9LMS

Crystal structure of Pichia pastoris-expressed FAST-PETase-N212A/K233C/S282C variant

Summary for 9LMS

• Entry DOI: 10.2210/pdb9lms/pdb
• Descriptor: Poly(ethylene terephthalate) hydrolase, 2-acetamido-2-deoxy-beta-D-glucopyranose (3 entities in total)
• Functional Keywords: hydrolase, pet hydrolase, pet degradation enzyme
• Biological source: Piscinibacter sakaiensis (Ideonella sakaiensis)
• Total number of polymer chains: 1
• Total formula weight: 28295.35

Authors

Li, X.,Ning, Z.Y.,Huang, S.Q.,Zeng, C.,Zeng, Z.Y.,Ji, R.,Huang, J.-W.,Chen, C.-C.,Guo, R.-T. (deposition date: 2025-01-20, release date: 2025-07-23)

Primary citation

Li, X.,Huang, J.W.,Ning, Z.,Huang, S.,Zeng, C.,Zeng, Z.,Ji, R.,Peng, R.,Liu, X.,Min, J.,Chen, C.C.,Guo, R.T.
Combined approaches to enhance the Pichia pastoris-expressed PET hydrolase.
Int.J.Biol.Macromol., :145862-145862, 2025

PubMed Abstract: Enzymatic degradation of polyethylene terephthalate (PET) provides a sustainable and promising strategy for the recycling of plastic waste. Herein, we employed site-directed mutagenesis and machine learning methods to further enhance the performance of an efficient mutant of IsPETase, FAST-PETase-N212A, and resulting in seven variants with enhanced activity. We also found that the α3-β5 loop containing the T140D mutation plays a significant role in both type I and type II cutinases. Subsequently, we determined the complex structures of two activity-elevated mutants with the PET analogue mono(2-hydroxyethyl)terephthalic acid, revealing a different binding mode. Finally, to facilitate the industrial application of PET hydrolases, we exploited the industrial strain Pichia pastoris to express the activity-enhanced mutants. Compared with E. coli-produced proteins, these mutants expressed by P. pastoris exhibited higher activity and thermal stability.

PubMed: 40653245
DOI: 10.1016/j.ijbiomac.2025.145862

Experimental method

X-RAY DIFFRACTION (1.71 Å)