Conformational stability and integrity of barley seed β-amylase: evidence of kinetic intermediate in urea-induced unfolding.

This research article rates the molecular phenomenon of the barley β-amylase unfolding in the presence of urea and its conformational stability under the influence of selected artificial chaperones (CTAHS, DTAB, CTAB, TTAB, and SHSO₃), with the aim to minimize the enzyme inactivation and aggregation rate in the presence of the chaotropic denaturant (urea). The enzyme was monomeric with a molecular mass of 64.64 kDa. The refolding kinetics was carried out in additive dilution process and artificially mediated chaperone manner. Here, the urea equilibrium unfolding curve assumed a three states conformation of native (N), intermediate (I) and unfolded (U), where the (I) state conformationally have more of secondary and less folded three dimensional structure, a state likened to a β-amylase molten globule conformation. The enzyme has free energy (ΔG) of unfolding of approximately 27 kJmol^-1. These selected chaperones prevented aggregation and stabilized the unfolded intermediates. The cationic artificial chaperones (CTAHS, DTAB, CTAB, and TTAB) observed to be an efficient stabilization of the enzyme against denaturant compared to the anionic chaperon (SHSO₃), characterized with poor refolding kinetics. CTAHS was the most effective chaperone having the percentage (%) refolded structure of 42 ± 2%. Using ANS as hydrophobic probe, β-amylase has a calculated surface hydrophobicity of 204.1 ± 5 and ANS-bound site of 0.29 ± 0.02. These values were compromised after refolding. Taken together, the high intrinsic surface hydrophobicity of β-amylase might be connected to poor recovery rate of the unfolded enzyme.