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

Abstract

BackgroundThe 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.ResultsProduction 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.ConclusionBy 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.

Natural microbial polysaccharides as effective factors for modification of the catalytic properties of fungal cellobiose dehydrogenase

“…Therefore, it is relevant to maintain the binary structure of the enzyme (Bollella et al 2018 ). The effective stabilization of CDH only with sugars as cryoprotectants during freezing and freeze-drying (Fischer et al 2014 ) has been investigated, but no studies are available on the CDH storage protection effect with polysaccharides.…”

“…Application of CDH in all proposed areas requires high stability of the enzyme and, in particular, maintenance of its intact form with a cytochrome domain. A review of previous literature focused on the stabilization of CDH shows only reports of the use of sugars as effective protective substances in the freeze-drying process (Fischer et al 2014 ) and stabilization of the enzyme with genetic engineering techniques (Balaž et al 2019 ). There are no available research regarding the possibilities of the use of naturally produced fungal and bacterial EPSs as protective agents and modulators of CDH catalytic activity.…”