Immobilization of Cyclodextrin glycosyltransferase onto three dimensional- hydrophobic and two dimensional- hydrophilic supports: A comparative study

Cyclodextrin glycosyltransferase (CGTase) degrades starch into cyclodextrin via enzymatic activity. In this study, we immobilize CGTase from Thermoanaerobacter sp. on two supports, namely graphene nanoplatelets (GNP) consisting of short stacks of graphene nanoparticles and a calcium-based two-dimensional metal organic framework (Ca-TMA). The uptakes of CGTase on GNP and Ca-TMA reached 40 and 21 mg g-1 respectively, but immobilized CGTase on Ca-TMA showed a higher specific activity (38 U mg-1) than that on GNP (28 U mg-1). Analysis of secondary structures of CGTase, shows that immobilization reduces the proportion of beta-sheets in CGTase from 56% in the free to 49% and 51.3% for GNP and Ca-TMA respectively, alpha-helix from 38.5% to 18.1 and 37.5%, but led to increased beta-turns from 5.5 to 40% and 11.2% for GNP and Ca-TMA, respectively. Lower levels of conformational changes were observed over the more hydrophilic Ca-TMA compared to hydrophobic GNP, resulting in its better activity. Increased beta-turns were found to correlate with lower beta-CD production, while more beta-sheets and alpha-helix favored more beta-CD. Reusability studies revealed that GNP retains up to 74% of initial CGTase activity, while Ca-TMA dropped to 33% after eight consecutive uses. The results obtained in this work provide insight on the effect of support's surface properties on CGTase performance and can assist in developing robust CGTase-based biocatalysts for industrial application. The surface properties of a support could lead to various effect on the enzyme immobilized on it. We used two supports with different hydrophobicities for cyclodextrin glycosyltransferase immobilization and studied the effects on protein uptake, specific activity, secondary structures of the enzyme and reusability. The results provides insight into developing better biocatalysts for starch conversion.image