Part:BBa_K5023002:Design

Enzymes that degrade PET

In the intricate world of molecular biology, enzymes play a pivotal role, acting as biocatalysts that drive countless biochemical reactions. Their specificity, efficiency, and adaptability make them invaluable tools in both natural biological processes and biotechnological applications. Among the vast array of enzymes, wild PETase, Fast-PETase, MHETase and PHL7 have garnered attention in our design for their properties and applications.

As we delve into the comparative analysis of these enzymes, the overarching goal is to understand their potential in biodegrading plastics and aiding the bioremediation of water bodies. By harnessing the power of these enzymes, we stand a chance to reverse the damage caused by microplastics and restore the health of our aquatic ecosystems. Below are 4 enzymes that we analyzed for use in the project:

PETase

PETase is an enzyme identified in the bacterium Ideonella sakaiensis 201-F6. This enzyme is capable of hydrolyzing poly(ethylene terephthalate) (PET), a common plastic material that is resistant to microbial degradation. PETase exhibits a distinct preference for PET over other substrates. It efficiently hydrolyzes PET, producing mono(2-hydroxyethyl) terephthalic acid (MHET) as the major product. PETase has prominent hydrolytic activity for PET, especially in its glassy state, which is critical for the growth of I. sakaiensis on PET in various environments. PETase also exhibits higher activity against commercial bottle-derived PET, which is highly crystallized. I. sakaiensis adheres to PET and secretes PETase to target this material. The exact binding mechanism of PETase is not detailed, but it's noted that without a three-dimensional structure determined for PETase, the exact binding mechanism remains unknown. The discovery of PETase offers a potential biological solution for PET degradation. The enzyme's ability to break down PET into its constituent monomers can pave the way for environmentally friendly remediation strategies.

Fast-PETase

FAST-PETase is a mutant variant derived from the PETase enzyme. It contains five mutations compared to the wild-type PETase. These mutations include N233K, R224Q, and S121E from prediction, along with D186H and R280A from the scaffold. This enzyme demonstrated superior PET-hydrolytic activity relative to both wild-type and other engineered alternatives between 30 and 50°C across a range of pH levels. At 50°C, FAST-PETase showed the highest overall degradation of all mutants tested, releasing 33.8 mM of PET monomers (the sum of terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalate (MHET)). The enzyme's activity against post-consumer PET (pc-PET) was substantially higher than that of other enzymes like WT PETase, ThermoPETase, DuraPETase, LCC, and ICCM under the same conditions. FAST-PETase was able to almost completely degrade untreated post-consumer PET from 51 different thermoformed products in just one week. The enzyme also demonstrated the capability to depolymerize untreated, amorphous portions of a commercial water bottle. A time-course analysis revealed an almost linear decay rate of PET degradation and a concomitant increase in crystallinity over time. The development of FAST-PETase offers a potential solution for the degradation of PET plastics, especially given its enhanced activity and stability across a range of conditions. This enzyme can play a pivotal role in addressing the environmental challenges posed by PET plastic accumulation.

MHETase

MHETase is an enzyme identified in the bacterium Ideonella sakaiensis. It is responsible for the hydrolysis of MHET (mono(2-hydroxyethyl) terephthalic acid). This enzyme hydrolyzes MHET to produce TPA (terephthalic acid) and EG (ethylene glycol). The gene encoding MHETase in I. sakaiensis is designated as ISF6_0224. This gene is located adjacent to the TPA degradation gene cluster. The ISF6_0224 protein sequence matches those of the tannase family, which is known to hydrolyze the ester linkage of aromatic compounds such as gallic acid esters, ferulic saccharides, and chlorogenic acids.The purified recombinant ISF6_0224 protein efficiently hydrolyzed MHET with a turnover rate (kcat) of 31 ± 0.8 s⁻¹ and a Michaelis constant (Km) of 7.3 ± 0.6 mM. The enzyme did not show any activity against PET, BHET, pNP-aliphatic esters, or typical aromatic ester compounds catalyzed by the tannase family enzymes. The discovery of MHETase in I. sakaiensis provides insight into the metabolic pathway of PET degradation by this bacterium. The enzyme plays a crucial role in breaking down PET into its simpler constituents, which can then be further metabolized by the bacterium. In our design we use BBa_K3002037 the basic part from Kaiser Collection, this part contains the introns 1 and two times intron 2 of RBCS2, that perfectly matches to pAR promoter.

PHL7

PHL7 is a recently discovered metagenomic-derived polyester hydrolase. This enzyme is capable of efficiently degrading amorphous polyethylene terephthalate (PET) found in post-consumer plastic waste. It Is stable in a temperature range suitable for PET hydrolysis between 65°C to 70°C. It hydrolyzes PET most effectively around 70°C, which is close to the glass transition temperature (Tg) of the polymer. PHL7 interacts with the terephthalic acid (TPA) moiety of its substrate in a lock-and-key mechanism, rather than an induced fit. This is similar to the enzyme LCC but different from IsPETase. The ligand-induced opening of the substrate-binding groove in PHL7 is restricted due to decreased flexibility of subsite I, which is influenced by the residue H185. This prevents the aromatic residue W156 from moving away from the active site. The aromatic π-stacking clamp, comprised of residues F63 and W156, is crucial for the binding and hydrolysis of BHET. The overall folds of PHL7 and its cocrystal structure with TPA (PHL7×TPA) are nearly identical, with minor deviations in the orientations of active site amino acids.