Poly(hydroxyalkanoate) Generation from Nonchiral Substrates Using Multiple Enzyme Immobilizations on Peptide...

Area of Science:

• Biotechnology
• Biocatalysis
• Enzyme Engineering

Background:

• Enzyme immobilization is crucial for biocatalysis, enabling cofactor recycling and cost-effective production.
• Current methods often struggle with preserving enzyme activity and broad applicability.

Purpose of the Study:

• To develop a rapid, two-step enzyme immobilization technique preserving activity.
• To enable cost-effective production of chiral products with cofactor recycling.
• To demonstrate the versatility of the method across different enzyme classes.

Main Methods:

• Enzyme immobilization via affinity tag-assisted binding to self-assembled β-sheet peptide scaffolds.
• Utilized (R)-specific poly(hydroxyalkanoate) synthase, (S)-specific dehydrogenase, and (R)-specific hydratase.
• Confirmed immobilization using fluorescence microscopy and enzyme activity via spectroscopic assays.

Main Results:

• Successfully immobilized multiple enzymes, including PhaCRe, FadB, and PhaJ4Pa.
• Demonstrated cofactor recycling (coenzyme A) for poly(3-hydroxybutyrate) production from β-butyrolactone.
• Preserved enzyme activity throughout the immobilization process under nondenaturing conditions.

Conclusions:

• The developed method offers a simple, cost-effective approach for immobilizing diverse enzymes.
• Facilitates the efficient production of chiral compounds with reusable cofactors.
• Has significant potential for both research and industrial biocatalytic applications.