Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity

Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2° ADH) was optimally active near 90°C displaying thermostability half-lives of 1.2 days, 1.7 h, 19 min, 9.0 min, and 1.3 min at 80°C, 90°C, 92°C, 95°C, and 99°C, respectively. Enzyme activity loss upon heating (90–100°C) was accompanied by precipitation, but the soluble enzyme remaining after partial inactivation retained complete activity. Enzyme thermoinactivation was modeled by a pseudo-first order rate equation suggesting that the rate determining step was unimolecular with respect to protein and thermoinactivation preceded aggregation. The apparent 2° ADH melting temperature (Tm) occurred at ∼115°C, 20°C higher than the temperature for maximal activity, suggesting that it is completely folded in its active temperature range. Thermodynamic calculations indicated that the active folded structure of the 2° ADH is stabilized by a relatively small Gibbs energy (▵Gstab.‡ = 110 kJ mol−1). 2° ADH catalytic activities at 37°C to 75°C, were 2-fold enhanced by guanidine hydrochloride (GuHCl) concentrations between 120 mM and 190 mM. These results demonstrate the extreme resistance of this thermophilic 2° ADH to thermal or chemical denaturation; and suggest increased temperature or GuHCl levels seem to enhance protein fixability and activity.