Rational Design of Disulfide Bridges in Bb PETaseCD for Enhancing the Enzymatic Performance in PET Degradation

Abstract

Polyethylene terephthalate (PET) is one of the most prevalent transparent thermoplastics. It is commonly utilized due to its low cost and high durability. With the massive accumulation of waste PET, however, serious environmental pollution has become a global problem. Compared to traditional chemical degradation, biodegradation of PET catalyzed by PET hydrolase (PETase) is more environmentally friendly and energy-efficient. BbPETase^CD from the Burkholderiales bacterium is a PETase that shows favorable properties for application in the biodegradation of PET. To enhance the enzymatic performance of this enzyme, this work focuses on the rational design of disulfide bridges in BbPETase^CD. We utilized two computational algorithms to predict the probable disulfide-bridge mutations in BbPETase^CD, and five variants were acquired from the computations. Among these, the N364C/D418C variant with one additional disulfide bond showed higher expression than the wild-type enzyme (WT) and the best enzymatic performance. The melting temperature (T_m) of the N364C/D418C variant presented an increase of 14.8 °C over that of WT (56.5 °C), indicating that the additional disulfide bond significantly raised the thermodynamic stability of the enzyme. Kinetic experiments at different temperatures also demonstrated the thermal stability increase of the variant. The variant also showed significantly increased activity over WT when using bis(hydroxyethyl) terephthalate (BHET) as the substrate. More remarkably, the N364C/D418C variant exhibited approximately an 11-fold increase over the WT enzyme in the long-term (14 days) degradation of PET films. The results prove that the rationally designed disulfide bond significantly improved the enzymatic performance of the enzyme for PET degradation.

Keywords: BbPETaseCD; PET degradation; disulfide bridge; rational design.

Figure 1

Activity of BbPETase^CD and its variants using BHET as substrate. The final concentration of enzyme was 50 nM. The final concentration of BHET was 200 mg/L.

Figure 2

Residual activity of BbPETase^CD and its variants against BHET after incubation at 40 °C (a), and 50 °C (b). The reaction temperature after incubation was 50 °C. The reaction buffer was basic reaction buffer at pH 9.0. The final concentration of enzyme was 50 nM. The final concentration of BHET was 200 mg/L. The T_m value of BbPETase^CD and its variants (c).

Figure 3

Hydrolytic activity of BbPETase^CD and its variants on PET films at 30 °C (a) and 40 °C (b). The final concentration of the enzyme was 200 nM. Two PET films of 6 mm diameter were placed in each centrifuge tube as substrates.

Figure 4

Hydrolytic activity of BbPETase^CD and its variants on PET films at 40 °C. The final concentration of the enzyme was 200 nM. Two PET films of 6 mm diameter were placed in each reaction centrifuge tube as substrates.