THERMOSTABILIZATION OF Bacillus subtilis GH11 XYLANASE BY SURFACE CHARGE ENGINEERING
Juliana Sanchez, Raquel Fonseca Maldonado, Richard J. Ward
International Journal of Biological Macromolecules
Aiming to improve thermostability of the mesophilic xylanase A from Bacillus subtilis(XynA), five single mutants (S22E, S27E, N32D, N54E and N181R) were used to construct a random combinatorial library, and screening of this library for thermostable XynA variants identified a double mutant (S22E/N32D). All 6 mutants were expressed inEscherichia coli (BL21) and purified. Xylanase activity showed all mutants have an optimum catalytic temperature (Topt) of 55 °C, and with the exception of the S27E mutant, a higher specific activity than the wild-type XynA. The time for loss of 50% activity at 55 °C (t50) decreased in the order S22E/N32D > N181R > S22E > Wild-type > S27E = N32D ≈ N54E. The values of the van’t Hoff denaturation enthalpy change (ΔHND), melting temperature (Tm) and heat capacity at constant pressure (ΔCp) between the native and denatured states were estimated from thermal denaturation curves monitored by circular dichroism ellipticity changes. The decreasing order of Gibbs free energy change at 328 K (ΔG328) S22E/N32D > N181R > S22E > Wild-type > S27E ≈ N54E > N32D correlates well with the thermotolerance results, and is dominated by changes in ΔHND which is consistent with increased in hydrogen bonding in the thermostable mutants.
Circular dichroism, Secondary structure, Protein denaturation, Thermodynamics, Biochemistry