Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Area of Science:

• Enzymology
• Protein Engineering
• Biocatalysis

Background:

• Cyclodextrin glycosyltransferase (CGTase) enzymes produce cyclodextrins from starch.
• Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase) shares structural similarity with CGTases but produces maltose from starch.
• Key structural differences in the substrate-binding clefts distinguish these enzymes.

Purpose of the Study:

• To engineer CGTase to exhibit the reaction and product specificity of G2-amylase.
• To investigate the functional impact of specific structural modifications on CGTase activity and specificity.

Main Methods:

• Comparative structural analysis of CGTase and G2-amylase to identify key differences.
• Site-directed mutagenesis to introduce G2-amylase-like structural features into Thermoanerobacterium thermosulfurigenes CGTase.
• Biochemical assays to evaluate the cyclization, hydrolysis, and product specificity of the engineered CGTase mutants.

Main Results:

• Exchanging the loop at acceptor subsite +3 reduced cyclization activity but minimally affected product specificity.
• Introducing a five-residue insertion at donor subsites -3/-4 significantly decreased cyclization activity, favoring hydrolysis.
• The modified CGTase produced linear starch products, primarily maltose, and exhibited increased exo-specificity.

Conclusions:

• Specific structural modifications can convert CGTase into a starch hydrolase.
• Altering substrate binding at donor subsites -3/-4 is a viable strategy to enhance CGTase exo-specificity.
• This engineered enzyme has potential applications in the food industry, such as in baking.