钩状木霉生物还原制备纳米银的研究
发布时间：2018-09-18 22:00
【摘要】：本实验采用在土壤悬液中添加Ag+的方法在东北林业大学帽儿山林场土壤中筛选出能合成纳米银的真菌菌株,通过形态学特征观察及分子生物学方法对菌株进行鉴定;利用真菌合成纳米银,采用UV-vis, TEM、XRD及FTIR等手段对纳米银进行表征；以大肠杆菌和枯草芽孢杆菌为受试菌株,对纳米银的杀菌性能进行初步测定；利用SDS-PAGE电泳技术分析真菌胞外滤液的蛋白变化及纳米银表面附着蛋白,对纳米银的合成机理进行初步探讨。(1)合成纳米银真菌的筛选及鉴定采用在土壤悬液中添加Ag+的方法筛选出能合成纳米银的真菌NYZJ03并对其进行鉴定。PDA固体培养基上的NYZJ03菌丝为白色,基质为黄色；菌落生长较缓慢,表面光滑,气生菌丝较少；菌丝具有明显的分枝,较为密集,间隔明显且很有规律,大约为5 μm左右；不能明显的看到细胞核。NYZJ03特异性ITS基因序列长度为577 bp,在GenBank中的Accession No.是KM054532；系统发育树显示菌株NYZJ03与钩状木霉形成一个族群,同源性达99.5%以上。根据菌株的菌落特征、菌丝形态和ITS基因序列分析,初步将该菌鉴定为钩状木霉(Trichoderma hamatum)。NYZJ03保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC No.9333。(2)纳米银的合成及表征采用菌体与AgNO3混合培养的方法合成纳米银并利用紫外全波长扫描(UV-vis)、X射线衍射分析(XRD)、透射电子显微镜(TEM)、能量色谱分析(EDS)及傅里叶变换红外分析(FTIR)等手段对纳米银进行表征；热重分析法和原子光谱吸收法分别对纳米银的产率和Ag+转化率进行测定和计算。结果显示纳米银纳为面心立方结构,结晶程度较好且很纯净；纳米银具有单分散性,无团聚现象,多数为近球形,绝大多数小于9nm,平均为6.69 nm; FTIR结果表明纳米银可能被蛋白或多肽等生物大分子包裹；热重分析结果显示纳米银的产率为67.12%,而原子光谱吸收的结果显示Ag+的转化率为84.41%。(3)纳米银杀菌性能研究及合成机理的探讨纳米银对大肠杆菌的MBC为10μg/mL, MIC为7μg/mL,而对枯草芽孢杆菌的MBC为5μg/mL, MIC为4μg/mL。10 μg/mL的纳米银对大肠杆菌的致死率开始时较低,40 min明显增大,达到86.70%,到70 min时,达到99.23%,已经基本被杀死。5μg/mL的纳米银对枯草芽孢杆菌的致死率在40 min时已经达到96.22%,到60 rmin时,已达到99.84%。纳米银对枯草芽孢杆菌的致死率要大于对大肠杆菌的致死率。添加AgNO3后菌体分泌的胞外蛋白明显减少,主要含有三种蛋白质,相对分子质量分别为40 kDa,55 kDa,70 kDa。利用尿素和SDS对纯化的纳米银进行变性及煮沸处理可以将附着于纳米银粒子表面的蛋白分离下来,而处理下来的蛋白相对分子质量约为40 kDa,说明该蛋白条带参与了纳米银的合成及稳定。
[Abstract]:In this experiment, we used the method of adding Ag to the soil suspension to screen the fungus strains which could synthesize nano-silver in the soil of Maoershan Forest Farm of Northeast Forestry University, and identified the strains by morphological observation and molecular biological method. Silver nanoparticles were synthesized by fungi and characterized by UV-vis, TEM,XRD and FTIR. The bactericidal properties of silver nanoparticles were preliminarily determined by Escherichia coli and Bacillus subtilis. The changes of protein in extracellular filtrate of fungi and the surface attachment protein of silver nanoparticles were analyzed by SDS-PAGE electrophoresis. The synthesis mechanism of silver nanoparticles was preliminarily discussed. (1) screening and identification of silver nanocrystalline fungi by adding Ag in soil suspensions to screen the fungi NYZJ03 which could synthesize silver nanoparticles and identify them on solid medium. The NYZJ03 hypha is white, The substrate is yellow, the colony grows slowly, the surface is smooth, the hyphae is less, the hyphae has obvious branching, dense, the interval is obvious and regular, about 5 渭 m; It can't be seen that the nucleolus. NYZJ03 specific ITS gene sequence length is 577 bp, in GenBank Accession No.. The KM054532; phylogenetic tree showed that the strain NYZJ03 and Trichoderma hook formed a population, the homology was more than 99.5%. According to the colony characteristics, mycelium morphology and ITS gene sequence analysis, Preliminary identification of this bacterium as Trichoderma hook (Trichoderma hamatum). NYZJ03 was deposited in the (CGMCC), preservation number of CGMCC No.9333. (2) nanocrystalline silver in the (CGMCC), preservation center of the Chinese microbial species preservation management committee. The synthesis and characterization of this bacterium were synthesized by the method of mixed cell culture and AgNO3 culture. The silver nanoparticles were characterized by UV-vis X-ray diffraction (XRD), transmission electron microscope (TEM), energy chromatographic analysis (EDS) and Fourier transform infrared analysis (FTIR). The yield and Ag conversion of silver nanoparticles were measured and calculated by thermogravimetric analysis and atomic absorption spectrometry, respectively. The results show that the nanocrystalline silver nanocrystalline has a face-centered cubic structure with a good degree of crystallization and is very pure, and the nanocrystalline silver has monodispersity and no agglomeration, most of which are nearly spherical. Most of them were smaller than 9 nm, with an average of 6.69 nm; FTIR. The results showed that silver nanoparticles might be encapsulated by biological macromolecules such as protein or polypeptide. Thermogravimetric analysis showed that the yield of silver nanoparticles was 67.12, while the conversion rate of Ag was 84.41. (3) the bactericidal properties of silver nanoparticles and the mechanism of synthesis were studied. The MBC of silver nanoparticles to Escherichia coli was 10 渭 g / mL, MIC was 7 渭 g / mL, and the MBC of silver nanoparticles to Escherichia coli was 7 渭 g / mL. The MBC of Bacillus graminearum was 5 渭 g / mL, and that of silver nanoparticles with MIC of 4 渭 g/mL.10 渭 g/mL was significantly higher than that of Escherichia coli at the beginning. By 70 min, 99.23% of silver nanocrystalline, which had been basically killed by 5 渭 g/mL, had reached 96.22% at 40 min and 99.84% at 60 rmin, and the mortality of silver nanoparticles against Bacillus subtilis was 96.22% at 40 min and 99.84% at 60 rmin. The mortality of silver nanoparticles to Bacillus subtilis was higher than that to Escherichia coli. The extracellular proteins secreted by the bacteria decreased significantly after adding AgNO3, mainly containing three proteins, with a relative molecular weight of 40 kDa,55 kDa,70 kDa.. The purified silver nanoparticles were denatured and boiled by urea and SDS to separate the proteins attached to the surface of the nanoparticles. The relative molecular weight of the treated protein was about 40 kDa, which indicated that the protein band was involved in the synthesis and stability of silver nanoparticles.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;O614.122
本文编号：2249205
[Abstract]:In this experiment, we used the method of adding Ag to the soil suspension to screen the fungus strains which could synthesize nano-silver in the soil of Maoershan Forest Farm of Northeast Forestry University, and identified the strains by morphological observation and molecular biological method. Silver nanoparticles were synthesized by fungi and characterized by UV-vis, TEM,XRD and FTIR. The bactericidal properties of silver nanoparticles were preliminarily determined by Escherichia coli and Bacillus subtilis. The changes of protein in extracellular filtrate of fungi and the surface attachment protein of silver nanoparticles were analyzed by SDS-PAGE electrophoresis. The synthesis mechanism of silver nanoparticles was preliminarily discussed. (1) screening and identification of silver nanocrystalline fungi by adding Ag in soil suspensions to screen the fungi NYZJ03 which could synthesize silver nanoparticles and identify them on solid medium. The NYZJ03 hypha is white, The substrate is yellow, the colony grows slowly, the surface is smooth, the hyphae is less, the hyphae has obvious branching, dense, the interval is obvious and regular, about 5 渭 m; It can't be seen that the nucleolus. NYZJ03 specific ITS gene sequence length is 577 bp, in GenBank Accession No.. The KM054532; phylogenetic tree showed that the strain NYZJ03 and Trichoderma hook formed a population, the homology was more than 99.5%. According to the colony characteristics, mycelium morphology and ITS gene sequence analysis, Preliminary identification of this bacterium as Trichoderma hook (Trichoderma hamatum). NYZJ03 was deposited in the (CGMCC), preservation number of CGMCC No.9333. (2) nanocrystalline silver in the (CGMCC), preservation center of the Chinese microbial species preservation management committee. The synthesis and characterization of this bacterium were synthesized by the method of mixed cell culture and AgNO3 culture. The silver nanoparticles were characterized by UV-vis X-ray diffraction (XRD), transmission electron microscope (TEM), energy chromatographic analysis (EDS) and Fourier transform infrared analysis (FTIR). The yield and Ag conversion of silver nanoparticles were measured and calculated by thermogravimetric analysis and atomic absorption spectrometry, respectively. The results show that the nanocrystalline silver nanocrystalline has a face-centered cubic structure with a good degree of crystallization and is very pure, and the nanocrystalline silver has monodispersity and no agglomeration, most of which are nearly spherical. Most of them were smaller than 9 nm, with an average of 6.69 nm; FTIR. The results showed that silver nanoparticles might be encapsulated by biological macromolecules such as protein or polypeptide. Thermogravimetric analysis showed that the yield of silver nanoparticles was 67.12, while the conversion rate of Ag was 84.41. (3) the bactericidal properties of silver nanoparticles and the mechanism of synthesis were studied. The MBC of silver nanoparticles to Escherichia coli was 10 渭 g / mL, MIC was 7 渭 g / mL, and the MBC of silver nanoparticles to Escherichia coli was 7 渭 g / mL. The MBC of Bacillus graminearum was 5 渭 g / mL, and that of silver nanoparticles with MIC of 4 渭 g/mL.10 渭 g/mL was significantly higher than that of Escherichia coli at the beginning. By 70 min, 99.23% of silver nanocrystalline, which had been basically killed by 5 渭 g/mL, had reached 96.22% at 40 min and 99.84% at 60 rmin, and the mortality of silver nanoparticles against Bacillus subtilis was 96.22% at 40 min and 99.84% at 60 rmin. The mortality of silver nanoparticles to Bacillus subtilis was higher than that to Escherichia coli. The extracellular proteins secreted by the bacteria decreased significantly after adding AgNO3, mainly containing three proteins, with a relative molecular weight of 40 kDa,55 kDa,70 kDa.. The purified silver nanoparticles were denatured and boiled by urea and SDS to separate the proteins attached to the surface of the nanoparticles. The relative molecular weight of the treated protein was about 40 kDa, which indicated that the protein band was involved in the synthesis and stability of silver nanoparticles.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;O614.122
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