Study Of Digestive Proteases Of Lizard Fish Saurida Elongata

Lizard fish( Saurida elongate) is a typical flesh-eating fish which contains large amount of proteases in its alimentary canal. In this study, the enzymic activities of digestive proteases in its stomach and intestine were studied first. And then the main proteases in stomach and intestine were purified and the enzymic characters were studied. Utilization of digestive proteases in hydrolyzing of the muscle of lizard fish was studied at last. The results are as follows:Enzyme activities of digestive proteases in lizard fish were studied in this paper first. According to the study, there are two protease species in alimentary canal of lizard fish, acid proteases and alkaline proteases. The results show that the most favorable reacting pH value and temperature for proteases of stomach and of intestine are 2.0, 10.0 and 40℃ 45℃, 50℃respectively. Both enzymes have high temperature tolerance and the high temperature (50℃) can activate intestine preteases. PMSF and Pepstatin A can inhibit the activities of proteases in stomach completely while EDTA and Trypsin inhibitor have no effect on them. PMSF and Trypsin inhibitor both can strongly inhibit the enzymic activities in intestine,while PCMB and EDTA have no effect on them. There are two protease isoenzymes in stomach and four in intestine on non-dissociating discontinuous polyacrylamide gel electrophoresis (PAGE) using casein as a substrate.After organic mashing, ammonium sulfate fractions, Q FF anion chromatography and Sephedax G-100 gel chromatography, the pure sample of pepsin from lizard fish was obtained .The result shows a single band by SDS-PAGE and the apparent molecular weight is 37.7kDa. The optimum temperature of pepsin is 45℃and the optimum pH value is 2.2 when cascein is used as a substrate. The activity almost has no change after being incubated at 40℃for 3h and as same as at 30℃which demonstrate that the pepsin has kind of temperature tolerance. Though the enzyme is stable in wide pH range (from pH 1 to pH 10), it becomes unstable at high alkali circumstance. Kinetic constants Km and Vmax are calculated as 2.25g/L and 8.772μg/min respectively when casein is used as substrate. Ca²⁺, Mn²⁺, Mg²⁺ and Ba²⁺ have no effect on the enzyme activty. Hg²⁺ can inhibit the enzymic activity slightly while Zn²⁺, Cu²⁺ and Fe³⁺ can inhibit it obviously. The results also show that EDTA and Trypsin inhibitor had little effect on the enzyme but PMSF and Pepstatin A can strongly inhibit its enzymic activity. The pepsin of lizard fish is a serine protease. PCMB and 2,3-butanedione can also inhibit the enzymic activity while other reagents such as 2-ME, BrAc and TNBS have no effect on it.A trypsin fraction was isolated from the intestine of lizard fish in three steps including defatted by CCl4, Sephadex G-100 gel filtration chromatography, Q FF ionic exchange chromatography. Purity was increased to 5.87 folds with approximately 11.58% yield for the enzyme. The apparent molecular weight of the trypsin is estimated to 23.3kDa by SDS-PAGE. The optimum pH and temperature of trypsin are 10.0 and 50℃respectively when cascein is used as a substrate. The trypsin is stable at pH range from 5 to 11 and is stable at 40℃and below for 3 h. Kinetic constants Km and Vmax are calculated as 3.32 g/L and 31.75μg/min respectively when casein is used as substrate. Cu²⁺, Fe3+ and Hg²⁺ can inhibit the enzymatic activity markedly while Zn²⁺ has a noticeable but not extreme inhibition. Trypsin inhibitor, PMSF and NBS can inhibit the activity of the enzyme while PCMB has no effect on it, which demonstrates that the protease was a serine-protease, specifically trypsin which has a tryptohpyl residue at the center of the active site.Digestive proteases of lizard fish were used to hydrolyze the muscle of lizard fish. From the results, the most suitable hydrolysis temperatures of stomach proteases and intestine proteases are respectively 40℃and 50℃. Compared with other commercial proteases such as pepsin, trypsin and papain, stomach proteases of lizard is not well in hydrolysis of the muscle of lizard fish while intestine proteases have a better hydrolysis effect which is almost 3-5 times higher than other enzymes. A certain concentration of Na+(0.1mol/L) can inhance the hydrolysis effect of digestive proteases while K+ and Ca²⁺ has little effect on it.