Immobilization of β-Galactosidase onto Functionalized Graphene Nano-sheets Using Response Surface Methodology and Its Analytical Applications

Background: beta-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction. Methodology/Principal Findings: Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (E-a) remains unchanged whereas optimum pH and K-m were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene. Significance: Immobilized beta-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.