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Cyclodextrin glucanotransferases (CGTases) 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 CGTases 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 CGTase mainly forming α‐ and β‐CD (CD6 and CD7) derived from Geobacillus stearothermophilus ET1/NO2 (GeoT), and (b) a CGTase with lower cyclization activity from the alkaliphilic Bacillus sp. G825‐6, which mainly synthesizes γ‐CD (CD8). The A1, B, A2, and CDE domains of the G825‐6 CGTase were replaced with corresponding GeoT CGTase 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 CD6 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 CGTase and it synthesized up to 3.3 g·L−1 CD9 to CD12, corresponding to a 1.8‐ and 2.3‐fold increase compared to GeoT and G825‐6 CGTase, respectively. In conclusion, GeoT‐A1/A2/CDE may be a candidate for the further development of CGTases specifically forming larger CD.

Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. Based on multiple protein alignments and protein structure information, a mutagenic megaprimer was designed to encode a partial randomization of eight amino acid residues within the loop region. The library obtained encoding amino acid sequences occurring in wild type CGTases in combination with a screening procedure yielded sequences displaying a changed CD product specificity. As a result, variants of the CGTase from the alkaliphilic Bacillus sp. G825-6 synthesizing mainly CD9 to CD12 could be obtained. When the mutagenesis experiment was performed with the CGTase G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created. In the presence of the amino acid residue R183, the synthesis of CD8 was suppressed and larger CD were obtained as the main products. The alterations not only affected the product specificity, but also influenced the thermal stability of some of the CGTase variants indicating the importance of the loop structure for the stability of the CGTase.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.