An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
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 re...
| Main Authors: | , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Springer Science and Business Media LLC
2021
|
| Online Access: | https://hdl.handle.net/1721.1/134195 |
| _version_ | 1826193610582786048 |
|---|---|
| author | Zhong-Johnson, En Ze Linda Voigt, Christopher A Sinskey, Anthony J |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Zhong-Johnson, En Ze Linda Voigt, Christopher A Sinskey, Anthony J |
| author_sort | Zhong-Johnson, En Ze Linda |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T09:41:50Z |
| format | Article |
| id | mit-1721.1/134195 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:41:50Z |
| publishDate | 2021 |
| publisher | Springer Science and Business Media LLC |
| record_format | dspace |
| spelling | mit-1721.1/1341952023-02-23T15:15:42Z An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films Zhong-Johnson, En Ze Linda Voigt, Christopher A Sinskey, Anthony J Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology 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. 2021-10-27T20:03:56Z 2021-10-27T20:03:56Z 2021 2021-08-02T18:12:01Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/134195 en 10.1038/s41598-020-79031-5 Scientific Reports Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf Springer Science and Business Media LLC Scientific Reports |
| spellingShingle | Zhong-Johnson, En Ze Linda Voigt, Christopher A Sinskey, Anthony J An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films |
| title | An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films |
| title_full | An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films |
| title_fullStr | An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films |
| title_full_unstemmed | An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films |
| title_short | An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films |
| title_sort | absorbance method for analysis of enzymatic degradation kinetics of poly ethylene terephthalate films |
| url | https://hdl.handle.net/1721.1/134195 |
| work_keys_str_mv | AT zhongjohnsonenzelinda anabsorbancemethodforanalysisofenzymaticdegradationkineticsofpolyethyleneterephthalatefilms AT voigtchristophera anabsorbancemethodforanalysisofenzymaticdegradationkineticsofpolyethyleneterephthalatefilms AT sinskeyanthonyj anabsorbancemethodforanalysisofenzymaticdegradationkineticsofpolyethyleneterephthalatefilms AT zhongjohnsonenzelinda absorbancemethodforanalysisofenzymaticdegradationkineticsofpolyethyleneterephthalatefilms AT voigtchristophera absorbancemethodforanalysisofenzymaticdegradationkineticsofpolyethyleneterephthalatefilms AT sinskeyanthonyj absorbancemethodforanalysisofenzymaticdegradationkineticsofpolyethyleneterephthalatefilms |