Immobilization of β-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications

• Published in: PloS one Vol. 7; no. 7; p. e40708
• Main Authors: Kishore, Devesh, Talat, Mahe, Srivastava, Onkar Nath, Kayastha, Arvind M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18-07-2012; Public Library of Science (PLoS)
• Subjects: Activation energy; Analysis of Variance; Aspergillus; beta-Galactosidase - metabolism; Biology; Biosensors; Biotechnology - methods; Carbon; Chemical bonds; Chemical interactions; Chemical reactions; Chemistry; Commodities; Consumption; Electron microscopy; Enzymatic activity; Enzyme kinetics; Enzyme Stability; Enzymes, Immobilized - metabolism; Food; Fourier transforms; Galactosidase; Graphene; Graphite; Graphite - metabolism; Hydrolysis; Immobilization; Infrared spectroscopy; Kinetics; Lactose; Lactose - metabolism; Lactose intolerance; Materials Science; Methods; Milk; Molecular biology; Nanoparticles - chemistry; Nanoparticles - ultrastructure; Nanotechnology; Optimization; Oxidation; pH effects; Proteins; Recycling; Response surface methodology; Scanning electron microscopy; Sheets; Solubility; Spectroscopy; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Thermal stability; Vibration; β-Galactosidase
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0040708

β-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.
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.
Immobilized β-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.