Thermostability of alcohol dehydrogenase: Evidence for distinct subunits with different deactivation properties

Abstract

Several independent experimental techniques, including nondenaturing and denaturing isoelectric focusing, spin labeling, and enzyme immobilization, indicate that four ethanol-active subunits of horse liver alcohol dehydrogenase (LADH) can be classified as one of two types, designated E(1) and E(2). Thermal inactivation studies of LADH in solution and immobilized to two different supports demonstrate that the first-order rate constants of deactivation of E(1) and E(2) differ by more than an order of magnitude. Furthermore, E(1), and E (2) can be distinguished by EPR spectroscopy, with the less stable subunit type, E(2), appearing to have the less compactly structured active-site environment. The less stable enzyme form also loses catalytic activity upon covalent attachment to CNBr-Sepharose but remains active when adsorbed to Octyl-Sepharose. Moreover, the immobilization results in conjunction with lysine modification studies suggest that E(2) immobilized to CNBr-Sepharose cannot bind coenyzme. Overall, these results illustrate how EPR measurements in concert with activity assays can provide insights into the molecular mechanisms of enzyme stabilization.

Prevention of Swelling Phenomenon of Alginate Beads To Improve the Stability and Recyclability of Encapsulated Horse Liver Alcohol Dehydrogenase

“…More precisely, the half-life of immobilized HLADH is three and more than four times higher than that of the free enzyme at 40 and 50 °C, respectively, while at 60 °C an increase of only 20 % is obtained. In a previous study, the rate constants of deactivation of free and covalently immobilized HLADH on CNBr-Sepharose performed at 60 °C were 0.0064 and 0.0039 min À 1 , respectively, [32] values very similar to those of this work. The enthalpy change, ΔH 0 , is often used to determine the energy required to inactivate or denature the enzyme.…”

Studies of NADP+-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii

“…The compactly structured active site is reported to be responsible for the thermal stability of a thermostable subunit of horse liver ADH (Skerker and Clark 1989) and the denaturant resistance of a thermostable malic enzyme (Guagliardi et al 1989). Our results indicate that TADH possesses a compact structure d f / f o = 1.17) and it is relatively insensitive toward sulfhydryl reagents.…”

Immobilization of α - amylase from locale bacteria isolate Bacillus subtilis ITBCCB148 with diethylaminoethyl cellulose (DEAE-Cellulose)

“…Diethylaminoethyl cellulose (DEAE-Cellulose) is one of the reagents that is frequently used in the enzyme purification process, as the use of this compound is able to increase the enzyme stability, while at the same time it minimizes the loss of enzyme activity. Previous investigation using immobilization process and chemical modification have successfully shown an increase in the enzyme stability toward pH and temperature [3][4][5][6][7][8] . Based on these facts, in this research the immobilization process was chosen in an attempt to increase the stability of α-amylase which was produced, isolated, and purified from local bacteria isolate B. subtilis ITBCCB148.…”