Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability

Abstract

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

“…There are now several examples of proteins that have been stabilized by the introduction of numerous mutations with cumulative small stabilizing effects (Table 1) [76,88–91]. However, a clear conclusion to be drawn from others is that very large stability differences in some cases are due to only one or a few point mutations [39,45,46,92–94]. Another method is to anchor the loops to the protein surface, either by non-covalent interactions or using a disulfide bridge.…”

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

“…This residue is also present in other CD8‐forming CGTases and could be responsible for the observed shift in CD product size specificity. The variant GeoT‐A1 maintained the thermostability of the parental enzyme, indicating that the A1 domain of the G825‐6 CGTase also confers thermostability properties .…”

Mutations at calcium binding site III in cyclodextrin glycosyltransferase improve β-cyclodextrin specificity

“…Most CGTases, like other ␣-amylases, have two or more calcium binding sites [15,19,20]. Studies have suggested that the calcium binding sites in CGTases contribute to their thermostability and product specificity [15,[21][22][23]. The enzyme used in our studies, the ␤-CGTase from Bacillus circulans STB01, possesses three calcium binding sites called CaI, CaII and CaIII [15].…”