Authors: Hajer Ben Hlima Nushin Aghajari Mamdouh Ben Ali Richard Haser Samir Bejar
Publish Date: 2011/12/04
Volume: 39, Issue: 4, Pages: 537-546
Abstract
The role of two amino acid residues linked to the two catalytic histidines His54 and His220 in kinetics and physicochemical properties of the Streptomyces sp SK glucose isomerase SKGI was investigated by sitedirected mutagenesis and molecular modeling Two single mutations F53L and G219D and a double mutation F53L/G219D was introduced into the xylA SKGI gene The F53L mutation increases the thermostability and the catalytic efficiency and also slightly shifts the optimum pH from 65 to 7 but displays a profile being similar to that of the wildtype enzyme concerning the effect of various metal ions The G219D mutant is resistant to calcium inhibition retaining about 80 of its residual activity in 10 mM Ca2+ instead of 10 for the wildtype This variant is activated by Mn2+ ions but not Co2+ as seen for the wildtype enzyme It does not require the latter for its thermostability but has its halflife time displaced from 50 to 20 min at 85°C The double mutation F53L/G219D restores the thermostability as seen for the wildtype enzyme while maintaining the resistance to the calcium inhibition Molecular modeling suggests that the increase in thermostability is due to new hydrophobic interactions stabilizing α2 helix and that the resistance to calcium inhibition is a result of narrowing the binding site of catalytic ionAll authors have agreed to submit this manuscript to the Journal of Industrial Microbiology and Biotechnology This work was funded by the Tunisian Ministry of Higher Education the FrenchTunisian CMCU programme no 09G 0801 and the French National Research Center CNRS
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