9QDE

Crystal structure of the non-glycosylated polyester hydrolase Leipzig 7 (PHL7) mut3 variant expressed in Pichia pastoris (P_PHL7mut3_ng)

Summary for 9QDE

• Entry DOI: 10.2210/pdb9qde/pdb
• Descriptor: Polyester Hydrolase Leipzig 7 (PHL-7), catalysis-deficient S131A mutant, CHLORIDE ION (3 entities in total)
• Functional Keywords: e. coli, lcc, k. phaffii, petase, p. pastoris, hydrolase
• Biological source: compost metagenome
• Total number of polymer chains: 1
• Total formula weight: 28763.35

Authors

Useini, A.,Strater, N.,Striedner, G.,Sonnendecker, C. (deposition date: 2025-03-06, release date: 2025-09-10, Last modification date: 2025-12-03)

Primary citation

Fohler, L.,Faschingeder, F.,Leibetseder, L.,Zhao, Z.,Useini, A.,Strater, N.,Sonnendecker, C.,Ewing, T.A.,Moers, A.P.H.A.,Werten, M.W.T.,van Vliet, D.M.,Julsing, M.K.,Zimmermann, W.,Striedner, G.
Trade-Offs between Stability and Activity of Glycosylated and Non-Glycosylated Polyester Hydrolases PHL7 and PHL7mut3.
Acs Es T Eng, 5:2781-2791, 2025

PubMed Abstract: Plastic pollution has become a global environmental challenge, driving interest in enzymatic polyethylene terephthalate (PET) recycling by using polyester hydrolases. In this study, we produced the PET-degrading enzyme PHL7 and its variant PHL7mut3 in and (syn. ) to investigate the impact of N-glycosylation on enzyme properties. While glycosylation upon expression in enhanced thermal stability, it reduced the catalytic activity of the enzymes, revealing a trade-off that adds complexity to the selection of the best-suited expression system. Additionally, we engineered to produce non-glycosylated enzyme variants by substituting the asparagine residues (N) at all three putative N-glycosylation sites with glutamine residues (Q). The non-glycosylated -produced enzymes showed a lower activity compared to those produced in , likely due to the differences in the amino acid sequence. The effects of glycosylation were less pronounced in PHL7mut3 than in PHL7, yet N-glycosylation strongly influenced the performance of both enzymes. We further demonstrate that the PET degradation performance of PHL7mut3 is less dependent on the buffer ionic strength than that of PHL7. The study emphasizes the need for the informed selection of a suitable expression host for polyester hydrolases to balance enzyme activity, thermostability, and production titer for applications in PET recycling.

PubMed: 41262865
DOI: 10.1021/acsestengg.5c00272

Experimental method

X-RAY DIFFRACTION (1.02 Å)