# Trade-Offs between Stability and Activity of Glycosylated and Non-Glycosylated Polyester Hydrolases PHL7 and PHL7mut3

## TL;DR

This study compares how glycosylation affects the stability and activity of two PET-degrading enzymes, PHL7 and PHL7mut3, in different expression systems.

## Contribution

The study reveals a trade-off between thermal stability and catalytic activity due to glycosylation in polyester hydrolases.

## Key findings

- Glycosylation in P. pastoris increased thermal stability but reduced catalytic activity.
- Non-glycosylated enzymes from P. pastoris had lower activity than those from E. coli.
- PHL7mut3 showed less dependence on buffer ionic strength for PET degradation than PHL7.

## 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 Escherichia
coli and Pichia pastoris (syn. Komagataella phaffii) to investigate
the impact of N-glycosylation on enzyme properties. While glycosylation
upon expression in P. pastoris 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 P. pastoris 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 P. pastoris-produced enzymes showed a lower activity
compared to those produced in E. coli, 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.