Molecular modifications of Serratia plymuthica sucrose isomerase in Bacillus subtilis to improve its catalytic properties

Sucrose isomerase (SI) has important potential for isomaltulose production. In this study, the SpSI from Serratia plymuthica, with a specific enzyme activity of 358.87 U/mg, was selected by heterologous expression in Bacillus subtilis WB800 and modified by a semirational design to improve its catalytic efficiency, thermal stability and isomaltulose conversion ratio. Four single-point mutants (T522S, D222H, K340D and G467N) and three combined mutants (T522S/K340D, D222H/K340D, G467N/K340D) with improved enzyme activity were screened from a mutant library generated via a semirational design and ordered recombination mutations. Notably, these mutants presented a 2.06- to 2.37-fold increase in specific enzyme activity compared with that of the wild-type enzyme. The T522S/K340D, D222H/K340D, and G467N/K340D mutants demonstrated increased thermostability. The optimal pH for the mutants T522S/K340D, D222H/K340D and K340D shifted to 5.5 from 6.0. Their activities are facilitated by Ca 2+, Ba 2+, and Mn 2+. The kinetic parameters suggested that the k cat values of the mutants G467N/K340D and T522S/K340D were 1.14–1.48 times greater than that of the original enzyme. Additionally, the isomaltulose conversion of the D222H/K340D mutant improved to 82.35%, whereas that of the original enzyme was 79.46%. Three-dimensional structural analysis revealed that reduced polar bonds and increased amino acid rigidity contributed to enhanced enzyme activity, isomaltulose conversion, and thermostability. These findings indicate the potential value of SpSI and its mutants for isomaltulose production.