Thermostability and unfolding behavior of the wild-type (1,3-1,4)-beta-glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12-M) delta F14 and H(A12-M) delta Y13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12-M) delta F14 is constructed by the fusion of 12 N-terminal amino acids of AMY with amino acids 13-214 of MAC, and by deletion of F14. In H(A12-M) delta Y13F14A, the N-terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala. The sequence of the N-terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes. The half transition temperatures T(m) are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca(2+)-free buffer, MAC, H(A12-M) delta F14 and H(A12-M) delta Y13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two-step unfolding was found. In the presence of Ca2+, the two-step unfolding of AMY is strengthened. Furthermore, for H(A12-M) delta F14, a two-step unfolding is induced by Ca2+. These data indicate a two-domain structure of AMY and H(A12-M) delta F14, in the presence of Ca2+. Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+. Study holds ProTherm entries: 7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767, 7768, 7769, 7770, 7771, 7772, 7773, 7774, 7775, 7776, 7777, 7778, 7779, 7780 Extra Details: MAC: Bacillus macerans (1,3-1,4)-beta-glucanase unfolding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||32|
|Proteins||Beta-glucanase ; Beta-glucanase ; Beta-glucanase ; Beta-glucanase|
|Assays/Quantities/Protocols||Experimental Assay: dG ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dG details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: dHcal details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: Tm details:Additives , ionic:CaCl2: 1 mM|
|Libraries||Mutations for sequence FDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYTGPAHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGYIKWYVDGVLKHTATANIPSTPGKIMMNLWNGTGVDDWLGSYNGANPLYAEYDWVKYTSNQTGGSFFEPFNSYNSGTWEKADGYSNGGVFNCTWRANNVNFTNDGKLKLGLTSSAYNA ; Mutations for sequence QTGGSFFEPFNSYNSGTWEKADGYSNGGVFNCTWRANNVNFTNDGKLKLGLTSSAYNKFDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYTGPAHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGYIKWYVDGVLKHTATANIPSTPGKIMMNLWNGTGVDDWLGSYNGANPLYAEYDWVKYTSN|