【作者】 赵兴华；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2008， 博士

【摘要】 疯草是豆科棘豆属和黄芪属有毒植物的总称,也是世界范围内危害草原畜牧业最严重的一类毒草。苦马豆素是疯草类植物中的主要有毒成分之一,能抑制细胞中α–甘露糖苷酶活性,影响糖蛋白的代谢,导致细胞空泡变性,失去正常功能,致使中毒动物死亡,造成巨大经济损失。目前在疯草毒性成分、中毒机理、化学防除和脱毒利用等研究方面取得了一些成绩,但迄今还没有有效控制疯草中毒的方法,致使疯草中毒问题日趋严重,成为草原畜牧业发展的大患。近年来应用微生物降解技术去除菜籽饼、棉籽饼和银合欢植物中的有毒成分获得重要进展,有些技术已应用于实践,可以为疯草解毒研究提供借鉴。本文主要对苦马豆素的提取及检测方法、苦马豆素降解菌的分离鉴定、降解特性、降解酶和质粒等方面进行研究。1.完善超声波协助的液/液萃取提取苦马豆素的方法以水为溶剂,采用20 KHz超声循环提取机处理变异黄芪草粉,获得的提取液浓缩后离心除去固体杂质,浓缩液用正丁醇反复萃取,正丁醇萃取液用稀酸水萃取,稀酸水萃取液用氨性氯仿萃取,浓缩氨性氯仿萃取液成浸膏,此浸膏经石蜡油油浴减压升华得到白色针状结晶,经鉴定确定为苦马豆素,提取率约为50 mg/kg。2.建立检测苦马豆素的气相色谱内标法分别采用甲醇和水为溶剂对植物样品进行预处理,选择甲基化–α–D–甘露糖苷和D–甘露醇作为气相色谱分析的内标物。在柱温为210℃时,甲基化–α–D–甘露糖苷的出峰时间为5.71 min,苦马豆素出峰时间为6.23 min,而D–甘露醇出峰时间为9.07 min,结果显示以甲基化–α–D–甘露糖苷为内标物分析效果更好。该方法在0.008～2.000 g/L范围内具有良好的线性关系,检出限为0.001 g/L,0.512,0.125,0.032 g/L三个浓度水平在24 h内测定相对标准偏差分别为1.9%、2.7%和0.7%,添加回收率分别为88.04%、90.18%和93.45%。本研究建立的分析方法重现线性好,精确度高,结果准确可靠,可以满足疯草植物样品中苦马豆素含量的分析,为检测生物降解过程中苦马豆素含量变化奠定了基础。3.苦马豆素降解菌的分离与鉴定通过富集驯化培养,从疯草生长环境中的土壤中分离出15株能够耐受高浓度苦马豆素的细菌。对其降解性能进一步研究发现,这15株菌对苦马豆素只具有较低的降解能力,在14 d内,对50 mg/L的苦马豆素降解率均在20%以下,且降解性能不稳定。将1 kg甘肃棘豆样品埋藏于土壤中,6个月后分离草样周围的土壤做为分离源,从中分离出8株细菌,该8株菌在48 h内对50 mg/L的苦马豆素的降解率均在65%以上,其中有4株菌降解能力最强,对苦马豆素降解率分别为100%、100%、99.03%和97.8%,分别命名为YLZZ-1、YLZZ-2、YLZZ-3和YLZZ-4。根据形态学特征观察、生理生化试验、16S rDNA序列测定与分析,以及基于16S rDNA序列的系统发育分析等方法对分离获得的降解菌进行鉴定。菌株YLZZ-1鉴定为醋酸钙不动杆菌,菌株YLZZ-2和菌株YLZZ-4鉴定为嗜麦芽寡养单胞菌,菌株YLZZ-3鉴定为多食鞘氨醇杆菌。菌株YLZZ-1和菌株YLZZ-2已保存于中国典型培养物保藏中心,保藏号分别为CCTCC M 207108和CCTCC M 207109,并申报了国家发明专利,专利申请号分别为200710018724.9和200710018722.X,该两株菌的部分16S rDNA序列已经登陆GeneBank,登陆号分别为EU022688和EU02268。4.降解特性分析醋酸钙不动杆菌YLZZ-1在12 h左右可以将50 mg/L的苦马豆素降解完全,能以苦马豆素为惟一碳源进行生长;在pH 6.0～8.0、温度25℃～35℃时适合菌株YLZZ-1的生长,菌株降解苦马豆素的活性较强,其中最适pH为7.0、最适温度为30℃;底物浓度增加对菌株YLZZ-1生长具有一定的抑制作用,增加细菌接种量可以提高底物的转化率,基础培养基中添加淀粉能促进菌株对苦马豆素的降解,而添加乳糖、甘露醇对降解率提高作用不大,添加葡萄糖对降解有抑制作用。嗜麦芽寡养单胞菌YLZZ-2能在12 h内将50 mg/L苦马豆素降解完全,能以苦马豆素为惟一碳源进行生长,且降解能力强,可以降解400 mg/L的苦马豆素。菌株YLZZ-2在pH 6.0～9.0、温度25℃～35℃时生长良好,降解苦马豆素的活性较强,其中最适pH为7.0、最适温度为30℃。5.降解菌的降解酶与质粒初步分析对醋酸钙不动杆菌YLZZ-1降解苦马豆素的酶进行提取和定位,并对酶促降解特性进行研究。结果显示,菌株YLZZ-1胞内粗酶液对SW具有较高的降解活性,而胞外粗酶液和细胞周质提取液的降解活性极低,菌株经过一定时间的非诱导培养后,提取的粗酶液中的酶活力显著降低,由此推断降解菌产生的苦马豆素降解酶属于胞内酶,且为诱导酶,底物苦马豆素对降解酶的降解活性或降解酶基因的表达具有诱导作用,为进一步分离和纯化降解酶提供了依据。降解酶在25℃～45℃有较高降解活性,酶的适宜pH为6.0～8.5,且有较好的pH稳定性,其酶促反应的最适温度为30℃,最适pH为8.0,酶的热稳定性结果表明,酶在35℃以下稳定性良好,在45℃、55℃、60℃、70℃下保温2 h,酶活力随着温度的升高而下降迅速,70℃下剩余最高酶活力的15.79%左右。粗酶液中蛋白含量为1.2198 mg/mL,粗酶对苦马豆素降解动力学参数米氏常数Km为0.07398 mmol/L,最大反应速率Vmax为0.1115μmoL/(mg·min)。菌株YLZZ-1具有氨苄青霉素和氯霉素抗性,含有两种质粒。更多还原

【Abstract】 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.更多还原