Microcalorimetric Determination of the Thermostability of Three Hybrid (1–3,1–4)-β-Glucanases

Abstract

Thermodynamic parameters of the three hybrid (1–3,1–4)-β-glucanases H(A12-M), H(A12-M)ΔY13, and H(A16-M) composed of short N-terminal regions derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the homologous Bacillus macerans enzyme were determined in 2mM sodium cacodylate pH 6.0,1.5 M guanidine hydrochloride, containing 1 mM CaCl₂ or 1 mM EDTA Melting of H(A12-M)ΔY13 and H(A16-M) in the presence of calcium ions is characterized by two subtransitions; only one transition is observed in the case of H(A12-M). In calcium-free buffer each of the three hybrid enzymes melts in one two-state transition. Transition temperatures T _m and molar enthalpy changes ΔH are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M)ΔY13 and H(A16-M) than for the less thermostable enzyme H(A12-M).