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−1 s−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.
Highlights
The sucrose isomer isomaltulose (6-O-α-d-glucopyranosyl-dfructose, referred to as palatinose) and the diastereomeric mixture of the corresponding sugar alcohols derived by catalytic hydrogenation, commonly known as isomalt,[1] have attracted significant interest as safe low-calorie sweeteners in the food industry
Isomaltulose is produced from sucrose using immobilized bacteria that naturally express an enzyme known as sucrose isomerase (SI; EC 5.4.99.11)
Reaction mechanism for the sucrose isomerase SmuA, which converts sucrose into isomaltulose as well as the by-products trehalulose, glucose and fructose
Summary
The sucrose isomer isomaltulose (6-O-α-d-glucopyranosyl-dfructose, referred to as palatinose) and the diastereomeric mixture of the corresponding sugar alcohols derived by catalytic hydrogenation, commonly known as isomalt,[1] have attracted significant interest as safe low-calorie sweeteners in the food industry. Isomaltulose is found in sugar cane juice as well as honey.[3] Currently, isomaltulose is produced from sucrose using immobilized bacteria that naturally express an enzyme known as sucrose isomerase (SI; EC 5.4.99.11). This enzyme catalyses the isomerization of sucrose to produce mainly isomaltulose and trehalulose, accompanied by hydrolysis of 42 sucrose to glucose and fructose as by-products (Scheme 1).[4]
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