Exploring the pH dependence of an improved PETase

Area of Science:

• Biochemistry
• Enzymology
• Materials Science

Background:

• Enzymatic recycling of polyethylene terephthalate (PET) offers a sustainable solution to plastic pollution.
• PET hydrolases (PETases) are crucial for this process, but their depolymerization mechanisms remain unclear.
• The alkaline pH optimum of PETases is often attributed to the catalytic histidine's protonation state.

Purpose of the Study:

• To elucidate the mechanistic details of PET depolymerization by an optimized PETase, LCC^ICCG.
• To determine the pKa values and tautomeric states of histidines in LCC^ICCG, particularly the catalytic H242.
• To investigate the influence of temperature and substrate phase (soluble vs. solid) on enzyme activity.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to identify histidine tautomeric structures and determine pKa values.
• Enzyme activity assays using soluble and solid PET substrates.
• Temperature-dependent kinetic studies.

Main Results:

• Five out of six histidines in LCC^ICCG exhibit unusually low pKa values (< 4.0).
• The catalytic H242 displays a temperature-dependent pKa ranging from 4.9 at 30°C to 4.7 at 50°C.
• Enzyme activity with soluble substrates aligns with protonation/deprotonation curves, but a discrepancy emerges with solid plastic substrates.

Conclusions:

• The catalytic histidine's protonation state is a significant factor in PETase activity, but not the sole determinant, especially with solid substrates.
• Further mechanistic studies are needed, focusing on the enzyme's behavior at the liquid-solid interface for effective plastic degradation.
• This research provides critical insights into PETase function, advancing enzymatic plastic recycling strategies.