Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot

Area of Science:

• Enzymology
• Protein Engineering
• Biocatalysis

Background:

• Cal-A lipase's triglyceride binding mode is poorly understood.
• Engineering enzyme selectivity requires a systematic approach.

Purpose of the Study:

• To engineer Cal-A lipase for altered triglyceride selectivity.
• To identify key regions and mutations responsible for substrate discrimination.

Main Methods:

• Region-focused saturation mutagenesis and randomization of Cal-A.
• Recombination of targeted libraries using Golden Gate assembly.
• High-throughput screening for triglyceride hydrolytic activity.
• Analysis of variants to identify key substitutions.

Main Results:

• Identified Cal-A variants with distinct selectivity for short-chain or long-chain triglycerides.
• The 217-245 helix-loop-helix motif was identified as a critical region for triglyceride recognition.
• Mutational load was localized to specific regions without affecting distal protein areas.

Conclusions:

• The study enhances understanding of Cal-A lipase's mechanism and substrate selectivity.
• A holistic protein engineering strategy focusing on specific regions is effective.
• This approach is broadly applicable to engineering other protein scaffolds.