Isolation And Characterization Of Swainsonine-degrading Bacteria Strain Hw 08

Locoweed is a type of toxic plant which spreads widely in the west of China. Animal intoxication by foraging locoweed brings great economic losses to livestock industry. Swainsonine (SW), the toxin in locoweed and an inhibitor ofα-mannosidase, can lead to cellular vacuolation resulting in nervous system disturbances of poisoning animals clinically and even death. In this study, a strain of SW-degrading bacteria with high-performance was isolated from soil where O.ochrocehala has been buried, furthermore, its degradation characteristics, degradation enzyme, degradation product's component and toxicity were investigated. Isolation and identification of SW-degrading bacteria and toxicity assay of its degrading products will lay the groundwork for the mechanism of SW-degrading and the construction of genctic engineering bacteria to degrade SW. The results are as follows.1. Isolation and characterization of SW-degrading bacteriaA strain of SW-degrading bacteria was isolated from soil and named as HW 08. The ability of degrading SW was confirmed by thin-layer chromatography and gas chroma- tography. Morphological investigation showed that HW08 was Gram positive, ball-rod shape, flagella-free. Physiological and biochemical results showed that HW08 was positive for oxidase and hydrogen peroxidase, gelatinolytic, indoltheticum, and negative for melanin, amylolysis. Chemotaxonomy identification indicated that fatty acids of HW 08 was FA3c type, and amine acid and saccharide in cell wall of HW08 was type I. 16S rDNA sequencing and Blasting indicated that HW 08 was taxonomically classified as Arthrobacter, Micrococcaceae, Actinomycetales. And the homology exhibited 100% compared to Arthrobacter nitroquajacolicus. So strain HW08 was named as Arthrobacter nitroquajacolicus HW 08.2. SW-degrading characteristics of HW 08HW 08 was characteristic of degrading 300 mg/L SW within 4 hrs after re-inoculating 100 times without stimulus of SW, no plasmid inside in charge of degrading SW, and taking advantage of different carbon sources with different proliferative rates, such as glucose, sucrose, lactose, xylose, galactose and mannitole. Regarding its capability of degrading SW, the inoculating amount and degrading time was reversely related, which manifested degrading rate of 38.2% within 4 hrs in MSM media with 1 000 mg/L SW and highest degrading rate when HW 08 was maintained in 100ml volume, at pH 7.0, 30℃and 210 r/min. In addition, HW 08 harbored broad degradation spectrum and it could also degrade pyrocatechol, O-phenylene diamine, Picric acid and phthalate besides SW.3. Determination of degrading enzyme and products from HW 08The enzyme extracted from HW 08 was an endoenzyme, showing highest degrading rate of SW when isolated from 36 hrs-cultured HW 08 by centrifuging, resuspending in Tris-Cl buffer and then treating with ultrasonic. Orthogonal design demonstrated that the highest degrading rate (97.2±3.25%) of SW-degrading enzyme was found at the condition that pH 8.0, 30℃, SW concentration was 300 mg/L and the reaction time was 40 min. Further analysis showed that the enzyme was an induced endoenzyme with SW degrading rate of 75% after lactose induction. The primary purified enzyme was enriched in the precipitation of 40%-60% NH₄SO₄ and characterized by solubility of (68.43±10.16) mg/L, specific activity of 2.76 U/mg, Km of 0.2μmol/ml, and Vmax of 6.2μmol/min. Furthermore, SDS-PAGE showed two specific bands with about 87.1 KD and 81.3 KD.Two specific products were found through thin layer chromatography of 0 h, 3 h, 4 h, 5 h, 6 h culture. GC-MS assay further confirmed that one of the products was palmitic acid, while the other was unknown.4. Toxicity of SW degradation productsThe toxicity of SW degradation products was analyzed by feeding test mice with sterilely filtered supernatant of HW 08-treated SW solution. Results showed that no clinical symptoms was found obviously in mice which took degrading products in different concentrations orally. And there were no significant differences (P>0.05) on weight and organ coefficient of interclass mice. No abnormal cells were found in blood smears from tested mice. Blood routine examination demonstrated that red blood cells, granular leukocytes and hemoglobin content decreased, while white blood cells, mononuclear cells, platelets increased in tested mice. Serum biochemical analysis displayed higher levels of ALT, AST, ALP, BUN and CRE in tested groups when compared to the normal controls, while the reverse results were found for Glu, and ALB. But the results of blood and serum biochemical indices had no differences significantly (P>0.05) compared to the normal controls. Histopathologic examination showed no significant lesions in the brain, cerebellum, ovary, testis of tested mice. However, pathological changes slightly were found in liver, spleen, kidney, lung, heart of tested mice. Alpha-mannosidase inhibition test in vitro showed significant difference between equal amount of SW and SW degradation products on the inhibitory rate (P<0.01), which manifested only (5±2.52)% inhibitory rate of the latter.