On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates

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 nanopartic les and protein molecules) results in the formation of the fungal protease- gold nanoparticle conjugates. The protein-gold nanoparticle bioconjugate wa s redispersed in buffer solution and indicated the formation of efficient b ioconjugates with intact native protein structures. The bioconjugates in so lution were characterized by UV-vis spectroscopy, fluorescence spectroscopy , and biocatalytic activity measurements while drop-dried bioconjugate film s 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 m aterial 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.