Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
© Fischer et al.; licensee BioMed Central Ltd. 2014
Received: 2 April 2014
Accepted: 30 October 2014
Published: 19 November 2014
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.
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.
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.
Cellobiose dehydrogenase (CDH, EC 220.127.116.11) is an extracellular enzyme produced by a number of wood-degrading fungi . One of them is the plant pathogen Sclerotium (Athelia) rolfsii which is common in the tropics and subtropics and attacks mostly crops and vegetables. It is mainly producing cellulolytic enzymes in order to enter the host organism . The biological function of CDH is controversial. For example, CDH increases the efficiency of cellulose degradation or reduces product inhibition of cellulases by oxidizing cellobiose to the corresponding lactone ,. This theory was also confirmed several years later .
CDH is a monomeric protein consisting of a flavin and a heme domain. Both domains are connected by a protease-sensitive linker. When CDH is cleaved by proteases, it results in an active flavin domain and an inactive heme fragment. It is possible to distinguish between the holoenzyme and the flavin domain by using both one and two electron acceptors. Two electron acceptors like 2,6-dichlorophenol-indophenol (DCIP) can be reduced either directly at the flavin domain or via internal electron transfer at the heme domain. One electron acceptors like cytochrome c can only be used at the heme domain. Thus, they are used to detect only the intact enzyme.
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.
Organism and culture conditions
Sclerotium rolfsii strain CBS 191.62 was obtained from the Centraalbureau voor Schimmelcultures (Baarn, The Netherlands). The fungus was maintained on glucose-maltose Sabouraud agar plates, which were inoculated with a piece (diameter 1 cm) of overgrown agar and then incubated at 30°C for 5 to 7 days.
A medium propagated by Sachslehner et al.  was used in some studies. It contained 43 g/L α-Cellulose, 80 g/L peptone from meat, 2.5 g/L NH4NO3, 1.5 g/L MgSO4 x 7H2O, 1.2 g/L KH2PO4, 0.6 g/L KCl , and 0.3 ml/L trace element solution (1.0 g/L ZnSO4 x H2O, 0.3 g/L MnCl2 x 4H2O, 3.0 g/L H3BO3, 2.0 g/L CoCl2 x 6H2O, 0.1 g/L CuSO4 x 5H2O, 0.2 g/L NiCl2x 6H2O and 4.0 ml/L conc. H2SO4). The natural pH of the medium was 5.5. For optimizing the culture conditions of Sclerotium rolfsii with regard to maximum production of cellobiose dehydrogenase, start pH was varied (5.5, 5.0 and 4.0) by adding appropriate amounts of phosphoric acid. Additionally, a pH-stat method for a start pH value of 5.5 was carried out by measuring and readjusting the pH to 5.5 with 5 M NaOH on a daily basis.
Further, the composition of the medium was modified. The medium of Sachslehner et al.  was used as a reference. First, single salts were omitted from the medium described above to evaluate their influence on enzyme production. In a second series, a basal medium containing 43 g/L α-Cellulose, 80 g/L peptone from meat and 0.3 ml/L trace element solution was used. NH4NO3, MgSO4, KH2PO4, KCl or none of them were added (amounts as in the reference medium).
All experiments were carried out in 300 ml unbaffled Erlenmeyer flasks containing 100 ml culture medium. Flasks were inoculated with 2 agar plugs (diameter 1 cm) of a freshly grown culture and incubated at 30°C and 150 rpm until enzyme activity remained constant. Samples of 1 ml were withdrawn starting after 7 days of incubation and analyzed for enzyme activity.
First, the mycelia were separated from the cultivation medium containing the CDH enzyme by filtration using a folded filter. This crude enzyme extract was desalted and concentrated to about one fifth of its initial volume using a Vivacell 250 filtration device (Sartorius AG, Germany) equipped with a PES membrane having a MWCO of 50 kDa. Within two dialysis steps and applying a pressure of 4 bar, conductivity was reduced from about 18 mS/cm to about 1 mS/cm indicating a successful salt removal. No enzyme loss was detected during this step.
Enzyme purification was carried out on an ÄKTA Prime Plus system (GE Healthcare) equipped with a 150 ml superloop for the application of large sample volumes. Different adsorbents for anion exchange (DEAE Sepharose FF as a weak and Q Sepharose XL as a strong ion exchanger, column volume: 5 ml, both by GE Healthcare) and hydrophobic interaction chromatography (Butyl-S FF, Butyl FF, Octyl FF, Phenyl HP, Phenyl FF (low sub), column volume: 1 ml, all by GE Healthcare) were tested to optimize purification procedure. With IEX, binding capacity was determined at various pH values (50 mM sodium acetate buffer) and buffer concentrations (20 – 200 mM sodium acetate, pH 4.5) by applying 110 ml of desalted and appropriately diluted crude enzyme extract (final concentration about 3 U/ml) onto the column. Fractions (2 ml) of the flow-through were collected and analyzed for CDH activity to determine a breakthrough profile. Also, elution conditions were optimized by analyzing the collected fractions for enzyme activity and protein content. Fractions having a higher specific activity than the applied sample were pooled. Ammonium sulfate precipitation was carried out by adding an appropriate amount of a 3.8 M solution to obtain the desired saturation before applying the sample to various HIC columns. Concentration and composition of the starting buffer were optimized as well.
Preservation of the enzyme
Seven sugars and two sugar alcohols were tested for their ability to serve as lyo- or cryoprotectants for CDH. Therefore 1 ml of purified enzyme solution (containing 11 U/ml) and 0.5 ml of lyoprotectant solution (to give a final concentration of 10 μmol per unit CDH) were mixed and either frozen at -20°C or freeze dried in 1.5 ml Eppendorf tubes. Freeze-drying was carried out in an Alpha 1-4 freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Plate temperature was set to -50°C and within 3 hours a product temperature of -28°C was obtained. Vacuum (0.005 mbar) was applied and plate temperature was set to 30°C. The process was stopped after 4 to 5 days when product temperature was positive. Samples were rehydrated with 1 ml water immediately after drying and analyzed for residual enzyme activity. Frozen samples were thawed after 60 and 160 hours and also assayed for remaining CDH activity.
Enzyme activity assay
For measuring the enzyme concentration, the DCIP assay as propagated by Baminger et al.  was used. Therefore, 100 μl 300 mM lactose, 20 μl 200 mM NaF and 760 μl 100 mM sodium acetate buffer pH 4.0 were incubated at 30°C for at least 10 minutes before analysis. A solution of 3 mM DCIP (containing 10% v/v ethanol) was tempered separately. For analysis, 100 μl of DCIP solution were added to the lactose/NaF/buffer mixture and 20 μl appropriately diluted sample solution was added. After mixing the reduction of DCIP was measured at 520 nm every 5 seconds for 3 minutes. The extinction coefficient for DCIP at 520 nm and pH 4.0 was determined to be 6.9 mM−1 cm−1. One unit was defined as the amount of enzyme that reduces 1 μmol DCIP per minute under the described assay conditions.
Protein content was determined according to the Bradford method . Therefore 100 μl appropriately diluted sample solution were mixed with 1 ml of Bradford reagent (Roti®-Quant, Carl Roth GmbH, Germany) and incubated for 10 minutes at room temperature. Extinction was measured at 595 nm. Bovine serum albumin was used as a standard.
Application of CDH for lactobionic acid synthesis
The partial purified enzyme as well as the crude enzyme extract were used in a 4.8% lactose solution to synthesise lactobionic acid. Enzyme substrate ratio was set to 70 DCIP-Units per gram lactose. DCIP (1 μmol/unit CDH) was used as a redox mediator and laccase from Trametes versicolor (Sigma Aldrich, Germany) was added in a 5-fold excess over CDH activity. Reaction was carried out in a water bath at 35°C and 200 rpm. Samples were analyzed for glucose, galactose, lactose and lactobionic acid using HPLC. The column was a Hi-Plex Na column (300 mm x 7.7 mm from Agilent Technologies Deutschland GmbH) which was used at 0.3 mLmin−1 (eluent 0.2% sodium azide in water) and 80°C.
Results and discussion
Influence of culture conditions on CDH yield
Influence of pH
Since the pH of the culture medium shifts to lower levels during cultivation, it was assumed using a pH-stat method to cultivate at a constant pH value of 5.5 might further enhance CDH production. Therefore, an appropriate amount of 5 M NaOH was added on a daily basis. However, a maximum enzyme concentration of only 2.6 U/ml was reached after 13 days. It then slightly dropped down to 2.3 U/ml and remained on that level until the end of the experiment after 21 days. CDH production using cultivation without pH adjustment was 3 times higher at that time. Consequently, control of pH is not favorable.
Influence of medium composition
KH2PO4 seems to have a negative impact on enzyme production as when omitting this salt, a significantly higher enzyme concentration could be reached as can be seen clearly from the data at days 14 to 17. Although, compared to the reference medium there seems to be a small decline in enzyme concentration with ongoing cultivation, so that at day 20 enzyme activity reached the same value (6.3 U/ml). The same can be conclude for NH4NO3 as there is significantly higher enzyme production from day 12 to 20 when this salt is omitted. In contrast to KH2PO4, enzyme concentration is enhanced until the end of the experiment resulting in about 15% higher CDH concentration compared to the reference medium. On the other hand, KCl and MgSO4 seem to have no impact on CDH production as when omitting these salts no significant difference in enzyme production was observed. However, there might be a tendency that CDH is depleted by the fungus if cultivation would be continued as indicated by considerably lower enzyme activity values at day 20.
Using the same cultivation method (flasks containing 100 ml medium), Sachslehner et al.  obtained an enzyme activity of 3.6 U/ml after 13 days of cultivation. Ludwig and Haltrich  achieved an enzyme concentration of 4.1 U/ml after 14 days. In 2003 they reported about 7 U/ml in the same medium after 13 days . With reducing peptone from meat to 20 g/L and adding 30 g/L leucine instead, 11 U/ml were obtained after 13 days. This good result could not be confirmed in the present study as enzyme production using this medium composition was very low (1.2 ± 0.9 U/ml after 16 days (n = 4)). The difference might be due to the usage of meat peptone from different suppliers therefore resulting in a different composition.
Optimization of ion exchange chromatography
dynamic binding capacity determined at 5% breakthrough and specific enzyme activity in the elute
Dynamic binding capacity (Units)
Specific activity (U/mg)
Q Sepharose XL
Q Sepharose XL
Q Sepharose XL
Q Sepharose XL
DEAE Sepharose FF
Optimization of hydrophobic interaction chromatography
To choose an appropriate hydrophobic interaction medium for the second purification step, the binding properties of several materials was determined by applying 30 units onto the columns and determining breakthrough curves. From the various columns tested, only Phenyl HP was suited for CDH purification as with the other columns CDH binding was weak indicated by high c/c0 values right from the beginning of sample application (data not shown). Using Butyl-S FF, Octyl FF and Phenyl FF low sub the entire enzyme was lost during sample application and washing. With the Butyl-FF column some of the enzyme was bound but loss was still high with 70%. Therefore all further experiments were carried out with a Phenyl HP column.
Next, ammonium sulfate concentration (0.5 M, 0.9 M, and 1.1 M) in the starting buffer was varied. Also, the effect of adding NaCl (0.2 M, 2.1 M) was studied. An ammonium sulfate concentration of 0.5 M is too low which could also be seen from the breakthrough profile as most of the enzyme was not bound therefore resulting in a high loss of nearly 70%. Raising the concentration of (NH4)2SO4 to 0.9 M (as was used by Baminger et al.  on a Phenyl Resource column) was also not sufficient as most of the target enzyme was eluting during the washing step (loss about 60%). Thus, 1.1 M ammonium sulfate was used as loss was only a third (19%). When adding 0.2 M NaCl to the starting buffer, enzyme loss could be minimized (about 10%). A further increase of NaCl to 2.1 M resulted in an enhanced binding of other proteins which in turn resulted in a worse separation during elution. To gain a certain specific activity more enzyme loss would have to be taken into account.
Tandem purification protocol
Purification of cellobiose dehydrogenase from Sclerotium rolfsii
CDH activity (Units)
Total protein (mg)
Specific activity (U/mg)
IEX (Q Sepharose XL)
HIC (Phenyl HP)
However, the obtained enzyme solution after hydrophobic interaction chromatography still contains some other proteins than CDH as could be detected by SDS-PAGE, i.e. the final enzyme solution is not 100% pure. To evaluate if the other proteins have a negative effect on the oxidation of lactose to lactobionic acid (i.e. hydrolysis into glucose and galactose catalyzed by enzymes like α-galactosidase or β-glucosidase which are also produced by the fungus ), the enzyme solution was used in a 4.8% lactose solution. For comparison, the crude enzyme extract was used in another experiment.
Preservation of purified CDH
Experiments for evaluating two possibilities (freezing and freeze-drying) to preserve CDH were carried out at high enzyme concentration (11,000 U/L) as residual activity is generally higher at higher enzyme concentration ,. Sugars are known to be effective in stabilizing enzymes during freeze-drying therefore acting as lyoprotectants ,, but no studies on CDH recovery during freeze-drying are known so far.
In another series, storage of the enzyme at -20°C was evaluated. In general, a solution of 50% (w/v) glycerol is often used when trying to make enzymes storable. Therefore, as expected, using high glycerol concentrations gave a residual activity of 100% after 60 hours of storage. However, after 160 hours a small decline is observed. Also, glucose seems to be very effective although enzyme stability might not be satisfying as indicated by the relatively high drop in CDH activity after 160 hours compared to stable activity values when using lactose, glycerol, mannitol or no lyoprotectant. On the other hand, cellobiose clearly has a negative effect in enzyme stability during freezing as residual activity values are below the ones when using no additives. Raffinose, similar to glucose, is stabilizing the enzyme at the beginning of the process but with prolonged storage it seems to be less effective and therefore being not suitable.
Generally, freezing at -20°C effects enzyme activity to a much lesser extent than freeze-drying as can be seen from the data when using no further agents. Although it is shown that with adding glucose or lactulose no activity is lost, the process of freeze-drying is obviously more cost and time consuming than simply freezing the solution at -20°C. Additionally, when adding the right amount of glycerol (no effect was observed when using only 10 μmol per unit) it is also possible to retain all of the enzyme activity. It has to be noted that the experiments carried out during this study are only considered to be preliminary. More research is necessary in studying enzyme behavior during storage, for example determining the half-life time.
To produce cellobiose dehydrogenase from Sclerotium rolfsii, it was possible to simplify a rather complex cultivation medium to a basic medium consisting only of α-cellulose, peptone from meat, and trace elements. Additionally, enzyme production was increased by 21%. Purification of the enzyme was studied systematically resulting in a yield of 56%. Although other proteins were detected in the resulting enzyme solution, its purity was considered to be sufficient for oxidizing lactose to lactobionic acid. With freeze-drying of CDH, glucose and lactulose could be identified to be good lyoprotectants. However, freezing at -20°C is preferred for storage as this method being much simpler and residual activity being 100% when using 50% (w/v) glycerol.
CF designed the study, carried out the experimental work and drafted the manuscript. AK participated in the design of the study and commented on the manuscript. TK conceived the study, participated in its design and commented on the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Julia Streithoff for her support during the experimental work.
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