Theoretical and Experimental Studies of the Effects of Heat, EDTA, and Enzyme Concentration on the Inactivation Rate of α-Amylase from Bacillus sp.

Abstract

The effects of calcium ion-chelating ligand (EDTA), heat, and enzyme concentration on the stability of a metalloenzyme (R-amylase, from Bacillus sources) solution have been examined by experimental as well as theoretical studies. A simple two-stage inactivation model has been presented that explicitly includes the calcium ion concentration and can explain all experimental results. The first stage involves a reversible inactivation process caused by the dissociation of a metal ion (Ca 2+ ) from the active enzyme molecule, followed by the second stage of inactivation in which the apoenzyme (protein without the metal) undergoes an irreversible thermal inactivation (denaturing). These studies also suggest that the diluted enzyme solution is more prone to inactivation than the concentrated enzyme solution.

Model for Inactivation of α‐Amylase in the Presence of Salts: Theoretical and Experimental Studies

“…As in our previous papers (14,16), if the rate constants in the absence of anions are considered to be k 1 , k -1 , and k 2 , in the presence of anions, the corresponding rate constants are reduced by 1/[1 + γ]. For example,the rate constant (k′ 1 ) in the presence of anions is given by k 1 /[1 + γ].…”

“…Secondly, for a better understanding of the role of anions (especially Cl -) in enzyme stabilization, the enzyme inactivation experiments were carried out (with NaCl) in the absence as well as presence of Na 2 EDTA solutions. Finally, a three-step inactivation model has been presented (an extension of our earlier model in the aqueous solution (14,16)) that explains all of the experimental results obtained for different salt solutions.…”

“…While the reversibly inactivated form (E 2-) can be quickly transformed into the active form (CaE) by adding calcium ions, the denatured form (EI 2-) cannot be readily reactivated. In the absence of anions, eqs 2 and 3 represent the two stages of enzyme inactivation (14,16). From the equilibrium eq 1, we can derive the following expressions:…”

Conformational stability and integrity of α-amylase from mung beans: Evidence of kinetic intermediate in GdmCl-induced unfolding

“…2B), one or two Ca 2+ ions are strongly bound to the enzyme with affinity differences of about one order of magnitude. As in case of the barley isoenzymes and several bacterial α-amylases [26,29,30], this inactivation is irreversible and suggests an essential function of the metal ion in stabilizing the active site structure. From the activation of VrAMY by Ca 2+ ions ( Fig.…”

Study on Some Properties of Calcium-dependent a -Amylase from Bacillus subtilis through Submerged Fermentation of Wheat Bran

“…Some amylases from Bacillus strains were extremely thermophilic having optimum temperature of 100 °C 41,42 . These Ca 2+ play an important role in the thermal inactivation of amylases produced by Bacillus species 43,44 .…”