Preliminary Study On Swainsonine-degrading Enzyme Of Arthrobacter Nitroquajacolicus HW08and Its Degration Characteristics

Locoweed refers to some poisonous plants of Oxytrcpis and Astragalus. In ourcountry, locoweed mainly distributes in northwest and southwest pastoral areas.Locoism characterized by neurological symptoms and reproductive barriers emergeswhen livestock ingest a lot of locoweeds; severe cases even lead to death. There isstill no special treatment of locoweed poisoning disease, which has brought greateconomic losses to livestock production in the whole world. Studies have determinedthe toxin in locoweed to be swainsonine (SW), an inhibitor of α-mannosidase.Microbiology or its enzyme degration of SW can be used to convert locoweed toforage resources.Arthrobacter nitroquajacolicus HW08was a strain of SW-degrading bacteriaisolated from soil. In this study, the optimal degration conditions of SW-degradingenzyme and factors affecting its activity were investigated. The amino acids ofSW-degrading enzyme were tested using TLC. The extraction and purification werealso studied to increase the biodegradation of SW, providing the foundation for theapplication of the HW08bacteria in actual production. The results are as follows:(1) Different gradients of pH and temperature were set to detect the stability anddegration activity of HW08SW-degrading enzyme. The SW-degrading enzyme canmaintain long-term stability under the condition of pH7.0～10.0,30℃. Under thecondition of pH8.0and30℃, HW08can achieve the optimal degration effect to SW.(2) Metal ions and chemicals were used to detect their effects on the degradingenzyme activity, Ag+, Zn2+, Fe2+showed strong inhibitory effect on theSW-degrading enzyme activity, Co2+showed promoting effect on SW-degradingenzyme activity at low dose while inhibitory effect at high dose. PMSF,β-mercaptoethanol, EDTA all showed inhibitory effect on the degrading enzymeactivity, while Triton X-100showed promoting effects on enzyme activity. (3) When the SW-degrading enzyme was preserved at4℃and－20℃, the enzymeactivity loss slowly at the first2d, and greatly reduced at d3, but the activity reachedpeak level again at d4. Compared with4℃, the SW-degrading enzyme showed betterpreservation effect under the condition of－20℃. Triton X-100and BSA couldreduce the loss of enzyme activity in the process of enzyme preservation, and TritonX-100showed stronger protecting effects than BSA.(4) SDS-PAGE analysis showed that there was a specific band in intracellular enzymefrom SW induction; the weight was roughly calculated to be91kD. Ammoniumsulfate precipitation and dialysis was applied to purify the SW-degrading enzyme.Enzyme treated by ammonium sulfate with saturation of40%~60%can achieve thestrongest degrading activity. The soluble protein content of enzyme salted out byammonium sulfate with saturation of40%,50%,60%were1.4326mg/mL,1.208mg/mL,0.9964mg/mL, respectively. The total enzyme activity was251.3U, and thebiggest specific activity was2.25U/mg.(5)Amino acids Gln, Hyp, Tyr, Thr, Lys, His Orn and Ser were detected inSW-degrading enzyme of HW08using TLC.