Abstract：The fluorescence spectroscopic characteristics of DhaA immobilized by amino-modified-mesocellular foam (MCF-NH2) were investigated during denaturation procedures with urea and DMSO as denaturant by using F-4600 fluorescence spectrometer. The unfolding process of DhaA in denaturants was analyzed by phase diagram of fluorescence, and thermodynamic parameters were calculated with residual activity ratio of DhaA. The difference between the non-immobilized and immobilized DhaA in aspect of unfolding process and thermodynamic parameters were compared. The results showed that the catalytic activity of DhaA declined with the increasing concentration of denaturants, and the catalytic activity of immobilized DhaA could be maintained better than that of free DhaA at the same concentration of denaturant. The stabilization effect of MCF-NH2 to DhaA was obvious before the denaturants concentration reached a critical concentration (5.5 mol·L-1 for urea, 7 mol·L-1 for DMSO). The unfolding procedure of DhaA induced by urea conformed to typical “two-state” model. The unfolding procedure of DhaA induced by DMSO conformed to “three-state” model, and the intermediate state of DhaA appeared at the DMSO concentration of 5.6 mol·L-1. Immobilization by MCF-NH2 didn’t change the degeneration process of DhaA, but could raise the thermodynamic parameters during the unfolding process of DhaA. When induced by urea, the ΔG(H2O) was 8.51 kcal·mol-1 for free DhaA and was increased to 9.55 kcal·mol-1 for the immobilized one. However, the solvent-accessible surface area (m) increased from 3.69 to 4.00 kcal·(mol·mol·L-1)-1 after immobilization, which might be caused by the convenient moving of urea molecules into MCF-NH2 with electrostatic attraction. The amino and hydroxyl groups in the channel of MCF-NH2 could enhance DhaA rigidity through hydrogen bonding effectively reduced the effect of increasing of urea-accessible surface area, and improved the urea tolerance of DhaA. When DhaA was induced by DMSO, the ΔG(H2O) values of process from fold state to intermediate state were always 12.12 kcal·mol-1 in the before and after immobilization. The solvent-accessible surface area of DhaA dropped from 3.39 to 2.30 kcal·(mol·mol·L-1)-1 after immobilization, which might be caused by the block effect of amino and hydroxyl groups in MCF-NH2 which effectively inhibiting the entry of non-polar DMSO molecules. From intermediate state to unfold state, the exposure of hydrophobic amino acids in DhaA leaded to an increase of m value, but the m value of immobilized DhaA [4.40 kcal·(mol·mol·L-1)-1] was still lower than the free DhaA [4.94 kcal·(mol·mol·L-1)-1] due to the polar microenvironment in the channel. Studying the unfolding process and thermodynamic parameters by fluorescence spectrometer is an effective technological mean for the research on DhaA stability, which can also offer a methodological guidance for stabilization mechanism study for other enzymes.
