Purification And Kinetic Properties Of Trypsin From Rice Field Eel (Monopterus Albus)

Trypsin is a sort of digestive enzyme, which belongs to the family of proteases and has important physiological action in proteinic assimilation of fish's diaet. Trypsin comes from pancreas, intestines and pyloric ceca of fish. 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. This research is to purify and characterize trypsin from rice field eel (Monopteras albus), detailed researchs are showed: But up to now, there are no reports about the purification and properties of trypsin from rice field ee l(Monopteras albus).Rice field eel(Monopteras albus) is a freshwater fish, which is nutritious and delicious, so its culture is widely developed. Research about trypsin from rice field eel (Monopteras albus) will offer correlative information to feed preparation.1. Study on the purification of trypsin from rice field eel (Monopteras albus)Fresh rice field eels(Monopteras albus) were anatomized on ice tray, and take out hepatopancreas, then washed with distilled water, and chopped and homogenized in fourfold (w/v) of 50mM Tris-HCl, pH8.0, containing 0.02% sodium azide with tissue grinder. The mixture was stirred overnight at 4℃ and centrifuged at 15000 g for 40min at 4℃ , the supernatant was collected .The crude enzyme solution was subjected to 30%⁶0% ammonium sulfate(AS) fractionation. The AS fraction was dissolved in an appropriate amount of 50mM Tris-HCl, pH8.0, and dialyzed against the same buffer.Ion exchange stuffing adopted DEAE-Sapharose^? Fast Flow, and incased to chromatography column (1.2×20cm).The dialyzate was loaded onto a DEAE-Sapharose? Fast Flow column equilibrated with 100ml pH7.0 50mM T ris—HC1 at a rate of 8-10 ml/min. Unabsorbed proteins were washed with the equilibration buffer. The trypsin-like enzyme was eluted from the column using 50m M T ris—HCl, p H7.0 containing 0.1M NaCl, 0.2M NaCl, 0.3M NaCl, 0.4M NaCl, 0.5M NaCl respectively, then collected elucted apex conteining trypsin for the next purifying step.Affinity chromatography adopted 5ml HiTrap Benzamidine FF (high sub) affinity equipped column. The dialyzate was loaded onto affinity equipped column until the effluent had no detectable absorption at...