Standard Curve

To quantify the enzymatic activity of PET-degrading enzymes in our experiment, we fabricated standard curves of TPA and MHET concentrations versus absorbance. The original data is shown in the following table.

The standard curve drawn is as follows.

To facilitate subsequent calculations, we swapped the X-axis and Y-axis and obtained a linear function of the integral of the product concentration with respect to the absorbance value.

 PET05

The Optimal Reaction Conditions of PET05

We tested the optimal enzymatic activity conditions for the degradation of low-crystalline PET by PET05 through two sets of orthogonal experiments. The data from the first orthogonal test are as follows.

We found that PET05 only had enzymatic activity at a near-saturated NaCl concentration of 4-5 meters. So we conducted the second orthogonal experiment, and the data are as follows.

We concluded that the optimal pH value for the PET05 reaction is 9, the optimal sodium chloride concentration is 5M, the optimal reaction temperature is 55℃, and the Enzyme Loading is 50nM, which can achieve enzyme saturation.

Long-term Reaction of PET05

Under the optimal conditions of enzyme activity, we conducted a long-term reaction using PET05 for up to 312 hours. Sampling was conducted at different times of the reaction, PET films were taken under a stereomicroscope, and the product concentration was determined using a UPLC. The state of PET sheets at different reaction durations is as shown in the figure below.

The product concentrations at different reaction durations are as shown in the table below.

From the data and line chart, it can be seen that the maximum reaction duration PET05 can achieve under the optimal conditions for enzyme activity is approximately 200 hours. This is another highly representative characteristic of PET05, in addition to its high salt tolerance.

 Mining of PET Degrading Enzymes from Marine Sources

We purified and subjected 40 protein sequences, which were mined and screened from in silico experiments, to enzyme activity assays. These sequences were cloned into the pET-32a(+) plasmid, as shown in the figure.

Ultimately, we identified four of these protein sequences that exhibit PET-degrading enzyme activity. Their orthogonal experiment results are shown in the figure below.

Below are the AlphaFold-predicted 3D structures of these four enzymes.

Upon validation through wet-lab experiments, three of the four obtained PET hydrolases were found to belong to Cluster 42_29. An evolutionary tree was constructed using these sequences together with the nine known PET hydrolases, as shown in the figure below. Cluster 42_29 can be considered a potential family of PET hydrolases, though this requires further wet-lab experimental validation.

 The Engineering Modification of PET05

Enzyme Active Site Pocket Mutagenesis Based on Structural Homology and Molecular Docking Results

Based on the mutation sites designed from in silico experiments, we first designed a pair of primers for each mutant, and performed PCR amplification using pET-32a(+)-PET05 as the template. The figure shows the results of agarose gel electrophoresis, where 10 μL was taken from the 50 μL PCR system for characterization.

After PCR, the target fragments were subjected to self-homologous recombination, and sequencing was performed to verify whether the mutant construction was successful. We cultured and purified 70 successfully constructed mutant proteins. The protein expression is shown in the following SDS-PAGE gel electrophoresis image.

They were reacted with low-crystallinity PET under the same conditions as the wild-type PET05, and the enzyme activity was detected by UPLC. The results are shown in the following figure (mutants with higher enzyme activity than the wild-type are marked with orange fluorescence in the figure).

We have completed the construction and testing of 16 double combinatorial mutants, with the results shown in the following figure. Among them, the top-performing one in characterization is D5 (PET05-I150A-S203T), whose enzyme activity has increased by 62% compared with the wild type. Although the enzyme activity—both before and after modification—is relatively low and lacks practical application value, the increase in the enzyme activity of the modified protein proves that our modification strategy is effective.

Enzyme Thermostability Engineering Based on Deep Learning and Physicochemical Algorithms

The construction and testing procedures for nine single-point mutations and two pairs of disulfide bond mutants were consistent with those described in Section 4.1. The enzymatic activity data are provided below.

We determined the Tm values of several proteins using a real-time quantitative PCR instrument. With the exception of protein B8, the Tm values of most proteins were higher than that of the wild-type PET05.This indicates that after our modification, the thermal stability of PET05 has achieved a significant improvement.