Counter‐intuitive enhancement of degradation of polyethylene terephthalate through engineering of lowered enzyme binding to solid plastic

Abstract

Degradation of solid polyethylene terephthalate (PET) by leaf branch compost cutinase (LCC) produces various PET‐derived degradation intermediates (DIs), in addition to terephthalic acid (TPA), which is the recyclable terminal product of all PET degradation. Although DIs can also be converted into TPA, in solution, by LCC, the TPA that is obtained through enzymatic degradation of PET, in practice, is always contaminated by DIs. Here, we demonstrate that the primary reason for non‐degradation of DIs into TPA in solution is the efficient binding of LCC onto the surface of solid PET. Although such binding enhances the degradation of solid PET, it depletes the surrounding solution of enzyme that could otherwise have converted DIs into TPA. To retain a subpopulation of enzyme in solution that would mainly degrade DIs, we introduced mutations to reduce the hydrophobicity of areas surrounding LCC's active site, with the express intention of reducing LCC's binding to solid PET. Despite the consequent reduction in invasion and degradation of solid PET, overall levels of production of TPA were ~3.6‐fold higher, due to the partitioning of enzyme between solid PET and the surrounding solution, and the consequent heightened production of TPA from DIs. Further, synergy between such mutated LCC (F125L/F243I LCC) and wild‐type LCC resulted in even higher yields, and TPA of nearly ~100% purity.

Efficient polyethylene terephthalate biodegradation by an engineered Ideonella sakaiensis PETase with a fixed substrate-binding W156 residue

“…We speculated that the strong adsorption of enzyme onto the PET surface was the main reason for the cessation of production increase, which hindered its accessibility to adjacent ester bonds. 41–43 The addition of fresh PA variant after 48 h led to a further increase in soluble monomer production, whose accumulation versus time trends were identical to those observed at the beginning of the reaction. The soluble monomer production after 96 h was nearly two times higher than that after 48 h, suggesting that no obvious product inhibition took place.…”

“…S6 †). 42 The atomic force microscopy (AFM) results depicted the surface roughness of PET gradually increasing as the enzymatic hydrolysis process took place (Fig. 2c).…”

Whether the wobbling W156 is a pre-requisite for efficient PET biodegradation by IsPETase

“…19). We speculate the strong adsorption of enzyme onto the PET surface was the main reason for the cessation of production increase, which hindered its accessibility to adjacent ester bonds [42][43][44] . Moreover, the release of the additive in the colored pcPET may harm the enzyme activity.…”

“…The results of HPLC showed the presence of a significant peak after BHET, which may correspond to oligo ethylene terephthalate that was unidentified and unquantified (Supplementary Fig. 20) 43 . With the large amounts of TPA released, its acidification effect on the reaction buffer was significant, causing the pH of the reaction solution to drop continuously 34 .…”

From Bulk to Binding: Decoding the Entry of PET into Hydrolase Binding Pockets

“…[25][26][27] In addition, a reduction in the hydrophobicity of LCC in proximal areas has been demonstrated to reduce the binding to a PET surface, resulting in increased enzyme activity due to increased conversion of degradation intermediates and consequently elevated TPA production. 60…”