Individual amino acids in the N‐terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases

• Published in: Protein science Vol. 5; no. 11; pp. 2255 - 2265
• Main Authors: Welfle, Karin, Politz, Oliver, Borriss, Rainer, Misselwitz, Rolf, Welfle, Heinz
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01-11-1996
• Subjects: Bacillus - enzymology; Bacillus amyloliquefaciens; Bacillus macerans; Calcium - chemistry; Calorimetry, Differential Scanning; circular dichroism; conformation; differential scanning calorimetry; Endo-1,3-beta-Glucanase; Enzyme Stability; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - metabolism; Guanidine; Guanidines; hybrid enzymes; Models, Molecular; Protein Denaturation; Protein Folding; stability, unfolding; Temperature; Thermodynamics; β‐glucanase
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560051112

Thermostability and unfolding behavior of the wild‐type (1,3–1,4)‐β‐glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12‐M)ΔF14 and H(A12‐M)ΔY13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12‐M)ΔF14 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)ΔY13F14A, 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 Tm are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca2+‐free buffer, MAC, H(A12‐M)ΔF14 and H(A12‐M)ΔY13F14A 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)ΔF14, a two‐step unfolding is induced by Ca2+. These data indicate a two‐domain structure of AMY and H(A12‐M)ΔF14 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+.