Designing Novel Aspartic Protease (NAP) Using Ancestral Sequence Reconstruction (ASR) for Cheesemaking

Area of Science:

• Enzymology and protein engineering
• Computational biology and bioinformatics
• Food science and technology

Background:

• Rhizomucor miehei protease (RMP) is a widely used milk coagulant enzyme (MCE) in cheese production.
• Current MCEs like RMP have limitations including non-specific proteolytic activity and high thermostability, negatively impacting cheese quality and yield.
• There is a need for improved MCEs with enhanced specificity and controlled stability for dairy applications.

Purpose of the Study:

• To computationally design a novel aspartic protease (NAP) with improved κ-casein affinity and reduced thermal stability.
• To address the limitations of existing milk coagulant enzymes through enzyme engineering.
• To develop next-generation coagulants for industrial dairy applications using ancestral sequence reconstruction (ASR).

Main Methods:

• Computational enzyme engineering using ancestral sequence reconstruction (ASR) to design NAP.
• Introduction of targeted amino acid modifications in catalytic and flap regions of the enzyme.
• Molecular docking, molecular dynamics (MD) simulations, and structural analyses to compare NAP with wild-type RMP.
• Binding affinity calculations (MM-GBSA) and structural metric analyses (SASA, Rg, RMSD, RMSF, FEL, PCA).

Main Results:

• The designed NAP exhibited enhanced substrate interaction and binding affinity compared to wild-type RMP.
• NAP demonstrated reduced thermal stability and increased flexibility, suggesting improved performance under milder conditions.
• New hydrogen bonds and salt bridges were formed in the NAP-κ-casein complex, indicating altered interaction dynamics.
• Computational analyses predicted improved specificity and reduced off-target proteolysis for NAP.

Conclusions:

• The novel aspartic protease (NAP) designed through ASR shows significant promise as a superior milk coagulant.
• NAP's enhanced specificity and controlled thermal stability offer potential solutions to current limitations in cheese production.
• Computational enzyme design, particularly ASR, is a powerful approach for developing tailored enzymes for industrial applications.
• Further experimental validation is required to confirm the efficacy of NAP in real-world dairy processes.