Engineering a highly active sucrose isomerase for enhanced product specificity using a 'battleship' strategy.

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyzes the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the hygroscopic byproduct trehalulose necessitates additional separation effort to obtain a crystalline product. Therefore, we improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but using a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving overall just 55 point mutations, the triple mutant Y219L/D398G/V465E was obtained, showing 2.3-fold lower trehalulose production still with high catalytic efficiency (kcat/KM = 11.8 mM-1.s-1). While this mutant SmuA appears attractive as industrial biocatalyst, our semi-rational protein engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.