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

BMC Biotechnology

Christin Fischer¹  Annett Krause¹  Thomas Kleinschmidt¹ 

[1] Anhalt University of Applied Sciences, Department of Applied Biosciences and Process Engineering, Bernburger Str. 55, Köthen, 06366, Germany

关键词: Lactobionic acid;    Cryoprotection;    Lyophilisation;    Enzyme purification;    Sclerotium rolfsii;    CDH;    Cellobiose dehydrogenase;   

Others  :  1084417
DOI  :  10.1186/s12896-014-0097-5

 received in 2014-04-02, accepted in 2014-10-30,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Fischer et al.; licensee BioMed Central Ltd.

【 图 表 】

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