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Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol
BACKGROUND: Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG pr...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025525/ https://www.ncbi.nlm.nih.gov/pubmed/33827582 http://dx.doi.org/10.1186/s12934-021-01569-4 |
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author | Schwaiger, Katharina N. Cserjan-Puschmann, Monika Striedner, Gerald Nidetzky, Bernd |
author_facet | Schwaiger, Katharina N. Cserjan-Puschmann, Monika Striedner, Gerald Nidetzky, Bernd |
author_sort | Schwaiger, Katharina N. |
collection | PubMed |
description | BACKGROUND: Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. RESULTS: Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h(−1); 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 10(4) U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 10(3) U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. CONCLUSIONS: Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01569-4. |
format | Online Article Text |
id | pubmed-8025525 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-80255252021-04-08 Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol Schwaiger, Katharina N. Cserjan-Puschmann, Monika Striedner, Gerald Nidetzky, Bernd Microb Cell Fact Research BACKGROUND: Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. RESULTS: Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h(−1); 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 10(4) U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 10(3) U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. CONCLUSIONS: Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01569-4. BioMed Central 2021-04-07 /pmc/articles/PMC8025525/ /pubmed/33827582 http://dx.doi.org/10.1186/s12934-021-01569-4 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Schwaiger, Katharina N. Cserjan-Puschmann, Monika Striedner, Gerald Nidetzky, Bernd Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol |
title | Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol |
title_full | Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol |
title_fullStr | Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol |
title_full_unstemmed | Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol |
title_short | Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol |
title_sort | whole cell-based catalyst for enzymatic production of the osmolyte 2-o-α-glucosylglycerol |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025525/ https://www.ncbi.nlm.nih.gov/pubmed/33827582 http://dx.doi.org/10.1186/s12934-021-01569-4 |
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