Role of methoxypolyethylene glycol on the hydration, activity, conformation and dynamic properties of a lipase in a dry film.
A combined approach based on the use of ATR-FT/IR and steady-state fluorescence spectroscopy allowed to shed light on the effects of the additive methoxypolyethylene glycol (MePEG) on the hydration, conformation and dynamic properties of lipase from Burkholderia cepacia dehydrated to form a film. Spectroscopic data show that the additive has little effect on the structure of the protein; however, H/D exchange kinetic and fluorescence anisotropy suggest a more flexible enzyme molecule when in the presence of MePEG. By infrared spectroscopy, we estimated that, after conditioning the films at water activity of 1, the water content in the lipase dehydrated with MePEG is 5.4- and 4.7-fold higher than in the absence of the additive and the additive alone, respectively. Additionally, our infrared data suggest that MePEG acts by hindering intermolecular protein-protein interactions and contributing to increase the accessibility and flexibility of the lipase in the dehydrated solid film. These factors also explain the enhancement of the enzyme catalytic activity (i.e., up to 3.7-fold in neat organic solvent) when in the presence of MePEG. The method and results presented might better address the use of additives for the preparation of enzymes employed in non-aqueous media or of proteins used in a dry form in different fields of biotechnology.
Secundo
Francesco
F
Istituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131 Milano, Italy. francesco.secundo@icrm.cnr.it
Barletta
Gabriel
G
Mazzola
Giorgio
G
D001426:Bacterial Proteins / Q000737:chemistry*; D016956:Burkholderia cepacia / Q000201:enzymology*; D002384:Catalysis; D003890:Desiccation; D005454:Fluorescence Polarization; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water
D001426:Bacterial Proteins; D012997:Solvents; D014867:Water; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D008049:Lipase