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 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.
Gole
A
A
Materials Chemistry Division, National Chemical Laboratory, Pune 411 008, India.
Dash
C
C
Soman
C
C
Sainkar
S R
SR
Rao
M
M
Sastry
M
M
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology; D002384:Catalysis; D004355:Drug Stability; D005656:Fungal Proteins / Q000737:chemistry; D017979:Gold Colloid / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D013194:Staining and Labeling; D014961:X-Ray Diffraction
D005656:Fungal Proteins; D017979:Gold Colloid; D016282:Aspartic Acid Endopeptidases