Calcium Ion Contribution to Thermostability of Cyclodextrin Glycosyltransferase Is Closely Related to Calcium-Binding Site CaIII

Abstract

In the study, we investigated the contribution of Ca²⁺ to the thermostability of α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans , which has two calcium-binding sites (CaI and CaII), and β-CGTase from Bacillus circulans , which contains an additional calcium-binding site (CaIII), consisting of Ala315 and Asp577. It was found that the contribution of Ca²⁺ to the thermostability of two CGTases displayed a marked difference. Ca²⁺ affected β-CGTase thermostability significantly. After Ca²⁺ was added to β-CGTase solution to a final concentration of 5 mM followed by incubation for 120 min at 60 °C, residual activity of β-CGTase was 88.3%, which was much higher than that without Ca²⁺. However, Ca²⁺ had a small contribution to α-CGTase thermostability. Furthermore, A315D and D577K mutations at CaIII could significantly change the contribution of Ca²⁺ to β-CGTase thermostability. These results suggested that the contribution of Ca²⁺ to CGTase thermostability was closely related to CaIII.

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].…”

“…The cgt gene (GenBank accession number: KJ660983) encoding the wild-type ␤-CGTase from B. circulans STB01 was previously used to construct plasmid cgt/pST [15]. This plasmid was used for site-directed mutagenesis, sequencing, and expression of CGTase proteins.…”

“…A major disadvantage of CGTases for the industrial production of cyclodextrins is that the reaction produces a mixture of ␣-, ␤-and ␥-cyclodextrins [15,16]. Two types of cyclodextrin production process can be used.…”

“…Therefore, mutants with improved cyclodextrin product specificity are of high industrial interest, which could reduce the use of organic solvents for selective complexation of the cyclodextrin of interest, lowering the costs of cyclodextrin production [8,17,18]. 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].…”

“…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]. Our earlier work demonstrated that mutations at Ala31, which is near CaI, enhanced the ␤-cyclodextrin specificity of this CGTase, which may be a result of stabilizing CaI [22] in stabilizing CaIII, we speculated that the identity of the amino acid residue at 315 at CaIII might also be related to the product specificity of this ␤-CGTase.…”