Purification and kinetic characterization of cyclodextrin glycosyltransferase from Alkalihalobacillus Trypoxylicola KRM.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9) is an enzyme with its unique capability to transform starch and related substrates into cyclodextrins (CDs). Cyclodextrins are widely used in various industries (food, pharmaceuticals, textiles, and cosmetics). The present study focuses on the purification method of native CGTase from Alkalihalobacillus trypoxylicola KRM and enzyme characterization. The CGTase was purified to homogeneity by ammonium sulfate precipitation followed by starch adsorption and gel filtration. The purified CGTase demonstrated a specific activity of 2733.46 U/mg, corresponding to a 33.2-fold purification. Optimal activity was found at pH 8.0 (Tris HCl buffer) and at 50 °C temperature. CGTase was strongly inhibited by Fe3 +, Zn2+, Pb2+ and Al3+, while some metal ions (Ca2+, Na+, K+ and Mg2+) exerted stimulating effect. A metal chelating agent had a strong inhibitory effect, suggesting that metal ions may contribute to enzyme activity or structural stability. The detergent Triton X-100 was found to be lethal to the CGTase. CGTase showed temperature-dependent loss of activity with increasing first-order deactivation rates and Arrhenius analysis indicated an activation energy of ∼78.7 kJ/mol. The Km (1.83 mg/ml), and Vmax (833.33 μmol/min/ml) values were measured using a Lineweaver-Burk plot. To the best of our knowledge, this is the first report of a CGTase purified from Alkalihalobacillus trypoxylicola KRM. The enzyme exhibited moderate thermal stability and favorable catalytic properties under alkaline conditions.