title: pH-dependent immobilization of urease on glutathione-capped gold nanoparticles.
abstract: Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Although the enzyme serves a significant role in several detoxification and analytical processes, its usability is restricted due to high cost, availability in small amounts, instability, and a limited possibility of economic recovery from a reaction mixture. Hence, there is a need to develop an efficient, simple, and reliable immobilization strategy for the enzyme. In this study, the carboxyl terminated surface of glutathione-capped gold nanoparticles have been utilized as a solid support for the covalent attachment of urease. The immobilization has been carried out at different pH conditions so as to elucidate its effect on the immobilization efficiency and enzyme bioactivity. The binding of the enzyme has been quantitatively and qualitatively analyzed through techniques like ultraviolet-visible spectroscopy, intrinsic steady state fluorescence, and circular dichorism. The bioactivity of the immobilized enzyme was investigated with respect to the native enzyme under different thermal conditions. Recyclability and shelf life studies of the immobilized enzyme have also been carried out. Results reveal that the immobilization is most effective at pH of 7.4 followed by that in an acidic medium and is least in alkaline environment. The immobilized enzyme also exhibits enhance activity in comparison to the native form at physiological temperature. The immobilized urease (on gold glutathione nanoconjugates surface) can be effectively employed for biosensor fabrication, immunoassays and as an in vivo diagnostic tool in the future.
authors.affiliation: Department of Chemistry, University of Delhi, Delhi, 110007, India.
authors.forename: Seema
authors.initials: S
authors.lastname: Garg
authors.forename: Arnab
authors.initials: A
authors.lastname: De
authors.forename: Subho
authors.initials: S
authors.lastname: Mozumdar
mesh_terms: D001672:Biocompatible Materials / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D000067493:Dynamic Light Scattering; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology*; D006046:Gold / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D010316:Particle Size; D017433:Protein Structure, Secondary; D012015:Reference Standards; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014510:Urease / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001672:Biocompatible Materials; D004800:Enzymes, Immobilized; D006046:Gold; D014510:Urease; D005978:Glutathione
keywords: covalent immobilization; glutathione; gold nanoparticles; urease