The additive stabilizing effect of E518I/S662R/Q706P may be
caused by the relatively distant location on the enzyme structure
of these residues without direct interaction among these three
sites [10,37]; therefore, the individual contribution of each mutation
was reflected in the combined mutant. Similar reports on B.
deramificans pullulanase also indicated that the stabilizing effect of
single-site mutation can be combined. The mutant (D437H/D503Y)
of B. deramificans pullulanase displayed a higher optimal temperature
and half-life than that of the WT enzyme and single-site
mutants