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. 2020 Aug 8;58(4):1585–1592. doi: 10.1007/s13197-020-04671-4

Effect of feeding habits of fish on the characteristics of collagenolytic proteases isolated from the visceral waste

Ankeeta Nayak 1, R K Majumdar 1,✉, Naresh K Mehta 1, Upasana Mohanty 1, Swapnarani Samantaray 1
PMCID: PMC7925775  PMID: 33746285

Abstract

In this study, influence of feeding habits of fish on the activity of collagenolytic proteases (CP) has been investigated. CP from the visceral waste of freshwater fish (Pangas, Rohu and Common carp) of different feeding habits was isolated and partially purified by 2-steps, (NH4)2SO4 fractionation and dialysis. Enzymatic activity and purification fold was determined in each step. The molecular mass of the enzymes were close to that of serine collagenases. Enzyme was assayed for temperature and pH optima, effect of sodium chloride and inhibitors. Optimum temperature and pH was 40 °C and 7–8 respectively. Soybean trypsin inhibitor inhibited the enzyme activity, whereas, EDTA exerted no effect, led to confirmation of serine collagenases. CP of carnivore was more active over a wide range of temperature and pH compared to herbivore and omnivore. The study revealed that the feeding habit of fish play decides the optimal physiological conditions for maximum activity of CP.

Keywords: Collagenolytic proteases, Enzyme purification, Enzyme inhibition, Pangasianodon hypophthalmus, Labeo rohita, Cyprinus carpio

Introduction

Fish processing waste is considered as a rich source of high quality protein and enzymes and these could be of high valued end products when extracted properly. Disposing of fish processing wastes to the natural water bodies without proper treatment can be life threatening to the aquatic animals and habitat including chocking of water bodies due to eutrophication (Bhaskar and Mahendrakar 2007). The digestive tract or the viscera which is normally wasted contributes 5–8% of the total fish weight (Mahendrakar 2000). Fish viscera include stomach, pyloric caeca, intestine, liver, pancreas etc. which contain several digestive enzymes such as pepsin, trypsin, chymotrypsin, elastase, and collagenase. However, quantitative and qualitative variations of digestive enzymes in fish is influenced by the factors such as feeding habit, age, season, maturation, diet, ambient temperature (Debnath et al. 2007). Moreover, different feeding habits of fish also contribute further diversification of different digestive enzymes and their characteristics.

Collagenases are present in epithelial, cartilaginous, bony tissues, and digestive tracts of fish and shellfish (Daboor et al. 2012). Collagenases are proteases capable of degrading the native triple helix of collagen under physiological conditions (Aoki et al. 2003) and are classified into two major groups, metallo-collagenases and serine collagenases, both play different physiological functions. However, the term collagenolytic proteases are used in a broader sense and refer to the enzymes with ability to degrade fibrillar or non-fibrillar collagen substrate (Liu et al. 2010). Metallo-collagenases are zinc-containing extracellular enzymes having molecular weight ranging from 30 to 150 kDa and are involved in remodelling the extracellular matrix. Whereas, serine collagenases having molecular weights < 60 kDa (Daboor et al. 2010) are probably involved in many physiological functions along with production of hormones and pharmacologically-active peptides, as well as in various cellular functions which include protein digestion, blood-clotting, fibrinolysis, complement activation and fertilization (Neurath 1984).

Enzymes from animal sources have advantages over chemical techniques and have wide industrial applications mostly in medicinal, detergent cosmetics, food, textiles, animal feed. Fish visceral enzymes find several industrial and pharmacological applications because of their unique properties and high activity over a wide range of pH and temperature conditions (El Hadj-Ali et al. 2011; Nasri et al. 2012). In the present study, collagenases from the visceral waste of three freshwater fish (Rohu, Labeo rohita; Pangas, Pangasianodon hypophthalmus and Common carp, Cyprinus carpio) having different habits has been characterized to assess the influence of feeding habits on enzymatic activity. Extraction and purification of such enzymes from the fish visceral wastes which are normally discarded could become a valued recycling of the waste and would improve the economy of the fisheries sector in general.

Materials and methods

Materials

Viscera of fish species such as Rohu (Labeo rohita), Pangas (Pangasianodon hypophthalmus), and Common carp (Cyprinus carpio), used in this study were collected from different fish markets of Agartala, Tripura. The samples were packed in polythene bags, placed in ice and transported to laboratory within 30 min. In laboratory, the viscera was washed with chilled water, kept in plastics bags and stored at -20 °C until used for enzyme extraction to prevent spoilage of the material and degradation of the enzymes due to bacterial action (Daboor et al. (2012). All the chemicals and reagents used in the study were obtained from HiMedia, India.

Preparation of crude enzyme extract

Crude enzyme extract was prepared according to the method suggested by Daboor et al. (2012). Thawed and chopped fish viscera was added to 50 mMTris-HCl buffer containing 5 mM CaCl2 (pH 7.4) at the ratio of 1:3 (w/v) and at 4 °C. The mixture was homogenized using tissue homogenizer (IKA® T25 digital TltraTurrax®) at a speed of 7000 rpm with mixing interval of 15 s and cooling in an ice bath for 30 s between mixing. The homogenate was centrifuged (REMI Compufuge) at 5000 rpm for 30 min at 4 °C and then filtered. Following the above procedure, the residues were re-extracted and the two supernatants were combined. The resultant supernatant was filtered through a 0.8 µ Millipore filter paper.

Partial purification of enzyme

The ammonium sulfate fractionation of crude enzyme extract was followed for partial purification as described by Daboor et al. (2012). Solid ammonium sulfate was added to the enzyme extract at a concentration of 40% and held for 1 h at 4 °C. Thereafter, the ammonium sulfate concentration was increased to 80% by adding required amount of ammonium sulfate and held overnight at 4 °C. Finally, the solution was centrifuged at 7000 rpm for 30 min at 4 °C and the pellets thus obtained was re-suspended in 1–2 mL of 50 mMTris-HCl buffer containing 5 mM CaCl2 (pH 7.4). Desalting of the solution was done through dialysis (Dialysis tube, MWCO 12-14 K) against the same buffer, for three consecutive times. The ammonium sulfate free solution was then centrifuged at 7000 rpm at 4 °C for 30 min and the supernatant was collected and stored at − 20 °C as described by Daboor et al. (2012) for further use.

Determination of protein

The protein content of the crude and the partially purified enzyme was determined by the Lowry’s method (Lowry et al. 1951) using crystalline bovine serum albumin as the standard.

Collagenolytic assay

The collagenolytic enzyme assay was done according to Kim et al. (2002) with slight modification. Collagen peptide was used as substrate for the assay. A reaction mixture was prepared by adding 0.1 mL of enzyme, 5 mg of collagen peptide and 1 mL of 50 mMTris-HCl buffer (pH 7.5), incubated at 40 °C for 30 min. The reaction was stopped by adding 0.2 mL of 50% trichloroacetic acid. After 10 min at room temperature, the solution was centrifuged at 1800xg for 20 min. The supernatant (0.2 mL) was mixed with 1.0 mL of ninhydrin solution, incubated at 100 °C for 20 min followed by cooling to room temperature. Subsequently, the mixture was diluted with 5 mL of 50% 1-propanol. The absorbance was taken at 570 nm. A blank was prepared by adding 5 mg of collagen peptide and 1 mL of 50 mMTris-HCl (pH 7.5) buffer without addition of any enzyme. The concentration of hydrolyzed-amino acids was determined by a standard curve based on a solution of l-leucine. One unit (U) of enzyme activity is defined as the amount of enzyme required for the hydrolysis of 1 μmol of substrate per min. Total activity, specific activity, purification fold and recovery percent were calculated using the formula suggested by Daboor et al. (2012).

Determination of molecular weight

The molecular weight of the crude as well as partially purified collagenolytic proteases was determined through SDS- polyacrylamide gel electrophoresis (SDS-PAGE) following the method described by Laemmli (1970). The molecular mass standard of 10-245 kDa was used. The protein bands were stained with Coomassie Brilliant Blue R-250 (0.1%) and bands were visualized with gel documentation system (Gel Doc™ EZ Imager, Bio-Rad, UK).

Determination of optimum temperature and pH

To determine the effect of temperature, the reaction mixture was incubated at various temperatures (10–100 °C) and tested for activity as described previously. The reaction mixture was prepared by adding 0.1 mL of enzyme to 5 mg of collagen peptide and 1 mL of 50 mMTris-HCl buffer (pH 7.5) containing 0.36 mM CaCl2. To determine thermal stability, the reaction mixture was incubated at 40 °C for 5–40 min. Aliquots were withdrawn at an interval of 5 min to test the remaining activity according to the procedure described previously.

Optimum pH for collagenolytic activity was determined by using reaction mixtures containing enzyme (0.1 mL) with 1 mL of different pH buffers along with 5 mg of collagen peptide. The buffers used were 50 mM citrate-Na2HPO4 (pH 3.0–6.0), 50 mMTris-HCl (pH 7.0–8.0), and 50 mM Na2CO3–NaHCO3 (pH 9.0–12.0) that contained 0.36 mM CaCl2. The activity was measured as described previously. To determine stability of the collagenases at a given pH, the enzyme extract was incubated with particular pH specific buffer and incubated for 30 min before estimating the remaining activity following the method as described previously.

Effect of NaCl on collagenolytic activity

To determine this, NaCl was added to reaction mixture as mentioned earlier at different concentrations, i.e., 20, 40, 60, 80 and 100 mM and then the activity for the treatments was measured as described previously. The reaction mixture without NaCl was taken as control.

Effect of inhibitors on collagenolytic activity

Two enzyme inhibitors soybean trypsin inhibitor (STI) and EDTA were added to the reaction mixture in different concentration. The concentrations used for STI was 5.5 mg/mL, 7.5 mg/mL and 10 mg/mL and for EDTA was 0.1 to 0.5 M. The inhibitor was added to the reaction mixture before the activity measurement. Then the activity for the treatments was measured as described previously. The blank was run simultaneously without addition of inhibitors.

Statistical analysis

All the experiments were carried out in triplicate and the results are presented as mean ± standard deviation. Significant divergences among mean values were determined with Duncan’s multiple range tests. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows version 16.0, SPSS, Inc., Chicago, IL).

Results

Purification of the collagenolytic proteases

Collagenolytic proteases (CP) from the visceral waste of the fishes was purified by two-step process. In the first step, crude enzyme extract was fractionated with ammonium sulphate (40–80%), followed by dialysis for removal of ammonium sulphate in the second step. The protein content of the crude CPs were found to be 6.265, 5.954 and 6.569 for Pangas, Rohu and Common carp respectively. The result of the purification of the collagenolytic proteases are presented in Table 1.

Table 1.

Purification of collagenolytic proteases from processing waste of Pangas, Rohu and Common carp*

Visceral samples Purification steps Protein content(mg/ml) Total activity(U/ml) Specific activity(U/mg) Recovery (%) Purification fold
Pangasianodon hypophthalmus Crude 6.265 ± 0.144a 29.67 ± 1.116a 4.73 ± 0.25a 100 1
(NH4)2SO4 (40–80%) 4.208 ± 0.056b 23.23 ± 1.06b 5.54 ± 0.34a 78.31 1.17
Dialysis 2.244 ± 0.091c 20.35 ± 1.120c 9.08 ± 0.69b 68.60 1.91
Labeo rohita Crude 5.954 ± 0.049a 16.87 ± 1.53a 2.83 ± 0.28a 100 1
(NH4)2SO4 (40–80%) 3.976 ± 0.068b 13.59 ± 0.32b 3.44 ± 0.12a 80.56 1.21
Dialysis 1.749 ± 0.143c 8.00 ± 0.881c 4.60 ± 0.70b 47.41 1.62
Cyprinus carpio Crude 6.569 ± 0.096a 20.17 ± 0.55a 3.07 ± 0.12a 100 1
(NH4)2SO4 (40–80%) 4.795 ± 0.065b 16.63 ± 0.96b 3.49 ± 0.19a 82.47 1.13
Dialysis 2.918 ± 0.203c 12.07 ± 1.24c 4.13 ± 0.22b 59.87 1.34
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*Values given in the table are mean ± SD, n = 3. Values bearing different superscripts (a, b, c) are significantly different (p < 0.05)

Soluble collagen peptide was used as substrate to determine enzymatic activity of CP. The total activity (U/ml) of crude CPs were found to be 29.67, 16.87 and 20.17 in case of Pangas, Rohu and Common carp respectively (Table 1). Total enzymatic activity of CPs showed reduction after both the purification steps, and exhibited a significant (p < 0.05) decrease at the rate of 31.4%, 52.5% and 40.1% in case of Pangas, Rohu and Common carp respectively.

On the other hand, the specific proteolytic activity (U/mg) of the proteases showed gradual increase along with the purification steps and post dialysis values were found to be 92%, 62.5% and 34.5% increase from the values of crude CPs in case of Pangas, Rohu and Common carp respectively. The purification folds along with recovery from the crude extract were 1.91 (68.6%), 1.62 (47.41%) and 1.34 (59.87%) after dialysis in case of Pangas, Rohu and Common carp respectively.

Figure 1 shows the gel images of SDS-PAGE for both crude and purified collagenolytic proteases from all the fishes. The molecular weights of crude collagenolytic proteases of Pangas, Rohu and Common carp were found to be in the range of 180 to < 11 kDa, 100 to < 11 kDa and 100 to < 20 kDa respectively. Whereas, the partially purified enzymes showed two to four bands in respect of all the samples.

Fig. 1.

Fig. 1

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Electrophoretic pattern of collagenolytic proteases from the processing waste of Pangas (a), Rohu (b) and Common carp (c) (A = Protein marker, B = Purified enzyme extract, C = Crude enzyme extract)

Effect of temperature on collagenolytic activity of CP

Collagenolytic activity of partially purified CPs at different temperatures (10–100 °C) and at pH 7.5 is shown in Fig. 2a. The optimum temperature of CPs from all the fishes was found to be 40 °C. Thermal stability of CPs incubated at 40 °C for different duration is given in Fig. 2b.

Fig. 2.

Fig. 2

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Enzymatic activity of collagenolytic proteases from visceral waste of Pangas, Rohu and Common carp at different temperatures (a) and thermal stability of enzyme (b)

Effect of pH on collagenolytic activity of CP

Figure 3a shows the pH requirement of partially purified CPs from all the fishes at temperature optima of 40 °C. The activity was measured on a pH range of 3.0–11.0. The optimum pH was recorded as 7.0, 7.0, and 8.0 in respect of Pangas, Rohu and Common carp respectively. The stability of enzyme at different pH (at fixed incubation period of 30 min at each pH) for maximum enzymatic activity was determined and presented in Fig. 3b.

Fig. 3.

Fig. 3

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Enzymatic activity of collagenolytic proteases from visceral waste of Pangas, Rohu and Common carp at different pH (a) and pH stability of enzyme (b)

Effect of NaCl on collagenolytic activity

In case of all the fishes, the activity of the CPs significantly decreased with the increase of the salt concentration at standard assay conditions and the result is presented in Fig. 4a.

Fig. 4.

Fig. 4

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Enzymatic activity of collagenolytic proteases from visceral waste of Pangas, Rohu and Common carp at different conc. of NaCl (a), STI (b) and EDTA (c)

Effect of inhibitors on collagenolytic activity of CP

Soybean trypsin inhibitor (STI) inhibited the activity of CPs in case of all the samples. Figure 4b shows the activity of CPs at varying concentrations of STI. But addition of ethylenediaminetetraacetic acid (EDTA) in the reaction mixture did not show any significant (p > 0.05) effect on the enzymatic activity of CP at standard assay conditions (Fig. 4c).

Discussion

In this study, collagenolytic proteases were isolated from the viscera of three freshwater fish of different feeding habits such as carnivore (Pangas), herbivore (Rohu) and omnivore (Common carp). The protein content of the crude CPs showed a significant (p < 0.05) decrease during purification steps, i.e., ammonium sulphate fractionation and dialysis and this may be due to removal of other proteins. The result revealed a lower protein content of CP of Rohu compared to carnivore (Pangas) and omnivore (Common carp), may be attributed to the feeding habit of Rohu which mostly feeds on plant materials. Influence of feeding habits and habitat temperature of the fishes on the diversity of digestive enzymes has been reported (Godfrey and Reichelt 1983). The protein content of the digestive proteolytic enzymes is dependent upon the species and different feeding behaviours (Klomklao et al. 2007).The observations recorded in this study corroborate well with the report of Daboor et al. (2010).

The study revealed that maximum reduction of total activity following different purification steps resulted in case of Rohu followed by Common carp and Pangas. Similar observations have also been reported by several authors while purifying enzymes (El Hadj Ali et al. 2010; Kim et al. 2012). Such behaviour of CP isolated from the viscera of Rohu may possibly be related with its low protein content, as decrease of total activity of CP at different purification steps depend on the recovery percentage of proteases in each step (Daboor et al. 2012; Kim et al. 2002).

A significant (p < 0.05) increase of specific activity of enzymes along the purification steps was observed and this may be due to removal of interfering proteins during (NH4)2SO4 fractionation and dialysis, as opined by other researchers (Daboor et al. 2012; Kim et al. 2002; Park et al. 2002; Souchet and Laplante 2010). Simultaneously the purification fold showed an increase (p < 0.05) during enzyme purification and the findings were consistent with the observation reported by Daboor et al. (2012), Kim et al. (2002), Park et al. (2002) and Souchet and Laplante (2010).

Number of clear bands while measuring molecular mass of collagenolytic proteases had been reported by several authors (Daboor et al. 2012; Ali et al. 2011; Kim et al. 2002). In the present study, more than one bands were noted in SDS-PAGE of crude CPs and this might be due to presence of other digestive enzymes having varying molecular weights. The partially purified CPs exhibited various molecular mass, such as Pangas (35 kDa and 48 kDa), Rohu (25 kDa, 35 kDa and 62 kDa) and Common carp (33 kDa, 35 kDa and 48 kDa). Although, a protein band of < 11 kDa and < 20 kDa was found in case of Rohu and Common carp respectively, showed similarity with that reported for serine collagenases from Sardinella aurita as 14.2 kDa (Hayet et al. 2011) and tuna pyloric caeca as 15.0 kDa (Byun et al. 2003). The result of this study, therefore, substantiate the reports of several authors who reported the MW of collagenolytic proteases from fish or fish processing waste as 24.1 kDa from Atlantic cod by Kristjansson et al. (1995), 25-36 kDa from snow crab’s by-product by Souchet and Laplante (2010), 29.5 kDa from Parasilurus asotus pancreas by Yoshinaka et al. (1986), 27.0 kDa from file fish (Kim et al. 2002).

The typical molecular weights of serine collagenases and metallo-collagenases were reported to be 24.0 to 36.0 kDa (Roy et al. 1996) and 30.0 to 150.0 kDa (Harris and Vater 1982). Although the lowest molecular weights of partially purified CPs in case of all the samples as observed in the present study fall within the range of serine collagenases, however, different molecular weights suggest that the serine collagenase does not have a single structure and also not from single source as reported by several authors (Kristjansson et al. 1995; Roy et al. 1996; Park et al. 2002). The variation in the MW of CPs amongst the fishes studied corroborate well with the explanation of Khantaphant and Benjakul (2010) that molecular mass of digestive proteases is a genetic factor and varies amongst the fish species depending on their feeding behaviour.

The temperature optima for the enzymatic activity of CPs of all the fishes was found to be 40 °C. A similar value was reported by Daboor et al. (2012), Souchet and Laplante (2010), and Aoki et al. (2003) for fish collagenases. This is lower than the value of 45–50 °C, that was reported for Atlantic cod (Kristjansson et al. 1995) and file fish (Kim et al. 2002), but higher than that of 25 °C, reported for marine crab (Scylla serrata) (Sivakumar et al. 1999). The enzymatic activity decreased at the rate of 78.5, 92.5 and 95.5% in case of Pangas, Rohu, and Common carp respectively after incubating the enzyme for 40 min at their optimum temperature, i.e., 40 °C, revealed that the serine collagenolytic proteases of true carnivore (Pangas) was less heat labile compared to the omnivore (Common carp) and herbivore (Rohu). It was reported that 45% of the enzymatic activity was retained after incubating the enzyme at 40 °C for 30 min (Aoki et al. 2003) and cod collagenolytic proteases lost their activity after incubating the enzymes for 30 min even at low temperature (25 °C) (Kristjansson et al. 1995). From this observation, it may be understood that collagenolytic proteases of freshwater fish are more heat stable and this may be due to the influence of habitat temperature on the enzymatic activity. Optimum temperature for enzymatic activity is species specific and usually governed by environmental and genetic factors (Klomklao et al. 2006) and also habitat temperatures (Villalba-Villalba et al. 2013), whereas, thermal stability of an enzyme is governed by the fish habitat, environment and genetic features (Sabtecha et al. 2014).

The optimum pH for total collagenolytic activity was recorded as 7.0 for both Pangas and Rohu and 8.0 for Common carp. Although, similar pH optima for collagenolytic activity had been reported for file fish (Kim et al. 2002), Green shore crab (Carcinus maenas) (Roy et al. 1996). In many of the studies, an optimum pH of 7.5 was reported for CP from fish processing waste (Daboor et al. 2012), mackerel (Park et al. 2002), catfish (Parasilurus asotus) (Yoshinaka et al. 1986). Whereas, higher pH (8- 9.5) was reported for Atlantic cod (Gadus morhua) (Kristjansson et al. 1995). The partially purified CPs maintained almost 75% of their original activity between pH 5–10 for Pangas, pH 6–7 for Rohu, and pH 6–8 for Common carp after incubating the enzymes for 30 min at different pH. Collagenolytic protease of Pangas showed stability over a wide range of pH compared to Rohu and Common carp, and this may be attributed to the carnivorous feeding habit of the species. Stability of proteases at a given pH depends on the differences in molecular properties, i.e., bonding and stability of the structure as well as enzyme conformation amongst various species and anatomical location (Klomklao et al. 2007). High pH stability of the collagenolytic proteases isolated from the processing waste of freshwater fish seems to be an additional advantage for their industrial application.

The enzymatic activity significantly (p < 0.05) decreased with the increase of NaCl concentration. The CP from Rohu viscera showed maximum decrease of its activity compared to Pangas and Common carp. Similar observation was reported by Kim et al. (2002) and Park et al. (2002) that addition of Na+ in the reaction mixture decreased the enzymatic activity. Addition of NaCl causes ‘salting out’ effect that leads to denaturation of enzyme and finally loss of enzymatic activity.

The soybean trypsin inhibitor (STI) exhibited a strong inhibitory effect on the collagenolytic activity of the isolated CPs, suggesting the possibility that the collagenases belonged to the serine collagenase family. On the basis of inhibitory effect of STI on collagenases, many authors suggested their isolated collagenolytic enzymes to be belonging to the family of serine collagenases (Yoshinaka et al. 1986; Kristjansson et al. 1995). Addition of EDTA showed no effect on collegenolytic activity and this may be due to either absence of Zn+2 in the reaction mixture or scavenged by EDTA. Non-effect of EDTA on the enzymatic activity led to confirm that the activity of CPs was non-Zn+2 dependent, as zinc is an effective inhibitor of serine collagenases (Park et al. 2002; Aoki et al. 2003; Yoshinaka et al. 1986). Generally, metallo-collagenase specifically requires zinc ion for optimum activity and stability. Isolation of many serine proteases have been reported from fish (Yoshinaka et al. 1986; Kristjansson et al. 1995).

Conclusion

The study revealed that the collagenases extracted from the viscera of the three fishes of different feeding habits belonged to the family of serine collagenases. The visceral collagenolytic protease of stomach bearing fish (carnivores) was found to be more active compared to the stomachless ones. It was also observed that the collagenase of Pangas (carnivore) was more active over a wide range of temperature and pH compared to other two fishes. Whereas, CPs of Rohu and Common carp viscera were more salt tolerant than that of Pangas. All these variations of the functionalities of the CPs could be related to the feeding habits of fish. However, future studies may be required for further purification by chromatographic or other technique and investigation on their specific industrial applications based on their characteristics.

Acknowledgements

Authors are greatly thankful to the Dean, College of Fisheries, Central Agricultural University, Lembucherra, Tripura for providing facilities and extending support to organize the study.

Footnotes

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