An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

中文翻译：

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由过氧化物酶催化的人工木质化细胞壁选择性地定位在培养细胞的微纤维网络上

人工木质化细胞壁的合成分三个步骤：（1）微纤维网络的分离；(2) 通过免疫反应定位过氧化物酶；(3) DHP聚合使细胞壁木质化。人工木质细胞壁合成是按照三个步骤以及在自然界中的实际形成使用从源自柳杉的愈伤组织中提取的纤维素微纤维进行的。首先，我们在透射电子显微镜 (TEM) 网格上构建了多糖网络。通过化学处理优化制备方法，然后进行机械原纤化以产生微纤化网络。通过 TEM 检查形态，通过傅里叶变换红外 (FTIR) 光谱法进行化学表征。第二，我们优化了通过免疫反应技术将过氧化物酶置于微纤维上的过程。使用木葡聚糖抗体，我们可以确保附着在二级抗体上的金颗粒与微纤维网络一起广泛且均匀地定位。第三，我们应用附着在二抗上的过氧化物酶，并通过同时添加松柏醇和过氧化氢开始聚合网格上的木质素。人工木质化 30 min 后，TEM 观察显示木质素类物质沉积在多糖网络上。此外，FTIR 光谱显示木质素特异性条带有所增加，表明成功人工形成了木质细胞壁。