Characterization Of A Gelatin Hydrolytic Enzyme From Riemerella Anatipestifer

Infectious serositis in duckling induced by Riemerella anatipestifer (RA) was one of the current most contagious diseases against duck industry. RA mainly infected susceptible animals against heart, liver, spleen, brain and other organizations. There were many celluloses exudation on serosa of the infected ducklings, forming cellulosal pericarditis, perihepatitis, air bladder inflammation and discharged meningitis. The diseased duck manifested eyes or noses had excretion, the neck crooked, ataxia and discharged exclusive brown or yellow or white manure. Survivors grown slowly or even lose their economic value. Many domestic and foreign researches showed extracellular proteases with gelatinlytic activity, as one important virulence factor, were closely related to the occurrence of a variety of bacterial diseases. Some RA strains have the characteristics of gelatin liquefaction and about 50% of the isolates as positive previously, but our preliminary studies are quite different. This research ascertained the epidemic state of the liquefing gelatin strains, the influencing factor of producted active substances in positive strains which liquefied gelatin, and the physicochemical properties of active substances. Huabian ducklings were artificially infected with active substances for study on histopathologic effects. This research laid the foundation for the further study of the RA extracellular protease in the pathogenesis and the virulence factors of RA.The main outcomes summarized as follows:1.The method of nutritional gelatin plate was established to survey epidemic of RA liquating gelatin strains. This method was convenient, quickly and direct. 60 strains of RA from different localities were detected by this method, 57 strains could liquate gelatin and the positive rate was 95%.2.Crude enzyme of gelatin hydrolytic. enzyme was extracted from RA liquid culture supernatant by ultrafiltration and 65% saturation of ammonium sulfate precipitation. Huanbian ducklings were injected 1mL (correspond to liquid culture supernatant 140mL) crude enzyme through jugular vein injection at 14 days of age. After infected, most of the ducklings appearance melancholy spirit, weakness, drowsiness, some accompanied neurological symptoms such as shakes, head and neck crooked, even emergence of sporadic deaths. There was liver bleeding, subdural hemorrhage, cerebral edema, air bladder thickening and many exudations on heart. Histopathologically, cardiac muscle fibers swelling, some showed severe granular degeneration and part of cardiac muscle cell showed karyolysis or karyo pyknosis. Cerebrum telangiectasia and hyperemia, capillary wall between the brain tissues showed a larger gap and endothelial cells of blood capillary slightly swollen. Hepatic cell showed serious vacuolar degeneration that vacuolus which inequality of size emerged in almost all hepatic cell endochylema. Spleen central artery vascular endothelial cell swelling, vessel wall structural disorder, acinus lienalis showed small amounts of acidophil leukocyte and more akaryocyte. Crude extract of RA which without gelatin liquefaction activity showed no obvious clinical symptoms and histopathologic influence.3.The capacity to produce gelatin hydrolytic enzyme has some diversity in different RA strains. Shaking cultivation demonstrated that the strain named AF possessed the highest gelatin hydrolytic activity. The optimal cultivation conditions for AF producing gelatin hydrolytic enzyme were pH7.4, 100mL flask containing 10mL medium, 1% amount of inoculation, containing 5mM Ca²⁺ and 37℃incubating for 24 hours at 150rpm in LB liquid medium. The production of gelatin hydrolytic enzyme was inhibited when added 1% glucose or sucros to the medium.4.The optimal condition for gelatin hydrolytic enzyme activity were pH8.0, temperature 37℃with gelatin as the substrate. The enzyme was stabilization below 50℃, loss activity above 50% at 60℃for 0.5 hours and the activity completely lost at 65℃for 0.5 hours. The activity of enzyme was actived in divalent metal ions Ca²⁺, Mg²⁺, 1mmol/L Zn²⁺and weakly inhibited in 5-10mmol/L Zn²⁺ ,but strongly inhibited by Cu²⁺. The activity of enzyme was inhibited in metal-chelator EDTA, EGTA and other inhibitors such as 2-ME, NBS, DEPC and acetic anhydride . Residual activity recovered about 20% when added 20mM Ca²⁺ after the activity of enzyme was inhibited by EDTA or EGTA.