A novel and thermostable cyclomaltodextrin glucanotransferase from Anoxybacillus rupiensis PSO2 isolated from...

Cyclomaltodextrin glucanotransferase (CGTase) converts starch into cyclic oligosaccharides (cyclodextrins) with broad industrial relevance in starch-saccharification and bioconversion processes. Here, we report cloning, expression, biochemical characterisation and in silico structural analysis of a novel, thermostable CGTase (CgtO₂) from Anoxybacillus rupiensis PSO2, isolated from Yumthang hot spring, Sikkim, India. The CgtO₂ gene was cloned and overexpressed in Escherichia coli BL21 (DE3), and the recombinant protein was purified to homogeneity via a single step using Ni^2 + -NTA chromatography, yielding a specific activity of 618.51 mg^-1. The enzyme exhibited a temperature and pH optima of 70 ℃ and 8.0, respectively, and a remarkable thermostability at 100 ℃, retaining over 50% residual activity even after 210 min of incubation. Kinetic studies revealed a high substrate affinity for branched amylopectin (V_max: 214 ± 2.18 μmol min^-1 mg^-1, K_m: 0.823 ± 0.053 mg mL^-1) and raw potato peels (V_max: 198.1 ± 4.41 μmol min^-1 mg^-1, K_m: 1.331 ± 0.087 mg mL^-1), underscoring its effective catalytic performance on both simple and complex natural starch matrices. CgtO₂ exhibited cyclization activity of 237.9 ± 0.923 mL^-1 on raw potato peel as substrate. Scanning Electron Microscopy displayed extensive degradation of raw potato peels, and HPLC analysis detected the presence of α- and β-cyclodextrins. These findings establish CgtO₂ as a catalytically efficient and robust CGTase, making it a promising candidate for cost-effective and sustainable biorefinery applications.