[Directed evolution of aflatoxin detoxifzyme in vitro by error-prone PCR]

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Evolution

Context:

• Aflatoxins are toxic secondary metabolites produced by fungi, posing significant risks to food safety and human health.
• Detoxifymes are crucial for mitigating aflatoxin contamination, but their efficiency and stability often require improvement.
• Directed evolution offers a powerful strategy for protein engineering to enhance enzyme properties.

Purpose:

• To improve the activity and stability of aflatoxin detoxifzyme using directed evolution.
• To identify optimal mutant strains with enhanced enzymatic properties through high-throughput screening.
• To understand the structure-function relationship of aflatoxin detoxifzyme.

Summary:

• Utilized error-prone PCR and a horse radish peroxidase and recessive brilliant green (HRP-RBG) screening system to generate a mutant library of approximately 10^4 aflatoxin detoxifzyme variants.
• Two rounds of error-prone PCR and HRP-RBG screening yielded three optimal mutant strains: A1773, A1476, and A2863, exhibiting significantly enhanced enzyme activity and stability.
• Mutant A1773 demonstrated a 6.5-fold increase in activity and 70°C temperature tolerance. Mutant A1476 showed a 21-fold activity increase at pH 4.0. Mutant A2863 exhibited a 12.6-fold activity increase at both pH 4.0 and 7.5.

Impact:

• The study successfully engineered aflatoxin detoxifzyme variants with superior performance characteristics, including enhanced thermal and pH stability and significantly higher catalytic activity.
• Identified specific amino acid substitutions (e.g., Glu127Lys, Gln613Arg in A1773) responsible for the improved enzyme functions, providing insights into protein structure-activity relationships.
• The developed mutants and findings offer valuable tools and knowledge for combating aflatoxin contamination in agriculture and food industries.