胶质芽孢杆菌SM-01胞外多糖结构、链构象以及流变行为研究

发布时间：2018-04-28 03:13

本文选题：胶质芽孢杆菌胞外多糖 + 提取　；参考：《江南大学》2017年博士论文

【摘要】：细菌胞外多糖是一类具有多元结构和独特性能的生物大分子,因其优良的理化性质和生物活性而被广泛地应用于食品、医药、化工等领域。尽管目前已发现的细菌胞外多糖种类很多,但真正具有实际应用价值和实现工业化生产的却很少,多糖产量、生产成本以及对构效关系研究的欠缺是制约多糖生产和应用的主要因素,因此寻找具有独特性能的新型高产多糖是当前研究的热点。胶质芽孢杆菌(Bacillus mucilaginosus)是土壤中一类特殊的硅酸盐菌,能够利用有限资源产生大量胞外多糖。目前对于B.mucilaginosus的研究主要集中在其解钾溶磷固氮功能以及污水处理上,B.mucilaginosus能够降解土壤中的矿物质(硅酸盐、磷灰石、磷钙石),使其转化为可供植物吸收的营养物质;同时还可以作为絮凝剂和吸附剂除去污水中的各种污染物质,B.mucilaginosus这些功能被认为和其产生的胞外多糖密切相关。此外,研究发现B.mucilaginosus胞外多糖还具有一定的生物活性,能够促进小鼠胃溃疡部位的修复以及黄仔鸡免疫器官的发育。然而对于B.mucilaginosu 胞外多糖基本信息的研究非常少,其化学结构还不为人所知,极大地限制了其应用发展。因此对B.mucilaginosus胞外多糖化学结构和理化性质进行基础性研究将有助于了解其构效关系,拓宽其潜在应用领域。本研究通过对B. mucilaginosus SM-01发酵液进行提取优化,得到酸性杂多糖BMPS,通过各种表征方法对BMPS的一级结构和高级结构进行了研究,为开发B.mucilaginosus胞外多糖提供了理论依据,同时基于生物活性对其作为伤口修复材料进行了初步探讨,拓宽了B.mucilaginosus胞外多糖的应用前景,主要结果如下:(1)通过考察稀释、升温、改变pH、加入盐的量对发酵液粘度的影响,选择稀释与加盐协同使用的方法降低发酵液粘度。在发酵液中加入3% (w/v) NaCl,比较不同稀释倍数下,离心和硅藻土抽滤对发酵液除菌和除蛋白的影响以及对抽滤通量的影响,发现抽滤去除菌体和杂蛋白的效果远高于离心,在稀释倍数为3倍的情况下抽滤,能保持较好的抽滤通量,除菌率为92.6%,除蛋白率为69.7%。以硅藻土作为吸附剂,比较不同添加量对除菌、除蛋白以及多糖回收率的影响,发现添加10 g/L硅藻土后经重复抽滤三次,镜检无菌体存在,蛋白含量接近于零,多糖回收率达76.4%。考察不同截留分子量的超滤膜以及操作压力对超滤浓缩的影响,发现采用200 kDa截留分子量的超滤膜在0.1 MPa操作压力下超滤基本能截留所有多糖组分,经过超滤浓缩除盐,多糖最终总回收率为73.2%,内毒素含量低于0.25 EU/mL,此提取工艺简单易行且适合工业生产。对浓缩得到的粗多糖经离子交换色谱分离纯化,再利用体积排阻色谱和光散射联用进行纯度鉴定和分子量测定,发现B.mucilaginosus SM-01胞外多糖为均一的酸性多糖,命名为BMPS,其重均分子量Mw为2.67×106Da。(2)通过对BMPS进行化学组成分析发现BMPS不含磷酸基团和硫酸基团,糖醛酸含量为14.3%,O-乙酰基含量为4.99%。通过单糖分析得到其单糖组成为葡萄糖(Glc)、甘露糖(Man)、葡萄糖醛酸(GlcA)和一个未知糖醛酸(AM),摩尔比为3.2: 2: 0.5: 0.3。对BMPS先进行超声降解再进行温和酸水解得到一个中性寡糖片段BMPS-H,通过基质辅助激光解吸飞行时间质谱得到其Mw为1771.43 Da,单糖组成为Glc和Man,摩尔比为1.5: 1。通过甲基化分析发现BMPS-H分子为线性结构,主链由1,4-Man和1,4-Glc以及少量1,3-Glc构成,通过核磁共振得到其糖基连接顺序以及乙酰基的连接位点,进而推断出BMPS-H的化学结构为:β-D-Glc (1→[4)-β-D-Man (1→4)-β-D-Glc (1→]→3)-β-D-Glc (1→[4)-β-D-Man (1→4)-β-D-Glc (l→]n-β-D-Glc 2 2 OAc OAc其中m和n代表不同的重复单元数。(3)通过粘度测定发现BMPS在纯水中显示典型的聚电解质行为,加入NaNO3能有效抑制其聚电解质效应,同时通过经验B值法(B = 0.018)推断BMPS在溶液中以半刚性链存在。通过超声降解得到不同分子量的BMPS级分,利用粘度法和光散射法测得各级分在0.1mol/LNaNO3溶液中的Mw、数均分子量Mn、均方根旋转半径s2z1/2、第二维利系数A2、流体力学半径Rh以及特性粘数[η]等参数。BMPS所有级分的A2均为正值,说明0.1 mol/LNaNO3为其良溶剂,在该体系中所有级分均无聚集产生,适合进行溶液性质分析。通过[η]和Mw的指数关系(α= 0.82)、[η]和ss2z1/2的指数关系(α' = 0.64)、s2z1/2与Rh的比值(ρ= 1.82)得出分子量范围为37.4×104-267×104Da的BMPS在0.1 mol/L NaN03溶液中以半刚性链构象存在。通过无扰蠕虫状圆筒模型进行链构象参数计算,得到单位围长摩尔质量ML、持续长度q以及分子链直径d分别为663.5 nm-1、9.5 nm、0.6 nm。通过AFM对其形貌进行表征,发现分子链以直链或无规弯曲的形态存在,测得平均链直径为0.68 nm,与理论模型计算结果相符合。(4)通过对BMPS的稳态流变行为研究发现BMPS溶液为典型的假塑性流体,即使在较低的浓度下(0.01%)也具有剪切变稀的行为,且其剪切变稀行为具有浓度依赖性。通过Williamson模型获得BMPS在浓度区间为0.0101%-1.2%的零剪切粘度,由零剪切粘度与浓度的关系得到BMPS的临界交叠浓度为0.6%,说明BMPS分子链比一般半刚性链高分子更易发生缠结。通过动态流变研究发现,BMPS溶液在低频区以粘性为主的粘弹性体存在,而在高频区随着振荡频率的增大则会形成瞬态的网络结构,且随着浓度的增大,其形成瞬态网络结构的能力越强。(5)通过考察聚乙烯醇(polyvinyl alcohol,PVA)浓度和纺丝电压对PVA纳米纤维的影响,选择8%的PVA溶液作为助纺剂,纺丝电压为14 kV;将BMPS与PVA以质量比为3/100、5/100、10/100、20/100比例制成混合溶液进行静电纺丝。随着BMPS比例的增大,混合溶液的电导率和粘度相应增大,纳米纤维的直径逐渐变小,纺丝过程逐渐困难,由红外图谱可以发现BMPS和PVA之间形成了微弱的氢键。将不同比例的BMPS/PVA混纺膜通过戊二醛交联10min,能够改变其水溶性同时保持纤维形态。通过细胞相容性实验发现PVA纤维膜和BMPS/PVA混纺膜均能促进NIH3T3细胞的增殖,且相比较PVA纤维膜,BMPS/PVA混纺膜更适合细胞的生长与黏附,因此通过BMPS与PVA混纺制得的纳米纤维膜有望成为一种新型伤口修复材料。
[Abstract]:Bacterial extracellular polysaccharide is a kind of biological macromolecule with multiple structure and unique properties. Because of its excellent physical and chemical properties and biological activity, it has been widely used in the fields of food, medicine, chemical industry and so on. Although there are many kinds of bacterial extracellular polysaccharide found at present, the real application value and industrialization production are very few. The production of polysaccharides, the cost of production and the lack of research on structure-activity relationship are the main factors that restrict the production and application of polysaccharides. Therefore, it is a hot spot to find a new high yield polysaccharide with unique properties. Bacillus mucilaginosus is a special kind of silicate bacteria in the soil, which can make large use of limited resources. At present, the research on B.mucilaginosus mainly focuses on its potassium solution and nitrogen fixing function and sewage treatment. B.mucilaginosus can degrade minerals (silicate, apatite, phosphonite) in the soil and convert it into nutrient substance for plant absorption; and it can also be used as flocculant and adsorbent to remove sewage. In addition, the study found that B.mucilaginosus extracellular polysaccharide also has a certain biological activity, which can promote the repair of gastric ulcer in mice and the development of the immune organs of Yellow Broilers in mice. However, the B.mucilaginosus extracellular polysaccharide group (B.mucilaginosu) The study of this information is very small, its chemical structure is not well known, and its application development is greatly limited. Therefore, the basic research on the chemical structure and physicochemical properties of B.mucilaginosus extracellular polysaccharide will help to understand its structure-activity relationship and broaden its potential application field. This research is carried out through the B. mucilaginosus SM-01 fermentation broth. The acid heteropoly BMPS was extracted and optimized. The primary structure and advanced structure of BMPS were studied by various characterization methods. It provided a theoretical basis for the development of B.mucilaginosus extracellular polysaccharide. At the same time, based on biological activity, it was discussed as a wound repair material, which widened the application of B.mucilaginosus extracellular polysaccharide. The main results are as follows: (1) by investigating the influence of dilution, heating up, changing pH, adding salt content to the viscosity of the fermentation broth, selecting the method to reduce the viscosity of the fermentation liquid with the synergistic use of dilution and salt, and adding 3% (w/v) NaCl in the fermentation liquid to compare the effect of centrifugation and diatomite filtration on the deproteinizing and deproteinizing of the fermentation liquid under different dilution times. As well as the effect on the filtration flux, it was found that the effect of the filtration and removal of the bacteria and the heteroprotein was much higher than that of the centrifuge. In the case of the dilution multiple of 3 times, the extraction filtration rate was better, the rate of bacteria removal was 92.6%, the protein rate was 69.7%. and the diatomite was used as the adsorbent, and the effects of different addition amounts on the removal of bacteria, protein and polysaccharide recovery were compared. After 10 g/L diatomite was added, it was repeatedly pumped three times, and the protein content was close to zero, and the recovery rate of polysaccharide was 76.4%. to investigate the effect of ultrafiltration membrane with different interception molecular weight and the effect of operating pressure on ultrafiltration concentration. It was found that ultrafiltration with 200 kDa intercepting molecular weight could be basically intercepted under the operating pressure of 0.1 MPa. The total total recovery rate of polysaccharide was 73.2% and the content of endotoxin was less than 0.25 EU/mL. The extraction process was simple and suitable for industrial production by ultrafiltration concentration and desalination. The extraction process was simple and suitable for industrial production. The purified polysaccharide was purified by ion exchange chromatography, and then the purity identification and molecular measurement were used by volume exclusion chromatography and light scattering. It is found that B.mucilaginosus SM-01 extracellular polysaccharide is a homogeneous acid polysaccharide named BMPS, and its weight average molecular weight Mw is 2.67 x 106Da. (2). By chemical composition analysis of BMPS, it is found that BMPS does not contain phosphoric acid group and sulphuric acid group, glucuronic acid content is 14.3%, O- acetyl content is 4.99%. through monosaccharide analysis to become grapes. Carbohydrate (Glc), mannose (Man), glucuronic acid (GlcA) and an unknown glucuronic acid (AM), the mole ratio is 3.2: 2: 0.5: 0.3., and a neutral oligosaccharide fragment BMPS-H is obtained by ultrasonic degradation and mild acid hydrolysis of BMPS, and a Mw 1771.43 Da is obtained by the matrix assisted laser desorption flight time mass spectrometry, and the monosaccharide composition is the molar ratio. A linear structure of BMPS-H molecules was found by methylation analysis for 1.5: 1.. The main chain was composed of 1,4-Man and 1,4-Glc and a small amount of 1,3-Glc. The connection sequence of glycosyl groups and the junction sites of acetyl groups were obtained by nuclear magnetic resonance. The chemical structure of BMPS-H was deduced to be: beta -D-Glc (1 to [4) - beta -D-Man (1 to 4) - beta -D-Glc (1 to] 3) - beta -D-Glc ( 1 - [4) - beta -D-Man (1 to 4) - beta -D-Glc (L to]n- beta -D-Glc 22 OAc OAc in which m and N represent the number of different repeating units. (3) it is found that BMPS in pure water shows typical polyelectrolyte behavior through viscosity measurement, and the addition of NaNO3 can effectively inhibit its polyelectrolyte effect, and the empirical values method (0.018) is used to deduce the semi rigidity of the solution in the solution. The BMPS grade of different molecular weights is obtained by ultrasonic degradation. The Mw of different levels in 0.1mol/LNaNO3 solution is measured by viscosity method and light scattering method. The number of molecular weights Mn, the mean square root rotation radius s2z1/2, the second VL coefficient A2, the Rh of the hydrodynamic radius Rh and the characteristic viscosity number [ETA] are all positive, indicating that 0 .1 mol/LNaNO3 is a good solvent in which all the fractions of the system are not aggregated and are suitable for the analysis of the properties of the solution. Through the exponential relationship between [ETA] and Mw (alpha = 0.82), the exponential relationship between [ETA] and ss2z1/2 (alpha '= 0.64), the ratio of s2z1/2 to Rh (P = 1.82), the molecular weight range of 37.4 * 104-267 * 104Da BMPS in 0.1 mol/L NaN03 solution The existence of a semi rigid chain conformation. Through the calculation of the chain conformation parameters by the unperturbed vermicular cylinder model, the unit length mole mass ML, the length Q and the molecular chain diameter D are 663.5 nm-1,9.5 nm respectively, and the 0.6 nm. is characterized by AFM, and the molecular chain is found to be in the form of straight chain or random bending, and the average chain straight is measured. The diameter is 0.68 nm, which is in accordance with the theoretical model calculation results. (4) through the study of the steady rheological behavior of BMPS, it is found that BMPS solution is a typical pseudoplastic fluid, even at lower concentration (0.01%) has shear thinning behavior, and its shear thinning behavior is concentration dependent. The concentration range of BMPS is obtained by Williamson model. The zero shear viscosity of 0.0101%-1.2% is derived from the relationship between the zero shear viscosity and the concentration of the BMPS to get the critical overlapping concentration of 0.6%, which indicates that the BMPS molecular chain is more easily entangled than the general semi rigid chain polymer. Through the dynamic rheological study, it is found that the BMPS solution is viscous mainly in the low frequency region, while the frequency is oscillating in the high frequency region. A transient network structure is formed by increasing the concentration, and with the increase of concentration, the stronger the ability to form a transient network structure. (5) by examining the effect of the concentration of polyvinyl alcohol (PVA) and the spinning voltage on the PVA nanofibers, the PVA solution of 8% is selected as a spinning aid, the spinning voltage is 14 kV, and the mass ratio of BMPS to PVA is 3/10 to 3/10. With the increase of the proportion of 0,5/100,10/100,20/100, the electrical conductivity and viscosity of the mixed solution increase, the diameter of the nanofibers gradually becomes smaller and the spinning process is gradually difficult. The weak hydrogen bonds between BMPS and PVA can be found from the infrared atlas. The BMPS/PVA blend membrane of different proportions can be found. Through crosslinking 10min with glutaraldehyde, it can change its water solubility and maintain fiber morphology. Through the cell compatibility test, it is found that both the PVA fiber membrane and the BMPS/PVA blend membrane can promote the proliferation of NIH3T3 cells, and the PVA fiber membrane and the BMPS/PVA blend membrane are more suitable for the cell growth and adhesion. Therefore, the nano fiber produced by the blending of BMPS and PVA is made. The membrane is expected to be a new type of wound repair material.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936

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