Turanose (α-d-glucopyranosyl-(1→3)-α-d-fructose) is a rare disaccharide that is a potential low-calorigenic sweetener. This novel sucrose isomer has been efficiently synthesized by the amylosucrase from Bifidobacterium thermophilum (BtAS). In this study, we aimed to enhance turanose biosynthesis by designing a BtAS variant (BtAS-G374S) with improved thermal stability. The BtAS variant was immobilized on porosity-controlled silica carrier, and its enzymatic properties were thoroughly investigated. Using response surface methodology with central composite design, optimal immobilization conditions were determined to significantly boost the biosynthetic efficiency. The BtAS-G374S showed 1.6-fold higher specific activity (2.2 U/mg) than the wild-type enzyme (1.4 U/mg). Additionally, the turanose production yield of BtAS-G374S was significantly enhanced, reaching 65 %, compared to 25 % for the wild-type enzyme when reacting with 2 M sucrose. Immobilization of BtAS-G374S was optimized on controlled porosity carrier (CPC) silica carrier using Response Surface Methodology, achieving an enzyme activity of 7.89 U and an immobilization efficiency of 68.98 % under optimal conditions. Immobilization of BtAS-G374S on CPC silica carriers enhanced its pH and thermal stability. The immobilized enzyme showed a half-life of 50.23 h at 55 °C and retained 68 % of its initial biosynthetic yield after 10 reuses. These properties suggest its potential for efficient industrial turanose production.
Keywords: Amyloscurase; Immobilization; Turanose.
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