My bachelor thesis

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 using
potassium phosphate solution 20mM (pH 7.5) and precipitated by using cold acetone (ratio protease
extract 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 optimal
temperature and pH of the cold acetone (1:2) precipitated were 50 oC and 8 respectively. The enzyme
was 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% when
incubated in 70oC for 30 minutes. The enzyme was more stable in pH 7 and less stable in pH 10. 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 powder (0.65%, w/v) substrate respectively. The presence of NaCl 4.6 mM
concentration increased the activity of enzyme (control activity was 100%) to 104.10% whereas the
presence of CaCl2 4.6 mM didn’t increase the activity of enzyme significantly. The presence of EDTA
4.6 mM concentration decreased the activity to 84.43%.
Keywords: Skipjack tuna, protease, extraction, characterization
INTRODUCTION
Skipjack/cakalang fish (Katsuwonus pelamis Linnaeus) is one of the most species tuna caught
in Indonesia. Fish viscera are rich of many kinds of enzyme (Venugopal, 2006). Enzyme is commonly
used in food or chemical industry and also as food supplement to help food digestion. Protease is the
highest production among the other kinds of enzyme widely used. The objectives of this research were
to study the efficiency of protease precipitation by using acetone (ratio protease extract to acetone) or
ammonium 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 METHODS
Materials
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), ammonium
sulfate, 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 60
mesh, and demin. water.
METHODS
1. 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.
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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 DISCUSSION
Crude 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 protease
extract to cold acetone, 1:2) and this cold acetone precipitation method was used to purify enzyme
extract henceforth. Extracted enzyme could be in the inactive form (zymogen) but activation of
enzyme could happen cause of other enzyme consisted in intestine mucosa. This enzyme could
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3. activate zymogen likes trypsinogen and trypsinogen that has been activated could activate the other
zymogen likes chymotrypsinogen. So, the presence of inactive form of enzyme in enzyme extract
could be minimized.
Protein concentration was decreased but the enzyme specific activity was increased after the
precipitation (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 about
protease from sardine viscera, and Yaneza et al. (2004) that studied about protease from Monterey
sardine viscera. Protein concentration might decrease caused by soluble protein that castaway with
supernatant after centrifugation. But, specific activity was increase caused by the increasing of
purification 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= 0.05
Crude Protease Characterization
Optimum 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=0.05
Different 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 with
the increasing of temperature (Figure 2, left). Enzyme activity was increased caused by the increasing
of reaction constanta when the temperature was increased but the activity was decrease after optimum
temperature caused by denaturation of protein at high temperature. Optimum temperature of each kind
of protease was varying according to its temperature endurance (Reed a, 1975). This protease has
higher 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 than
protease 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 caused
by ionic group in active site that more active in neutral to alkalis condition. Similar result was reported
for Colossoma macropomum protease (Esposito et al., 2008). Different results were reported for
Monterey sardine viscera which has optimum pH 2.5 (Yaneza et al., 2004), bolti fish viscera which has
optimum pH 2.5 (El-Beltagy et al., 2004), and hepatopancreas of Jumbo squid which has optimum pH
4.5 (Lopez and Norman, 2007). Protease that was produced by papaya and pineapple were more active
in acid to neutral pH condition. Generally, proteases application at 50 oC and pH 8 were used in
production of protein hydrolysates from soybean (Suhartono, 1989), production of gelatin hydrolysates
for 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), and
extraction of astaxanthin pigment from shrimp waste (Armenta and Isabel, 2008). Besides that,
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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
100.00
Residual 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 5.041
Different notation indicate value have significant difference at α=0.05 0 30 60 90 180
Keterangan: notation a, b, c, and d for temperature 40o C (♦) Time (minutes)
Different notation indicate value have significant difference at α=0.05
Keterangan: 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.
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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 (Yanez
et al., 2004). The enzyme activity was decrease because EDTA could chelate the ion required for
activity of enzyme. The presence of NaCl 4.6mM could increase enzyme activity. The activity of
protease from skipjack tuna spleen decreased with NaCl addition (Klomklaoa et al, 2006). But, similar
result was reported for protease from bolti fish viscera (El-Beltagy et al., 2004). Addition of 4.6mM
CaCl2 was not significantly effect the enzyme activity. The activity of protease from bolti fish viscera
and true sardine viscera increase when CaCl2 was added to enzyme extract (El-Beltagy et al. 2004) and
Klomklaob et al. 2008). Protease from skipjack tuna viscera might be categorized to serine or metalo
protease 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 8
and 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 activity
on 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% whereas
the presence of CaCl2 4.6 mM didn’t increase the enzyme activity. The presence of EDTA 4.6 mM
decreased the enzyme activity. This study needs to study about identification of protease, effect of
activator and inhibitor, storage stability, application to food production, effect of extraction methods
and maximizing activation of inactivated form of enzyme in viscera.
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