Cycle 1：Single-point Mutation

Design：

To directly enhance the catalytic activity of 05PET enzyme, we successively utilized structural homology and molecular docking to locate its pocket, selected hot spot amino acids and conducted mutation and wet experiments for verification.

We first used AutoDock Vina to perform molecular docking of 05PET enzyme with trimeric PET long-chain molecules. By comparing with the structure of LCC, we excluded some non-active pocket areas. Based on this, we delineated a more reliable docking region and re-performed molecular docking. After identifying the sequence sites corresponding to the active pocket space, we took 05PET enzyme as the basis and conducted mutations based on the sequences of other PET enzymes, determining the following single-point mutation schemes:

G55A T56A T56Y T56F T57A T58G T62S T62A Y63I Y63L Y63A H123A H123W S124A Q125M Q125A Y148A Y148W I150A I150T I150L I150S G151A G152A D174A I176A I186V L199A L199I S200N S200A S200D S200R G201A G201N A202G S203A S203T H204A F205A F205L F205S E206A E206C E206V V208N V208A G209A G209I G209T G209S S200A S200N S200G S200P A211N A211D G212A G212T G212K G212S D213A D213I D213N D213T D234A D242A D246A D248A D248E

Build：

We designed forward and reverse primers for each mutant, and performed PCR using 32a-PET05 (i.e., the wild-type PET05 gene constructed in the pET-32a(+) plasmid vector) as the PCR template. The PCR products were subjected to agarose gel electrophoresis and gel extraction. The gel-extracted products were then subjected to homologous recombination. The homologous recombination products were used for slow transformation of E. coli DH5α. After transformation, the Ampicillin-resistant (Amp-resistant) plate medium was incubated at 37°C for 12 hours, and then sent to a sequencing company for sequencing. The sequencing and assembly results were checked. The detailed construction procedure can be found in notebook here.

Plasmids with correct sequencing results were returned to the laboratory by the company. We transformed the plasmids into E. coli BL21(DE3), followed by bacterial culture and protein purification. We determined the concentration (mg/mL) of the purified protein solution and the A260/280 ratio.

Test：

We constructed the same reaction system for both PET05 mutants and PET05 as follows:
Reaction buffer (600 μL): 50 mM Glycine, 5 M NaCl, pH = 9
Reaction time: 18 hours
Reaction temperature: 55 ℃
Rotation speed: 300 rpm
Reactant: flaky lcPET (with a uniform diameter)

According to the formula:
Mass (ng) = Enzyme Loading (nM) × Reaction Volume (μL) × Relative Molecular Mass of the Enzyme (g/mol) × 1000
we can calculate the mass of the enzyme solution to be added in a 600 μL reaction system.
According to the formula:
Volume of the Enzyme Solution to be Added (μL) = Mass (ng) × Extinction Coefficient / Concentration of the Enzyme Solution (ng/μL)
we can calculate the volume of the enzyme solution to be added.

We completed experiments for several mutants and PET05, with 3 parallel replicates in each group. The release amounts of the products TPA and MHET after 18 hours of reaction were determined via ultra performance liquid chromatography (UPLC). The detailed experimental data results can be found in here.

Learn：

After testing, we identified 10 mutants with higher enzymatic activity than the wild-type:

We will combine these mutations for the next round of engineering cycle.

Cycle 2：Double-Point Combinatorial Mutation

Design：

Based on the 70 sequences in section 2.3, we selected 10 mutants with enzymatic activity higher than that of the wild-type PET05. These 10 mutants were combined in pairs to form 38 types of double-point combinatorial mutants. We then designed the PCR templates and primers required for constructing these mutants:

Build：

The method is the same as Cycle 1.

Test：

The method is the same as Cycle 1.

Learn：

We have completed the construction and testing of a total of 16 double mutants:The mutants D2, D3, D4, D5, D6, D9, D11, D14, D16, D17, D21, D25, D30, D33, D36, and D38 exhibit significantly higher enzymatic activity than the wild-type. Among them, the D5 mutant achieves a product release amount of 1.28 μM within 18 hours, representing a 62% increase compared to wild-type PET05.