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Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosph...

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Autores principales: Usvalampi, Anne, Li, He, Frey, Alexander D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206812/
https://www.ncbi.nlm.nih.gov/pubmed/34150734
http://dx.doi.org/10.3389/fbioe.2021.678038
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author Usvalampi, Anne
Li, He
Frey, Alexander D.
author_facet Usvalampi, Anne
Li, He
Frey, Alexander D.
author_sort Usvalampi, Anne
collection PubMed
description Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.
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spelling pubmed-82068122021-06-17 Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis Usvalampi, Anne Li, He Frey, Alexander D. Front Bioeng Biotechnol Bioengineering and Biotechnology Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose. Frontiers Media S.A. 2021-06-02 /pmc/articles/PMC8206812/ /pubmed/34150734 http://dx.doi.org/10.3389/fbioe.2021.678038 Text en Copyright © 2021 Usvalampi, Li and Frey. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Usvalampi, Anne
Li, He
Frey, Alexander D.
Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis
title Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis
title_full Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis
title_fullStr Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis
title_full_unstemmed Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis
title_short Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis
title_sort production of glucose 6-phosphate from a cellulosic feedstock in a one pot multi-enzyme synthesis
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206812/
https://www.ncbi.nlm.nih.gov/pubmed/34150734
http://dx.doi.org/10.3389/fbioe.2021.678038
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