An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
Name
s41598-020-79031-5.pdf
Description
Published version
Size
2.82 MB
Format
Adobe PDF
Checksum (MD5)
d61317ca5b91510b7a0eef8589a4b02c
Author(s)
Zhong-Johnson, En Ze Linda
Voigt, Christopher A
Sinskey, Anthony J
Date Issued
2021
Journal
Scientific Reports
Publisher
Springer Science and Business Media LLC
Version
Final published version
Abstract
Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method’s ability to determine relative enzyme kinetics across variants and reveal structure–function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution 4.0 International license
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DOI of Published Version
https://doi.org/10.1038/s41598-020-79031-5
