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Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis
Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides c...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9113129/ https://www.ncbi.nlm.nih.gov/pubmed/34124750 http://dx.doi.org/10.1093/jimb/kuab031 |
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author | Xue, Yemin Xue, Mengke Xie, Fang Zhang, Mengchen Zhao, Hongyang Zhou, Tao |
author_facet | Xue, Yemin Xue, Mengke Xie, Fang Zhang, Mengchen Zhao, Hongyang Zhou, Tao |
author_sort | Xue, Yemin |
collection | PubMed |
description | Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a β-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-β-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent k(cat), respectively; N222F shows the highest k(cat)/k(m) value for pNPG. The substrate selectivity altered from pNPG to pNP-β-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-β-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis. |
format | Online Article Text |
id | pubmed-9113129 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-91131292022-06-08 Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis Xue, Yemin Xue, Mengke Xie, Fang Zhang, Mengchen Zhao, Hongyang Zhou, Tao J Ind Microbiol Biotechnol Biocatalysis> Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a β-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-β-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent k(cat), respectively; N222F shows the highest k(cat)/k(m) value for pNPG. The substrate selectivity altered from pNPG to pNP-β-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-β-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis. Oxford University Press 2021-06-14 /pmc/articles/PMC9113129/ /pubmed/34124750 http://dx.doi.org/10.1093/jimb/kuab031 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Biocatalysis> Xue, Yemin Xue, Mengke Xie, Fang Zhang, Mengchen Zhao, Hongyang Zhou, Tao Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
title | Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
title_full | Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
title_fullStr | Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
title_full_unstemmed | Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
title_short | Engineering Thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
title_sort | engineering thermotoga maritima β-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis |
topic | Biocatalysis> |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9113129/ https://www.ncbi.nlm.nih.gov/pubmed/34124750 http://dx.doi.org/10.1093/jimb/kuab031 |
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