【作者】 王妍；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 博士

【摘要】 疯草是一类能够导致动物中毒的植物,在我国西部地区广泛分布,严重危害当地畜牧业生产。疯草的毒性成分是苦马豆素,能抑制动物细胞中溶酶体α-甘露糖苷酶的活性,导致组织细胞空泡变性。中毒动物的临床表现以神经系统紊乱为特征,中毒严重者甚至导致死亡。本研究应用生物降解苦马豆素的方法,从埋置黄花棘豆的土壤中分离得到一株能够高效稳定降解苦马豆素的菌,并对该降解菌的特性、降解酶、降解产物及其毒性进行研究。苦马豆素降解菌的分离鉴定及降解产物毒性试验,将为该微生物降解苦马豆素的降解机理以及降解基因工程菌构建奠定基础。结果如下。1.苦马豆素降解菌的分离鉴定从埋置黄花棘豆的土壤中经富集筛选分离出了一个菌株,薄层色谱和气相色谱检测该菌能够降解苦马豆素,命名为HW 08菌。经形态学观察,HW 08菌革兰氏染色阳性,呈球-杆状生长,无鞭毛;经生理生化检测,氧化酶和过氧化氢酶阳性,明胶液化,可产吲哚,不产黑色素,不水解淀粉;经化学分类鉴定,该菌体脂肪酸成分为FA3c型,细胞壁中氨基酸与糖成分属于Ⅰ型;经分子生物学16S rDNA鉴定,HW 08菌属于放线菌目微球菌科中的节杆菌属,序列比对发现与解硝基邻甲氧基苯酚节杆菌（Arthrobacter nitroquajacolicus）同源性最高,最终命名为Arthrobacter nitroquajacolicus HW 08。2.苦马豆素降解菌的特性研究HW 08菌连续在无苦马豆素刺激的条件下转接100次,仍能在4 h内降解300 mg/L的苦马豆素;对HW 08菌未能提取出质粒;HW 08菌在不同的碳源培养基中增殖速度不同,能够利用葡萄糖、蔗糖、木糖、乳糖、半乳糖和甘露醇为碳源;HW 08菌降解等量的苦马豆素,接菌量与降解时间成反比关系;HW 08菌能够在1 000 mg/L苦马豆素MSM溶液中生长,4 h降解率为38.2%;当装液量为100 ml,pH为7.0,温度为30℃,摇床转速为210 r/min时,降解苦马豆素的能力最高;HW 08菌能降解苦马豆素外,还可以降解邻苯二酚、邻苯二胺、邻苯二甲酸、2,4,6-三硝基苯酚。3. HW 08菌降解酶定域及其降解产物培养36 h的HW 08菌,离心收集菌体,重悬于Tris-Cl缓冲液,经超声波破壁提取的胞内酶降解苦马豆素能力最高;采用正交设计优化降解酶的条件,发现在pH 8.0、30℃、底物苦马豆素浓度为300 mg/L时,反应40 min,降解率最高,为（97.2±3.25）%;研究证实该酶属于诱导型胞内酶,经乳糖诱导HW 08菌提取的胞内酶具有降解苦马豆素能力,降解率为（75±3.65）%。初步纯化苦马豆素降解酶,主要集中在40%～60%饱和硫酸铵沉淀的蛋白部分,此时可溶性蛋白含量为（68.43±10.16） mg/L,比活力为2.76 U/mg,米氏常数K_m为0.2μmol/ml,最大反应速率V_max为6.2μmol/min;经SDS-PAGE分析有两条特异条带,分子量约87.1 kD和81.3 kD。培养0 h,3 h,4 h,5 h,6 h的样品经薄层色谱紫外检测,出现两个特异点;经GC-MS检测,发现存在两个特异峰,通过谱库检索,一种物质为棕榈酸,另一种物质尚待证实。4.苦马豆素降解产物的毒性研究将HW 08菌完全降解苦马豆素后的上清液过滤除菌,进行小鼠毒性试验。结果表明,灌服不同浓度降解产物的试验动物均无明显临床症状,试验组间动物的体质量和脏器系数均差异不显著（P>0.05）。血涂片瑞氏染色未见异常细胞;血常规检测红细胞和粒细胞减少,白细胞、单核细胞、血小板增多,血红蛋白含量偏低;血清生化指标ALT、AST、ALP、BUN和Cre含量比正常对照值偏高,Glu和ALB含量偏低,但差异均不显著（P>0.05）。病理组织学检查发现,降解产物对小鼠的大脑、小脑、卵巢、睾丸等器官无明显病变,但对肝脏、脾脏、肾脏、肺脏、心脏等器官有轻度病理改变;体外α-甘露糖苷酶抑制试验证实,苦马豆素与等量苦马豆素的降解产物对α-甘露糖苷酶的抑制率差异极显著（P<0.01）,降解产物对α-甘露糖苷酶的抑制率仅为（5±2.52）%。更多还原

【Abstract】 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.更多还原