鉤狀木霉生物還原制備納米銀的研究
發(fā)布時(shí)間：2018-09-18 22:00
【摘要】：本實(shí)驗(yàn)采用在土壤懸液中添加Ag+的方法在東北林業(yè)大學(xué)帽兒山林場(chǎng)土壤中篩選出能合成納米銀的真菌菌株,通過形態(tài)學(xué)特征觀察及分子生物學(xué)方法對(duì)菌株進(jìn)行鑒定;利用真菌合成納米銀,采用UV-vis, TEM、XRD及FTIR等手段對(duì)納米銀進(jìn)行表征；以大腸桿菌和枯草芽孢桿菌為受試菌株,對(duì)納米銀的殺菌性能進(jìn)行初步測(cè)定；利用SDS-PAGE電泳技術(shù)分析真菌胞外濾液的蛋白變化及納米銀表面附著蛋白,對(duì)納米銀的合成機(jī)理進(jìn)行初步探討。(1)合成納米銀真菌的篩選及鑒定采用在土壤懸液中添加Ag+的方法篩選出能合成納米銀的真菌NYZJ03并對(duì)其進(jìn)行鑒定。PDA固體培養(yǎng)基上的NYZJ03菌絲為白色,基質(zhì)為黃色；菌落生長(zhǎng)較緩慢,表面光滑,氣生菌絲較少；菌絲具有明顯的分枝,較為密集,間隔明顯且很有規(guī)律,大約為5 μm左右；不能明顯的看到細(xì)胞核。NYZJ03特異性ITS基因序列長(zhǎng)度為577 bp,在GenBank中的Accession No.是KM054532；系統(tǒng)發(fā)育樹顯示菌株NYZJ03與鉤狀木霉形成一個(gè)族群,同源性達(dá)99.5%以上。根據(jù)菌株的菌落特征、菌絲形態(tài)和ITS基因序列分析,初步將該菌鑒定為鉤狀木霉(Trichoderma hamatum)。NYZJ03保藏在中國(guó)微生物菌種保藏管理委員會(huì)普通微生物中心(CGMCC),保藏編號(hào)為CGMCC No.9333。(2)納米銀的合成及表征采用菌體與AgNO3混合培養(yǎng)的方法合成納米銀并利用紫外全波長(zhǎng)掃描(UV-vis)、X射線衍射分析(XRD)、透射電子顯微鏡(TEM)、能量色譜分析(EDS)及傅里葉變換紅外分析(FTIR)等手段對(duì)納米銀進(jìn)行表征；熱重分析法和原子光譜吸收法分別對(duì)納米銀的產(chǎn)率和Ag+轉(zhuǎn)化率進(jìn)行測(cè)定和計(jì)算。結(jié)果顯示納米銀納為面心立方結(jié)構(gòu),結(jié)晶程度較好且很純凈；納米銀具有單分散性,無團(tuán)聚現(xiàn)象,多數(shù)為近球形,絕大多數(shù)小于9nm,平均為6.69 nm; FTIR結(jié)果表明納米銀可能被蛋白或多肽等生物大分子包裹；熱重分析結(jié)果顯示納米銀的產(chǎn)率為67.12%,而原子光譜吸收的結(jié)果顯示Ag+的轉(zhuǎn)化率為84.41%。(3)納米銀殺菌性能研究及合成機(jī)理的探討納米銀對(duì)大腸桿菌的MBC為10μg/mL, MIC為7μg/mL,而對(duì)枯草芽孢桿菌的MBC為5μg/mL, MIC為4μg/mL。10 μg/mL的納米銀對(duì)大腸桿菌的致死率開始時(shí)較低,40 min明顯增大,達(dá)到86.70%,到70 min時(shí),達(dá)到99.23%,已經(jīng)基本被殺死。5μg/mL的納米銀對(duì)枯草芽孢桿菌的致死率在40 min時(shí)已經(jīng)達(dá)到96.22%,到60 rmin時(shí),已達(dá)到99.84%。納米銀對(duì)枯草芽孢桿菌的致死率要大于對(duì)大腸桿菌的致死率。添加AgNO3后菌體分泌的胞外蛋白明顯減少,主要含有三種蛋白質(zhì),相對(duì)分子質(zhì)量分別為40 kDa,55 kDa,70 kDa。利用尿素和SDS對(duì)純化的納米銀進(jìn)行變性及煮沸處理可以將附著于納米銀粒子表面的蛋白分離下來,而處理下來的蛋白相對(duì)分子質(zhì)量約為40 kDa,說明該蛋白條帶參與了納米銀的合成及穩(wěn)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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：TB383.1;O614.122
本文編號(hào)：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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：TB383.1;O614.122
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