Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrate...

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Main Authors: Sana, Barindra, Ding, Ke, Siau, Jia Wei, Pasula, Rupali Reddy, Chee, Sharon, Kharel, Sharad, Lena, Jean-Baptise Henri, Goh, Eunice, Rajamani, Lakshminarayanan, Lam, Yeng Ming, Lim, Sierin, Ghadessy, John F.
Other Authors: School of Materials Science and Engineering
Format: Article
Language:English
Published: 2024
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Online Access:https://hdl.handle.net/10356/174342
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-1743422024-03-29T15:45:40Z Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches Sana, Barindra Ding, Ke Siau, Jia Wei Pasula, Rupali Reddy Chee, Sharon Kharel, Sharad Lena, Jean-Baptise Henri Goh, Eunice Rajamani, Lakshminarayanan Lam, Yeng Ming Lim, Sierin Ghadessy, John F. School of Materials Science and Engineering School of Chemistry, Chemical Engineering and Biotechnology Engineering Hydrolase Thermostability Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity. National Research Foundation (NRF) Published version This work was funded by the National Research Foundation, Singapore (NRF‐CRP22‐2019‐0005). 2024-03-27T00:16:54Z 2024-03-27T00:16:54Z 2023 Journal Article Sana, B., Ding, K., Siau, J. W., Pasula, R. R., Chee, S., Kharel, S., Lena, J. H., Goh, E., Rajamani, L., Lam, Y. M., Lim, S. & Ghadessy, J. F. (2023). Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches. Biotechnology and Bioengineering, 120(11), 3200-3209. https://dx.doi.org/10.1002/bit.28523 0006-3592 https://hdl.handle.net/10356/174342 10.1002/bit.28523 37555384 2-s2.0-85167355148 11 120 3200 3209 en NRF-CRP22-2019-0005 Biotechnology and Bioengineering © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Engineering
Hydrolase
Thermostability
spellingShingle Engineering
Hydrolase
Thermostability
Sana, Barindra
Ding, Ke
Siau, Jia Wei
Pasula, Rupali Reddy
Chee, Sharon
Kharel, Sharad
Lena, Jean-Baptise Henri
Goh, Eunice
Rajamani, Lakshminarayanan
Lam, Yeng Ming
Lim, Sierin
Ghadessy, John F.
Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches
description Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.
author2 School of Materials Science and Engineering
author_facet School of Materials Science and Engineering
Sana, Barindra
Ding, Ke
Siau, Jia Wei
Pasula, Rupali Reddy
Chee, Sharon
Kharel, Sharad
Lena, Jean-Baptise Henri
Goh, Eunice
Rajamani, Lakshminarayanan
Lam, Yeng Ming
Lim, Sierin
Ghadessy, John F.
format Article
author Sana, Barindra
Ding, Ke
Siau, Jia Wei
Pasula, Rupali Reddy
Chee, Sharon
Kharel, Sharad
Lena, Jean-Baptise Henri
Goh, Eunice
Rajamani, Lakshminarayanan
Lam, Yeng Ming
Lim, Sierin
Ghadessy, John F.
author_sort Sana, Barindra
title Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches
title_short Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches
title_full Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches
title_fullStr Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches
title_full_unstemmed Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches
title_sort thermostability enhancement of polyethylene terephthalate degrading petase using self- and nonself-ligating protein scaffolding approaches
publishDate 2024
url https://hdl.handle.net/10356/174342
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