A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis

Area of Science:

• Enzymology
• Biotechnology
• Microbial biochemistry

Background:

• Cyclomaltodextrinases are enzymes with diverse hydrolytic and transglycosylation activities.
• These enzymes are crucial in carbohydrate metabolism and have industrial applications.

Purpose of the Study:

• To clone, overexpress, and characterize a cyclomaltodextrinase from Geobacillus thermopakistaniensis.
• To investigate the enzyme's substrate specificity, optimal conditions, and stability.

Main Methods:

• Gene cloning and overexpression in Escherichia coli.
• Protein purification and biochemical characterization.
• Enzyme activity assays with various substrates (cyclodextrins, pullulan, maltotriose, acarbose).

Main Results:

• The recombinant enzyme, CDaseGt, showed optimal activity at 55°C and pH 6.0.
• CDaseGt exhibited the highest specific activity against α-cyclodextrin (1200 μmol min⁻¹ mg⁻¹), which is notably higher than previously reported enzymes.
• The enzyme demonstrated significant activity towards pullulan (105 μmol min⁻¹ mg⁻¹) and was thermostable up to 65°C.
• CDaseGt also showed hydrolysis and transglycosylation activities on maltotriose and acarbose.
• Enzyme activity was enhanced by Mn²⁺, and EDTA improved thermostability.
• The enzyme exists in active dimeric and less active monomeric forms, with equilibrium dependent on concentration.

Conclusions:

• CDaseGt is a highly active and thermostable cyclomaltodextrinase with potential for biotechnological applications.
• Its superior activity against α-cyclodextrin and stability make it a promising candidate for industrial processes involving carbohydrate modification.