Studies on the Thermodenaturation Behavior of Bacillus subtilisα‐Amylase on Chromatographic Media

Abstract

The thermodenaturation behavior of Bacillus subtilis α-amylase on some chromatographic media was studied by determining their adsorption parameters with frontal analysis. The experimental results show that on a RP-C 18 reversed-phase medium, a Chelating Sepharose Fast-Flow chelated by Zn 2＋ affinity medium and a WCX-1 cation-exchange medium, a stable conformation of α-amylase molecule separately exists below or over 30 ; while ℃ on a PEG-400 hydrophobic medium and a modified PEG-400 medium, a stable conformation of α-amylase molecule separately exists below 40 and 30 , and w ℃ hen the experimental temperatures are separately over 40 and 30 , ℃ a drastically conformational change of α-amylase molecules can continuously take place. And by combining the intrinsic fluorescence emission spectrum and thermal inactivation profile of α-amylase in free solution and on the PEG-400 and modified PEG-400 hydrophobic media, it can be concluded that in liquid chromatographic procedure, chromatographic media can induce the conformational change of α-amylase molecules and promote their thermodenaturation; and in hydrophobic interaction chromatography, the higher the hydrophobicity of chromatographic medium, the lower the conformational change temperature of α-amylase molecules on the chromatographic medium.