Genetically-modified R-ω-transaminase: purification and self-assembly facilitating interaction with substrate...

Area of Science:

• Biocatalysis
• Protein Engineering
• Enzyme Immobilization

Background:

• Enzyme engineering is crucial for developing efficient biocatalysts.
• Elastin-like polypeptides (ELPs) offer unique properties for enzyme fusion and purification.
• D-amino acid oxidase and R-omega-transaminase are key enzymes in chiral amine synthesis.

Purpose of the Study:

• To develop an easy-to-operate method for using R-omega-transaminase.
• To enhance the catalytic efficiency of R-omega-transaminase through fusion and complex formation.
• To create a self-assembling two-enzyme system for improved biocatalysis.

Main Methods:

• Genetic engineering to fuse R-omega-transaminase (R-ω-TA) with elastin-like polypeptide (ELP).
• Purification of the fusion enzyme using reversible phase transition.
• Formation of self-assembled enzyme clusters and substrate droplets for a single-enzyme system.
• Assembly of two-enzyme complexes (ELP-R-ω-TA and ELP-D-amino acid oxidase) for a two-enzyme system.

Main Results:

• ELP-R-ω-TA self-assembled into micrometer-sized enzyme clusters.
• Substrate (R)-1-phenylethylamine formed micrometer-sized droplets, creating a high substrate concentration microenvironment.
• Two-enzyme complexes formed significantly larger clusters, leading to a 99.6% efficiency in product formation.

Conclusions:

• Two-enzyme complexes substantially improve catalytic efficiency.
• The self-assembling nature of these complexes facilitates enzyme immobilization.
• Non-covalent methods, such as entrapment in porous supports, can be used for enzyme immobilization.