Synergistic engineering PETase reveals loop-region mutations for enhanced catalytic activity and thermal stability

Area of Science:

• Biotechnology
• Environmental Science
• Enzyme Engineering

Background:

• Plastic pollution, particularly from Polyethylene Terephthalate (PET), is a global crisis.
• Current PET biodegradation methods are limited by the efficiency of PET hydrolases.

Purpose of the Study:

• To enhance the catalytic activity and thermostability of Ideonella sakaiensis PETase (IsPETase) for improved PET biodegradation.
• To identify novel mutation sites for optimizing PET hydrolase performance.

Main Methods:

• Employed an integrated strategy of semi-rational design and directed evolution.
• Discovered and combined four new mutation sites (N114, N205, N233, S269) into a quadruple mutant (QM-PETase-2).
• Utilized molecular dynamics simulations to analyze structural changes and substrate binding.

Main Results:

• Developed QM-PETase-2 with a 4.9-fold increase in catalytic efficiency and a +12.4 °C increase in melting temperature (Tm).
• Identified that the beneficial mutations are located in the enzyme's loop region, enhancing structural stability.
• Demonstrated that these mutations improve performance when introduced into other high-performance PETase variants (FAST-PETase, PA-PETase, DepoPETase).

Conclusions:

• The newly discovered mutations are effective and universal for improving PETase performance.
• Engineered PETases show enhanced activity and stability, advancing plastic biodegradation technology.
• This research provides a foundation for developing more efficient enzymes to combat PET plastic pollution.