation (Balti et al., 2008). Generally, proteases that were produced from fish viscera were stable at temperature 30 until 50oC (Klomklaoa et al. (2007), Yaneza et al. (2004), El-Beltagy et al. (2004), Bougatef et al. (2007), dan Yanezb et al. (2008)). 120.00 120.00 100.00E 100.00 100.00d 100.00e 100.00Residual activity (%) 100.005 100.005 100.00 E Residual activity (%) 80.00 80.00 64.63c 60.00 D b b 50.43 53.99 53.06 60.00 48.25a 53.56d 40.00 50.27D 25.54C 40.00 17.77B 31.98c 20.00 4 A 28.071C 23.15b 11.29 3 5.65 20.00 20.98B 12.67a 6.11 16.844 0.00 2.692 1.921 3 10.29 7.402 8.041A 0 30 60 90 180 Time (minute s) 0.00 5.041Different notation indicate value have significant difference at α=0.05 0 30 60 90 180Keterangan: notation a, b, c, and d for temperature 40o C (♦) Time (minutes) Different notation indicate value have significant difference at α=0.05Keterangan: notation A, B, C, D, and E for temperature 50o C (■) Keterangan: notation a, b, c, d, and e for pH 7 (♦)Keterangan: notation 1, 2, 3, 4, and 5 for temperature 70o C (▲) Keterangan: notation A, B, C, D, and E for pH 8 (■) Keterangan: notation 1, 2, 3, 4, and 5 for pH 10 (▲) Figure 3. Temperature (left) and pH (right) stability of skipjack tuna viscera crude protease Protease skipjack tuna viscera had less stability for longer incubation time in neutral to alkaline pH condition (Figure 5). Incubation for 30 min in pH 7 and 8 still remained more than 50% residual activity. Protease was not stable in pH 10. Similar results were reported for Monterey sardine viscera (Yanez et al., 2004) and bolti fish viscera (El-Beltagy et al., 2004). Substrate Specificity 120.00 c 100.00 100.00 Relative activity (%) 80.00 60.00 40.00 20.00 b 5.34 2.85a 0.00 Kasein BSA Bulu ayam Substrate Different notation indicate value have significant difference at α=0.05 Figure 4. Substrate specificity of skipjack tuna viscera crude protease The enzyme showed the highest activity on casein substrate 0.65% (w/v) at 50oC and pH 8. The enzyme activity was 5.40, 0.29, and 0.15 units/mg protein in casein (0.65%, w/v), BSA (0.65%, w/v) and chicken feather (bulu ayam) powder (0.65%, w/v) substrate respectively. Protease from Thunnus obesus and Thunnus albacore stomach had high activity on casein substrate (Daulay et al., 1996) and Hartono, 1994). Alkaline protease from Bacillus sp bacteria was reported can be used in production of protein hydrolysates and gelatin hydrolysates (Suhartono, 1989). This protease from skipjack tuna viscera might be used in those production processes too. 4
  5. 5. Effect of EDTA, NaCl, and CaCl2 120.00 104.10* 101.48 100.00 100.00 84.43* Relative activity (%) 80.00 60.00 40.00 20.00 0.00 Control EDTA NaCl CaCl2 * indicate value have significant difference with control at α=0.05 Figure 5. Effect of EDTA, NaCl, dan CaCl2 on protease activity The activity of skipjack tuna viscera protease was decrease by the addition of EDTA 4.6 mM.Soybeans trypsin inhibitor and EDTA did not affect Monterey sardine viscera enzyme activity (Yanezet al., 2004). The enzyme activity was decrease because EDTA could chelate the ion required foractivity of enzyme. The presence of NaCl 4.6mM could increase enzyme activity. The activity ofprotease from skipjack tuna spleen decreased with NaCl addition (Klomklaoa et al, 2006). But, similarresult was reported for protease from bolti fish viscera (El-Beltagy et al., 2004). Addition of 4.6mMCaCl2 was not significantly effect the enzyme activity. The activity of protease from bolti fish visceraand true sardine viscera increase when CaCl2 was added to enzyme extract (El-Beltagy et al. 2004) andKlomklaob et al. 2008). Protease from skipjack tuna viscera might be categorized to serine or metaloprotease but generally serine protease was the commonly protease that found in fish viscera.CONCLUSION The enzyme showed the highest activity when precipitated by using cold acetone 1:2, at pH 8and temperature 50oC. The enzyme was stable in 40oC for 3 hours of incubation but less stable in 70 oC.The enzyme was more stable in pH 7 and less stable in pH 10. The enzyme showed the highest activityon casein substrate (0.65%, w/v) compare to BSA and chicken feather powder substrate (0.65%, w/v).The presence of NaCl 4.6 mM increased the enzyme activity (control was 100%) to 104.10% whereasthe presence of CaCl2 4.6 mM didn’t increase the enzyme activity. The presence of EDTA 4.6 mMdecreased the enzyme activity. This study needs to study about identification of protease, effect ofactivator and inhibitor, storage stability, application to food production, effect of extraction methodsand maximizing activation of inactivated form of enzyme in viscera.REFERENCESArmenta, Roberto E dan Isabel Guerrero Legarreta. 2008. “Amino Acid Profile and Enhancement of the Enzymatic Hydrolysis of Fermented Shrimp Carotenoproteins,” J. Foodchem 112: 310-315.Aurand, Leonard W., A. Edwin Woods, dan Marion R. Wells. “Enzymes.” Food Composition and Analysis. New York: The AVI Publishing Company, Inc., 1987.Balti, Rafik, Ahmed Barkia, Ali Bougatef, Naourez Ktari, dan Moncef Nasri. 2008. “A Heat-Stable Trypsin from the Hepatopancreas of the Cuttlefish (Sepia officinalis): Purification and Characterization,” J. Foodchem 113: 146-154.Bougatef, Ali, Nabil Souissi, Nahed Fakhfakh, Yosra Ellouz-Triki, dan Moncef Nasri. 2007. “Purification and Characterization of Trypsin from the Viscera of Sardine (Sardina pilchardus) ,” J. Foodchem 102: 343-350.Bradford, M.M. 1976. “A Rapid and Sensitive Method for the Quantization of Microgram Quantities of Protein Utilizing the Principle of Dye Binding,” Analytical Biochem 72: 248-254.Daulay, Djundjung, Made Astawan, dan Aep Hidayat. 1996. “Assessment of Potency and Characterization of the Gastric Proteases of Tuna (Thunnus obesus) As a Rennet Substitute in Cheese-Making,” Bul. Tech and food Industry VII (2): 23-30. 5
  6. 6. El-Beltagy, A.E, T.A. El-Adawy, E.H. Rahma, and A.A. El-Bedawey. 2004. “Purification and Characterization of an Acidic Protease from the viscera of Bolti fish (Tilapia nilotica),” J. Foodchem 86: 33-39.Esposito, T.S., Ian P.G. Amaral, Diego S. Buarque, Givanildo B. Oliveira, Luiz B. Carvalho Jr, and Ranilson S. Bezerra. 2008. “Fish Processing Waste as a Source of Alkaline Proteases for Laundry Detergent,” J. Foodchem 112: 125-130.Gomez, M.J. Garcia, S. Huerta Ochoa, O. Loera Corral, dan L.A. Prado Barragan. 2007. “Advantages of Proteolytic Extract by Aspergillus oryzae from Fish Flour over a Commercial Proteolytic Preparation,”J. Foodchem 112: 604-608.Guangrong, Huang, Ying Tiejing, Huo Po, dan Jiang Jiaxing. 2006. “Purification and Characterization of a Protease from Thermophilic Bacillus strain HS08,” AJB 5 (24): 2433-2438.Hartono, A.L. “Characteristic evaluation of The Gastric Proteases of Tuna (Thunnus albacore) As a Calf Rennet Substitute,” Thesis, Institute of Pertanian Bogor, 1994.Klomklaoa, Sappasith, Soottawat Benjakul, Wonnop Visessanguan, Hideki Kishimura, dan Benjamin K.Simpson. 2007. “Purification dan Characterisation of Trypsins from the Spleen of Skipjack Tuna (Katsuwonus pelamis),” J. Foodchem 100: 1580-1589.Klomklaob, Sappasith, Hideki Kishimura, dan Soottawat Benjakul. 2008. “Endogenous Proteinases in True Sardine (Sardinops melanostictus),” J. Foodchem 107: 213-220.Lopez, Jose Luis Cardenas dan Norman F. Haard. 2007. “Identification of a Cysteine Proteinase from Jumbo Squid (Dosidicus gigas) Hepatopancreas as Cathepsin L,” J. Foodchem 112: 442-447.Reed, Gerald. “Effect of Temperature and pH.” Food Science and Technology a series of monographs. 2nd ed., 1975.Suhartono, Maggy T. “Sumber dan Peranan.” Enzim dan Bioteknologi. Bogor: Departement of Education and Culture General Directorate of High Education between University of Biotechnology Institute of Pertanian Bogor, 1989.Syed, D.G, Jae Chan Lee, Wen Jun Li, Chang Jin Kim, and Dayanand Agasar. 2008. “Production, Characterization and Application of Keratinase from Streptomyces gulbargensis,” J. Biortech 100: 1868-1871.Venugopal, V. “Application of Enzymes in Fish Processing and Quality Control.” Seafood Processing. United State Of America: Taylor&Francis Group, LLC CRC Press, 2006.Yaneza, Francisco Javier Castillo, Ramon Pacheco-Aguilar, Fernando Luis Garcia-Carreno, and Maria de Los Angeles Navarrete-Del Toro. 2004. “Characterization of Acidic Proteolytic Enzymes from Monterey sardine (Sardinops sagax caerulae) viscera,” J. Foodchem 85: 343-350.Yanezb, Fransisco Javier Castillo, Ramon Pacheco Aguilar, Maria Elena Lugo Sanchez, Guillermina Gracia Sanchez, Idania Emedith Quintero Reyes. 2008. “Biochemical Characterization of an isoform of Chymotrypsin from the Viscera of Monterey sardine (Sardinops sagax caerulea), and comparison with Bovine Chymotrypsin ,” J. Foodchem 112: 634-639. 6

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