Introduction
Fish farming industry relies largely on the development of formulated compound diets for promoting growth (Tacon, 2020). However, the ability of fish to digest compound diets is mostly related to the existence of the digestive enzymes in different parts of the gastrointestinal tract (Yúfera and Darias, 2007; Nolasco‐Soria, 2021). In gastric fish species, protein is digested along the gastrointestinal tract by several proteases like pepsin, trypsin, and chymotrypsin. Trypsin, as an alkaline protease, has a key role in the digestion of protein, hydrolyzing them to free amino acids and small peptides for intestinal absorption; therefore, the activity of trypsin has been widely used as a valuable indicator of digestive capacity in fish (Nazdar et al., 2018). Hence, a better understanding of the properties of trypsin is necessary to generate valuable information for protein degradation in the fish digestive tract. In the present work, it was aimed to evaluate the effect of some physicochemical factors on the trypsin activity from pyloric caeca and intestine of rainbow trout (Oncorhynchus mykiss).
Methodology
Viscera from 10 specimens of rainbow trout with an average weight of 800 ± 31 g were obtained from a local market. Those samples were packed in polyethylene bags, placed in ice with the sample/ice ratio of approximately 1:2(w/w), and directly transported to the laboratory. Upon arrival, the pyloric caeca and intestine were removed from the rest of the collected viscera, washed with cold distilled water (4°C), pooled and stored at -80°C for further analysis (Zamani et al., 2023). The frozen samples were partially thawed in the refrigerator at 4°C for 2h and then cut into small pieces. Those pieces were homogenized in 50mM Tris–HCl buffer, filtered with a cheese cloth for separation of the floating fat phase and then centrifuged at 4°C (Zamani et al., 2014). The resulting supernatant from each sample was collected and finally used for assessment of the physicochemical factors including optimum temperature and thermostability (from 10 to 70 °C), optimum pH and pH stability (from 4.0 to 11.0), inhibitors (SBTI, TLCK, TPCK, PMSF, pepstatin A, iodoacetic acid, and EDTA) and metal ions (K+, Na+, Ca2+, Mg2+, Cu2+, Ba2+, Zn2+, and Al3+) on the trypsin activity according to the method described by Zamani et al. (2023).
Results
According to the obtained results, optimum temperature and pH of the trypsin from pyloric caeca and intestine were recorded at 55°C and 8.5, respectively. The stability of the trypsin from the both samples was well preserved at temperatures of up to 50°C and pH from 5.0 to 11.0. The enzyme activity from the both samples was significantly inhibited in presence of SBTI, TLCK, PMSF, pepstatin A and iodoacetic acid (p<0.05), while TPCK and EDTA showed no inhibitory effect on the enzyme activity. The enzyme activity from the both samples was significantly increased in the presence of Ca2+ and Mg2+ and decreased by Cu2+, Ba2+, Zn2+, and Al3+ (p<0.05). However, Na+ and K+ did not show any significant effect on the activity of both samples.
Discussion and conclusion
Enzymes are one of the main biological macromolecules that their maximum activity depend on an optimum temperature to make them functional. The trypsin from pyloric caeca and intestine of rainbow trout had optimum temperature of 55°C. However, an obvious decrease in the trypsin activity of both samples was observed at temperatures above 60°C, probably due to thermal inactivation of this enzyme caused by protein unfolding (Zamani et al., 2014). Similar optimum temperature (55°C) was recorded for trypsins in silver mojarra, cuttlefish, unicorn leatherjacket, and beluga and sevruga (Silva et al., 2011; Balti et al., 2012; Zamani and Benjakul, 2016; Zamani et al., 2023). The optimal temperature of trypsin is in the range of 30-60°C and the differences could be attributed to the temperature of fish habitat or experimental conditions used in assessments (Klomklao and Benjakul, 2018). The stability of trypsin from both samples was highly maintained up to 50°C. These results were in accordance with those of mandarin fish, grey triggerfish, mrigal carp, catfish, albacore tuna, common dolphinfish, and beluga and sevruga (Lu et al., 2008; Jellouli et al., 2009; Khangembam and Chakrabarti, 2015; Dos Santos et al., 2016; Klomklao and Benjakul, 2018; dos Santos et al., 2020; Zamani et al., 2023). In general, thermostability of the trypsin enzyme might vary by some factors such as fish species and experimental conditions (Kanno et al., 2010).
Trypsin from both samples had a maximal activity at pH 8.5. The optimum pH (8.5) recorded for the enzyme in both samples was similar with data reported from the brownstripe red snapper, masu salmon, albacore tuna, and Asian seabass (Kanno et al., 2010; Khantaphant and Benjakul, 2010; Klomklao and Benjakul, 2018; Patil et al., 2023). The stability of trypsin from both samples was highly preserved at pH values comprised between 5.0 and 11.0. Similar results were reported for trypsins from grey triggerfish, masu salmon, zebra blenny, catfish, albacore tuna, common dolphinfish, and beluga and sevruga (Jellouli et al., 2009; Kanno et al., 2010; Ktari et al., 2012; Dos Santos et al., 2016; Klomklao and Benjakul, 2018; dos Santos et al., 2020; Zamani et al., 2023). The high ranges of pH may change the net charge and conformation of an enzyme and inhibit to bind to substrate properly, resulting in the abrupt loss of enzymatic activity (Klomklao and Benjakul, 2018). Trypsins are mainly known to be more activity within a range of pH values comprised between 7.5 and 10.5 (Simpson, 2000). The sensitivity of protease enzymes to various inhibitors is a valuable tool for their proper functional characterization (Dos Santos et al., 2020). The results of this study showed that specific inhibitors including SBTI and TLCK had a complete inhibitory effect on the enzyme activity from both samples while the other inhibitors partially inhibited the enzyme activity or had no inhibitory effect on the enzyme activity. Our findings was in agreement with data reported in the brownstripe red snapper, masu salmon, silver mojarra, zebra blenny, mrigral carp, albacore tuna, common dolphinfish, Asian seabass, and beluga and sevruga (Kanno et al., 2010; Khantaphant and Benjakul, 2010; Silva et al., 2011; Ktari et al., 2012; Khangembam and Chakrabarti, 2015; Klomklao and Benjakul, 2018; dos Santos et al., 2020; Zamani et al., 2023; Patil et al., 2023). SBTI is a single polypeptide chain that acts as a reversible competitive inhibitor of trypsin and forms a stable, enzymatically inactive complex with trypsin, resulting in reduction of the enzyme availability (Senphan et al., 2015). TLCK is an irreversible inhibitor of trypsin and trypsin-like serine protease that deactivates these enzymes through the formation of a covalent bond with histidine residue in the catalytic site of the enzyme and blocks the active center of the enzyme for binding to substrate (Eun-Sil et al., 1998). The obtained results from the metal ions showed that Ca2+ and Mg2+ increased the enzyme activity from the both samples while Cu2+, Ba2+, Zn2+, and Al3+ decreased the enzyme activity and Na+ and K+ had no effect on the enzyme activity. Our results were in agreement with data reported in mandarin fish, zebra blenny, common dolphinfish, and beluga and sevruga (Lu et al., 2008; Ktari et al., 2012; dos Santos et al., 2020; Zamani et al., 2023). Metal ions can affect enzyme-catalyzed reactions by changing the electron flow in a substrate or enzyme and play a key role in binding to the substrate, depending on the functional groups present in the active site of enzyme. Abita et al. (1969) showed that Ca2+ can bind to the N-terminal of trypsinogen and, without changing its structure, increase the affinity of the lysine-isoleucine bond for hydrolysis by trypsin. Of course, the effect of metal ions on the activity of trypsin enzyme may depend on fish species as well as dietary and environmental adaptations (Khangembam and Chakrabarti, 2015). Based on the obtained results, the trypsin enzyme activity from the pyloric caeca and intestine of rainbow trout was significantly affected by the tested physicochemical factors which can affect the optimal activity of the enzyme in digestive physiology.
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgment
The author would like to thank the staff of the Fisheries Laboratory at Malayer University for their helpful assistance during the research process. |
