On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates
On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates
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DOI：
10.1021/bc0001241
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发表时间：
2001-09-01
影响因子：
4.7
通讯作者：
Sastry, M
中科院分区：
文献类型：
--
作者：
Gole, A;Dash, C;Sastry, M
我们在此提出的细节有关的制备胶体金与天冬氨酸蛋白酶的真菌斋藤曲霉（F-蛋白）和他们的表征和酶活性的生物共轭物。在蛋白质友好的条件（pH = 3）下简单混合胶体金和蛋白质溶液，然后离心（以除去未复合的金纳米颗粒和蛋白质分子）导致真菌蛋白酶-金纳米颗粒缀合物的形成。将蛋白质-金纳米颗粒生物缀合物再分散在缓冲溶液中，并表明形成了具有完整天然蛋白质结构的有效生物缀合物。通过紫外可见光谱、荧光光谱和生物催化活性测量表征溶液中的生物缀合物，而通过扫描电子显微镜（SEM）、X射线能量色散分析（EDAX）和X射线衍射（XRD）测量表征在Si（111）衬底上的滴干生物缀合物膜。显微镜图像确实显示了一些聚集体的形成，但是生物缀合物材料中酶的天然结构的完整性通过荧光和生物催化活性测量来验证。该酶在生物缀合物材料中保留了大量的生物催化活性，并且与溶液中的游离酶相当。
We present herein details pertaining to the preparation of bioconjugates of colloidal gold with aspartic protease from the fungus Aspergillus saitoi (F-prot) and their characterization and enzymatic activity. Simple mixing of the colloidal gold and protein solutions under protein-friendly conditions (pH = 3) followed by centrifugation (to remove uncomplexed gold nanoparticles and protein molecules) results in the formation of the fungal protease-gold nanoparticle conjugates. The protein-gold nanoparticle bioconjugate was redispersed in buffer solution and indicated the formation of efficient bioconjugates with intact native protein structures. The bioconjugates in solution were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while drop-dried bioconjugate films on Si (111) substrates were characterized by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), and X-ray diffraction (XRD) measurements. Microscopy images do show some aggregate formation, but the intactness of the native structure of the enzyme in the bioconjugate material was verified by fluorescence and biocatalytic activity measurements. The enzyme retains substantial biocatalytic activity in the bioconjugate material and was comparable to that of free enzyme in solution.