Engineering a Highly Active Sucrose Isomerase for Enhanced Product Specificity by Using a “Battleship” Strategy

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.

Development of an engineered Bacillus subtilis strain for antibiotic‐free sucrose isomerase production

“…[8] This enzyme catalyzes the hydrolysis and isomerization of sucrose into isomaltulose as the main product, along with minor quantities of trehalulose, glucose, and fructose. [8,9] Using a food-grade expression system for SIase could enhance isomaltulose production and its widespread applications. For this purpose, several microbial strains with food-safe backgrounds, including Yarrowia lipolytica, Corynebacterium glutamicum and Bacillus subtilis, have been used for the heterologous expression of SIase.…”

Construction and Catalytic Study of Affinity Peptide Orientation and Light Crosslinking Immobilized Sucrose Isomerase

“…Isomaltulose is less rapidly cleaved than sucrose, providing a healthier option for individuals with obesity, diabetes, or cardiovascular disease. 17 Thus, isomaltulose has been commonly used in all kinds of food and drinks, and its increasing demand has promoted the immobilization of sucrose isomerase (SI). 18 In this study, photo-crosslinking and affinity adsorption were combined to achieve an efficient and environmentally friendly preparation of oriented immobilized sucrose isomerase.…”

“…Isomaltulose is less rapidly cleaved than sucrose, providing a healthier option for individuals with obesity, diabetes, or cardiovascular disease . Thus, isomaltulose has been commonly used in all kinds of food and drinks, and its increasing demand has promoted the immobilization of sucrose isomerase (SI) .…”

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

“…Currently, isomaltulose is produced commercially from sucrose using a bacterial strain that naturally expresses sucrose isomerase (SIase) [ 5 ]. SIase uses sucrose as a substrate and converts it mainly to isomaltulose, together with a small proportion of trehalulose, and trace byproducts, including glucose and fructose [ 5 , 6 ].…”