摘要
本文通过微生物发酵原理、细胞结构学和生物矿化原理多学科交叉,提出以天然营养型酵母活体细胞为模板反应器,用人体所需或能够代谢的磷酸盐来复制生物分子本身特有的复杂纳米结构新合成思路。合成了系列复杂形貌的磷酸盐纳米复合材料,并对其合成机理、结构组成和性能进行了系统的研究。
以酵母细胞为催化模板,合成了CHA/酵母蛋白纳米复合材料,利用正交实验法对合成条件进行优化研究和放大实验,采用现代测试技术对合成材料的结构组成和性能进行了综合测试分析。结果表明:合成材料的结构组成重复性好,可实现批量合成,且反应条件温和,工艺简单,无污染,易产业化。在酵母细胞的生理环境中,能快速形成低结晶度碳酸羟基磷灰石(CHA)纳米颗粒,其晶胞参数α=9.4924?,c=6.9092 ?。与直接沉淀法合成的纳米HA(α=9.2329?,c=6.7707 ?)对比,纳米CHA的晶格结构产生了明显的膨胀畸变现象,富钙层钙原子和填隙CO32-基团在CHA颗粒表面和晶界上偏析聚集,形成呈正电性的亚稳态富钙层。CHA纳米颗粒在酵母蛋白的网状结构中均匀分布,并对酵母细胞中的复杂纳米结构进行了复制。
CHA/酵母蛋白纳米复合材料由于其特殊的颗粒间隙孔结构和纳米复合结构及CHA的晶格结构膨胀畸变现象,表现出独特的荧光增强效应,对大鼠骨髓基质干细胞、洋葱细胞、面包酵母、鲁氏酵母和米曲霉菌细胞都有一定的荧光转染性能。体外细胞和溶血实验结果表明, L929细胞可以在其表面上很好地黏附增殖,无细胞毒性,无溶血作用。且对难溶药物的负载缓释效果明显,阿莫西林难溶药物的装载量可达112.16 mg/g,药物主要包埋负载在孔结构中,释放速率均匀,没有突释现象,6天释放出总载药量的55 %左右,缓释性能优良,其释药动力学行为属于费克扩散控制型,主要受扩散和降解两种机理控制。多孔结构和残留的各种蛋白酶的催化作用,提高了其可降解性能,因此,CHA/酵母蛋白纳米复合材料,是一种可降解的纳米生物活性材料。并具有特有的疏水亲水两亲性(接触角θ=120°)和左旋光性能(比旋光度为-23°)。同时,酵母蛋白中含有17种人体所需氨基酸(75mg/g),是补充优质完全蛋白质和钙的多功能材料,并有望成为一种新型的荧光标识材料。
以酵母细胞为催化模板,分别合成了其他磷酸盐(锌、铁和镁)/酵母蛋白纳米复合材料,并在不同温度热处理2h,合成了不同含碳量的磷酸盐(钙、锌、铁和镁)/活性碳纳米复合材料。结果表明,四水磷酸锌晶粒和富镁型白磷镁石晶粒都沿(020)晶面方向择优生长,而无定形磷酸铁(核)则与球蛋白自组装成具有核壳结构的纳米颗粒。磷酸锌和磷酸镁/酵母蛋白纳米复合材料在不同激发波长下都能够发射不同颜色的荧光,并对洋葱细胞、鲁氏酵母、米曲霉菌和大肠杆菌细胞都有一定的荧光转染性能,也有望成为一种新型的荧光标识材料。不同的磷酸盐/活性碳纳米复合材料都具有优异的氧还原电催化活性,且远高于已经商业化的MnO2催化剂。并从孔结构、原位复合活性碳和晶格结构三个方面解释了不同温度合成样品的氧还原电催化活性的差异,这些材料可作为氧电极催化剂材料应用于生物传感器、金属空气电池、燃料电池等研究中
合成机理研究表明:不同金属离子在酵母细胞内的吸附对蛋白质结构组成的影响不同,其氨基酸总含量Mg>Ca>Fe>Zn,对蛋白质结构规整度的影响Ca>Fe>Mg>Zn。在酵母细胞基因调控下,不同的金属离子吸附络合在不同的蛋白质分子上,并沿着其特定方向按照层生长理论择优生长,超分子自组装形成具有不同形貌的磷酸盐/酵母蛋白纳米复合材料,但都具有颗粒间隙孔结构,其孔径分布范围Fe(2～60nm)>Ca(2～40nm)>Mg(2～24nm)>Zn(2～12nm),并且异相快速成核位点都是酵母细胞代谢网络中螺旋沟槽上有序排列的极性亲水基团。酵母细胞的生物矿化过程都包括离子吸附、传输、沉积矿化和超分子自组装四个阶段,并分为酵母细胞壁上的生物诱导矿化和细胞内的生物控制生长。
By means of the multi-crossed disciplines among the principle of microbial fermentation, cytoarchitectonics and the principle of biomineralization, this paper presented a novel synthetic method which its own peculiar multilevel nanostructure of yeast cell tissues was copied with phosphate required or metabolized by human, by taking living yeast cells as templets and reactors. A series of phosphate nano-composite materials with complex surface shapes were synthesized, and synthesis mechanism, structure, composition and performance were researched systematically.
CHA/yeast protein nanocomposite has been synthesized successfully by using yeast cells as the catalytic templates. The orthogonal test was used to optimize the synthetic parameters and its scale-up experiment was carried out. The results showed that its structure and composition had a good repetition and can be synthesized in batch. The present technology has many advantages including the mild reaction conditions, simple process, no pollution and easy to industrial production. The modern testing technologies were used to characterize the structure and composition of the synthetic samples. The synthetically analysed results indicated that the hydroxyl-carbonate-apatite (CHA) crystal nano-grains with the lower crystallinity were rapidly formed in the physiological environment of yeast calls, which has the unit cell parametersα=9.4924?, c=6.9092 ?. Compared with HA nanopowders (α=9.2329?,c=6.7707 ?) obtained by direct precipitation method, distortion phenomenon existed in the lattice structure of nano-CHA and metastable rich Ca layer with positive electrical property were formed by the aliquation of Ca atoms and CO32- groups on the surface and grain boundary of CHA particles. Nano-CHA grains were dispersed evenly in the herarchical network structure of yeast proteins, and copied the complex nanostructure in yeast cell.
The CHA/yeast protein nanocomposite exhibited a unique fluorescence enhancement effect, because of the unique multi-level porous and nano-composite structures, and the distorting lattice structure of nano-CHA. It has the performance of transfection fluorescence to bone marrow stem cells of rat, onion cells, Saccharomyces cerevisiae, Aspergillus oryzae and Escherichia coli. The cell proliferation and hemolytic experiments in vitro show that L-929 fibroblasts had well adherence and proliferation properties on the sample surface , the toxic degree of the samples was in zero grade standard and it had no hemolysis. CHA/yeast protein nanocomposite has the visible effects loaded and slowly released insoluble drug, the quantity loaded insoluble drug amoxicillin is up to 112.16 mg/g, the drug was embedded in the porous structure of the carrier, which have sustained release rate and no burst phenomenon, and the total amount of drug releasing is up to 55% after 6 days. The dynamic equation released amoxicillin drug from the carrier belongs to Fick's diffusion control model, which mainly were control by the two mechanisms of the drug diffusion and the carrier degradation. The hierarchical porous structure and catalysis of residual proteases in CHA/yeast protein nanocomposite improved its degradation property. The CHA mineralization with heterogeneous nucleation is easy in the electric double layer formed on its grain surface. Therefore, CHA/yeast protein nanocomposite is degradable bioactivity nanomateriale and had unique amphiphilic property (contact angleθ=120°), left optical activity (specific rotation[α] 25℃D =-23°) and good affinity to hydrophobic drug. Specially, beacause there are 17 kinds of amino acids needed by human in the yeast protein, it is also a multifunctional drug carrier material supplemented with complete proteins and calcium trace element and a fluorescence-labelled material.
Phosphates (Zn, Fe and Mg)/yeast protein nanocomposites have been synthesized successfully by using yeast cells as the catalytic templates, respectively. Phosphates/yeast protein nano-composites were calcined at different temperatures for 2h to synthesize various phosphates/active C nano-composites. The results indicated that both Zn3(PO4)2 4H2Oand rich Mg bobierite crystal grains have (020) preferred growth. The amorphous iron phosphate (core) with globulin in yeast cell can self assemble to form the grains with core-shell structure. These particles were dispersed in the herarchical network structure of yeast protein. The synthetic samples can emit different color fluorescences under different excitation wavelengths, and have the performance of transfection fluorescence to onion cells, Saccharomyces cerevisiae, Aspergillus oryzae and Escherichia coli. The synthetic samples possess better electro-catalytic activity than commercialized MnO2 catalyst. The differences of catalytic activity of samples obtianed at different temperatures in the oxidation reduction were attributed to porous structures and in situ-composite activated carbon. These materials can be used as a catalyst for oxygen electrode materials used in bio-sensors, metal-air battery, fuel cell, etc. These synthetic hybrid materials can also be used as fluorescent labeling materials.
The study results of the synthetic mechanism shown that different metal ions adsorbed in yeast cell have different effects on the structure and composition of proteins. The order of amino acid content in the synthetic samples is Mg>Ca>Fe>Zn, the effect order on the ordered degree of protein structure is Ca>Fe>Mg>Zn. Different metal ions were adsorbed on the surface of different protein molecules by gene regulation in yeast cell, have preferred growth along the special direction according to layer growth theory. Phosphates/yeast protein nanocomposites with different morphologies were formed by supramolecular self assembly of protein molecules. These materials have herarchical porous structure with vermiform morphology, the range order of their pore size distribution is Fe(2～60nm)>Ca(2～40nm)>Mg(2～24nm)>Zn(2～12nm), and the sites of fast heterogeneous nucleation are all polar hydrophilic groups arranged orderly on helical grooves in metabolic network of yeast cell. The biomineralization of yeast cells involves four processes: ion adsorption, ion transmission, deposition mineralization and supramolecular self-assembly, which were divided into mineralization induced by organism on cell wall and growth controlled by organism in cell.
引文
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