Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface
•Eleven mutants with different novel disulfide bridges were constructed.•Mutant DS255 exhibited significantly enhanced stability.•Engineered disulfide bonds led to increase in ΔG* and decrease in ΔS* of DS255.
Rational design was applied to glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10 to improve its thermal stability by introduction of disulfide bridges between subunits. One out of the eleven mutants, designated as DS255, displayed significantly enhanced thermal stability with considerable soluble expression and high specific activity. It was extremely stable at pH ranging from 4.5 to 10.5, as it retained nearly 100% activity after incubating at different buffers for 1 h. Mutant DS255 also exhibited high thermostability, having a half-life of 9900 min at 50 °C, which was 1868-fold as that of its wild type. Moreover, both of the increased free energy of denaturation and decreased entropy of denaturation of DS255 suggested that the enzyme structure was stabilized by the engineered disulfide bonds. On account of its robust stability, mutant DS255 would be a competitive candidate in practical applications of chiral chemicals synthesis, biofuel cells and glucose biosensors.
Journal: Enzyme and Microbial Technology - Volume 53, Issues 6–7, 10 December 2013, Pages 365–372