Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability

Cyclodextrin glucanotransferases ( CGT ases) convert α‐1,4‐glucans to cyclic oligosaccharides (cyclodextrins, CD ), which have found applications in the food and the pharmaceutical industries. In this study, we used two CGT ases with different cyclization activities, product specificities, and pH and temperature optima to construct chimeric variants for the synthesis of large‐ring CD . We used (a) a synthetic thermostable CGT ase mainly forming α‐ and β‐ CD ( CD 6 and CD 7) derived from Geobacillus stearothermophilus ET 1/ NO 2 (GeoT), and (b) a CGT ase with lower cyclization activity from the alkaliphilic Bacillus sp. G825‐6, which mainly synthesizes γ‐ CD ( CD 8). The A1, B, A2, and CDE domains of the G825‐6 CGT ase were replaced with corresponding GeoT CGT ase domains by utilizing a megaprimer cloning approach. A comparison of the optimum temperature and pH , thermal stability, and CD products synthesized by the variants revealed that the B domain had a major impact on the cyclization activity, thermal stability, and product specificity of the constructed chimera. Complete suppression of the synthesis of CD 6 was observed with the variants GeoT‐A1/B and GeoT‐A1/A2/ CDE . The variant GeoT‐A1/A2/ CDE showed the desired enzyme properties for large‐ring CD synthesis. Its melting temperature was 9 °C higher compared to the G825‐6 CGT ase and it synthesized up to 3.3 g·L −1 CD 9 to CD 12, corresponding to a 1.8‐ and 2.3‐fold increase compared to GeoT and G825‐6 CGT ase, respectively. In conclusion, GeoT‐A1/A2/ CDE may be a candidate for the further development of CGT ases specifically forming larger CD.