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Jun 3, 2011 - Abstract BglG, a Stachybotrys microspora b-glucosi- dase produced in the presence of glucose and cellobiose, was used individually as sole ...

World J Microbiol Biotechnol (2012) 28:23–29 DOI 10.1007/s11274-011-0787-2

ORIGINAL PAPER

Cellobiose dehydrogenase influences the production of S. microspora b-glucosidase Walid Saibi • Ali Gargouri

Received: 23 February 2011 / Accepted: 9 May 2011 / Published online: 3 June 2011 Ó Springer Science+Business Media B.V. 2011

Abstract BglG, a Stachybotrys microspora b-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5 min at 100°C) of the aliquots, which supported the nonproteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a d-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of d-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.

W. Saibi (&)  A. Gargouri Laboratoire de Valorisation de la Biomasse et Production de Prote´ines chez les Eucaryotes, Centre de Biotechnologie de Sfax (CBS), Universite´ de Sfax, B.P’1177’, 3018 Sfax, Tunisia e-mail: [email protected]

Keywords b-Glucosidase  Glucose  Cellobiose  Cellobiose dehydrogenase  d-Gluconolactone  Stachybotrys microspora

Introduction Cellulose, a major cell-wall constituent in higher plant, is a linear polysaccharide consisting of b-1, 4-linked glucose residues. The first step in the saccharification of cellulosic materials involves the synergistic action between endoglucanases (E.C 3.2.1.4) and exoglucanases (E.C 3.2.1.91), yielding short cello-oligosaccharides and mainly cellobioses, which are cleaved by b-glucosidases (EC 3.2.1.21) (Amouri and Gargouri 2006; Brini et al. 2010; Saibi et al. 2007; Smaali et al. 2004). By degrading these products into glucose, b-glucosidases contribute to overcoming the inhibition of endoglucanases and exoglucanases through cellulose hydrolysis end-products. In fact, b-glucosidases are widely distributed in the living world and play pivotal roles in various biological processes other than the biological conversion of cellulose to glucose in fungi and bacteria (Davies 2010; Pozzo et al. 2010; Saibi et al. 2007). They are employed in various biotechnological applications, such as biofuel production (for example, ethanol) (Buaban et al. 2010; Hardiman et al. 2010; Lever et al. 2010), and food production processes, such as the hydrolysis of bitter compounds during juice extraction and the liberation of aroma from wine grapes. b-glucosidases have also found applications in the synthesis of alkyl- and aryl-glycosides from natural polysaccharides or their derivatives and alcohols through reversed hydrolysis or trans-glycosylation reactions (Baldrian and Valaskova 2008; Brini et al. 2010; Saibi et al. 2007; Smaali et al. 2004). These products are,

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in fact, highly valued in the pharmaceutical, cosmetic, and detergent industries. The determination and understanding of the mechanisms involved in the oxidation of cellobiose has attracted much attention in recent years. Of particular interest, cellobiose dehydrogenase (CDH; EC 1.1.99.18), an extracellular redox enzyme produced by various wood degrading (Henriksson et al. 1997) and ascomycete fungi (Harreither et al. 2011) has often been reported for its ability to catalyze the oxidation of cellobiose, higher water soluble cellodextrins, lactose, and mannobiose, to their corresponding lactones (Henriksson et al. 1997; Li et al. 1997). Stachybotrys microspora is a filamentous fungus having various b-glucosidases. Two of those b-glucosidases were previously characterized (Amouri and Gargouri 2006; Saibi et al. 2007). A third one, named bglG, was produced in the presence of glucose or cellobiose, used individually as a unique carbon source at the concentration of 1%, purified, and then characterized from the glucose culture medium (data not shown). The thermo-activity, thermo-stability, and re-folding recovery of bglG were, on the other hand, studied in the presence of wheat dehydrin (DHN-5). The latter was previously described as a Late Embryogenesis Abundant protein with a cryo-protective effect molecule (Brini et al. 2007). In fact, and for the first time, wheat dehydrin DHN-5 was proved to exert a heat-protective effect on distinct enzymatic activities, particularly the case of bglG (Brini et al. 2010). The present study was undertaken to investigate and report on the response elements so as to further validate our proposed model explaining the difference observed during time course production of bglG in the presence of glucose and cellobiose used individually as sole carbon sources.

Materials and methods

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trace element solution composed of 1.6 g l-1 MnSO4, 2 g l-1 ZnSO4, 0.5 g l-1 CuSO4, and 0.5 g l-1 CoSO4). During the pre-culture, glucose, at a concentration of 1%, was used as a sole carbon source. The fungal strain was cultivated in the presence of 1% concentrations of glucose or cellobiose and compared to the modified Mandels medium used as control during the time courses. Enzyme assays The activity of b-glucosidase was monitored using p-NPGlc as a substrate. In fact, 0.2 ml of 1 mM p-NP-Glc (in 0.1 M sodium acetate buffer pH 5) was incubated with bglG at an appropriate dilution at 50°C for 15 min. The reaction was stopped by adding 0.6 ml of 0.4 M glycin– NaOH buffer pH 10.8; the liberated p-nitrophenol (p-NP) was measured at 400 nm. The molecular extinction coefficient of the p-NP was 18,000 M-1 cm-1. One unit of enzymatic activity was determined as the amount of enzyme required to release 1 lmol of p-NP per min under the described assay conditions (Brini et al. 2010). The cellobiose dehydrogenase (CDH) assay was performed as follows: 2.5 ml cold substrate solution (4 mM potassium ferricyanide and 2.5 mM cellobiose in 50 mM sodium acetate buffer, pH 5) were added to screw-cap test-tubes containing the enzyme sample diluted in 50 mM sodium acetate buffer, pH 5, to a final volume of 5 ml. The mixture was incubated at 40°C. Samples of 0.5 ml were taken at regular intervals, beginning at time zero, when CDH was added, and then immediately mixed with 0.5 ml of DNS reagent that stopped the reaction. When all samples were collected, the DNS tubes were boiled for five minutes, and then cooled; their absorbencies were read at 575 nm. One unit of CDH was defined as the amount of enzyme oxidizing 1 nmol of cellobiose per minute under the above mentioned conditions (Henriksson et al. 1997).

Biological strain Zymogram analysis The biological strain used in this work is a filamentous fungus that was identified as Stachybotrys microspora by the Centraalbureau voor Schimmel cultures- the Netherlands (Amouri and Gargouri 2006). b-glucosidase production Stachybotrys microspora was grown on Potato Dextrose Agar medium at 30°C for 4 days. Spores were harvested in 0.1% Tween 80 solution and used to inoculate a modified Mandels medium (Mandels and Reese 1957) that consisted of the following ingredients per litre: 2 g KH2PO4, 1.4 g (NH4)2SO4, 1 g yeast extract, 0.69 g urea, 0.3 g CaCl22H2O, 0.3 g MgSO47H2O, 1 ml Tween 80, and 1 mL

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Proteins were mixed with the same loading buffer as in SDS–PAGE, but they were not heated before loading on SDS-gel (Laemmli 1970). After electrophoresis, the gel was incubated for two hours in 20 mM Tris–HCl pH 8 to get rid of SDS, allowing the renaturation of proteins. After 15 min of equilibration in 0.1 M sodium acetate buffer pH 5, the gel was superposed against 1% agar gel containing 100 ll of 4-methylumbelliferyl-b-D-Glucoside (MU-Glc) at 25 lg ml-1. Following a suitable period of incubation, the system was observed under UV light, with excitation at 366 nm and emission at 445 nm. The zone of activity was indicated by the fluorescence emitted by methylumbelliferol (MU) released via enzyme action.

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The inhibitory effect of d-gluconolactone during bglG assessment

metabolite in food biotechnology, after the pentose phosphate pathway (Leonowicz et al. 2001).

The d-gluconolactone stock solution used in this study was prepared in ultra pure water at 1 M. d-gluconolactone added to the reaction mixture at different concentrations, varying from 0 to 10 mM. The relative enzymatic activity was determined as described above.

BglG kinetic production as carbon source dependent

During bglG time course in presence of cellobiose as sole carbon source, aliquots were tracked at regular intervals (2nd, 4th, 6th and 8th day from the cellobiose culture medium), and, during these studies, the amount of sugar was followed using a chromatographic column (Aminex HPX 42A, 300 9 7.8 mm) coupled to an HPLC system. Determination of the cellobiase capacity of bglG BglG was added to 5 g l-1 of cellobiose solution (prepared in 0.1 M sodium acetate buffer pH 5) at final volume of 5 ml and incubated at 50°C. Aliquots (50 ll) were taken during various time intervals and analyzed by HPLC (Aminex Fast carbohydrate 100 9 7.8 mm, 0.5 ml min-1); the peak areas were determined and converted into percentages of glucose formed and cellobiose hydrolyzed. The results were confirmed by the GOD method (Lin et al. 1999). Determination of protein concentration The protein amount of the samples used during these studies was determined using the Bradford assay (Bradford 1976). Bovine serum albumin was used as a standard. The protein concentration was determined to normalize the amount of protein used for zymogram analysis.

Results and discussion b-Glucosidase represents one of the glycoside hydrolases, enzymes responsible for the hydrolysis of the glycosidic bond in disaccharides, oligosaccharides, polysaccharides, and glycoconjugates (Davies 2010; Ishida et al. 2009; Saibi et al. 2007). They are characterized by the critical role they play during the depolymerisation of the cellulosic polymer through the cleaving of cellobiose towards glucose units. Cellobiose represents a natural substrate of CDH, which converts it into cellobionolactone, also a potential substrate of b-glucosidases that can split it into glucose and dgluconolactone. Cellobionolactone is considered the 2nd biological source of d-gluconolactone, an important

p-NPGase Activity (U/mL)

Chromatographic analysis

It is worth recalling that bglG is an S. microspora b-glucosidase that was induced by glucose and cellobiose which were used individually as unique carbon sources at a concentration of 1%. The enzyme was purified, subjected to biochemical property characterization studies (data not shown), and then used in subsequent studies and investigations (Brini et al. 2010; Saibi et al. 2010). The data obtained from zymogram assays revealed that while the time course production of bglG followed an exponential curve in the presence of the glucose Mandels medium, it showed a growing bell-shaped curve in the case of cellobiose (Fig. 1a). The zymogram profile, shown in (Fig. 1b), demonstrates that the b-glucosidase activity produced was the same in the presence of the two carbohydrates (glucose and cellobiose) described. In fact, the time course production of bglG, in the presence of cellobiose, the natural substrate of various, but not all, b-glucosidases, showed that this diholoside induced the synthesis of bglG, with an optimum level of production at the 4th day. After that, and as the zymogram profile in Fig. 1 illustrates, bglG production would decrease throughout the 6th, 8th and 10th days of culture. These findings raise speculation about a potential factor that was released during mycelium growth in the cellobiose culture medium, but not in the glucose one, and that had the ability to reduce bglG activity. The explanatory model presented in Figs. 2 and 3 was, accordingly, proposed to account for this hypothesis. 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0,05 0 0

2

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Time (Day)

A

B

Fig. 1 Kinetic production of bglG and zymogram analysis. a Time course of bglG production in the presence of glucose (closed square) or cellobiose (open square) at a concentration of 1%, used as sole carbon source, compared to modified Mandels medium (open circle) that was used as a control. b Zymogram analysis showing bglG activity, using MU-Glc as substrate: Glc refers to glucose, Celb to cellobiose, and BC to biological control (Mandels medium)

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Fig. 2 Summary of the tree chemical equations describing the reactions catalyzed by bglG (E1, E3) and CDH (E2)

CDH

(E1)

Cellobiose

Cellobionolactone

bglG

(E2)

Cellobiose

Glucose

bglG

+

(E3)

Cellobionolactone

Cellobiose

Bgl Glucose

CDH

Delta-gluconolactone Bgl

Cellobionolactone

Glucose

Fig. 3 Proposed model explaining the data given after the analysis of the time course of bglG production in the presence of cellobiose or glucose, used as unique carbon sources, at a concentration of 1%

Description of the proposal model We suggest that, during the time course of bglG production in the cellobiose Mandels medium, cellobiose, the substrate of both enzymes (CDH and b-glucosidases), would be converted by CDH and bglG on cellobionolactone and glucose residues, respectively (Fig. 2E1, E2). Afterwards, and as described in (Fig. 2E3), bglG would be trapped by this acidic lactone, considered as its potential substrate, and then cleaved into glucose and d-gluconolactone. This released acidic lactone, known as a specific and strong inhibitor of b-glucosidases, would, then, inhibit bglG.

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Glucose

+

-Gluconolactone

Figure 3 summarizes and illustrates these propositions involved in the explanatory model presented in the current study. In fact, several points need to be proved so as to strengthen the validity and reliability of this probing mode. The latter include providing evidence for the following: (1) the presence of CDH in the cellobiose Mandels medium; (2) the capacity of bglG to cleave cellobiose into glucose units; (3) the inhibitory effect of the aliquots in the cellobiose Mandels medium during bglG activity assay, i.e. the presence of a bglG inhibitory factor in the cellobiose culture medium, and; (4) The inhibitory effect of d-gluconolactone with regards to bglG. Validation of the proposed model The first point that needed confirmation in the model proposed in the present study was the presence of CDH in the cellobiose Mandels medium. In fact, the findings provided ample evidence for CDH activity in the culture medium containing glucose or cellobiose. As Fig. 4 illustrates, CDH was produced by S. microspora grown on the cellobiose culture medium, but not on the glucose one. This particular result provided support to the plausibility of equation E2 illustrated in Fig. 2, which represents a significant part of the model described in Fig. 3. The second point that required validation related to the ability of bglG to cleave cellobiose into glucose units. The

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27 100

0,35

0,3

Residual activity (%)

Optical Density (575 nm)

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0 0

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Time (min) Fig. 4 Evidence for the presence or absence of CDH activity in the Stachybotrys microspora culture, in the presence of glucose (open square) or cellobiose (closed diamond) as sole carbon sources

Fig. 6 The effect of aliquots taken from the cellobiose culture medium on the activity of bglG (produced in the presence of glucose). The same result was proved before and after the heat treatment of these aliquots for 5 min at 100°C

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Fig. 5 The ability of bglG to split cellobiose into glucose units. The amount of glucose released during the reaction was determined using God kit

data presented in Fig. 5 clearly demonstrated this ability, showing that bglG is a cellobiase that has the capacity to split cellobiose into glucose with a recovery of 74%. Accordingly, this finding corroborates the plausibility of the second equation described in Fig. 2E2. So far, then, both the presence of CDH in the cellobiose culture medium and the ability of bglG to cleave cellobiose into glucose units were confirmed. The third, and ultimate, condition that needed to be proved so to further confirm the model proposed in this work pertained to providing evidence for the inhibitory effect that the aliquots from the cellobiose Mandels medium exerted during the pure bglG activity assays. In fact, 50 ll from the supernatant of S. microspora, cultivated on cellobiose Mandels medium and tracked at different time courses (2nd, 4th, 6th, 8th and 10th days), were added to the bglG activity assay medium. The histogram illustrated

bglG residual activity (%)

[Glucose] (g/l)

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[&-gluconolacone] (mM) Fig. 7 The effect of d-gluconolactone on bglG activity. The curve illustrates the inhibition effect of d-gluconolactone, used at various concentrations, during bglG assessment

in Fig. 6 shows that immediately following the addition of the described aliquots, bglG activity underwent a decrease of up to 50% with and without heat treatment (incubation of aliquots at 100°C for 5 min). In fact, this result gave solid support to the assumption that the cellobiose culture medium contains a non-proteinaceous inhibitor of the bglG activity. Once the structural features of the model proposed in the present study were confirmed, further studies were performed to evaluate the effect of d-gluconolactone on the

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Fig. 8 Chromatographic profiles illustrating the rate of consumption of cellobiose by S. microspora during mycelium growth in the cellobiose Mandels medium. a, b, c Summarize the evolution of the amount of cellobiose at the 2nd, 4th and 8th days of culture, respectively, and show the appearance of a novel metabolite on the 8th day of culture

activity of bglG. In fact, Fig. 7 shows that bglG was strongly inhibited by d-gluconolactone, and that the IC50 values were obtained at low molarity (at the concentration of 0.15 mM), which imply that bglG was very sensitive to very small quantities of d-gluconolactone. These findings corroborate the rapid decrease of bglG activity during the time course of its production in the cellobiose culture medium, which, in turn, endorses the fact that this acidic lactone played an inhibitory role observed during bglG assessment (Fig. 3). The range of action of BglG was investigated in the presence of two substrates, namely cellobiose and cellobionolactone. The first is split into two glucose molecules (Fig. 2E1) and the second into glucose and d-gluconolactone (Fig. 2E3), with the latter being the first and strongest inhibitor of b-glucosidases (Davies 2010), particularly bglG (Fig. 7). The ensuing question that arose touched upon the ability of glucose to inhibit bglG. In fact, this inhibition was presupposed to be emanating not from glucose, on the grounds that the measurements of the amounts of glucose during the time course of bglG production showed that the quasi-totality of glucose in the culture medium was consumed during mycelium growth and that the residual amount of glucose could not inhibit bglG (Fig. 8). Furthermore, Fig. 8 clearly shows that a glycosidic metabolite appeared from the 8th day of mycelium growth, which was, presumably, an acidic lactone. In fact, the elution time of glucose was 19.52 min,

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and a novel molecule was detected 17.54 min after the injection of the sample.

Conclusion Overall, the findings presented above provide ample evidence in support for the validity of the explanatory model proposed in the present study and for the plausibility of its underpinning hypothesis. Accordingly, the evolution of kinetic behaviour during enzyme production depends on various parameters, such as carbon source, which promotes the valorisation of biomasses and the production of high added value molecules, such bioethanol, aroma, and d-gluconolactone etc. Acknowledgments This work was supported by grants from the Ministry of Higher Education, Scientific Research and Technology, Tunisia. The authors wish to express their gratitude to Pr. ANOUAR Smaoui from the Sfax Faculty of Science for his constructive proofreading and language polishing services.

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