新型胞外多糖产生菌Phyllobacterium sp. nov. 921F及其多糖结构的研究
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摘要
细菌胞外多糖由于其化学结构的多样性和新颖性,以及逐渐被发掘的多种新型生物学活性,受到了越来越多的科研工作者的关注。目前所报道的细菌胞外多糖主要有黄原胶、热凝胶和结冷胶三类。其结构不同,功能各异,分别应用于各个不同的领域,如食品工业、纺织工业、石油化工等方面。本论文以获得具有新型、高效且形状稳定的胞外多糖产生菌为基本目的和出发点,期望能够获得一株具有工业生产及应用的新型细菌胞外多糖。
本文通过定向筛选技术获得一株胞外多糖产生菌。结合生理、生化鉴定和16SrRNA技术确定该菌株为叶杆菌属新种(Phyllobacterium sp.nov.921F)。现已申请国家专利保护——专利申请号:200710167753.1。该产糖菌株已保存于中国典型微生物培养保藏中心。由于目前尚未有相关菌种的产糖的报道,我们采用定向诱导技术和条件优化后,使得该菌株经84 h摇瓶培养后,产糖量达1.8%。在此基础上,进行10 L全自动发酵罐试验,产糖量提升至2%以上。胞外多糖提取工艺的优化方面,通过引入金属盐溶液而有效的减少了有机溶剂的加入量,显著减少了提取工艺的生产成本。以上结果表明:该菌株具有产糖量能力高、产糖性状稳定、多糖提取工艺简单且相比其它细菌胞外多糖提取能耗低等特点。达到了上游发酵工业对菌种的要求水平,具有潜在开发价值。
胞外多糖理化性质和基本组成分析结果表明该多糖易溶于水,其水溶液呈透明粘稠状;多糖由半乳糖和葡萄糖组成,二者摩尔比为1.07:1;有丙酮酸基团存在;通过GPC分析得到多糖重均分子量为1082 kDa。由于该胞外多糖表现出很强的粘度特性。因此本文考察了多糖溶液浓度、剪切力、pH值、温度、放置时间、恒温加热、无机盐及蔗糖浓度对多糖溶液表观粘度的影响。结果表明该胞外多糖表观粘度受剪切速率、蔗糖浓度等因素影响较小且稳定性好,在pH 3-12之间变化不明显。随着温度的升高粘度下降。多糖的表观粘度受无机盐的影响有一定的下降,但变化程度不大。因此该多糖的粘度特性也展示出较好的应用前景。
在糖链结构解析方面,本研究采用专一性酶解技术系统、准确的对糖链结构进行解析。以胞外多糖为特异性底物筛选一株降解菌进而制备专一性工具酶,以获得酶解寡糖终产物。经分离纯化制备出四、六、八糖,并通过一级质谱确定了各片段的分子量大小,结合二级质谱、核磁谱图及多糖基本性质,综合对该酶解寡糖进行了结构分析。结果证明该方法制备的寡糖具有保留糖链结构完整、准确,且结果重复性强等特点,为准确解析多糖结构提供保障。本文还对胞外多糖进行了分子水平的表征,通过轻敲模式的原子力显微技术观察到该细菌胞外多糖溶液在云母片上的构象信息,展示了单个胞外多糖分子的立体构象。该胞外多糖的水溶液在低浓度条件下,多糖分子均以单分子链的形态出现,单个分子链的高度为0.7 nm左右,链长约600 nm。
最后,本文利用专一性酶解技术制备得到的具有简单重复单元的的葡半寡糖进行了一些潜在活性方面的研究,发现该寡糖在吸湿、保湿、草莓保鲜及促进种子萌发具有一定的活性。由于目前尚未有相关寡糖活性方面的报道。通过该研究不仅丰富了当前寡糖的应用前景,而且这种新型寡糖结构在理论研究方面为今后构效关系的研究提供了新的平台资源
Bacteria derived exo-polysaccharides are new kinds of products made by biology technology recently.Their different applications attribute to their different structures. According to currently reports,there are majorly three kinds of products,Xanthan,Gellan and Curdlan applied in industry,for example food,textile or petroleum and chemical indurstry.On the basis of finding and isolating the microb with properties of a new kind of exo-polysaccharide,high yield and stabilization,we tried many ways to isolate and culture it which we hope to hold high productivity and be applied widely in industry.
A new kind of bacteria holding high exo-polysaccharides yield and stabilization was isolated through the compound screening methods of qualitative and quantitative analysis according to previous reports.The strain 921F was identified as Phyllobacterium sp.by micro-biochemical identification and 16S ribosomal RNA gene sequencing analysis.The medium components and culture conditions are optimized.Results show that the optimal culture medium is as follows:sucrose,3%;beef extract,0.45%;(NH_4)_2SO_4,0.04%; MgCl_2,0.03%.The optimal culture conditions are as follows:initial pH 7.0,temperature 30℃,medium volume 75 ml/250 ml,inoculum volume 8%(v/v),duration,72～96 h.The maximal exo-polysaccharide yield could reach 1.8%.In another hand,an experiment of 10 liter scaled fermentation was performed with the purpose of amplifying culture volume towards industry scale in the coming future.
Exo-polysaccharides were precipitated with the use of ethanol and salt according to the optimal extracting process.Characters of exo-polysaccharides were investigated, including molecular weights,saccharides composition et al.IR,UV and AFM of exo-polysaccharides were performed to obtain more information about the molecular structure. The exo-polysaccharides we precipitated show highly viscous and translucent.So the experiment of apparent viscosity of exo-polysaccharides under different conditions were assayed,including the effects of substrate concentration,shear velocity,pH,temperature, durations,salts concentration et al.
Method of enzyme hydrolysis was introduced to produce the final product from exo-polysaccharides. Tetrasaccharides,hexasaccharides and octasaccharides were separated and purified by passing through Bio-Gel P4 column several times.Molecular weights of the purified sections were determinate with MS.With the help of the information from upper charpter,ESI-CID-MS/MS and NMR of oligosaccharides were conducted to analysis its structure.So the structure of exo-polysccharides produced from Phyllobacterium sp.could be primarily illustrated.
In the final area,we conducted some potential study about bioactivity of the related oligosaccharides,such as moisture absorption,moisture retention and fresh keeping of strawberry.The influence of oligosaccharide was also tested on the seed germination at different concentrations.Compared with the control,results indicate that the rate of seed germination was enhanced by increasing the activities of several enzymes beneficial for germination.
本文通过定向筛选技术获得一株胞外多糖产生菌。结合生理、生化鉴定和16SrRNA技术确定该菌株为叶杆菌属新种(Phyllobacterium sp.nov.921F)。现已申请国家专利保护——专利申请号:200710167753.1。该产糖菌株已保存于中国典型微生物培养保藏中心。由于目前尚未有相关菌种的产糖的报道,我们采用定向诱导技术和条件优化后,使得该菌株经84 h摇瓶培养后,产糖量达1.8%。在此基础上,进行10 L全自动发酵罐试验,产糖量提升至2%以上。胞外多糖提取工艺的优化方面,通过引入金属盐溶液而有效的减少了有机溶剂的加入量,显著减少了提取工艺的生产成本。以上结果表明:该菌株具有产糖量能力高、产糖性状稳定、多糖提取工艺简单且相比其它细菌胞外多糖提取能耗低等特点。达到了上游发酵工业对菌种的要求水平,具有潜在开发价值。
胞外多糖理化性质和基本组成分析结果表明该多糖易溶于水,其水溶液呈透明粘稠状;多糖由半乳糖和葡萄糖组成,二者摩尔比为1.07:1;有丙酮酸基团存在;通过GPC分析得到多糖重均分子量为1082 kDa。由于该胞外多糖表现出很强的粘度特性。因此本文考察了多糖溶液浓度、剪切力、pH值、温度、放置时间、恒温加热、无机盐及蔗糖浓度对多糖溶液表观粘度的影响。结果表明该胞外多糖表观粘度受剪切速率、蔗糖浓度等因素影响较小且稳定性好,在pH 3-12之间变化不明显。随着温度的升高粘度下降。多糖的表观粘度受无机盐的影响有一定的下降,但变化程度不大。因此该多糖的粘度特性也展示出较好的应用前景。
在糖链结构解析方面,本研究采用专一性酶解技术系统、准确的对糖链结构进行解析。以胞外多糖为特异性底物筛选一株降解菌进而制备专一性工具酶,以获得酶解寡糖终产物。经分离纯化制备出四、六、八糖,并通过一级质谱确定了各片段的分子量大小,结合二级质谱、核磁谱图及多糖基本性质,综合对该酶解寡糖进行了结构分析。结果证明该方法制备的寡糖具有保留糖链结构完整、准确,且结果重复性强等特点,为准确解析多糖结构提供保障。本文还对胞外多糖进行了分子水平的表征,通过轻敲模式的原子力显微技术观察到该细菌胞外多糖溶液在云母片上的构象信息,展示了单个胞外多糖分子的立体构象。该胞外多糖的水溶液在低浓度条件下,多糖分子均以单分子链的形态出现,单个分子链的高度为0.7 nm左右,链长约600 nm。
最后,本文利用专一性酶解技术制备得到的具有简单重复单元的的葡半寡糖进行了一些潜在活性方面的研究,发现该寡糖在吸湿、保湿、草莓保鲜及促进种子萌发具有一定的活性。由于目前尚未有相关寡糖活性方面的报道。通过该研究不仅丰富了当前寡糖的应用前景,而且这种新型寡糖结构在理论研究方面为今后构效关系的研究提供了新的平台资源
Bacteria derived exo-polysaccharides are new kinds of products made by biology technology recently.Their different applications attribute to their different structures. According to currently reports,there are majorly three kinds of products,Xanthan,Gellan and Curdlan applied in industry,for example food,textile or petroleum and chemical indurstry.On the basis of finding and isolating the microb with properties of a new kind of exo-polysaccharide,high yield and stabilization,we tried many ways to isolate and culture it which we hope to hold high productivity and be applied widely in industry.
A new kind of bacteria holding high exo-polysaccharides yield and stabilization was isolated through the compound screening methods of qualitative and quantitative analysis according to previous reports.The strain 921F was identified as Phyllobacterium sp.by micro-biochemical identification and 16S ribosomal RNA gene sequencing analysis.The medium components and culture conditions are optimized.Results show that the optimal culture medium is as follows:sucrose,3%;beef extract,0.45%;(NH_4)_2SO_4,0.04%; MgCl_2,0.03%.The optimal culture conditions are as follows:initial pH 7.0,temperature 30℃,medium volume 75 ml/250 ml,inoculum volume 8%(v/v),duration,72～96 h.The maximal exo-polysaccharide yield could reach 1.8%.In another hand,an experiment of 10 liter scaled fermentation was performed with the purpose of amplifying culture volume towards industry scale in the coming future.
Exo-polysaccharides were precipitated with the use of ethanol and salt according to the optimal extracting process.Characters of exo-polysaccharides were investigated, including molecular weights,saccharides composition et al.IR,UV and AFM of exo-polysaccharides were performed to obtain more information about the molecular structure. The exo-polysaccharides we precipitated show highly viscous and translucent.So the experiment of apparent viscosity of exo-polysaccharides under different conditions were assayed,including the effects of substrate concentration,shear velocity,pH,temperature, durations,salts concentration et al.
Method of enzyme hydrolysis was introduced to produce the final product from exo-polysaccharides. Tetrasaccharides,hexasaccharides and octasaccharides were separated and purified by passing through Bio-Gel P4 column several times.Molecular weights of the purified sections were determinate with MS.With the help of the information from upper charpter,ESI-CID-MS/MS and NMR of oligosaccharides were conducted to analysis its structure.So the structure of exo-polysccharides produced from Phyllobacterium sp.could be primarily illustrated.
In the final area,we conducted some potential study about bioactivity of the related oligosaccharides,such as moisture absorption,moisture retention and fresh keeping of strawberry.The influence of oligosaccharide was also tested on the seed germination at different concentrations.Compared with the control,results indicate that the rate of seed germination was enhanced by increasing the activities of several enzymes beneficial for germination.
引文
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