A highly active alpha amylase from Bacillus licheniformis: directed evolution, enzyme characterization and structural...

Area of Science:

• Enzyme Engineering
• Protein Engineering
• Biotechnology

Background:

• Bacillus licheniformis alpha-amylase (BLA) is crucial for industrial applications.
• Improving enzyme stability under acidic conditions is a key challenge.
• Direct evolution offers a powerful strategy for enzyme optimization.

Purpose of the Study:

• To enhance the stability and activity of Bacillus licheniformis alpha-amylase (BLA) under acidic conditions.
• To identify specific mutations responsible for improved enzyme performance.
• To elucidate the structural basis for enhanced enzyme function.

Main Methods:

• Employing direct evolution via error-prone polymerase chain reaction (PCR) for BLA mutagenesis.
• Screening mutant libraries for enhanced activity and stability at low pH.
• Characterizing the kinetic and stability properties of the selected mutant (H281I).
• Utilizing structure modeling to analyze the impact of mutations on enzyme structure-function relationships.

Main Results:

• A beneficial mutation, H281I, was identified in BLA, significantly increasing specific activity.
• The H281I mutant exhibited 62.6% higher specific activity at pH 4.5 and 27.5% higher at pH 6.5 compared to wild-type (WT) at 95°C.
• The H281I mutant showed a 1.7-fold and 1.4-fold increase in kcat/Km at pH 4.5 and 6.5, respectively.
• Structural analysis suggested the H281I mutation optimizes the local environment for substrate binding, leading to increased catalytic efficiency.

Conclusions:

• The H281I mutation enhances the catalytic efficiency and specific activity of BLA under acidic conditions.
• Direct evolution is an effective method for improving enzyme properties for industrial applications.
• Understanding mutation-induced structural changes provides insights into enzyme design principles.