A mutant alpha-amylase with enhanced activity specific for short substrates

Area of Science:

• Enzymology
• Protein Engineering
• Biochemistry

Background:

• Saccharomycopsis fibuligera alpha-amylase is a key enzyme in carbohydrate metabolism.
• The active site of alpha-amylase contains critical residues that influence substrate binding and catalytic activity.
• Lysine 210 (K210) is located at the active site and its role in substrate interaction requires further elucidation.

Purpose of the Study:

• To investigate the role of the 210th lysine (K210) residue in the active site of Saccharomycopsis fibuligera alpha-amylase.
• To determine how mutations at K210 affect substrate binding affinities and catalytic activity.
• To understand the contribution of K210 to the enzyme's subsites.

Main Methods:

• Site-directed mutagenesis was employed to alter K210 to arginine (R) and asparagine (N).
• The affinities of the 7th and 8th subsites were assessed for the mutant enzymes.
• Catalytic activity, specifically for maltose (G2) hydrolysis, was measured and compared to the native enzyme.

Main Results:

• Replacement of K210 with R strengthened the 7th subsite affinity and weakened the 8th subsite affinity.
• The K210R mutant exhibited a three-fold increase in catalytic activity for maltose hydrolysis, attributed to enhanced 7th subsite affinity.
• The K210N mutant showed a significant decrease in catalytic activity (to 1% of native) due to reduced 7th subsite affinity.

Conclusions:

• The K210 residue plays a crucial role in both the 7th and 8th subsites of Saccharomycopsis fibuligera alpha-amylase.
• Modifying K210 can significantly alter substrate binding and catalytic efficiency.
• Targeted mutations at K210 offer a strategy for engineering alpha-amylase activity for specific applications.