Startseite Biochemical characterization of trypsin from Indonesian skipjack tuna (Katsuwonus pelamis) viscera
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Biochemical characterization of trypsin from Indonesian skipjack tuna (Katsuwonus pelamis) viscera

  • Faiza A. Dali , Nurjanah Nurjanah , Hanifah N. Lioe und Maggy T. Suhartono EMAIL logo
Veröffentlicht/Copyright: 22. Juni 2024
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Open Agriculture
Aus der Zeitschrift Open Agriculture Band 9 Heft 1

Abstract

Trypsin production from skipjack tuna (Katsuwonus pelamis) viscera is one significant way to increase the value of fish’s industrial waste. The present work reports the biochemical properties of trypsin from skipjack tuna viscera. The trypsin was fractionated using 0–60% ammonium sulfate and dialyzed. The enzyme was characterized to find the optimum temperature and pH for the substrate N-α-benzoyl-dl-arginine-p-nitroanilide. The 40–50% ammonium sulfate fractionation showed the highest activity at a specific activity of 1.66 U/mg and yield of 69.91%. Specific activity increased after dialysis to 2.17 U/mg with 4.49 times purity and yield of 39.20%. The molecular weights of the enzymes were estimated as 25, 29, and 35 kDa based on the enzyme activity separated by electrophoresis. The enzyme worked optimally at a temperature and pH of 50–60°C and 8.0, respectively. Metal ions (Ca2+, K+, Na+, Mg2+) at a concentration of 20 mM showed no influence on the activity. Enzyme activity was inhibited by Zn2+ at 20 mM, phenyl methyl sulfonyl fluoride (PMSF), benzamidine, and soybean trypsin inhibitor (SBTI), which confirmed the characteristics of a serine protease.

Keywords: biochemical characteristics; fish viscera; fractionation; trypsin

1 Introduction

Protease enzymes for industrial applications dominate approximately 60% of the total enzyme sales worldwide [1,2]. Trypsin is a serine protease that hydrolyzes peptide bonds between the arginine and lysine of protein substrates into simple peptide compounds. Trypsin is in great demand, particularly in food industries [3,4,5,6] and non-food industries, such as tissue culture and vaccine manufacture [7] and medicine [8]. Applications in the agricultural sector include food, as food is one of the major products of agriculture. Trypsin is used to hydrolyze protein into peptides, which can be developed into functional food or functional food ingredients. By far, the most widely utilized protease in proteomics is trypsin. The price of commercial trypsin is heavily influenced by the enzyme’s quality, which is determined by its purity and activity [9].

Trypsin from animals is commonly isolated from the pancreas, such as from pigs and cattle. However, these two sources have shortcomings due to religious restrictions and fears of causing disease transmission. For such reasons, it is essential to find alternative raw materials. Fish viscera is reported to substitute the present source of commercial trypsin (pig or cattle). The characteristics of the obtained trypsin depend on the type of fish and its living habitat. Studies of trypsin sources from various types of fish have been reported, including from the intestinal part of Euthynnus affinis [10], Coryphaena hippurus [11], and the viscera of Sardinella longiceps [12].

Recovery and characterization of proteolytic enzymes from the internal organs of fish have been described in recent years, leading to some intriguing new applications for these enzymes. Trypsins are alkaline proteases with fundamental properties that can benefit industrial processes because of their strong stability and enzymatic activity in adverse conditions such as high temperatures and alkaline pH. Fish enzymes are similar to mammalian trypsin with respect to molecular weight, cleavage specificities, pH stability, and inhibitor response [11,13].

Skipjack tuna (K. pelamis) is an essential pelagic fishery consumed by locals and is considered an important Indonesian non-oil and gas export commodity. In 2018, it contributed to a foreign exchange of USD 713.9 million (14.69%) of the total export value of Indonesian fishery products. Skipjack tuna is very popular among Indonesian people as a source of good animal protein: usually, the fish is processed into various dishes and fried products [14], and most of the meat parts are used and preserved by canning, smoking (smoked skipjack), and freezing (frozen filets). Fish processing activities in the fishing industry will produce byproducts. The byproduct of skipjack tuna can possibly reach 1.09–1.64 tons/day [15]. Fish viscera is one of the byproducts that contain spleen, pancreas, liver, stomach, gallbladder, intestines, heart, and gonads. Fish viscera weigh about 20% of fish biomass (depending on the type of fish) [4]. Fish viscera, if not used, is disposed of as low-value waste. The dumping of fish visceral waste poses a significant hazard to the fishery sectors and the environment. It is crucial to handle and utilize fish viscera byproducts to help reduce the burden of environmental problems and provide added value by using the viscera to produce useful products, such as enzymes, that can later be used in the industry. The first step in evaluating the potential of trypsin enzymes from fish viscera is to study the extraction and characterization. Laboratory experiments are required for future industrial-scale production to save time and money. This research aimed to extract trypsin enzyme from a mixture of skipjack tuna viscera and obtain basic information on its biochemical properties.

2 Materials and methods

2.1 Materials

Skipjack tuna viscera was obtained from the fishing industry in Bogor, West Java, Indonesia (Figure 1). In the first week of October 2021, skipjack tuna were caught and kept in a freezer at −20°C for 3 days. Then, the viscera were collected in a plastic pack, stored in a freezer at −20°C for 1 day, and taken to the laboratory within 1 h before enzyme extraction. When the viscera were prepared, they were brought to 4°C and cleaned using ice water. The skipjack viscera used weighed 263.2 ± 43.16 g with a length of 19.3 ± 0.67 cm and a width of 9.7 ± 1.44 cm. The yield of skipjack tuna viscera was 8.565 ± 8.2%. Next, the viscera were cut into small pieces to a size of about 1–2 cm. All chemicals used were of analytical grade.

Figure 1 Skipjack tuna viscera.
Figure 1

Skipjack tuna viscera.

2.2 Crude enzyme extract

Enzyme extraction was performed using the method of Bougatef et al. [16] with slight modifications. About 100 g of skipjack tuna viscera was homogenized at 1:2 (w/v) using buffer A (concentration 10 mM of Tris-HCl, pH 8.0, containing 10 mM of CaCl2). Homogenization was performed using a homogenizer instrument (Armfield L4R, Armfield Ltd., Hampshire, UK) at a speed of 11,000 rpm for 1 min and at a temperature of 4°C. The homogenate was centrifuged (Himac CR 21 G, Hitachi Co., Ltd., Tokyo, Japan) for 15 min at 10,000×g and at a temperature of 4°C. The supernatant was produced and reported as a crude enzyme extract.

2.3 Fractionation of trypsin

The supernatant was precipitated using ammonium sulfate (NH4)2SO4 at 0–30%, 30–40%, 40–50%, and 50–60% (w/v). Ammonium sulfate was gradually added to the enzyme solution and then agitated continuously with a magnetic stirrer (Thermolyne Cimarec 3: SP47235, Barnstead International, Dubuque, Iowa, USA) for 45 min at 4°C. The fractions were centrifuged at 15,000×g for 10 min at 4°C. Furthermore, each fraction (0–30%, 30–40%, 40–50%, 50–60%) was tested for protein concentration [17] and enzyme activity [18], with some modifications. The selected precipitate was based on the fraction with the highest enzyme specific activity.

2.4 Dialysis

The precipitate was added to a dialysis membrane (Sigma-Aldrich D9277, Merck KGaA, Darmstadt, Germany) with a molecular weight cut-off (MWCO) of 14 kDa, with a width of 10 mm and a diameter of 6 mm. Then, the membrane was placed in 1 mM pH 8.0 Tris-HCl buffer, containing 1 mM CaCl2 with a 100× the sample volume, and stirred for 30 min at 4°C. The dialysate was analyzed for its enzyme activity and protein levels.

2.5 Trypsin activity assay

The measurement of trypsin enzyme activity was performed according to the modified method of Erlanger et al. [18]. About 1 mL of dimethyl sulfoxide was added to 43.5 mg of BAPNA. This mixture was then dissolved in 50 mM Tris HCl (pH 8.0), containing 20 mM CaCl2·2H2O to a volume of 100 mL. BAPNA solution was used as a substrate. Then, 50 μL of the enzyme sample was mixed with 2.5 mL of BAPNA solution and then incubated in an incubator (Gravity convection incubator economy model 2EG, GCA Corp., Chicago, IL, USA) at 37°C for 10 min. Following this phase, 1 mL of 30% acetate solution was added to the sample mixture, and then the mixture was incubated for 10 min at 37°C. The absorbance was measured using a spectrophotometer (UV-Vis 2450, Shimadzu Corp., Kyoto, Japan) with a wavelength of 410 nm. Enzyme activity was calculated according to the following equation (1):

(1) Enzyme activity ( U / mL ) = ( Sample absorbance − Blank absorbance ) × total volume after being reacted ( mL ) × 1 , 000 8 , 800 × incubation time × volume of the enzyme being reacted ( μ L ) .

The molar coefficient of p-nitroaniline is 8,800. One unit of activity is the amount of enzyme required to release 1 μmol of p-nitroaniline/minute. Values are represented as the mean of three experimental replicates.

2.6 Protein determination

Protein concentration was measured using Bradford’s method [17], in which bovine serum albumin served as the standard. The Bradford solution consisted of 10 mg of Coomassie Brilliant Blue (CBB) G-250 in 5 mL ethanol (95%) and 10 mL phosphoric acid solution (85%) in 500 mL. The experiment was carried out with three replicates for each measurement. The mixture was monitored using a Shimadzu spectrophotometer (UV-Vis 2450) at 595 nm.

2.7 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography

SDS-PAGE was carried out following Laemmli's protocol [19] with slight modifications. About 10–20 μL of crude extracts, precipitated samples, and dialysate were injected separately into the electrophoresis gel. A 12% separator gel (v/v) and a 4% retaining gel (v/v) were prepared before sample addition. The sample was diluted in a 5× sample buffer containing 60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, and 0.1% bromophenol blue and then heated for 5 min at 100°C before being injected into the gel. The electrophoresis process was performed using a Hoefer instrument (Hoefer Scientific Instrument, Hoefer Inc., San Francisco, USA) at 70 and 20 A for 4 h until bromophenol blue reached the bottom of the gel. The protein was stained with a dye solution (0.25% CBB R-250 on 50% methanol and 10% acetic acid) and de-stained using a softening solution (7.5% acetic acid and 5% methanol). Markers were used to estimate the molecular weight of proteins. The protein marker used had a molecular weight ranging from 10 to 180 kDa (PM1500, Smobio Technology Inc., Hsinchu, Taiwan).

Zymography was performed with the addition of 0.5% casein to a 12% separator gel. After the electrophoresis process, the gel was soaked in Triton X-100 solution (2.5%) for 1 h at room temperature and lightly agitated with a 60 rpm shaker using a digital water bath (model LWB-322DS, Daihan Labtech Co., Ltd., Namyangju, Kyonggi-do, South Korea). The gel was washed with distilled water to remove Triton X-100 and then incubated with 10 mM Tris-HCl (pH 8.0) at 60°C for 30 min. The gel was stained with a staining solution for 1 h and then discolorated for 24 h, so that a clear zone was formed on the background of the blue gel, indicating the presence of protease activity.

2.8 Effect of temperature and pH on trypsin activity

The optimum temperature of the enzyme was determined at various temperatures (20, 30, 40, 50, 60, and 70°C) for 10 min using the BAPNA substrate. Enzyme activity was also determined at pH (5.0–10.0) using the same procedure at the optimum temperature. The buffers used were 100 mM sodium acetate (pH 5.0–6.0), 100 mM phosphate (pH 7.0), 100 mM Tris-HCl (pH 8.0), and 100 mM glycine-NaOH (pH 9.0–10.0).

2.9 Inhibitory effect of metal ions on trypsin activity

The influence of various metal ions (concentration 20 mM) on the activity of dialyzed enzymes was determined by mixing enzymes in a solution of monovalent (K+, Na+) or divalent (Ca2+, Mg2+, Zn2+) metal ions in a ratio of 1:1 (v/v). The enzyme was pre-incubated with metal ions at room temperature for 30 min. Then, the enzyme activity was tested using the BAPNA substrate at optimum temperature and pH for 10 min. The activity of enzymes in the absence of metal ions was assumed to be 100%. The activity of the trypsin was also determined in the presence of various inhibitors. The enzyme inhibitors used were PMSF, benzamidine (5 mM each), and SBTI (0.05 mM). The activity of enzymes in the absence of inhibitors was assumed to be 100%.

2.10 Statistical analysis

Data were analyzed using an analysis of variance (ANOVA) followed by Duncan using IBM SPSS Statistics Version 25 for Windows software (SPSS Inc., Chicago, IL, soft Corp., Washington, USA). Data are presented as mean ± standard deviation (SD), calculated using Microsoft Excel 2016 (Microsoft Corp., Washington, USA).

3 Results and discussion

3.1 Partial purification

The enzyme was purified by determining the concentration of (NH4)2SO4 suitable for the precipitation of the trypsin protease enzyme from skipjack offal. The concentration of (NH4)2SO4 that corresponds to the protein characteristics of the enzyme-induced enzyme precipitation. Crude enzyme extracts were precipitated with ammonium sulfate, which was carried out in a stratified manner as a preliminary step to the removal of other proteins (organic and inorganic impurity compounds) in the crude extracts. The properties of the trypsin protease enzyme are very well extracted with ammonium sulfate concentrations between 40 and 60% [11]. Data from several researchers correspond to the results obtained, namely the concentration of (NH4)2SO4 (40–50%) (Figure 2). The specific activity in the pellets of enzymes precipitated using (NH4)2SO4 40–50% was 1.66 U/mg, which was higher than that using (NH4)2SO4 at fractions 0–30% (1.08 U/mg), 30–40% (1.31 U/mg), and 50–60% (1.27 U/mg). Fraction 3 (40–50%) showed the highest value of 1.66 U/mg compared to other fractions. Statistically, there is a significant difference (P < 0.05) between fraction 3 and the other fractions.

Figure 2 Trypsin activity after fractionation with ammonium sulfate. All values are reported as mean ± SD, n = 3. Values with different letters revealed significant differences with Duncan’s test (P < 0.05).
Figure 2

Trypsin activity after fractionation with ammonium sulfate. All values are reported as mean ± SD, n = 3. Values with different letters revealed significant differences with Duncan’s test (P < 0.05).

Two steps of semi-purification were carried out against the trypsin enzyme obtained from skipjack viscera, including precipitation with ammonium sulfate and dialysis (Table 1). Enzyme extracts precipitated with ammonium sulfate 40–50% showed a specific activity of 1.66 U/mg with a purification factor of 3.44 times and a 69.91% yield. Dialysis increased the purity to 4.49 times and 39.20% yield. The purification and characteristics of the enzyme structure are related to variations in the recovery of trypsin from fish. Zamani and Benjakul [6] reported a dialysate of 40–60% ammonium sulfate deposits from pyloric caeca Aluterus monoceros showed 8.08-fold purification (yield: 40.34%), while trypsin from mackerel tuna intestine without dialysis exhibited 1.3-fold purification with a yield of 29.66% [10]. Ammonium sulfate salt is best for concentrating proteins [20].

Table 1

Enzyme activity and purification fold of trypsin from skipjack tuna viscera

Purification steps Total activity (U) Total protein (mg) Specific activity (U/mg) Purification fold Yield (%)
Crude extract 54.80 ± 0.94 113.52 ± 4.51 0.48 ± 0.02 1 100
Fraction (40–50%) 38.32 ± 1.34 23.04 ± 0.05 1.66 ± 0.06 3.44 69.91
Dialyzed 21.48 ± 0.11 9.90 ± 0.01 2.17 ± 0.01 4.49 39.20

Values are reported as mean ± SD, n = 3.

Table 2 compares the characteristics of enzymes derived from skipjack tuna viscera after partial purification and those reported by others. The findings reveal similarities in optimal temperature, pH, inhibitors, and activators. The inhibitors prevent the substrate from interacting with the enzyme’s active site, thus preventing the enzyme from catalyzing the process. The majority of activators are inorganic ions, particularly metal ions or cations. Our enzyme is distinct because its specific activity is relatively high compared to that in reports on A. monoceros fish [6] and E. affinis [10]. This implies that it has the potential to be a replacement for commercial enzymes. Our enzyme yield is also relatively high (39.20%), with a viscera percentage of 13.07%. This industry, where we took our sample, leaves 8.50% of the skipjack fish as viscera. The dialyzed enzyme that can be produced from viscera amounted to 39.20%. The problem of fish industry byproducts, particularly digestive organs, offers good possibilities as a source of enzymes.

Table 2

Comparison of the semi-purified trypsin from fish

Characteristics K. pelamis (this work) E. affinis [10] A. monoceros [6]
Optimum pH 8.0 9.0 8.0
Optimum temperature (°C) 50–60 60 55
Inhibitor (mM) SBTI (0.05), Benzamidine (5), PMSF (5), Zn2+ (20) Zn2+ dan Ca2+ (5) SBTI (0.05), TLCK (5)
Molecular weight (kDa) 25, 29, 35 — 23.5
Specific activity (U/mg) 2.17 0.38 1.86

Furthermore, the ammonium sulfate fractionation precipitation technique can protect enzyme molecules and separate the target enzyme from other protein components. Enzyme extracts are precipitated with ammonium sulfate salt as they are non-toxic, inexpensive, and straightforward. Excessive salt consumption can disrupt the enzyme’s protein structure, resulting in a decrease in activity. The solubility of proteins that interact with water-polar molecules, ionic interactions of proteins with salt, and the repulsion of proteins with the same charge all contribute to protein precipitation with ammonium sulfate salt. Salt molecules increase the solubility of protein enzymes during the salting process by diminishing electrostatic interactions between protein molecules. Protein–solvent interactions become more energetically beneficial as salt concentration increases, and the protein precipitates from the solution. Ammonium sulfate can also be eliminated using dialysis. Dialysis separates the dissolved molecules according to their size. The MWCO of the dialysis membrane was 14 kDa. Smaller ions move quickly through the membrane, whereas larger molecules are trapped. When the solution reaches equilibrium, the ions disperse uniformly throughout the solution while the proteins remain concentrated in the membrane, which lowers the overall salt concentration of the suspension.

3.2 SDS-PAGE and zymogram

SDS-PAGE 12% was effective for determining the approximate molecular weight of the enzyme (Figure 3). Enzyme extracts and precipitate of ammonium sulfate at a concentration of 40–50% have a complex protein profile (Figure 3, lanes 1 and 2). The removal of contaminants in 40–50% fraction through dialysis did not reduce the number of protein bands (Figure 3, lane 3). The molecular weights of trypsin are estimated at 25, 29, and 35 kDa. The trypsin activity of skipjack offal was analyzed using zymography. The substrate used was 0.5% casein, and the protein concentration introduced into the gel well was 0.13–0.29 mg/mL with an injected volume of 5 μL. Clear bands were detected, indicating the presence of enzymes in the enzyme extract in the 40–50% fraction of ammonium sulfate and dialysate. Impure enzymes are evidenced by the large number of visible clear bands with molecular weight ranges of 25, 29, and 35 kDa. In our zymogram analysis, the enzyme appeared active during the electrical movement. Consequently, the zymogram appeared as faint continuous bands at higher molecular weights. Trypsin isoforms from albacore tuna (Thunnus alalunga) liver have molecular weights of 21 and 24 kDa [21], 27.5 kDa for fish Pterygoplichthys disjunctivus [22], and 40 kDa for fish Barbus callensis [5].

Figure 3 (a) SDS-PAGE and (b) zymography of partially purified trypsin from skipjack viscera. Lanes (M) of molecular weight marker: (1) crude extract, (2) ammonium sulfate fraction 40–50%, and (3) dialyzed enzyme.
Figure 3

(a) SDS-PAGE and (b) zymography of partially purified trypsin from skipjack viscera. Lanes (M) of molecular weight marker: (1) crude extract, (2) ammonium sulfate fraction 40–50%, and (3) dialyzed enzyme.

The findings of our study are intriguing in that the presence of electricity during the zymography process did not inactivate this enzyme. Casein zymography is a sensitive and fast technique for detecting the presence of enzymes. The enzyme was active during electrophoresis, as demonstrated by the clear zone from the start. The zymogram typically produces prominent bands that are spread in certain areas.

The enzyme activity was assessed using the BAPNA substrate. After partial purification, it revealed a specific activity of 2.17 U/mg, which was higher than those of other fish species (1.86 U/mg [6] and 0.38 U/mg [10]).

3.3 Temperature and pH profile

The effect of temperature and pH on the activity of the enzyme extract, 40–50% ammonium sulfate precipitate, and dialyzed enzyme from skipjack tuna viscera were analyzed in the temperature range of 20–70°C and the pH range of 4.0–10.0 (Figure 4). Figure 4a illustrates the optimum temperature of the crude extract of trypsin from skipjack tuna viscera. The maximum enzyme activity toward the BAPNA substrate was found at 60°C. Relative activities at 40, 50, and 70°C were about 21.30, 30.90, and 83.94%, respectively. The partially purified trypsin (ammonium sulfate fraction 40–50%) has an optimum temperature of 60°C (Figure 4b), while the enzyme undergoing the dialysis process shows an optimum temperature of 50°C. Dialysis might separate some of the minerals and the small molecules from the enzyme. Therefore, the enzyme became more sensitive to the temperature, as shown in Figure 4c. Further, there is a decrease in activity at temperatures above 70°C. High temperatures induced changes in the enzyme structure. Enzyme molecules have a delicate structure and are usually damaged by thermal treatment. The decrease in enzyme activity due to heating is caused by the change in the conformation of the enzyme active site [23], which leads to less capability of enzyme–substrate association. The enzyme active site that cannot bind to the substrate will not catalyze the conversion of the substrate to the product. Trypsin purified from the liver of albacore tuna has optimum temperatures of 60 and 55°C [21]. Nevertheless, this optimum temperature is higher than the one being reported from purified trypsin from C. hippurus intestine (40°C) [11].

Figure 4 Effect of temperature and pH on the activity enzyme from skipjack tuna viscera using BAPNA as substrate: (a) temperature profile of the crude extract; (b) temperature profile of the ammonium sulfate fraction; (c) temperature profile of the dialyzed enzyme; (d) pH profile of the crude extract; (e) pH profile of the ammonium sulfate fraction; and (f) pH profile of the dialyzed enzyme. Values are shown as mean ± SD, n = 3.
Figure 4

Effect of temperature and pH on the activity enzyme from skipjack tuna viscera using BAPNA as substrate: (a) temperature profile of the crude extract; (b) temperature profile of the ammonium sulfate fraction; (c) temperature profile of the dialyzed enzyme; (d) pH profile of the crude extract; (e) pH profile of the ammonium sulfate fraction; and (f) pH profile of the dialyzed enzyme. Values are shown as mean ± SD, n = 3.

Figure 4d displays the optimum pH of the crude extract of trypsin from skipjack tuna viscera. Enzyme activity increases with an increase in the pH up to 8.0. However, its activity decreases at pH 10.0. This study discovers the optimum activity as pH 8.0. The relative activity at pH 9.0 was about 48.63% of that at pH 8.0. After fractionation and dialysis, trypsin from skipjack tuna viscera showed an optimum activity at pH 8.0 (Figure 4e and f). The relative activities of a fraction of 40–50% at pH 9.0 and 10.0 were about 50.31 and 39.53%, and those of dialysate were about 86.65 and 77.04%, respectively. In our work, the dialysis enzyme was more resistant to higher pH. Dialysis may rearrange the folding to a favorable state to interact with the substrate at a higher pH. The change in pH influences the rate of enzyme reaction because the ionization of the acid and base groups of the enzyme also change. Trypsin is classified as an alkaline protease, indicating that it is more stable at alkaline pH and less stable at acidic pH. At acidic pH, the load distribution and conformational change of the enzyme make the enzyme unable to bind optimally to the substrate [24]. Trypsins purified from C. hippurus intestine [11], S. longiceps viscera [12], and A. monoceros pyloric caeca [6] have an optimum pH of 8.0. Bougatef [4] reported that trypsin has an optimum activity in the pH range of 8.0–11.

3.4 Effect of ions and inhibitors

Several metal ions at a concentration of 20 mM were tested to determine their effect on trypsin dialysate activity from skipjack viscera, and the results are summarized in Table 3. Ca2+ (99.59%) and K+ (98.97%) ions showed no effect on enzyme activity. Similar results were reported by Villalba-Villalba et al. [22] and Sila et al. [5] from trypsin of P. disjunctivus and B. callensis with 5 mM Ca2+. On the other hand, Ktari et al. [25] and Silva et al. [26] reported that 5 mM Ca2+ may increase trypsin enzyme activity in fish. The use of Ca2+ improved protease production [27]. The presence of 10 mM Ca2+ activates trypsinogen to trypsin and increases the thermal stability of the enzyme incubated for 8 h at a temperature of 30°C [28]. Stabilization was achieved by changing the conformation of trypsin molecules into a more compact structure [29]. Enzyme activity started to decrease in the presence of Mg2+ (87.34%) and Na+ (95.04%). When incubated with Zn2+, the trypsin activity of the dialysate was inhibited by approximately 57.81%. It is known that a decrease in the activity of the pure trypsin enzyme occurred with the addition of 5 mM Zn2+ from the fish species C. hippurus [11], Engraulis encrasicholus [30], Sepia officinalis [31], and Sardina pilchardus [16].

Table 3

Influence of various metal ions on the dialysate activity of trypsin from skipjack viscera

Ions Concentration (mM) Relative activity (%)
Control — 100a
K+ 20 98.97 ± 7.82a
Na+ 20 95.04 ± 7.18ab
Ca2+ 20 99.59 ± 9.07a
Mg2+ 20 87.34 ± 7.11b
Zn2+ 20 42.19 ± 5.01c

All values are reported as mean ± SD, n = 3. Values with different letters in the same column revealed significant differences with Duncan’s test (P < 0.05).

The influence of some synthetic inhibitors on the dialysate enzyme activity of skipjack offal is shown in Table 4. The enzyme in this study was inhibited at 62.11% by PMSF (5 mM). PMSF can sulfonate serine residues on the active sites of the protease enzyme, thereby inhibiting its activity. Serine, aspartate, and histidine form the catalytic site of the serine protease group. PMSF reacts with the OH groups of the amino acid serine, causing an irreversible inhibitory response. Trypsin inhibition is similar to that observed in other types of fish, for example, S. longiceps [12], Thunnus alalunga [32], Luphiosilurus alexandri [24], Salaria basilisca [25], P. disjunctivus [22], Paralichthys olivaceus [33], Boops boops [34], S. officinalis [31], and Sardinella aurita [35]. The trypsin dialysate enzyme from skipjack offal is also inhibited by trypsin-specific inhibitors, namely benzamidine (5 mM) and SBTI (0.05 mM), 78.21 and 100%, respectively. Benzamidine inhibits the activity of the enzyme trypsin [11,16,26,36,37]. SBTI binds strongly to the active sites of the enzyme to inhibit catalysis. SBTI inhibition of trypsin has been reported in previous studies [21]. Trypsin inhibitors are classified as serine protease inhibitors, which can decrease the action of the trypsin enzyme. The positive charges on the side chain of the Arg63 residue in SBTI formed electrostatic interactions with the negative charges on the side chain of Asp189 in trypsin, contributing to inhibitor binding to the active sites of trypsin. Interactions of SBTI Arg63 with the trypsin active domain contain three catalytic sites, His57, Asp102, and Ser195, and one binding site, Asp189. The positive charge of Arg63 residues in SBTI is important to attract the negative charge of Asp189 of trypsin, which is the mechanism of strong binding of these two amino acid residues, which is responsible for the strong inhibition of SBTI to trypsin enzyme. It effectively prevented substrate binding to trypsin by blocking the active center of the enzyme [38]. These reported characteristics confirm that the purified enzyme belongs to serine proteinase identified as trypsin.

Table 4

Influence of various inhibitors on the dialysate activity of trypsin from skipjack viscera

Inhibitors Concentration (mM) Relative activity (%)
Control — 100a
PMSF 5 37.89 ± 2.67b
Benzamidine 5 21.79 ± 1.90c
SBTI 0.05 0d

All values are reported as mean ± SD, n = 3. Values with different letters in the same column revealed significant differences with Duncan’s test (P < 0.05).

PMSF, benzamidine, and SBTI are typical inhibitors of serine protease. As these inhibitors reduced our trypsin enzyme, this confirms that trypsin extracted from the fish viscera belongs to a serine protease class.

4 Conclusions

The partially purified trypsin protease enzyme from skipjack tuna viscera showed a specific activity of 2.17 U/mg. The activity of trypsin extract increased after fractionation of the ammonium sulfate 40–50%, followed by dialysis. The optimum temperature and pH are 50–60°C and 8.0. The proposed molecular weights were 25, 29, and 35 kDa and were inhibited by trypsin-specific inhibitors. Skipjack tuna viscera as a source of trypsin enzyme may be useful in industries.

Acknowledgements

The authors express their gratitude to the Ministry of Education, Culture, Research and Technology, Republic of Indonesia, for supporting their PhD study.

  1. Funding information: This study was supported by the Doctorate Program of BPPDN 2019 from the Ministry of Education, Culture, Research, and Technology, Republic of Indonesia.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and consented to its submission to the journal, reviewed all the results and approved the final version of the manuscript. Conceptualization, formal analysis, investigation, methodology, writing – original draft, writing – review and editing: FAD, NN, HNL, MTS. Supervision: MTS.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-12-11
Revised: 2024-05-05
Accepted: 2024-05-21
Published Online: 2024-06-22

© 2024 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/opag-2022-0308
Schlagwörter für diesen Artikel
biochemical characteristics; fish viscera; fractionation; trypsin
Creative Commons
BY 4.0

Artikel in diesem Heft

  1. Regular Articles
  2. Supplementation of P-solubilizing purple nonsulfur bacteria, Rhodopseudomonas palustris improved soil fertility, P nutrient, growth, and yield of Cucumis melo L.
  3. Yield gap variation in rice cultivation in Indonesia
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  23. Growth, yield, and secondary metabolite responses of three shallot cultivars at different watering intervals
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  25. Technical feasibility and economic benefit of combined shallot seedlings techniques in Indonesia
  26. Control of Meloidogyne javanica in banana by endophytic bacteria
  27. Comparison of important quality components of red-flesh kiwifruit (Actinidia chinensis) in different locations
  28. Efficiency of rice farming in flood-prone areas of East Java, Indonesia
  29. Comparative analysis of alpine agritourism in Trentino, Tyrol, and South Tyrol: Regional variations and prospects
  30. Detection of Fusarium spp. infection in potato (Solanum tuberosum L.) during postharvest storage through visible–near-infrared and shortwave–near-infrared reflectance spectroscopy
  31. Forage yield, seed, and forage qualitative traits evaluation by determining the optimal forage harvesting stage in dual-purpose cultivation in safflower varieties (Carthamus tinctorius L.)
  32. The influence of tourism on the development of urban space: Comparison in Hanoi, Danang, and Ho Chi Minh City
  33. Optimum intra-row spacing and clove size for the economical production of garlic (Allium sativum L.) in Northwestern Highlands of Ethiopia
  34. The role of organic rice farm income on farmer household welfare: Evidence from Yogyakarta, Indonesia
  35. Exploring innovative food in a developing country: Edible insects as a sustainable option
  36. Genotype by environment interaction and performance stability of common bean (Phaseolus vulgaris L.) cultivars grown in Dawuro zone, Southwestern Ethiopia
  37. Factors influencing green, environmentally-friendly consumer behaviour
  38. Factors affecting coffee farmers’ access to financial institutions: The case of Bandung Regency, Indonesia
  39. Morphological and yield trait-based evaluation and selection of chili (Capsicum annuum L.) genotypes suitable for both summer and winter seasons
  40. Sustainability analysis and decision-making strategy for swamp buffalo (Bubalus bubalis carabauesis) conservation in Jambi Province, Indonesia
  41. Understanding factors affecting rice purchasing decisions in Indonesia: Does rice brand matter?
  42. An implementation of an extended theory of planned behavior to investigate consumer behavior on hygiene sanitation-certified livestock food products
  43. Information technology adoption in Indonesia’s small-scale dairy farms
  44. Draft genome of a biological control agent against Bipolaris sorokiniana, the causal phytopathogen of spot blotch in wheat (Triticum turgidum L. subsp. durum): Bacillus inaquosorum TSO22
  45. Assessment of the recurrent mutagenesis efficacy of sesame crosses followed by isolation and evaluation of promising genetic resources for use in future breeding programs
  46. Fostering cocoa industry resilience: A collaborative approach to managing farm gate price fluctuations in West Sulawesi, Indonesia
  47. Field investigation of component failures for selected farm machinery used in small rice farming operations
  48. Near-infrared technology in agriculture: Rapid, simultaneous, and non-destructive determination of inner quality parameters on intact coffee beans
  49. The synergistic application of sucrose and various LED light exposures to enhance the in vitro growth of Stevia rebaudiana (Bertoni)
  50. Weather index-based agricultural insurance for flower farmers: Willingness to pay, sales, and profitability perspectives
  51. Meta-analysis of dietary Bacillus spp. on serum biochemical and antioxidant status and egg quality of laying hens
  52. Biochemical characterization of trypsin from Indonesian skipjack tuna (Katsuwonus pelamis) viscera
  53. Determination of C-factor for conventional cultivation and soil conservation technique used in hop gardens
  54. Empowering farmers: Unveiling the economic impacts of contract farming on red chilli farmers’ income in Magelang District, Indonesia
  55. Evaluating salt tolerance in fodder crops: A field experiment in the dry land
  56. Labor productivity of lowland rice (Oryza sativa L.) farmers in Central Java Province, Indonesia
  57. Cropping systems and production assessment in southern Myanmar: Informing strategic interventions
  58. The effect of biostimulants and red mud on the growth and yield of shallots in post-unlicensed gold mining soil
  59. Effects of dietary Adansonia digitata L. (baobab) seed meal on growth performance and carcass characteristics of broiler chickens: A systematic review and meta-analysis
  60. Analysis and structural characterization of the vid-pisco market
  61. Pseudomonas fluorescens SP007s enhances defense responses against the soybean bacterial pustule caused by Xanthomonas axonopodis pv. glycines
  62. A brief investigation on the prospective of co-composted biochar as a fertilizer for Zucchini plants cultivated in arid sandy soil
  63. Supply chain efficiency of red chilies in the production center of Sleman Indonesia based on performance measurement system
  64. Investment development path for developed economies: Is agriculture different?
  65. Power relations among actors in laying hen business in Indonesia: A MACTOR analysis
  66. High-throughput digital imaging and detection of morpho-physiological traits in tomato plants under drought
  67. Converting compression ignition engine to dual-fuel (diesel + CNG) engine and experimentally investigating its performance and emissions
  68. Structuration, risk management, and institutional dynamics in resolving palm oil conflicts
  69. Spacing strategies for enhancing drought resilience and yield in maize agriculture
  70. Composition and quality of winter annual agrestal and ruderal herbages of two different land-use types
  71. Investigating Spodoptera spp. diversity, percentage of attack, and control strategies in the West Java, Indonesia, corn cultivation
  72. Yield stability of biofertilizer treatments to soybean in the rainy season based on the GGE biplot
  73. Evaluating agricultural yield and economic implications of varied irrigation depths on maize yield in semi-arid environments, at Birfarm, Upper Blue Nile, Ethiopia
  74. Chemometrics for mapping the spatial nitrate distribution on the leaf lamina of fenugreek grown under varying nitrogenous fertilizer doses
  75. Pomegranate peel ethanolic extract: A promising natural antioxidant, antimicrobial agent, and novel approach to mitigate rancidity in used edible oils
  76. Transformative learning and engagement with organic farming: Lessons learned from Indonesia
  77. Tourism in rural areas as a broader concept: Some insights from the Portuguese reality
  78. Assessment enhancing drought tolerance in henna (Lawsonia inermis L.) ecotypes through sodium nitroprusside foliar application
  79. Edible insects: A survey about perceptions regarding possible beneficial health effects and safety concerns among adult citizens from Portugal and Romania
  80. Phenological stages analysis in peach trees using electronic nose
  81. Harvest date and salicylic acid impact on peanut (Arachis hypogaea L.) properties under different humidity conditions
  82. Hibiscus sabdariffa L. petal biomass: A green source of nanoparticles of multifarious potential
  83. Use of different vegetation indices for the evaluation of the kinetics of the cherry tomato (Solanum lycopersicum var. cerasiforme) growth based on multispectral images by UAV
  84. First evidence of microplastic pollution in mangrove sediments and its ingestion by coral reef fish: Case study in Biawak Island, Indonesia
  85. Physical and textural properties and sensory acceptability of wheat bread partially incorporated with unripe non-commercial banana cultivars
  86. Cereibacter sphaeroides ST16 and ST26 were used to solubilize insoluble P forms to improve P uptake, growth, and yield of rice in acidic and extreme saline soil
  87. Avocado peel by-product in cattle diets and supplementation with oregano oil and effects on production, carcass, and meat quality
  88. Optimizing inorganic blended fertilizer application for the maximum grain yield and profitability of bread wheat and food barley in Dawuro Zone, Southwest Ethiopia
  89. The acceptance of social media as a channel of communication and livestock information for sheep farmers
  90. Adaptation of rice farmers to aging in Thailand
  91. Combined use of improved maize hybrids and nitrogen application increases grain yield of maize, under natural Striga hermonthica infestation
  92. From aquatic to terrestrial: An examination of plant diversity and ecological shifts
  93. Statistical modelling of a tractor tractive performance during ploughing operation on a tropical Alfisol
  94. Participation in artisanal diamond mining and food security: A case study of Kasai Oriental in DR Congo
  95. Assessment and multi-scenario simulation of ecosystem service values in Southwest China’s mountainous and hilly region
  96. Analysis of agricultural emissions and economic growth in Europe in search of ecological balance
  97. Bacillus thuringiensis strains with high insecticidal activity against insect larvae of the orders Coleoptera and Lepidoptera
  98. Technical efficiency of sugarcane farming in East Java, Indonesia: A bootstrap data envelopment analysis
  99. Comparison between mycobiota diversity and fungi and mycotoxin contamination of maize and wheat
  100. Evaluation of cultivation technology package and corn variety based on agronomy characters and leaf green indices
  101. Exploring the association between the consumption of beverages, fast foods, sweets, fats, and oils and the risk of gastric and pancreatic cancers: Findings from case–control study
  102. Phytochemical composition and insecticidal activity of Acokanthera oblongifolia (Hochst.) Benth & Hook.f. ex B.D.Jacks. extract on life span and biological aspects of Spodoptera littoralis (Biosd.)
  103. Land use management solutions in response to climate change: Case study in the central coastal areas of Vietnam
  104. Evaluation of coffee pulp as a feed ingredient for ruminants: A meta-analysis
  105. Interannual variations of normalized difference vegetation index and potential evapotranspiration and their relationship in the Baghdad area
  106. Harnessing synthetic microbial communities with nitrogen-fixing activity to promote rice growth
  107. Agronomic and economic benefits of rice–sweetpotato rotation in lowland rice cropping systems in Uganda
  108. Response of potato tuber as an effect of the N-fertilizer and paclobutrazol application in medium altitude
  109. Bridging the gap: The role of geographic proximity in enhancing seed sustainability in Bandung District
  110. Evaluation of Abrams curve in agricultural sector using the NARDL approach
  111. Challenges and opportunities for young farmers in the implementation of the Rural Development Program 2014–2020 of the Republic of Croatia
  112. Yield stability of ten common bean (Phaseolus vulgaris L.) genotypes at different sowing dates in Lubumbashi, South-East of DR Congo
  113. Effects of encapsulation and combining probiotics with different nitrate forms on methane emission and in vitro rumen fermentation characteristics
  114. Phytochemical analysis of Bienertia sinuspersici extract and its antioxidant and antimicrobial activities
  115. Evaluation of relative drought tolerance of grapevines by leaf fluorescence parameters
  116. Yield assessment of new streak-resistant topcross maize hybrids in Benin
  117. Improvement of cocoa powder properties through ultrasonic- and microwave-assisted alkalization
  118. Potential of ecoenzymes made from nutmeg (Myristica fragrans) leaf and pulp waste as bioinsecticides for Periplaneta americana
  119. Analysis of farm performance to realize the sustainability of organic cabbage vegetable farming in Getasan Semarang, Indonesia
  120. Revealing the influences of organic amendment-derived dissolved organic matter on growth and nutrient accumulation in lettuce seedlings (Lactuca sativa L.)
  121. Identification of viruses infecting sweetpotato (Ipomoea batatas Lam.) in Benin
  122. Assessing the soil physical and chemical properties of long-term pomelo orchard based on tree growth
  123. Investigating access and use of digital tools for agriculture among rural farmers: A case study of Nkomazi Municipality, South Africa
  124. Does sex influence the impact of dietary vitD3 and UVB light on performance parameters and welfare indicators of broilers?
  125. Design of intelligent sprayer control for an autonomous farming drone using a multiclass support vector machine
  126. Deciphering salt-responsive NB-ARC genes in rice transcriptomic data: A bioinformatics approach with gene expression validation
  127. Review Articles
  128. Impact of nematode infestation in livestock production and the role of natural feed additives – A review
  129. Role of dietary fats in reproductive, health, and nutritional benefits in farm animals: A review
  130. Climate change and adaptive strategies on viticulture (Vitis spp.)
  131. The false tiger of almond, Monosteira unicostata (Hemiptera: Tingidae): Biology, ecology, and control methods
  132. A systematic review on potential analogy of phytobiomass and soil carbon evaluation methods: Ethiopia insights
  133. A review of storage temperature and relative humidity effects on shelf life and quality of mango (Mangifera indica L.) fruit and implications for nutrition insecurity in Ethiopia
  134. Green extraction of nutmeg (Myristica fragrans) phytochemicals: Prospective strategies and roadblocks
  135. Potential influence of nitrogen fertilizer rates on yield and yield components of carrot (Dacus carota L.) in Ethiopia: Systematic review
  136. Corn silk: A promising source of antimicrobial compounds for health and wellness
  137. State and contours of research on roselle (Hibiscus sabdariffa L.) in Africa
  138. The potential of phosphorus-solubilizing purple nonsulfur bacteria in agriculture: Present and future perspectives
  139. Minor millets: Processing techniques and their nutritional and health benefits
  140. Meta-analysis of reproductive performance of improved dairy cattle under Ethiopian environmental conditions
  141. Review on enhancing the efficiency of fertilizer utilization: Strategies for optimal nutrient management
  142. The nutritional, phytochemical composition, and utilisation of different parts of maize: A comparative analysis
  143. Motivations for farmers’ participation in agri-environmental scheme in the EU, literature review
  144. Evolution of climate-smart agriculture research: A science mapping exploration and network analysis
  145. Short Communications
  146. Music enrichment improves the behavior and leukocyte profile of dairy cattle
  147. Effect of pruning height and organic fertilization on the morphological and productive characteristics of Moringa oleifera Lam. in the Peruvian dry tropics
  148. Corrigendum
  149. Corrigendum to “Bioinformatics investigation of the effect of volatile and non-volatile compounds of rhizobacteria in inhibiting late embryogenesis abundant protein that induces drought tolerance”
  150. Corrigendum to “Composition and quality of winter annual agrestal and ruderal herbages of two different land-use types”
  151. Special issue: Smart Agriculture System for Sustainable Development: Methods and Practices
  152. Construction of a sustainable model to predict the moisture content of porang powder (Amorphophallus oncophyllus) based on pointed-scan visible near-infrared spectroscopy
  153. FruitVision: A deep learning based automatic fruit grading system
  154. Energy harvesting and ANFIS modeling of a PVDF/GO-ZNO piezoelectric nanogenerator on a UAV
  155. Effects of stress hormones on digestibility and performance in cattle: A review
  156. Special Issue of The 4th International Conference on Food Science and Engineering (ICFSE) 2022 - Part II
  157. Assessment of omega-3 and omega-6 fatty acid profiles and ratio of omega-6/omega-3 of white eggs produced by laying hens fed diets enriched with omega-3 rich vegetable oil
  158. Special Issue on FCEM - International Web Conference on Food Choice & Eating Motivation - Part II
  159. Special Issue on FCEM – International Web Conference on Food Choice & Eating Motivation: Message from the editor
  160. Fruit and vegetable consumption: Study involving Portuguese and French consumers
  161. Knowledge about consumption of milk: Study involving consumers from two European Countries – France and Portugal
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Heruntergeladen am 30.9.2025 von https://www.degruyterbrill.com/document/doi/10.1515/opag-2022-0308/html
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