No abstract

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by-product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have 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 with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (k cat /K M = 11.8 mM À 1 s À 1). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein-engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.

The sucrose isomerase SmuA from Serratia plymuthica catalyses the production of isomaltulose, an artificial sweetener used in the food industry. To suppress the formation of the hygroscopic by-product trehalulose we applied a semirational protein engineering strategy inspired by the “battleship” board game. After seven cycles of introducing amino acid exchanges around the active site and investigating their influence on the enzymatic product profile we obtained a triple mutant that showed 2.3 times less trehalulose formation but had retained high catalytic efficiency.