Gholami-Shabani Mohammadhassan, Akbarzadeh Azim, Norouzian Dariush, Amini Abdolhossein, Gholami-Shabani Zeynab, Imani Afshin, Chiani Mohsen, Riazi Gholamhossein, Shams-Ghahfarokhi Masoomeh, Razzaghi-Abyaneh Mehdi
Department of Mycology, Pasteur Institute of Iran, Tehran, 13164, Iran.
Appl Biochem Biotechnol. 2014 Apr;172(8):4084-98. doi: 10.1007/s12010-014-0809-2. Epub 2014 Mar 9.
Nanostructures from natural sources have received major attention due to wide array of biological activities and less toxicity for humans, animals, and the environment. In the present study, silver nanoparticles were successfully synthesized using a fungal nitrate reductase, and their biological activity was assessed against human pathogenic fungi and bacteria. The enzyme was isolated from Fusarium oxysporum IRAN 31C after culturing on malt extract-glucose-yeast extract-peptone (MGYP) medium. The enzyme was purified by a combination of ultrafiltration and ion exchange chromatography on DEAE Sephadex and its molecular weight was estimated by gel filtration on Sephacryl S-300. The purified enzyme had a maximum yield of 50.84 % with a final purification of 70 folds. With a molecular weight of 214 KDa, it is composed of three subunits of 125, 60, and 25 KDa. The purified enzyme was successfully used for synthesis of silver nanoparticles in a way dependent upon NADPH using gelatin as a capping agent. The synthesized silver nanoparticles were characterized by X-ray diffraction, dynamic light scattering spectroscopy, and transmission and scanning electron microscopy. These stable nonaggregating nanoparticles were spherical in shape with an average size of 50 nm and a zeta potential of -34.3. Evaluation of the antimicrobial effects of synthesized nanoparticles by disk diffusion method showed strong growth inhibitory activity against all tested human pathogenic fungi and bacteria as evident from inhibition zones that ranged from 14 to 25 mm. Successful green synthesis of biologically active silver nanoparticles by a nitrate reductase from F. oxysporum in the present work not only reduces laborious downstream steps such as purification of nanoparticle from interfering cellular components, but also provides a constant source of safe biologically-active nanomaterials with potential application in agriculture and medicine.
天然来源的纳米结构因其广泛的生物活性以及对人类、动物和环境较低的毒性而备受关注。在本研究中，使用真菌硝酸还原酶成功合成了银纳米颗粒，并评估了其对人类致病真菌和细菌的生物活性。该酶是在麦芽提取物 - 葡萄糖 - 酵母提取物 - 蛋白胨（MGYP）培养基上培养后从尖孢镰刀菌IRAN 31C中分离得到的。通过超滤和在DEAE Sephadex上的离子交换色谱相结合对该酶进行纯化，并通过在Sephacryl S - 300上的凝胶过滤估计其分子量。纯化后的酶最大产率为50.84%，最终纯化倍数为70倍。其分子量为214 kDa，由125、60和25 kDa的三个亚基组成。纯化后的酶成功用于以依赖于NADPH的方式合成银纳米颗粒，使用明胶作为封端剂。通过X射线衍射、动态光散射光谱以及透射和扫描电子显微镜对合成的银纳米颗粒进行了表征。这些稳定的非聚集纳米颗粒呈球形，平均尺寸为50 nm，zeta电位为 - 34.3。通过纸片扩散法评估合成纳米颗粒的抗菌效果，结果显示对所有测试的人类致病真菌和细菌均具有很强的生长抑制活性，抑菌圈范围为14至25 mm。在本工作中，利用尖孢镰刀菌的硝酸还原酶成功实现了具有生物活性的银纳米颗粒的绿色合成，这不仅减少了诸如从干扰细胞成分中纯化纳米颗粒等繁琐的下游步骤，还提供了一种安全的生物活性纳米材料的恒定来源，在农业和医学领域具有潜在应用价值。
