Isolation And Identification Of Swainsonine Degrading Bacteria And Degradation Character

Locoweed are toxic plants of the genera Astragalus sp. and Oxytropis sp. Locoweed poisoning is one of the most widespread poisonous plant problems. Locoweeds contain swainsonine, which is the principal agent responsible for locoism in animals. Swainsonine is an inhibitor of mannosidase in cell, which can alter metabolism of glycoprotein, leads to cellular vacuolation and cellular death, finaly can results in animal death and cause tremendous economic losses. Up to now, the toxicology, pathology of locoism have been detailed described, but it is remain a great problem to protect animals from this poisonous disease, locoism has become the most important disease on the vast rangeland. Recently detoxification the toxin of rapeseed cake, cottonseed and Leucaena plant using biodegradation method has maken advancement, many techniques have applied to practice, these can provid inspiration to locoweed detoxification.This paper mainly concern on extraction and detection method of swainsonine, isolation and identification of swainsonine degrading bacterium, and theirs degradation characteristics, enzyme and plasmid.1. Improved liquid / liquid extraction method of swainsonine with Ultrasound-assisted. Deal locoweed powder with 20 KHz ultrasound in water. The liquid fraction was then condensed and removed impurity, then extracted with n-butanol for several times, the n-butanol extraction was extracted by sparse acid. Extraction then was concentrated and extracted by ammoniated chloroform. Concentrat ammoniated chloroform. Sublimate the residue and got white crystal. The crystal was preliminary determined as swainsonine. The extraction rate was about 50mg/kg.2. Founded internal gas chromatographic method for determination of swainsonine. Pretreat the locoweed plant samples with methanol and water respective, methylation–α–D–mannoside and D–manicol were selected as internal standard substances. When the column temperature was 210℃, the retention time of methylation–α–D–mannoside was 5.71 min, the retention time of SW was 6.23 min, and the retention time of D–manicol was 9.07 min. the GC diagram showed that the methylation–α–D–mannoside as internal standard substance was better. The linearity range from 0.008 to 2.000 g/L, and the limit of quantitation was 0.001 g/L. In 24 h, relative standard deviation of three contents were 1.9%, 2.7% and 0.7% respectively. The recoveries for the standards were 88.04%,90.18% and 93.45%. The method is better reproducibility, rapid accurate, high degree of accuracy, and is useful to carry out qualitative and quantitative analyses of SW content in locoweed samples. This lays the foundation for detection of swainsonine changes in the progress of biodegradation.3. Isolation and identification of swainsonine degrading bacterial strain 15 stains were found can resist to high concentration swainsonine from soil where locoweed grow, using traditional enrichment procedure. Further research of degradation ability found that them only have very low ability of swainsonine degradation. The degradation rates were lower than 20% within 14 days, and the degradation ability is unstable. 1 Kg ground plant materials of Oxytropis kansuensis Bunge were buried under ground with 10～20 cm deep for six months. Then soil samples were collected around those plants, eight isolates were obtained that can grow on with swainsonine as sole carbon source, the degradation rate were over than 65% within 48 h to 50 mg/L swainsonine, four of them designated as strain YLZZ-1, YLZZ-2, YLZZ-2 and YLZZ-4 were remarkable, the degradation rate were 100%, 100%, 99.03% and 97.8%.The four strains were identificated based on morphology, physiologic tests, 16S rDNA sequence, and phylogenetic characteristics. Strain YLZZ-1 was identificated as Acinetobacter calcoaceticu, strain YLZZ-2 and YLZZ-4 were identificated as Stenotrophomonas maltophilia, and YLZZ-3 as Sphingobacterium multivorum. Strain YLZZ-1 and YLZZ-2 were conserve by China Center for Type Culture Collection, the numbers were CCTCC M 207108 and CCTCC M 207109 they both have been applied for nation nvention patent, apply order is 200710018724.9和200710018722.X, 16S rDNA sequences of the two strains were submited to GenBank database and accession numbers were EU022688 and EU02268.4. Degradation characterization analysis When the swainsonine concentration was 50 mg/L, Acinetobacter calcoaceticu YLZZ-1 could degrade swainsonine completely about 12 hours. The growth of bacteria was depended on the decrement of swainsonine. The temperature and initial pH suitable for the strain was 25～35℃and 6.0～8.0respectively. The optimum temperature was 30℃and the optimum pH was 7.0. Addition of glucose could inhibit swainsonine degradation obviously. Addition of starch could enhance swainsonine degradation, and addition of lactose,manicol and cane sugar could enhance swainsonine degradation but not obvious.Stenotrophomonas maltophilia YLZZ-2 could completely degradated 400 mg/L swainsoine within 24 h. The temperature and initial pH suitable for the strain was 25～35℃and 6.0～9.0 respectively. The optimum temperature was 30℃and the optimum pH was 7.0. The growth of stain YLZZ-2 and degradation rate were faster, There was a linear relationship between the growth of stain YLZZ-2 and degradation of swainsonine.5. Research on enzymes and plasmid of biodegradation stain. Degradation characteristics of swainsonine were determined by the crude enzyme extracted from the isolated strain Acinetobacter calcoaceticus YLZZ-1. The results showed that intracellular enzyme of YLZZ-1 had high degradation activity but the activity of extracellular enzyme and cell fragment were low. When the stain was cultivated in the condition of noninducement, most of its enzyme activity was lost. This enzyme was an intracellular and induced enzyme. Swainsonine showed induction effect on the express of degradation activity and catabolic enzymes gene. It provided reliable evidences for extract of catabolic enzymes. In enzymatic degradation, The optimum temperature was 30℃, the optimum pH was 8.0. Higher degradation activity was express at 25℃～45℃and pH 6.0～8.5. In the thermal stability of enzyme, the results indicated that it had stability under 35℃, the activity of enzyme decreased velocity with the increase of temperature. at 70℃, the remain activity of enzyme was about 15.79%. The solubility protein of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the solubility protein of intracellular crude enzyme was 1.2198mg/L. The crude enzyme showed Km value for swainsonine was 0.07398 mmol/L,and the maximal enzymatic degradation rate was 0.1115μmoL/(mg?min). Strain YLZZ-1 has resistance to ampicillin and chloramphenicol, and has two type plasmids.