Thermal stabilization of an endoglucanase by cyclization.
An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.
Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
Johan F T
JF
van Lieshout
Odette N
ON
Pérez Gutiérrez
Wietse
W
Vroom
Antoni
A
Planas
Willem M
WM
de Vos
John
J
van der Oost
Sotirios
S
Koutsopoulos
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D057005:Bioengineering; D002118:Calcium / Q000494:pharmacology; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003500:Cyclization / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays; D004795:Enzyme Stability / Q000187:drug effects; D047668:Inteins; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D058767:Protein Unfolding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002480:Cellulase; D002118:Calcium