In the existing part BBa_K2982005, we added Scanning Electron Microscope (SEM) photos of PET films after being digested by S245I PETase mutant. The usage of SEM can accurately visualize how serious the PET film is digested by the enzyme. Besides, we added HPLC profile of products released from PET film digested with S245I PETase to show the extent of PET hydrolysis.
We performed PET film digestion to study the PET hydrolytic activity of S245I PETase. After incubating 9μg of purified proteins with PET film at 30°C for 96 hours, we viewed the digested PET film under a SEM.
The pitting resulting from the digestion of S245I PETase mutant can be visualized, and the buffer-only SEM photo acts as a control.
(B) HPLC profile of the products released from the PET films
Figure 2a. HPLC spectrum of TPA standard.
Figure 2b. HPLC spectrum of MHET standard.
Figure 2c. HPLC spectrum of products released from the PET film digested with S245I PETase.
HPLC profiles demonstrated that the detection peaks representing TPA monomer and MHET intermediate product formed during PET film digestion have retention times at 4.64 minutes (Figure 2a) and 5.17 minutes (Figure 2b) respectively. HPLC profiles of products released from PET film digestions using S245I PETase revealed incomplete PET hydrolysis as considerable amounts of intermediate product, MHET which showed a peak at 5.07 minutes were detected (Figure 2c). Therefore, we can conclude that S245I PETase exhibits hydrolytic activity for PET depolymerization.
