MICROCALORIMETRIC DETERMINATION OF THE THERMOSTABILITY OF 3 HYBRID (1-3,1-4)-BETA-GLUCANASES

Thermodynamic parameters of the three hybrid (1-3,1-4)-beta-glucanases H(A12-M), H(A12-M)Delta Y13, and H(A16-M) composed of short N-termina l regions derived from the Bacillus amyloliquefaciens enzyme and a C-t erminal region of the homologous Bacillus macerans enzyme were determi ned in 2 mM sodium cacodylate pH 6.0, 1.5 M guanidine hydrochloride, c ontaining 1 mM CaCl2 or 1 mM EDTA. Melting of H(A12-M)Delta Y13 and H( A16-M) in the presence of calcium ions is characterized by two subtran sitions; only one transition is observed in the case of H(A12-M). In c alcium-free buffer each of the three hybrid enzymes melts in one two-s tate transition. Transition temperatures T-m and molar enthalpy change s Delta H are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M)Delta Y13 and H(A16-M) than for t he less thermostable enzyme H(A12-M).