Levan-type
fructooligosaccharides (LFOs) and levan can potentially
be used as ingredients in prebiotics, skincare products, and antitumor
agents. The Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide-producing levansucrase, OPL)
was reported to productively synthesize LFOs; however, OPL’s
thermostability is low at high temperatures. To enhance OPL structural
stability, this study employed molecular dynamics (AMBER) to identify
a highly flexible region, as measured by its average root-mean-square
fluctuation (RMSF) value, on the OPL surface and computational protein
design (Rosetta) to rigidify and increase favorable interactions to
increase its structural stability. AMBER identified region nine (residues
277–317) as a highly flexible region that was selected for
design because it has the highest number of residues and the second-highest
average RMSF, and it is farthest from the active site. Rosetta designed
14 mutants with the best ΔΔG value in
each position, where three mutants have better ΔG than OPL. To determine whether their flexibilities and stabilities
are lower than those of OPL, all 14 designed mutants were simulated
at high temperature (500 K), and we found that K296E, G309S, and A310W
mutants were predicted to be more stable and could retain their native
structures better than OPL. Our results suggest that enhanced structural
stabilities of these mutants are caused by increased hydrogen bond
strengths of the designed residues and their neighboring residues.
This study designed K296E, G309S, and A310W mutants of OPL with high
potential for stability improvement, and they could potentially be
used for the effective production of LFOs.