Comparative Study On Proteolytic Activity, Purification And Characterization Of Trypsins From The Hepatopancreas Of Crucian Carp (Carassius Auratus) And Rice Field Eel (Monopterus Albus)

One of the main digestive proteases, which is detected in the pancreas, pyloric ceca and intestine of fish, is trypsin (EC 3.4.21.4). Trypsin is a member of a large family of serine proteases, specifically hydrolyze proteins and peptides at the carboxyl side of arginine and lysine residues and play the major roles in biological process including digestion, activation of zymogens of chymotrypsin and other enzymes. Trypsin exists in fish's pancreas with inactive zymogen, which is activated by enterokinase or trypsin, and its molecular structure has changed from trypsinogen to trypsin. But up to now, there are no reports about the comparative of purification, characterization and proteolytic activity of trypsins from rice field eel (Monopterus albus) and crucian carp (Carassius auratus). Research about trypsins will offer correlative information to feed preparation. Therefore, the main objectives of this study were to extract and purify trypsin from the hepatopancreas of fresh rice field eels (Monopterus albus) and crucian carp (Carassius auratus) and generate basic information about its main characteristics and proteolytic activity that might contribute to the commercial application of this by-product.1. Study on the purification of trypsin from rice field eel (Monopterus albus) and crucian carp (Carassius auratus)Rice field eel (Monopterus albus)and crucian carp (Carassius auratus) were washed twice with water and the hepatopancreas were separated, and then homogenized with 50 mM (pH 8.0) Tris–HCl at a ratio of 1:5 (w/v).The mixture was stirred continuously overnight at 4°C and centrifuged .The supernatant was considered the crude enzyme extract. The crude enzyme extract was subjected to ammonium sulphate fractionation and the precipitate in the 30–70% saturation range was collected by centrifugation. The precipitate was suspended in buffer and dialyzed 24 h at 4℃against repeated changes in the same buffer. The crude trypsin of Rice field eel(Monopterus albus)and crucian carp (Carassius auratus) were applied to the HiTrap Benzamidine FF(high sub)affinity equipped column(5ml) (Amersham Pharmacia Biotech),the proteins were eluted with 50mM Gly-HCl buffer (pH 3.0). Fractions showing protease activities were collected and accommodated its pH value by 1M Tris-HCl (pH9.0) for holding trypsin activity.Then the fractions were loaded into a DEAE-Sepharose TM Fast Flow column (Amersham Pharmacia Biotech) and equilibrated with 50 mM Tris-HCl (pH7.5) buffer. Unabsorbed protein was washed with equilibration buffer, and the column was eluted with a 500ml linear gradient ranging from 0 to 0.5 M NaCl. Through determine activities of trypsin by TAME on each step, The final preparations from Rice field eel(Monopterus albus)and crucian carp (Carassius auratus) were about 821-fold and 775-fold, with the recovery of 14.1% and 17.6%, respectively, from the crude trypsin.2. Comparative study on the characterization of trypsin from rice field eel (Monopterus albus) and crucian carp (Carassius auratus)Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions utilizing a 5% stacking gel and a 12% separating gel,trypsin from rice field eel (Monopterus albus) and crucian carp (Carassius auratus) show a single band and The molecular weight of the purified enzyme were estimated by SDS–PAGE as 25.4KDa and 26.8KDa respectively.The Michaelis constant (Km) of rice field eel and crucian carp trypsins determined to be 0.035mM and 0.023mM by using BAPNA (Nα-benzoyl-DL–arginine -p - nitroanilide) method.The optimum temperature for rice field eel and crucian carp trypsins for the hydrolysis of TAME(N-p-tosyl-L-arginine methyl ester)hydrochloride were 55℃,The thermal stability profile of the purified enzyme showed that the enzyme is stable below 50℃.The optimum pH of rice field eel and crucian carp trypsins were 8.0 and 8.5, respectively. Trypsin from crucian carp was stable at pH5.0-10.0 and trypsin from rice field eel was stable at pH6.0-9.0. The two trypsins were unstable at acid pH.The enzyme activity was not affected by Na+,Ca2+ , but inhibited slightly by Mg2+ and strongly by Cu2+. The trypsin activity was lightly increased at 10mM of K+ and then gradually decreased with the increase of K+ concentration.The enzyme was effectively inhibited by serine protease inhibitors, such as PMSF and benzamidine, but was lightly inhibited byβ-mercaptoethanol and EDTA.3. Comparative study on the proteolytic activity of trypsin from rice field eel (Monopterus albus) and crucian carp (Carassius auratus)The experiments were conducted to study enzymolysis kinetics and digestive rate in vitro of trypsin from rice field eel (Monopterus albus) and crucian carp (Carassius auratus) to casein, bovine albumin, lactalbumin hydrolysate, cottonseed meal, soybean meal, rapeseed meal and fish meal. The rates of amino acids produced from the enzymolysis fluid and the digestibility of those protein were determined respectively (P<0.05).The proteolytic activity of fish meal by trypsin from rice field eel was the hightest, and the proteolytic activity of cottonseed meal by trypsin from crucian carp was the hightest in the four fed materials. The activity of trypsin from crucian carp was higher than that of trypsin from rice field eel.