Sucrose Isomerase Mutants' Expression in Bacillus subtilis for Isomaltulose Production

Isomaltulose is produced via sucrose isomerase catalysis by Serratia plymuthica A30. The enzyme was expressed in Bacillus subtilis using surface display, in combination with Bacillus subtilis spore coat CotC. The promoter was further selected and optimized to determine P_amyE as the most suitable promoter, while the spore coat protein assay reveals CotC as optimal. By scanning and analyzing the catalyst motifs with single-point mutation construction, a maximum isomaltulose yield of 27.21 mg/mL was recorded in the F181I-mutant enzyme. Another strain encoding the H363P-mutant reached a maximum yield of 20.84 mg/mL, while the k_cat value also increased from 17.64 to 24.80. Structural analysis showed that the F181I-mutant had higher thermostability, whereas the H363P-mutant had increased k_cat. Both mutants displayed a 5-fold increase in isomaltulose yield with relatively simple construction procedures, making them suitable for high-level isomaltulose production.