Cargando…

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-bat...

Descripción completa

Detalles Bibliográficos
Autores principales: Humer, Diana, Ebner, Julian, Spadiut, Oliver
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369975/
https://www.ncbi.nlm.nih.gov/pubmed/32610584
http://dx.doi.org/10.3390/ijms21134625
_version_ 1783560893415555072
author Humer, Diana
Ebner, Julian
Spadiut, Oliver
author_facet Humer, Diana
Ebner, Julian
Spadiut, Oliver
author_sort Humer, Diana
collection PubMed
description Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.
format Online
Article
Text
id pubmed-7369975
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-73699752020-07-21 Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies Humer, Diana Ebner, Julian Spadiut, Oliver Int J Mol Sci Article Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications. MDPI 2020-06-29 /pmc/articles/PMC7369975/ /pubmed/32610584 http://dx.doi.org/10.3390/ijms21134625 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Humer, Diana
Ebner, Julian
Spadiut, Oliver
Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
title Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
title_full Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
title_fullStr Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
title_full_unstemmed Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
title_short Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
title_sort scalable high-performance production of recombinant horseradish peroxidase from e. coli inclusion bodies
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369975/
https://www.ncbi.nlm.nih.gov/pubmed/32610584
http://dx.doi.org/10.3390/ijms21134625
work_keys_str_mv AT humerdiana scalablehighperformanceproductionofrecombinanthorseradishperoxidasefromecoliinclusionbodies
AT ebnerjulian scalablehighperformanceproductionofrecombinanthorseradishperoxidasefromecoliinclusionbodies
AT spadiutoliver scalablehighperformanceproductionofrecombinanthorseradishperoxidasefromecoliinclusionbodies