- Addressing the global plastic waste crisis, particularly hard-to-recycle blended PET fibers, demands environmentally friendlier recycling methods.
- Researchers engineered a novel PET hydrolase PET2-21M and established large-scale production in yeast. This enzyme dramatically boosted PET bottle-grade PET breakdown.
- In parallel, its direct precursor PET2-14M-6Hot successfully degraded challenging blended fibers (PET/cotton, PET/PU) at moderate temperatures.
- This breakthrough offers a promising, energy-efficient path for a circular plastics economy, accelerating industrial-scale recycling of diverse polymer wastes.
A research team led by Professor Akihiko Nakamura of the Research Institute of Green Science and Technology, Shizuoka University (also a cross-appointment professor at the Institute for Molecular Science until March 2025), in collaboration with Researchers Takashi Matsuzaki and Toshiyuki Saeki of Kirin Holdings Co., Ltd., Professor Ryota Iino of the Institute for Molecular Science, and Professor Nobuyasu Koga of the Institute for Protein Research, The University of Osaka, have successfully engineered a novel PET hydrolase enzyme, PET2-21M, achieving a remarkable improvement in the biodegradation of bottle-grade polyethylene terephthalate (PET) plastics. High activity toward PET/cotton and PET/polyurethane (PU) textile blends was also demonstrated separately with the closely related variant PET2-14M-6Hot. This significant breakthrough addresses the urgent global challenge of recycling PET waste by offering a sustainable and efficient alternative to conventional recycling processes.
PET is a widely utilized synthetic polymer prominent in bottles, textiles, and packaging materials, representing approximately 83% of the synthetic fiber market. Despite its intrinsic recyclability, traditional mechanical recycling methods frequently result in material quality degradation and exhibit limited effectiveness for complex blended materials such as PET/cotton and PET/PU. Chemical recycling, while capable of producing high-purity materials, typically demands harsh conditions and environmentally hazardous reagents, thus limiting its practical sustainability.
In response, enzymatic recycling has emerged as an attractive alternative due to its capability to depolymerize PET into its original monomeric constituents under milder aqueous conditions. To enhance the PET-degrading efficiency of the enzyme PET2, researchers adopted an extensive engineering strategy. They systematically employed both random and targeted mutagenesis, combining seven newly identified beneficial mutations with a previously-reported engineered variant PET2-7M, resulting in the highly active PET2-14M enzyme. Additional surface modifications, which introduced positive charges to improve substrate binding, and strategic alterations in the substrate-binding cleft based on another enzyme HotPETase as a structural template, led to the creation of PET2-14M-6Hot. Further optimization produced the final engineered variant PET2-21M. Furthermore, large-scale productions of the PET2-14M-6Hot and PET2-21M were achieved in the yeast host, Komagataella phaffii. Notably, PET2-14M-6Hot reached yields of up to 691 mg L⁻¹ after 137 hours of cultivation, demonstrating high expression efficiency without glycosylation-induced heterogeneity.
The PET2-21M demonstrated significantly enhanced catalytic activity compared to the original enzyme wild-type PET2, with initial small-scale assays revealing a total product yield approximately 28.6 times greater. Subsequent scaled-up experiments in 300 mL reactors further validated these improvements; notably, PET2-21M depolymerized approximately 95% of commercial bottle-grade PET powder (20 g L⁻¹) within 24 hours at 60 °C, while the benchmark enzyme LCC-ICCG required its optimal temperature of 72 °C to reach a comparable conversion of 91%.
The superiority of PET2-21M was particularly evident under reduced enzyme loading conditions. Even when enzyme concentration was halved to 2.5 mg L⁻¹, PET2-21M maintained around 50% degradation efficiency, nearly doubling the performance of LCC-ICCG, which achieved only 26% conversion under identical conditions. This highlights PET2-21M's substantial potential to lower catalytic requirements and associated costs.
Importantly, PET2-21M retained its competitive advantage under higher substrate loading conditions (40 g L⁻¹). At an enzyme dosage of 10 mg L⁻¹, PET2-21M achieved a 79% conversion at 60 °C, closely rivaling LCC-ICCG's 95% conversion at its higher optimal temperature (72 °C). Furthermore, upon reducing enzyme dosage to 5 mg L⁻¹, PET2-21M still outperformed LCC-ICCG, demonstrating a 44% conversion compared to 29% for LCC-ICCG. This robust performance at moderate temperatures and reduced enzyme-to-substrate ratios positions PET2-21M as a highly promising candidate for industrial PET recycling processes, potentially enabling substantial reductions in both energy consumption and catalyst expenditure.
To evaluate the recycling potential of engineered PET hydrolases for textile waste, the PET2-14M-6Hot was compared with the benchmark enzyme LCC-ICCG on pure PET fibers and textile blends. At 60 °C, PET2-14M-6Hot generated 75.7 mM total degradation products from pure PET fibers within 24 hours, representing a 1.4-fold improvement over LCC-ICCG tested at its optimal 70 °C. Similarly, PET2-14M-6Hot achieved higher catalytic efficiency on PET/cotton (65/35 wt%) blends, producing 62.8 mM products versus 46.7 mM by LCC-ICCG, with minimal interference from cotton fibers.
For the challenging PET/PU textile blends (85/15 wt%), both enzymes exhibited reduced activity above PU's glass-transition temperature (Tg ≈ 55 °C). Nevertheless, at a lower reaction temperature of 50 °C, PET2-14M-6Hot maintained substantial catalytic activity, yielding 19.2 mM degradation products—more than double the 8.2 mM obtained by LCC-ICCG under identical conditions. This underscores PET2-14M-6Hot's superior capacity for processing complex blended textiles, which have traditionally resisted enzymatic degradation.
These results confirm the engineered PET2 enzyme family's significant potential for industrial-scale enzymatic recycling. Their ability to efficiently degrade diverse PET waste streams, including challenging textile blends at moderate temperatures, strongly supports broader applicability and sustainability benefits in PET recycling processes.
These findings represent a substantial advance towards realizing a more sustainable and economically viable circular plastics economy. The engineered PET2 enzymes' superior ability to depolymerize PET and complex fiber blends at moderate temperatures holds significant promise for practical industrial recycling operations, particularly in handling difficult-to-process blended textile waste. Future research efforts target further optimization of enzyme efficiency at even lower reaction temperatures and in the blended materials, ultimately facilitating broader industrial adoption and minimizing the environmental footprint of global plastic recycling efforts.
Information of the paper
Authors: Takashi Matsuzaki, Toshiyuki Saeki, Fuhito Yamazaki, Natsuka Koyama, Tatsunori Okubo, Daiki Hombe, Yui Ogura, Yoshihito Hashino, Rie Tatsumi-Koga, Nobuyasu Koga, Ryota Iino, Akihiko Nakamura
Journal Name: ACS Sustainable Chemistry & Engineering
Journal Title: "Development and Production of Moderate-Thermophilic PET Hydrolase for PET Bottle and Fiber Recycling"
DOI: 10.1021/acssuschemeng.5c01602
