Cargando…

Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii

BACKGROUND: The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. T...

Descripción completa

Detalles Bibliográficos
Autores principales: Fischer, Christin, Krause, Annett, Kleinschmidt, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241217/
https://www.ncbi.nlm.nih.gov/pubmed/25407159
http://dx.doi.org/10.1186/s12896-014-0097-5
_version_ 1782345819673853952
author Fischer, Christin
Krause, Annett
Kleinschmidt, Thomas
author_facet Fischer, Christin
Krause, Annett
Kleinschmidt, Thomas
author_sort Fischer, Christin
collection PubMed
description BACKGROUND: The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated. RESULTS: Production of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20°C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives. CONCLUSION: By simplifying the cultivation medium of Sclerotium rolfsii, the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20°C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme.
format Online
Article
Text
id pubmed-4241217
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-42412172014-11-24 Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii Fischer, Christin Krause, Annett Kleinschmidt, Thomas BMC Biotechnol Research Article BACKGROUND: The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated. RESULTS: Production of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20°C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives. CONCLUSION: By simplifying the cultivation medium of Sclerotium rolfsii, the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20°C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme. BioMed Central 2014-11-19 /pmc/articles/PMC4241217/ /pubmed/25407159 http://dx.doi.org/10.1186/s12896-014-0097-5 Text en © Fischer et al.; licensee BioMed Central Ltd. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.
spellingShingle Research Article
Fischer, Christin
Krause, Annett
Kleinschmidt, Thomas
Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
title Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
title_full Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
title_fullStr Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
title_full_unstemmed Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
title_short Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
title_sort optimization of production, purification and lyophilisation of cellobiose dehydrogenase by sclerotium rolfsii
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241217/
https://www.ncbi.nlm.nih.gov/pubmed/25407159
http://dx.doi.org/10.1186/s12896-014-0097-5
work_keys_str_mv AT fischerchristin optimizationofproductionpurificationandlyophilisationofcellobiosedehydrogenasebysclerotiumrolfsii
AT krauseannett optimizationofproductionpurificationandlyophilisationofcellobiosedehydrogenasebysclerotiumrolfsii
AT kleinschmidtthomas optimizationofproductionpurificationandlyophilisationofcellobiosedehydrogenasebysclerotiumrolfsii