Study On Digestive Ability, Purification And Characterization Of Trypsins From Hybrid Tilapia (Oreochromis Niloticus × O.aureus)

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. Tilapia's culture is widely developed. Research about the Hybrid Tilapia trypsin is the very important content of digestive circadian and 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 tilapia and investigate basic information about its main characteristics and digestive ability.1. The purification of trypsin from Hybrid Tilapia (Oreochromis niloticus×O.aureus)The hepatopancreas of tilapia were separated and washed twice with water and, 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 tilapia 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 tilapia were about 87021-fold, with the recovery of 33.9% from the crude trypsin.2. Comparative study on the characterization of trypsin from Hybrid Tilapia (Oreochromis niloticus×O.aureus)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 tilapia show a single band and The molecular weight of the purified enzyme was estimated by SDS–PAGE as 24.3KDa.The Michaelis constant (Km) of tilapia trypsins determined to be 0.087mM by using BAPNA (Nα-benzoyl-DL–arginine -p - nitroanilide) method.The optimum temperature for tilapia trypsins for the hydrolysis of TAME(N-p-tosyl-L-arginine methyl ester)hydrochloride were 65℃,The thermal stability profile of the purified enzyme showed that the enzyme is stable below 60℃.The optimum pH of tilapia trypsins was 8.0. Trypsin from tilapia carp was stable at pH6.0-10.0.The enzyme activity was not affected by Na+,Mg2+ , but inhibited slightly by Zn2+ and strongly by K+. The trypsin activity was lightly increased at 5mM of Cu2+ and then gradually decreased with the increase of Cu2+ concentration. The enzyme was effectively inhibited by serine protease inhibitors, such as PMSF and benzamidine, but was lightly inhibited byβ-mercaptoethanol and EDTA.3. The study on the digestive ability of trypsin from Hybrid Tilapia (Oreochromis niloticus×O.aureus)The experiments were conducted to study enzymolysis kinetics and digestive rate in vitro of trypsin from tilapia to casein, bovine albumin, lactalbumin hydrolysate, cottonseed meal, soybean meal, rapeseed meal and fish meal. The results showed: The digestive ability of fish meal by trypsin from tilapia was the lowest, and the digestive ability of cottonseed meal was the highest in the four fed materials.