Molecular Engineering L-Aspartate-Alpha-Decarboxylase to Enhance Catalytic Stability and Performance.

Area of Science:

• Biochemistry
• Protein Engineering
• Enzymology

Background:

• L-aspartate-alpha-decarboxylase (ADC) is crucial for beta-alanine biosynthesis.
• Current ADC variants exhibit limited catalytic stability and efficiency, hindering industrial use.

Purpose of the Study:

• To engineer Bacillus subtilis ADC for improved catalytic efficiency and stability.
• To identify key amino acid substitutions enhancing ADC performance.

Main Methods:

• Random mutagenesis and high-throughput screening.
• Saturation mutagenesis and computational analysis.
• Molecular dynamics (MD) simulations and hydrophilicity analysis.

Main Results:

• Identified four key substitutions (S7N, K63N, A99T, K113R) improving ADC.
• Developed two recombined variants (N3 and Y1) with up to 95% increased catalytic efficiency and 89% enhanced stability.
• Variant Y1 showed a 3.37-fold longer half-life and a 2-fold higher total turnover number.

Conclusions:

• Protein engineering strategies successfully improved ADC's industrial applicability.
• Enhanced hydrophilicity and steric hindrance in variants contribute to increased activity and stability.
• Optimized ADC variants show potential for efficient beta-alanine production.