【作者】 钱方；

【导师】 安利佳；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 功能性寡糖多来自于细胞壁多糖水解,在植物诱抗、抑制病原菌、增强免疫力、改善胃肠功能等方面具有重要生理活性。黄原胶是植物致病菌野油菜黄单胞菌所分泌的胞外多糖,其主链类似纤维素很难降解,除作为生物胶集增稠、悬浮、乳化、稳定于一体外,还是植物毒素因子,能引起十字花科植物黑腐病。本论文研究目的是通过生物方法降解黄原胶,进而去除十字花科植物黑腐病的毒素因子,同时探讨黄原胶降解后所产寡糖是否具有特殊的生理活性。从土壤中分离出黄原胶降解菌株XT11。经菌体表观特征、菌落形态、生理生化鉴定等传统细菌分类鉴定,以及G+C含量、16S rRNA基因序列相似性比对和系统发育分析等现代分子生物学鉴定,最终确定菌株XT11是微杆菌属Microbacterium一新种。与M.hydrocarbonoxydans亲缘关系最近,相似性97.8%,遗传距离0.008。黄原胶降解菌株XT11的16S rRNA基因序列GenBank登陆号为DQ350882。同时对微杆菌属的进化遗传关系进行聚类分析,微杆菌属共分两大簇群,其中黄原胶降解菌XT11与近缘菌M.hydrocarbonoxydans构成一子分支,置信度为85%。降解菌Microbacterium sp.XT11分泌胞外黄原胶降解酶,降解黄原胶主链使其粘度降低,产黄原胶寡糖。黄原胶降解酶为底物诱导酶。黄原胶、CMC、淀粉、麦芽糖等结构相似,均是其诱导剂,其中黄原胶诱导作用最佳。黄原胶降解菌XT11适宜发酵产酶条件为:0.3%黄原胶、0.3%酵母浸粉的基础培养基,初始pH7.0,28℃160r/min溶氧培养。培养基外加葡萄糖,虽促进XT11菌体自身生长,但却抑制黄原胶诱导产酶。蔗糖、乳糖、麦芽糖、淀粉和纤维素等碳源,也不同程度抑制黄原胶降解酶的诱导合成。从黄原胶降解菌XT11发酵产酶特性来看,随着发酵培养时间延长,降解菌不断增长,发酵液粘度迅速下降,黄原胶降解酶活力逐渐上升。发酵28h时酶活达到最高23U/mL。黄原胶降解酶酶学性质初步分析表明,作用黄原胶最适反应温度40℃,最适反应pH值6.0,最适底物浓度0.4%(w/v)。当葡萄糖浓度高于0.06%时,对黄原胶降解酶的酶促反应具有抑制作用。且葡萄糖浓度越高,抑制作用越强。黄原胶降解酶使酶解反应液粘度和还原糖含量呈现良好的对应关系,说明黄原胶类似纤维素的主链发生降解,这正是黄原胶降解产生寡糖所必需的。黄原胶降解菌XT11降解黄原胶可得到不同粘度/还原糖比值的黄原胶寡糖。黄原胶降解寡糖生理活性研究表明,黄原胶自身无清除自由基能力,其降解寡糖却具有清除羟基自由基活性,且活性与寡糖浓度基本呈正相关。寡糖聚合度是影响活性的主要因素,其中黄原胶寡糖S6(粘度/还原糖比40.9)羟基自由基清除能力最强。黄原胶寡糖只抑制植物病原菌野油菜黄单胞菌Xanthomonas campetris生长,而对大肠杆菌Esherichiacoli、胡萝卜软腐欧文氏菌Erwinia carotovora,伤寒沙门氏菌Salmonella typhi、绿脓杆菌Pseudomonas aeruginosa、变形链球菌Streptococcus mutans、金黄色葡萄球菌Staphylococcus aureus、枯草芽孢杆菌Bacillus subtilis等其它供试病原菌均无抑制作用。黄原胶寡糖使大豆子叶呈现诱抗活性,诱导植物产生防御反应,提高植物抗病性。进一步研究发现,黄原胶寡糖S4、S5(粘度/还原糖比分别为232.5和101.4)对野油菜黄单胞菌野油菜致病变种(Xcc)抑菌活性尤为显著。黄原胶寡糖不仅抑制野油菜黄单胞菌野油菜致病变种(Xcc)生长,还阻止其合成毒素因子黄原胶。同时,黄原胶寡糖还部分抑制野油菜黄单胞菌野油菜致病变种(Xcc)所分泌的作为致病因子的某些酶的活性,如纤维素酶、几丁质酶、果胶酶和蛋白酶等。总之,黄原胶寡糖有明显的防治野油菜黄单胞菌引起十字花科植物黑腐病的生理活性,有开发为生物农药的潜在优势。更多还原

【Abstract】 Oligosaccharides have been proved to have some bioactivities such as defense response, antimicrobe,immunity reinforcement,etc.,which were degraded mainly from constituent polysaccharides of plant or pathogen cell wails.Xanthan is exopolysaccharide produced by phytopathogen Xanthomonas campestris,which was defined as a virulence factor of X. campestris inducing black rot of cruciate flower.Xanthan could not be easily degraded by most microorganisms because xanthan consists of a main chain similar to cellulose.The aim of this article is to search for a microorganism degrading xanthan to form bioactive oligosaccharides and study the special physiological activity of the formed oligosaccharides.One strain XT11 capable of degrading xanthan was isolated from soil samples.Strain XT11 should be assigned to the genus Microbacterium because the determinative taxonomic properties of strain XT11 were consistent with that of the genus Microbacterium.This agrees with the phylogenetic analysis via 16S rRNA gene sequence similarity,in which the strain XT11 is clustered in the genus Microbacteriurn.The closest genetic distance occurred between XT11 and M.hydrocarbonoxydans,the similarity of which is 97.8%.Access number of 16S rRNA gene in GenBank is DQ350882.According to the cluster analysis of the heredity relation of Microbacterium,the Microbacteriurn is divided to two cluster groups. Strain XT11 and peri-edge bacterium M.hydrocarbonoxydans is classified as a son branch. The confidence level is 85%.Strain Microbacteriurn sp.XT11 produced exoenzyme degrading glycoside linkage in the backbone of xanthan,leading to the decrease in viscosity and produce xantho-oligosaccharides.The xanthan-degrading enzyme was induced by xanthan and its glycosidic anaiogus CMC,starch and maltose.The optimal medium constituent for xanthan-degrading strain XT11 is 0.3%xanthan and 0.3%yeast extract at the initial pH7.0. The optimum culture temperature is 28℃.The glucose added to the medium promotes the cell growth of the strain XT11,but inhibits the enzyme production induced by xanthan.The carbon source sucrose,lactose,maltose,starch and starch also inhibit the induction of xanthan-degrading enzyme by xanthan.The culture time course of xanthan-degrading strain XT11 showed that the viscosity of fermentation broth was quickly descent during cell growth,while the activities of xanthan-degrading enzymes was increased gradually.The highest enzyme activity was obtained at 28-h incubation time,which is 23 U/mL.The properties of xanthan-degrading enzyme showed that the optimal reaction temperature was 40℃,the optimal pH was 6.0,the optimal concentration of substrate was 0.4%(w/v).The concentration of glucose greater than 0.06%inhibits the enzymatic reaction of xanthan-degrading enzyme.A good correlation occurred at between the viscosity decrease and reducing sugar release when xanthan was degraded by xanthan-degrading enzyme,which suggested that the backbone of molecules xanthan was degraded endogenously.This is just necessary for xantho-oligosaccharide production.Xantho-oligosaccharides with different ratio of viscosity and reducing sugars were produced from xanthan by xanthan-degrading enzyme.The xantho-oligosaccharides showed the ability to scavenging hydroxy free radicle.The activity is correlated to the concentration of oligosaccharide.The drgree of polymerization is the key factor affecting the activities of oligosaccharide.The xantho-oligosaccharide S6(the value of viscosity and reducing sugar is 40.9) has the strongest ability to discard hydroxyl free radical.Xantho-oligosaccharide could induce defense response in soybean cotyledon,which can protect plants against microbial disease.The xanthan-degrading oligosaccharides inhibit the growth of pathogen Xanthomonas campestris which is the pathogenic bacteria variety,but not inhibit other supplied pathogenic bacteria such as Esherichia coli,Erwinia carotovora,Salmonella typhi,Pseudomonas aeruginosa,Streptococcus mutans,Staphylococcus aureus and Bacillus subtilis. Xantho-oligosaccharide S4,S5(the ratio of viscosity and reducing sugar is 232.5 and 101.4 respectively) showed the great activity of bacteriostasis to pathogen Xanthomonas campestris. The xantho-oligosaccharide could inhibit the cells growth of Xanthomonas campestris and prevent the synthesis of virulence factor xanthan.Also it can inhibit partially the production of of cellulase,chitinase,pectinase and proteinase by Xanthomonas campestris.The results showed that the xantho-oligosaccharide had potential application in protection of plants against black rot of cruciferae caused by Xanthomonas campestris.更多还原