Presentation Information
[1ENZ-18]Improving an enzymatic PET degradation system using a heterotrimeric PCNA protein
○Mirian Abad Rodriguez1, Hidehiko Hirakwa1 (1. Tsukuba University (Japan))
Keywords:
Polyethylene terephthalate (PET),PET degradation,PET hydrolase,MHET hydrolase,PCNA
Polyethylene terephthalate (PET) is one of the most widely used polymers due to its superior physical properties and low cost, resulting in immense demand. Recently, microbial PET hydrolases (PETases) have gained attention for thedegradation of PET into terephthalic acid (TPA) and ethylene glycol (EG). The hydrolysis of PET also generates the soluble intermediates, mono (2-hydroxyethyl terephthalate) (MHET), and bis (2-hydroxyethyl terephthalate) (BHET). The accumulation of these intermediates is known to inhibit the reaction. Interestingly, the mesophilic bacteriaIdeonella sakaiensis possesses an accessory enzyme MHET hydrolase (MHETase), which catalyzes the degradation of MHET to TPA and EG, along with a highly active PETase (IsPETase). The melting temperature of the wild typeIsPETase (48.7ºC) is significantly lower than the glass transition temperature of PET (70ºC), driving the development of thermostable variants such as ThermoPETase (S121E, D186H, R280A; Tm: 58.6ºC) or FASTPETase (S121E, D186H, R224Q, N233K, R280A; Tm: 67.1ºC).
For polymer substrates, the simultaneous action of multiple catalytic sites is expected to enhance reactivity through cooperative binding and increased local enzyme concentration. In this study, to improve the enzymatic PET degradationwe assembled multiple molecules of the PET degradation-related enzymes a heterotrimeric protein scaffold.Metallosphaera sedula’s PCNA is a heterotrimeric DNA sliding clamp, composed of the distinct subunits, PCNA1, PCNA2 and PCNA3. By fusion to the subunits, multiple proteins can be co-localized on the PCNAring.
FASTPETase was genetically fused to the N-termini of M.sedula’s PCNA subunits PCNA1, PCNA2 and PCNA3 respectively through a flexible Gly-Ser linker. The equimolar mixture of PETase-PCNA1 (200 nM) and PETase-PCNA2 (200 nM) showed higher PET-degradation activity than free FASTPETase (400 nM). HPLC analysis showed peaks of the intermediate product MHET and the final product TPA in both cases after a 52-hour reaction at 50ºC. In the presence of free MHETase, even though the TPA yield increased, the MHET was not completely converted to TPA. However, in the case of an equimolar mixture of PETase-PCNA1 (200 nM) and MHETase-PCNA2 (200 nM) the MHET peak was almost indetectable. The equimolar mixture of PETase-PCNA1 (200 nM), PETase-PCNA2 (200 nM) and MHETase-PCNA3 (200nM) showed higher activity than the above mixture and the MHET peak was still almost indetectable. It can be suggested that the co-localization of PETase and MHETase has a positive effect in the TPA yield during PET degradation.
References:
- Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H. Y., Choi, S. Y., Lee, S. Y., & Kim, K. J. (2019). Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catalysis, 9 (4), 3519-3526.
- Lu, H., Diaz, D. J., Czarnecki, N. J., Zhu, C., Kim, W., Shroff, R., Acosta, D. J., Alexander, B. R., Cole, H. O., Zhang, Y., Lynd, N. A., Ellington, A. D., & Alper, H. S. (2022). Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 604(7907), 662–667.
For polymer substrates, the simultaneous action of multiple catalytic sites is expected to enhance reactivity through cooperative binding and increased local enzyme concentration. In this study, to improve the enzymatic PET degradationwe assembled multiple molecules of the PET degradation-related enzymes a heterotrimeric protein scaffold.Metallosphaera sedula’s PCNA is a heterotrimeric DNA sliding clamp, composed of the distinct subunits, PCNA1, PCNA2 and PCNA3. By fusion to the subunits, multiple proteins can be co-localized on the PCNAring.
FASTPETase was genetically fused to the N-termini of M.sedula’s PCNA subunits PCNA1, PCNA2 and PCNA3 respectively through a flexible Gly-Ser linker. The equimolar mixture of PETase-PCNA1 (200 nM) and PETase-PCNA2 (200 nM) showed higher PET-degradation activity than free FASTPETase (400 nM). HPLC analysis showed peaks of the intermediate product MHET and the final product TPA in both cases after a 52-hour reaction at 50ºC. In the presence of free MHETase, even though the TPA yield increased, the MHET was not completely converted to TPA. However, in the case of an equimolar mixture of PETase-PCNA1 (200 nM) and MHETase-PCNA2 (200 nM) the MHET peak was almost indetectable. The equimolar mixture of PETase-PCNA1 (200 nM), PETase-PCNA2 (200 nM) and MHETase-PCNA3 (200nM) showed higher activity than the above mixture and the MHET peak was still almost indetectable. It can be suggested that the co-localization of PETase and MHETase has a positive effect in the TPA yield during PET degradation.
References:
- Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H. Y., Choi, S. Y., Lee, S. Y., & Kim, K. J. (2019). Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catalysis, 9 (4), 3519-3526.
- Lu, H., Diaz, D. J., Czarnecki, N. J., Zhu, C., Kim, W., Shroff, R., Acosta, D. J., Alexander, B. R., Cole, H. O., Zhang, Y., Lynd, N. A., Ellington, A. D., & Alper, H. S. (2022). Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 604(7907), 662–667.
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