Switched reaction specificity in polyesterases towards amide bond hydrolysis by enzyme engineering

Area of Science:

• Biotechnology
• Polymer Science
• Enzyme Engineering

Background:

• Polyamides like nylon are environmentally persistent and difficult to recycle due to their chemical stability.
• Existing amidases exhibit limited activity on polyamides, hindering biotechnological degradation approaches.

Purpose of the Study:

• To engineer polyesterases for enhanced amidase activity, enabling the hydrolysis of synthetic amide bonds in polymers.
• To investigate the role of enzyme design, specifically water network reallocation, in improving catalytic efficiency for polyamide degradation.

Main Methods:

• Enzyme engineering of *Humicola insolens* cutinase and *Thermobifida cellulosilytica* cutinase 1 to create variants with modified water networks.
• Assessing catalytic efficiency on soluble amide-containing substrates and the insoluble substrate 3PA 6,6.
• Utilizing molecular dynamics (MD) simulations to analyze transition state stabilization and water-molecule interactions.

Main Results:

• Designed enzyme variants showed increased catalytic efficiency towards amide-containing substrates.
• Enhanced hydrolysis of the insoluble polyamide substrate 3PA 6,6 was observed, correlating with improved transition state stabilization.
• A specific water-molecule network in the variants led to reduced activity on polyethylene terephthalate (PET).

Conclusions:

• Enzyme engineering can successfully enhance the amidase activity of polyesterases for synthetic polymers.
• The developed strategy of reallocating water networks offers a novel approach for designing biocatalysts for recalcitrant polymer degradation.
• This research opens avenues for biotechnological solutions to plastic pollution by enabling the breakdown of polyamides.