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1 Introduction

Prussian Blue (PB), or iron (II,III) hexacyanofer-rate (III,II), is an insoluble complex used in differ-ent areas, including detoxification of Tl+ [1] or ra-dioactive 137Cs+ [2]. The enzymatically controlledsynthesis of PB-like structures is of particular in-terest for various nanotechnological applications.Since the structure of PB (Fe(III)4[Fe(II)(CN)6]3) isindistinguishable from Turnbull’s Blue (Fe(II)3[Fe(III)(CN)6]2) [3], principally any enzyme capa-

ble of either reduction of ferricyanide to ferro-cyanide in the presence of Fe3+ (pathway 1), or thereduction of Fe3+ to Fe2+ in the presence of an ex-cess of ferricyanide (pathway 2), would induce for-mation of PB-like pigment in situ.

Ferricyanide can be reduced by FAD- and PQQ-dependent oxidoreductases by oxidation of theirspecific co-substrates (e.g., sugars), whereas lac-cases and peroxidases, in turn, oxidize ferrocyanideby dioxygen or H2O2 [4–6]. The reduction of Fe3+ istypical for the enzymes of anaerobes [7], whereasthe enzymes reducing Fe3+ salts under aerobic con-ditions are rare. Because of that, an enzymaticallyinduced in situ PB synthesis typically involvespathway 1, as it was shown for glucose oxidase(GOD) [8, 9] or yeast flavocytochrome b2 [10] in the

Research Article

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation

Liliya G. Vasilchenko1,*, Roland Ludwig2,*, Olga P. Yershevich1, Dietmar Haltrich2 and Mikhail L. Rabinovich1

1 A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia2 BOKU – University of Natural Resources and Life Sciences, Vienna, Austria

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme forboth bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assayfor CDH in the presence of various fungal oxidoreductases was developed. It is based on Pruss-ian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferri-cyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting withan excess of Fe3+ (pathway 1) and reduction of ferric ions to Fe2+ reacting with the excess of ferri-cyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domainlacking the cytochrome domain formed PB only via pathway 1 and was less active than the parentenzyme. The assay can be applied on active growing cultures on agar plates or on fungal culturesupernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreduc-tases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) norlaccase interfered with CDH activity in this assay. Applicability of the developed assay for the se-lection of new ascomycetous CDH producers as well as possibility of the controlled synthesis ofnew PB nanocomposites by CDH are discussed.

Keywords: Cellobiose dehydrogenase · Ferricyanide · Fe3+ reduction · High-throughput assay · Prussian Blue

Correspondence: Prof. Mikhail L. Rabinovich, A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, RussiaE-mail: mirabi@mail.ru

Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid);CDH, cellobiose dehydrogenase; cyt, cytochrome; DCIP, 2,6-dichloroin-dophenol; DH, dehydrogenase; GOD, glucose oxidase; HRP, horseradishperoxidase; PB, Prussian Blue * Both the authors contributed equally to this work.

Received 29 NOV 2011Accepted 26 JAN 2012Accepted article online 31 JAN 2012

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presence of ferricyanide and an Fe3+ or a substitut-ing Me3+ ion.

A rare example of enzyme-catalyzed Fe3+ re-duction under aerobic conditions is displayed bythe extracellular fungal flavocytochrome cellobiosedehydrogenase (EC 1.1.99.18, cellobiose:[acceptor]1-oxidoreductase; CDH) [11], which consists of anFAD-containing dehydrogenase (DH) domain anda cytochrome (cyt) domain bearing heme b. CDH iscapable of reducing both ferricyanide and Fe3+

salts, although with different reaction rates [11–13].Therefore, formation of PB in the presence of CDHand a specific substrate (cellobiose or lactose) mayproceed via both pathways. Contrary to that, thecatalytically active DHs lacking the cyt domain,which are usually formed by limited proteolysis ofCDH in the course of fungal cultivation [12], reducequinones, 2,6-dichloroindophenol (DCIP), and fer-ricyanide [14], but not Fe3+ salts.

Several CDHs are studied in bioelectrodevices,which involve direct electron transfer (DET) fromthe cyt domain of CDH to the anode of an electro-chemical cell [15, 16]. Recently, a novel oxidativepathway of lignocellulose decomposition involvingelectron transfer between CDH and a copper-de-pendent polysaccharide monooxygenase (Cel61)[17, 18] was also discovered. Until now, the only re-action to distinguish intact CDHs from their trun-cated DH domains, which do not support the DET-based applications, was the ability of the CDHholoenzyme to reduce cyt c3+ with cellobiose or lac-tose. However, this assay is expensive and relative-ly complicated in an extensive screening. Further-more, a routine screening of CDH activity in thefungal strains with DCIP or quinone reduction canbe completely masked by contaminating laccases,which immediately re-oxidize the reduced prod-ucts. This makes formation of PB by CDH in thepresence of cellobiose, ferricyanide, and Fe3+ ions apromising alternative to the existing high-through-put screening techniques for this enzyme.

PB and its analogs are also of interest as “artifi-cial peroxidases” catalyzing electrochemical H2O2reduction. This has stimulated application of PB to-gether with GOD or other H2O2-forming oxidases incorresponding biosensors [19]. Controllable deposi-tion of PB nanoparticles might substantially im-prove the quality of screen-printed electrodes forbiosensors [20, 21]. A controlled deposition of PBanalog neodymium (III) ferrocyanide on a GOD/chi-tosan-modified glassy carbon electrode was, e.g.,performed by the enzymatic reduction of ferri-cyanide with glucose in the presence of Nd3+ [8].

In another example [10] PB formation was in-duced by the oxidation of L-lactate with L-lac -tate:cyt c-oxidoreductase (EC 1.1.2.3; flavocyto -

chrome b2) in the presence of ferricyanide andFe3+. Controlled preparation of PB or its hetero-bimetallic analogs of narrow (nano)size distribu-tion, desired shape, surface texture, and organiza-tion also opens new areas of PB application in mo-lecular magnets [22, 23], ferromagnetic Lang-muir–Blodgett films [24], nanowires [25], contrastagents for magnetic resonance imaging, and mate-rials for drug delivery [26, 27].

This research was undertaken to study the abil-ity of different FAD-containing carbohydrate oxi-doreductases to form PB in the presence of bothFe3+ and ferricyanide and to develop a selective, re-liable, and cheap high-throughput screening assayfor CDH.

2 Materials and methods

2.1 Reagents

D-Glucose, D-xylose, L-arabinose, D-galactose, D-man -nose, cellobiose, α-cellulose, NH4Fe(SO4)2 ⋅ 12H2O,K3[Fe(CN)6], 2,2′-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS), DCIP, and salts for bufferpreparation were obtained from Sigma-Aldrich (St. Louis, USA) and were of analytical or higherpurity grade.

2.2 Organisms and culture conditions

The ascomycetes Diplodia pinea F-1176 and F-1177, D. pruni F-1481, D. zeae F-3611, Melanocarpusalbomyces F-1737 and F-1738, Hypoxylon nummu-larium F-1247, Papulaspora biformospora F-1635,Wallrothiella subiculosa F-3029 were obtained fromthe VKM (All-Russian Collection of Microorgan-isms). Chaetomium sp. INBI 2-26(−) was isolatedand identified as described previously [28]. Thecultures were maintained on potato dextrose agarslants.

To induce carbohydrate oxidoreductase activity,solid agar-plate cultivations of these fungi wereperformed on 2% agar containing (g/L): (NH4)2SO4(1.5), NaNO3 (1.5), KH2PO4 (1.0), MgSO4 ⋅ 7H2O (0.5),α-cellulose (20.0), and were enriched with either amixture of xylose (2.0), arabinose (2.0), galactose(2.0), mannose (2.0) and cellobiose (2.0) (medium1), milled sugar-beet pulp (15.0) and malt sprouts(6.0) (medium 2), or milled sugar-beet pulp (15.0),malt sprouts (6.0) and Bacto Tryptone (Difco,5.0)(medium 3). After 7 days of cultivation, ca. 5 mm ×5 mm agar slabs with developed mycelia were cutout with a sterile scalpel and used in the PB screen-ing assay.

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Inocula for the submerged cultivation ofChaetomium sp. INBI 2-26(−), M. albomyces F-1737and F-1738 were grown for 10 days on solid 2% agarmedium containing (g/L): KH2PO4 (1.0), MgSO4 ⋅7H2O (0.5), KCl (0.5); NH4Cl (0.45) α-cellulose(20.0), Bacto Tryptone (5.0) and trace element solu-tion (0.5 mL) consisting of (g/L): H3BO3 (1.0), FeSO4⋅ 7H2O (0.5), CuSO4 ⋅ 5H2O (0.2), CoCl2 ⋅ 6H2O (1.0),ZnSO4 ⋅ 7H2O (0.8), NH4)6Mo7O24 ⋅ 7H2O (0.1), andconcentrated H2SO4 (2 mL). Cultivations were per-formed in 750-mL shaken flasks containing 200 mLof the same medium and inoculated with10 mm × 10 mm inoculum slabs. Chaetomium sp.and M. albomyces were grown for 14 days at220 rpm at 28 and 37°C, respectively. CDH activityin the culture broth was measured with cellobioseand DCIP as substrates [28].

2.3 Enzymes

Highly purified CDH (Rz 0.53) and DH from S. rolf-sii (SrCDH and SrDH), as well as CDH fromChaetomium sp. INBI 2.26(−) (ChCDH, Rz 0.53),Myriococcum thermophilum (Rz 0.50, MtCDH), andCorynascus thermophilus (Rz 0.60, CtCDH) wereprepared as described in [29–32]. Pyranose DHfrom Agaricus meleagris (EC 1.1.99.29, AmPDH)was produced and purified as described in [33, 34].Purified GOD of Penicillium adamezii (PaGOD,[35]) and P. funiculosum (PfGOD, [36, 37]) were ob-tained from the Institute of Microbiology, NationalAcademy of Sciences, Belarussian Republic, Minsk.Laccase from Trametes pubescens (TpLacc) waspurified as described in ref. [5]. Horseradish per-

oxidase (HRP) salt-free preparation with 150 U/mg(Rz 1.4) was purchased from Sigma–Aldrich.

2.4 Enzyme reactions

Reduction of Fe3+ acetate by SrCDH and SrDH wasstudied spectrophotometrically in 0.1 M Na-acetatebuffer, pH 3.0–5.0 and an absorption coefficient forFe3+ of 1.33 mM–1cm–1 at 340 nm (25°C) [11]. Re-duction of ferricyanide by SrCDH and SrDH inthe present of a saturating cellobiose concentration(5 mM) was studied in sodium acetate buffer,pH 2.0–5.0 and 30°C using an absorption coefficientfor [Fe(CN)6]

3– of 1.04 mM–1cm–1 at 420 nm [11].Formation of PB by SrCDH and ChCDH in the pres-ence of 0.5–2 mM NH4Fe(SO4)2 and 2.5–10 mMK3[Fe(CN)6] was monitored at 700 nm in 0.1 M Na-acetate buffer, pH 3.0–5.5 and 25°C. In all casessaturating (5 mM) cellobiose concentration wasused. Further details are given in the legends to theFigs. 1–3. All kinetic experiments were performedin triplicates.

2.5 Data analysis

The Microcal Origin 6.1 Software was used to fit theinitial velocity data by non-linear least-squares re-gression to an equation for a Ping-Pong mecha-nism by using Eq. (1). All kinetic constants werecalculated by fitting the data to the Michaelis-Menten equation using Origin 6.1. This softwarewas also used for fitting substrate/time progresscurves to the integrated form of the Michaelis-Menten equation.

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A B

Figure 1. pH-Dependencies of ferricyanide reduc-tion by SrCDH (squares, solid lines) and SrDH (circles, dotted lines). (A) Catalytic constants at saturating ferricyanide concentrations. (B) Apparentfirst order rate constants at 50 μM ferricyanide con-centration. Cellobiose concentration 5 mM, 50 mM Na-citrate buffer, 30°C. Each experimental point is a mean of triplicate (SD < 5%). Theoretical curveswere built by means of Microcal Origin 6.1 softwarepackage.

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2.6 Microtiter plate assays

Microtiter plate assays were performed in standard96-well microtiter plates. In situ and ex situ forma-tion of the PB-like pigment was studied under theconditions described in Table 1, rows 1–7. Enzymat-ic DCIP reduction was elucidated as described inTable 1, rows 8–15. Oxidation of ABTS in the pres-ence of HRP (5 pM) coupled with H2O2 productionby carbohydrate oxidases was studied as describedin the rows 16–18 of Table 1. The plates were incu-bated at 25°C for 30–120 min and then scanned.

2.7 Solid agar plate screening assay

Agar slabs (5 mm × 5 mm) of the fungal culturesgrown on solid media 1, 2, or 3 were placed on aplate and then 20 mL of molten 2% agar containing0.15 mM cellobiose (or 0.21 mM of D-glucose, D-xy-lose, L-arabinose, D-galactose, or D-mannose),0.78 mM K3[Fe(CN)6] and 0.16 mM NH4Fe(SO4)2 in0.1 M Na-acetate buffer, pH 4.0 cooled to 42–43°Cwas added. After solidifying, the plates were incu-bated for 2–3 h at room temperature and monitoredfor the development of a blue halo around themycelial slabs.

3 Results

3.1 Kinetics of ferricyanide and Fe3+ reduction and PB formation by CDH and DH

Figure 1 shows the effect of the pH value on the re-duction of ferricyanide by both intact SrCDH andits truncated form, SrDH. Both enzyme forms showwell comparable reaction rates of the ferricyanidereduction with cellobiose at saturating concentra-tions of both substrates over the entire pH rangestudied (Fig. 1A), while a substantially lower reac-tion rate and lower catalytic efficiency (kcat/Km)was found for SrDH compared with the parent Sr-CDH (Fig. 1B, see also Table 2) at ferricyanide con-centration below its Km,app. In addition, SrDHshowed a shift of its pH optimum to a more acidicregion compared with SrCDH, particularly at lowferricyanide concentrations (Fig. 1B).

Since the difference between kcat/Km values ofSrCDH and SrDH is much higher than between thekcat of both species, the application of lower ferri-cyanide concentrations at pH 4.5 is very useful todistinguishing these enzyme species.

Yet another difference between the two enzymeforms was the inability of SrDH to reduce ferric ac-

Table 1. Reaction mixtures in the wells of the microtiter plates shown in Fig. 4

Column A B C D E F G HRow SrCDH SrDH MtCDH CtCDH ChCDH AmPDH PaGOD PfGOD

1 0.1 M Na-acetate buffer pH 4.5; 0.16 mM NH4Fe(SO4)2 + 0.78 mM K3[Fe(CN)6] + 0.15 mM cellobiose2 0.1 M Na-acetate buffer pH 4.5; 0.16 mM NH4Fe(SO4)2 + 0.15 mM cellobiose; after 2 h incubation 0.78 mM K3[Fe(CN)6]

was added3 0.1 M Na-acetate buffer pH 4.5; 0.78 mM K3[Fe(CN)6] + 0.15 mM cellobiose; after 2 h incubation 0.16 mM NH4Fe(SO4)2

was added4 0.1 M Na-acetate buffer pH 4.5; 0.16 mM NH4Fe(SO4)2 + 0.78 mM K3[Fe(CN)6] + 0.15 mM cellobiose + TpLacc5 0.1 M Na-acetate buffer pH 4.5; 0.16 mM NH4Fe(SO4)2 + 0.78 mM K3[Fe(CN)6] + 0.21 mM glucose6 0.1 M Na-acetate buffer pH 4.5; 0.16 mM NH4Fe(SO4)2 + 0.78 mM K3[Fe(CN)6]7 0.1 M Na-acetate buffer pH 4.5; 0.16 mM NH4Fe(SO4)2 + 0.78 mM K3[Fe(CN)6] + 0.15 mM cellobiose + 0.21 mM glucose,

no carbohydrate oxidoreductase added8 0.1 M CPB pH 4.5; 0.1 mM DCIP + 0.15 mM cellobiose9 0.1 M CPB pH 4.5; 0.1 mM DCIP + 0.15 mM cellobiose + TpLacc

10 0.1 M CPB pH 4.5; 0.1 mM DCIP + 0.21 mM glucose11 0.1 M CPB pH 4.5; 0.1 mM DCIP + 0.15 mM cellobiose + 0.21 mM glucose; no carbohydrate oxidoreductase added12 0.1 M CPB pH 7.0; 0.1 mM DCIP + 0.15 mM cellobiose13 0.1 M CPB pH 7.0; 0.1 mM DCIP + 0.15 mM cellobiose + TpLacc14 0.1 M CPB pH 7.0; 0.1 mM DCIP + 0.21 mM glucose15 0.1 M CPB pH 7.0; 0.1 mM DCIP + 0.15 mM cellobiose + 0.21 mM glucose; no carbohydrate oxidoreductase added16 0.1 M Na-acetate buffer pH 4.5; 0.53 mM ABTS + HRP + 0.21 mM glucose17 0.1 M Na-acetate buffer pH 4.5; 0.53 mM ABTS + HRP + TpLacc18 0.1 M Na-acetate buffer pH 4.5; 0.53 mM ABTS + HRP + 0.21 mM glucose; no carbohydrate oxidoreductase added

SrCDH, intact CDH from S. rolfsii; SrDH (also SrCBQR or cellobiose: quinone oxidoreductase, see column B in Fig. 4), flavin domain of CDH from S. rolfsii; MtCDH,CDH from M. thermophilum; CtCDH, CDH from C. thermophilus; ChCDH, CDH from Chaetomium sp. INBI 2.26 (−); AmPDH, pyranose dehydrogenase from A. melea-gris; PaGOD, glucose oxidase from P. adamezii; PfGOD, GOD from P. funiculosum; TpLacc, laccase from T. pubescens; HRP, horseradish peroxidase. CBP, citrate-phos-phate buffer. Rows 7, 11, 15, 18: no carbohydrate oxidoreductases added.

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etate (0.1–1 mM) with saturating amounts of cel-lobiose even by 100-fold more concentrated en-zyme compared with ferricyanide reduction, whileparent SrCDH readily reduced Fe3+ in the presenceof cellobiose, although with lower reaction ratesthan those for ferricyanide (Fig. 2). The kcat of thisreaction decreases more than 30 times from pH 3.5to 5.0, whereas the bimolecular constant kcat/Km –corresponding to the reaction rate at low substrateconcentrations – seems to be almost independenton pH in this range (Table 2). This differs Fe3+ fromferricyanide, where kcat/Km decreases with increas-ing pH.

Figure 3 illustrates the pH-dependence of the insitu PB formation in the reaction mixture of ferri-cyanide and Fe3+ reduced by cellobiose or glucose inthe presence of intact SrCDH or ChCDH. Althoughthe spectrum of PB slightly changes with pH (datanot shown), it is obvious that acidic SrCDH formsPB most actively at pH ∼ 3.5, whereas the neutralChCDH is most active at a pH near 4.5 and retains ameasurable activity up to at least pH 5.0. The same

optimums as for cellobiose were obtained by bothenzymes with glucose as substrate, although the ac-tivity of the basidiomycetous SrCDH in this reactionwas two orders of magnitude lower than that ofneutral ascomycetous ChCDH (cf. [38]).

3.2 Comparison of different carbohydrateoxidoreductase assays

In Fig. 4 different assays and assay conditions forvarious carbohydrate oxidizing enzymes, based onthe formation of PB, or the ABTS cation radical, orthe reduction of DCIP are compared. To distinguish

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Table 2. Catalytic efficiencies (kcat/Km) for the electron transfer from thereduced enzyme to the electron acceptor at 30°C

pH kcat/Km,A (kmin1,2) (mM–1s–1)

SrCDH SrDH

Fe(CH3COO)3a) [Fe(CN)6]

3– [Fe(CN)6]3–

2.5 n.d. 11 400 ± 1000 1050 ± 503.5 127 ± 17 3210 ± 500 260 ± 204.0 133 ± 18 n.d. n.d.4.5 153 ± 13 729 ± 50 16 ± 34.75 217 ± 27 n.d. n.d.

a) 25°C; n.d., not determined.

Data are the average of triplicate measurements ± the SD.

Figure 2. pH-Dependence of the catalytic constant of Fe3+ reduction bySrCDH in the presence of 5 mM cellobiose in 0.1 M Na-acetate buffer at25°C. Error bars denote SD.

A

B

Figure 3. PB formation induced by the oxidation of 5 mM cellobiose(squares, triangles) or 50 mM glucose (circles) with two different CDHs atdifferent pH (0.1 M Na-acetate buffer, 25°C). Activity of corresponding enzyme toward cellobiose at its pH-optimum was taken as 100%. (A)Acidic SrCDH in the presence of a mixture of 2.5 mM Fe(CN)6

3– + 0.5 mMFe3+ (triangles) or 10 mM Fe(CN)6

3– + 2 mM Fe3+ (squares). Inset: magni-fication for the relative activities by glucose oxidation in the presence of10 mM Fe(CN)6

3– + 2 mM Fe3+. (B) Neutral ChCDH in the presence of10 mM Fe(CN)6

3– + 2 mM Fe3+. Each experimental point is a mean of tripli-cate (SD < 5%). The solid and dotted lines were obtained by fitting sig-moid (A) and Gaussian multipeaks (B) equations to the data for SrCDHand ChCDH, respectively, using Microcal Origin 6.1 software.

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DHs from oxidases, which can also reduce ferri-cyanide or DCIP after dissolved oxygen depletion,concentration of reducing carbohydrate was takenbelow that of dissolved oxygen (0.25 mM) in all as-says. SrCDH, MtCDH, CtCDH, ChCDH, and AmPDHwere diluted to a nearly equal activity toward DCIPreduction with cellobiose at pH 4.5 (Fig. 4, row 8 forCDHs, row 10 for AmPDH), whereas SrDH wasused in a threefold excess of its DCIP activity overthe parent SrCDH. This higher activity is even vis-ible at pH 7.0 (Fig. 4, rows 12, 13), where the acidicSrCDH was almost not detectable because of high-

er dilution. The activity of PaGOD in the coupledABTS-HRP assay was taken twice as high as that ofPfGOD (cf. Fig. 4, wells G16, H16). The activity ofthe acidic TpLacc was adjusted to reach almostcomplete reversion of DCIP reduction by the test-ed CDHs at acidic pH (cf. Fig. 4 rows 8, 9 and theblank row 11, where no carbohydrate oxidoreduc-tases were added).

As follows from Fig. 4 row 9, laccase strongly in-terferes with the DCIP assay of carbohydrate DHsat pH 4.5, although it has no effect at pH 7.0 whereTpLacc is not active (cf. rows 12 and 13). The cou-pled ABTS-HRP assay for H2O2-forming oxidasesalike PaGOD or PfGOD (Fig. 4, row 16) is also notapplicable at pH 4.5 in the presence of laccase,which immediately oxidizes ABTS with dissolvedoxygen regardless of the H2O2 production (Fig. 4,row 17).

In the presence of Fe3+/ferricyanide and cel-lobiose, all CDHs form PB at pH 4.5 (Fig. 4, row 1),although ascomycetous ChCDH, MtCDH, andCtCDH are more active at this pH than the acidicbasidiomycetous SrCDH (cf. Fig. 3). SrDH alsoforms PB, although substantially slower despite ofits threefold higher DCIP activity. This results fromlower activity of SrDH toward ferricyanide at thispH (Fig. 2). Other tested enzymes including Am-PDH displayed no activity in this assay in the pres-ence of cellobiose.

Incubation of the aforementioned DHs with Fe3+

acetate and cellobiose (Fig. 4 row 2) and subse-quent staining with ferricyanide results in a less in-tense PB formation. This is particularly obvious forSrDH, where almost no color is developed in spiteof the threefold higher DCIP activity compared to SrCDH. Ascomycetous ChCDH, MtCDH, andCtCDH are, in contrast, quite active under theseconditions because of their ability of Fe3+ reduction(cf. Fig. 3 for ChCDH). In the inverse sequence ofreagent addition, namely incubation of the en-zymes with ferricyanide and cellobiose and subse-quent staining with the Fe3+ salt (Fig. 4, row 3), theobtained color intensities are close to those ob-tained by the incubation of the enzyme directlywith the Fe3+/ferricyanide mixture. This confirms a domination of pathway 1 (reduction of ferri-cyanide) over pathway 2 (reduction of Fe3+) in theblue pigment formation.

The interfering TpLacc activity, which com-pletely abolishes the discoloration of DCIP byCDHs at acidic pH (cf. Fig. 4, rows 12 and 13), onlymoderately inhibited PB formation (Fig. 4, row 4).TpLacc can inhibit the pathway 1 by re-oxidizingthe product ferrocyanide under aerobic conditions[5]; however, PB can still be formed by CDHholoenzymes via pathway 2. Apparently, laccase re-

Figure 4. Comparison of different carbohydrate oxidoreductase assays in96-well plates. Rows 1–7: PB formation in the presence of ferricyanide andFe3+ salt; rows 8–15: enzymatic reduction of DCIP; rows 16–18: coupledABTS/HRP test. For details see Section 2 and Table 1.

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directs the formation of the pigment from pathway1 to pathway 2, thus inhibiting PB formation bySrDH but not by CDH holoenzymes (cf. rows 2 and4). Therefore, this assay can be selectively used forthe screening of fungal producers of CDH holoen-zymes even in the presence of laccase.

At low glucose concentrations, neither CDHsnor AmPDH induced PB formation, although Am-PDH reduced DCIP with either 0.21 mM glucose or0.15 mM cellobiose over a wide pH-range (cf. rows10 and 14). Under aerobic conditions, PaGOD wasalso inactive toward ferricyanide or DCIP reduc-tion with glucose. Contrary to that, PfGOD trans-ferred electrons from 0.21 mM glucose on dioxygen(row 16), DCIP at both acid (row 10) and neutral(row 14) pH, or ferricyanide (row 5) with compara-ble efficiencies.

3.3 Screening of ascomycetes capable of PB formation

To demonstrate the applicability of the developedassay, we screened several ascomycetes for CDHbiosynthesis when grown in the presence of α-cel-lulose and different carbohydrates (media 1, 2, and3). PB formation was tested in agar plate screeningassays in the presence of either cellobiose or a mix-ture of monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, L-arabinose). No PB formationwas observed with either culture in the presence ofcarbohydrates other than cellobiose. However, sev-eral strains induced PB formation when cellobiosewas present (Fig. 5), indicating tentative CDH pro-duction. The CDH-positive reference organism,Chaetomium sp. INBI 2-26(−) formed blue halos re-gardless of the solid medium (1, 2, or 3; slants10–12) used for its growth and induction. Both test-ed M. albomyces strains formed PB only aftergrowth on the solid medium containing α-cellulosesupplemented with sugar-beet pulp and maltsprouts (medium 2; slants 2, 4, 7, 8)1. An enrichmentof this medium with Bacto Tryptone (medium 3)has, in turn, suppressed PB formation (slant 3).Hypo xylon nummularium F-1247, Diplodia pineaF-1176, D. pruni F-1481, and Papulaspora biformo-spora F-1635 did not induce PB formation aftergrowth on either of solid media (1, 2, or 3; slants 1,5, 6, 9). The ability of the M. albomyces strainsF-1737 and F-1738 to produce CDH was further

confirmed by submerged cultivation in cellulose-containing medium. Both selected strains formedca. 150 U/L of CDH activity with DCIP as electronacceptor at pH 6.0 and 20°C in compliance with therecently reported ability of another M. albomycesstrain to produce CDH [31].

4 Discussion

4.1 Activity of CDH toward reduction of ferric complexes

From the results of Table 2 the following observa-tions can be made:(i) The electron transfer to ferricyanide from the

reduced flavocytochrome SrCDH proceedssubstantially faster than from its truncatedFAD-domain (SrDH). This confirms an impor-tant role of the cyt domain of CDH in the one-electron transfer from the reduced FADH2 toiron complexes [12] and may be explained bybetter accessibility of the exposed heme b edgein the reduced cyt domain compared withFADH2 in CDH.

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Figure 5. PB plate screening assay. Ascomycetes from the All-Russian Microbial Collection VKM as well as the reference strain Chaetomium sp.INBI 2-26(−) producing neutral ChCDH were cultivated on solid agarmediums 1, 2, or 3 (details see in Section 2) and ca. 5 mm × 5 mm slabs cut from the developed cultures were transferred to another plateand poured with molten 2% agar containing cellobiose (0.15 mM),K3[Fe(CN)6] (0.78 mM), and FeNH4(SO4)2 (0.16 mM) in 0.1 M Na-acetatebuffer, pH 4.5. After solidification of agar the plates were incubated for 3 hat room temperature. Numeration of the agar slants: 1 – Hypoxylon num-mularium F-1247 (medium 2); 2, 3, 8 – Melanocarpus albomyces F-1738(mediums 2, 3, and 2 respectively); 4, 7 – M. albomyces F-1737 (both onmedium 2); 5 – Diplodia pinea F-1176 (medium 2); 6 – D. pruni F-1481(medium 2); 9 – Papulaspora biformospora F-1635 (medium 2); 10–12 –Chaetomium sp. INBI 2-26(−) (mediums 1, 2, and 3, respectively).

1 Upon extended incubation M. albomyces strains can cause partial PB dis-coloration around the colonies, which results in a formation of an uncol-ored halo between colony and the blue halo (see Fig. 5) apparently be-cause of excessive PB reduction with Prussian White (Fe2(FeCN)6) for-mation.

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(ii) The electron transfer from the reduced flavo-cytochrome to Fe3+ acetate differs from thatwith ferrocyanide by substantially lower cat-alytic efficiency and its pH-dependence.

Broad specificity of FAD-dependent DHs toward avariety of electron acceptors as well as the absenceof product inhibition of CDH with the reduced elec-tron acceptors [30] assume a bimolecular en-zyme–acceptor interaction rather than a kinetical-ly significant complexation of acceptors. Therefore,the catalytic cycle of CDH in the reaction with aone-electron acceptor may be expressed as follows(Eq. 1, the products are omitted for simplicity):

(1)

where S denotes a specific carbohydrate substrateand A is a non-specific one-electron acceptor. Thecatalytic efficiency, kcat/Km,A of CDH toward an ac-ceptor A is defined by the slowest of the two rateconstants characterizing interaction of A with ei-ther the two-electron-reduced enzyme Er or a one-electron-reduced intermediate Er1. Changes in thepH may exert different effects on approachingelectron acceptors by affecting both the net chargeof A and the local charge distribution near the re-dox center of Er or Er1. This may explain differencesin the pH-dependence of kcat/Km,A between thenegatively charged ferricyanide anion and the ironcation (Table 2), which should form predominantlyFe(OAc)3 in the case of Fe3+ or [Fe(OAc)]+ in thecase of Fe2+ in 0.1 mM Na-acetate buffer at pH 4.0[11].

The midpoint redox-potential of the coupleFe3+/Fe2+ in anaerobic 0.1 M Na-acetate buffer, pH4 (517–540 mV [11]) is substantially higher than theredox-potential of the ferri/ferrocyanide couple(370–436 mV vs. NHE, depending on pH and potas-sium ion concentration). Therefore, according to[39], a slower electron transfer from reduced CDHto an Fe3+ salt compared with ferricyanide is obvi-ously explained by contribution of the larger innerand outer sphere reorganization energy in the re-action (Eq. 2):

(2)

compared with the reaction (Eq. 3):

(3)

Understanding the kinetics of enzymatic Fe3+ ac-etate and ferricyanide reduction provides an in-strument for controlled production of the desirablePB forms. CDH or DH can principally induce both

E S ES E A E A Er r1+ ⇔ → + → + →

Fe(OAc) e [Fe(OAc)] 2AcO3++ → +− −

[Fe(CN) ] e [Fe(CN) ]63

64+ →− − −

insoluble PB (Eqs. (4, 6) for DH, (4–7) for CDH) andthe potassium-rich “soluble” PB formation (Eq. 8):

(4)

(5)

(6)

(7)

(8)

The presence of an acidic laccase prevents accu-mulation of ferrocyanide (Eq. 9):

(9)

and hinders formation of PB with DH but not withCDH. Thus, varying pH, relative concentrations offerricyanide and ferric ions, and, if necessary, lac-case, one can aerobically obtain a desirable form ofPB with CDH or DH.

4.2 Distinguishing different types of carbohydrateoxidoreductases with the PB assay

Based on our observations, all tested FAD-depend-ent oxidoreductases can be divided into two nega-tive (N) and three positive (Y) groups according totheir ability to aerobically induce PB formationfrom low (<0.8 mM) concentrations of ferricyanideand an Fe3+ salt in the presence of a specific sugarsubstrate, namely:

(1N) Oxidases similar to PaGOD, which reduceoxygen to H2O2 rather than ferricyanide and there-fore do not induce PB formation at carbohydrateconcentration below 0.25 mM.

(2N) DHs similar to AmPDH, which reduceDCIP, but neither reduce ferricyanide nor depositPB under the assay conditions.

(1Y) Mixed-type oxidases/DHs similar to PfGOD,which reduce either 0.78 mM ferricyanide or

4Fe CN 4H O

4Fe CN 2H O64 +

2(laccase, rapidly)

63

2

( )

( )

+ + ⎯ →⎯⎯⎯⎯⎯⎯

→ +

−

−

cellobiose 2Fecellobiono- -lactone 2Fe + 2H

3+ (CDH, slowly)

2++ ⎯ →⎯⎯⎯⎯⎯

→ δ + ++

3Fe CN 4Fe Fe Fe CN

“insoluble” PB64 3+

4 6 3( ) ( )

( )

+ → ⎡⎣

⎤⎦

−

2Fe CN 3Fe Fe Fe CN

“Turnbull's Blue”63 2+

3 6 2( ) ( )

( )

+ → ⎡⎣

⎤⎦

−

Fe(CN) Fe (or Fe(CN) Fe ) + KKFe[Fe(CN) ] (“soluble” PB)

64 3+

63 2+ +

6

+ + →→

− −

cellobiose 2Fe CN

cellobiono- -lactone 2Fe CN + 2H63 CDH, rapidly or DH, slowly

64

( )

( )

+ ⎯ →⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯

→ δ + ++

( )−

−

© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 9

0.1 mM DCIP in the presence of 0.25 mM O2, butalso reduce oxygen itself.

(2Y) DHs similar to SrDH, which reduce either0.78 mM ferricyanide or 0.1 mM DCIP.

(3Y) DHs similar to holoenzyme CDH, which re-duce either quinones and DCIP or one-electron ac-ceptors including both ferricyanide and Fe3+ salts.

Although typical GODs (group 1N) can reduceferricyanide by glucose oxidation [40, 41], aerobicformation of PB with these enzymes in the pres-ence of trivalent transition metal cations requireshigh concentrations of ferricyanide (20 mM) [8] be-cause of higher reactivity of most GODs towarddioxygen compared to ferricyanide. At low glucose(0.21 mM) and ferricyanide (0.78 mM) concentra-tions the typical GODs such as PaGOD [35] do notinduce PB formation in the presence of Fe3+ (Fig. 4,column g).

Contrary to GODs, FAD-dependent glucose de-hydrogenases (GDH) from Aspergilli [42–44] orGlomerella cingulata [44, 45] transfer electronsfrom glucose to quinones or ferricyanide ratherthan to dioxygen. PfGOD [36, 37] (group “1Y”) alsodemonstrates such a GDH activity by reducingbenzoquinone [36], DCIP, or ferricyanide (Fig. 4) ataerobic conditions, although with concomitant H2O2production. PB-assay distinguishes this mixed-type enzyme from the true GODs by the ability ofPB formation under aerobic conditions in the pres-ence of 0.2 mM glucose.

An opposite example of a true FAD-containingDH is AmPDH (group “2N”; Fig. 4, column f), whichreduces DCIP by oxidizing either glucose (prefer-ably) or cellobiose at pH 4.5 or 7.0. Slow reductionof ferricyanide and even of Cu2+ cation was also re-ported for a related PDH from Agaricus xanthoder-ma at pH 2.0 [46]. However, in the optimized PB as-say conditions AmPDH does not induce PB forma-tion from 0.78 mM ferricyanide + 0.16 mM Fe3+ saltwith either of the carbohydrates.

Disaccharide DHs of the groups “2Y” (e. g. SrDH)and “3Y” (CDH holoenzyme) can be distinguishedby the ability of PB formation in the presence of0.15 mM cellobiose via only pathway 1 (group “2Y”)or pathways 1 and 2 (group “3Y”). At equal activitytoward DCIP reduction, DH is substantially less ac-tive than parent CDH in ferricyanide reduction andPB formation at low ferricyanide concentrations.Moreover, formation of PB by enzymes such as DHis suppressed by acidic laccases, while CDH holo -enzymes still induce PB synthesis under these cir-cumstances, by using pathway 2.

The pH value of 4.5 selected for the optimizedPB assay is the optimum for PB formation by neu-tral ascomycetous enzymes, whereas the optimumfor acidic CDH is shifted in a more acidic region.

Neutral and acidic CDH enzymes can be visuallydistinguished by the discoloration of DCIP at pH 7(see Fig. 4). A combination of DCIP- and PB-assaysis therefore sufficient to distinguish neutral flavo-cytochrome CDH from other types of carbohydrateoxidoreductases including DH, even in the pres-ence of laccases. Hence, the PB assay at pH 4.5 cansubstitute the rather expensive ferricytochrome creduction assay for primary identification of prom-ising producers of neutral CDH holoenzymes. M.albomyces F-1737 and F-1738 strains selected bythis assay are examples of potential producers ofsuch neutral CDHs. Although this ascomycete is aknown laccase producer [47], this fact did not in-terfere with its identification as CDH producer bythe developed assay.

4.3 Possible applications of in situ PB depositionby carbohydrate oxidoreductases

Application in biosensors or nanocomposites in-cludes deposition of PB-like structures on differentsurfaces, scaffolds, and templates. The electrore-ductive deposition of PB from aqueous mixtures offerric and ferricyanide ions on conductive surfaces[48], including graphene [49], usually requires pH ∼1.0. This restricts entrapment of acid-labileenzymes into PB films [50] and decreases the over-all sensitivity of H2O2-biosensors for specificmetabolites [48]. To overcome this drawback, aGOD/chitosan film on the surface of a glassy car-bon electrode was doped with neodymium ferro-cyanide nanoparticles deposited in situ by immobi-lized GOD from an aqueous mixture of glucose,Nd3+, and 20 mM ferricyanide [8].

Chitosan and other (bio)polymeric 1D-, 2D-,and 3D-templates were also proposed for con-trolled deposition of PB nanoparticles of uniqueshape, size, and structure (PB-nanoparticle-dopednanocages, magnetic water-soluble PB-polymernanoparticles, 3D-network structured cyanide-based magnets, 1D PB nanowires) for optoelec-tronic, magnetic, and electrocatalytic applications[51–54]. A controllable synthesis of PB by the hy-drolysis of chitosan in a strongly acidic solution ofK3[Fe(CN)6] was described in [51]. Oligosaccha-rides – products of chitosan hydrolysis reduced fer-ricyanide to ferrocyanide, which then formed PB-nanocubes of controlled size with ferric ions ap-pearing by acidic decomposition of ferricyanide, al-though under rather severe conditions withconcomitant HCN production.

We have earlier applied a multienzyme systemcontaining CDH and cellulolytic enzymes absorbedby a cellulosic carrier for a “pseudo-reagent-less”reduction of electron acceptors in a biosensor with

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signal amplification [55]. This system is capable ofcontrollable reduction of ferricyanide with endoge-nously produced cellobiose in very mild (pH3.5–4.5) aerobic conditions and the in situ deposi-tion of PB on cellulosic template in the presence ofFe3+ ions. Compared to other polymers, celluloseoffers a fascinating diversity of templates (nano -crystallites of native Iα (triclinic), Iβ (monoclinic),or mercerized II, III, or IV polymorphous modifica-tions, whiskers, elementary fibrils, nanoporousamorphous structures, nanogels, nanofilms, sepa-rate molecules). This, together with application ofcellulases of different molecular mass and actionpatterns (cellobiose- or glucose-forming, randomor endwise-acting, processive or nonprocessive,specific for reducing or nonreducing end of poly-saccahride [56]), and a ferricyanide-reducing car-bohydrate DH, provides a background for a con-trollable synthesis of PB nanostructures of any de-sirable size and dimension.

PB-cellulosic nanocomposites might be, e.g.,used as molecular sieves permeable for ions with asmall Stokes radius (Cs+, K+, Cl–) and excludinglarge hydrated ions (Na+, Li+, Mg2+, SO4

2–). Suchsieves can be prepared upon multiple sequentialadsorption of metal cations and hexacyanometa-late anions in the form of densely packed particleswith diameters of 10–100 nm on an appropriatehighly porous polymeric supports [57].

Ascomycetous CDHs deposit PB optimally at pH4.5, where commercial cellulases from Trichodermaor Penicillium are the most active [58]. Like the fun-gal cellobiose-producing cellulases (cellobiohydro-lases), ascomycetous CDHs carry a so-called fami-ly I cellulose-binding module (CBMI; [31, 56]). Thismakes possible a specific absorption of cellobiohy-drolases and CDH by the nanoscale pores of (lig-no)cellulose, thus providing there necessary condi-tions for PB deposition. Cellulase and CDH pene-tration into different lignocellulosic materials maytherefore be monitored by PB deposition usingTEM. Variation of the cellobiohydrolase to CDHrelative activity provides a tool for control of theseeding creation and growth processes of PB struc-tures.

Compared to GDH and other ferricyanide-re-ducing enzymes, CDHs do not require high con-centrations of ferricyanide or transition trivalentmetal ions, which can inhibit enzyme activity. CDHsdeposit PB in the pH-range, where the PB-likestructures are quite stable. Unlike GODs, in the aer-obic conditions CDHs do not form substantialamounts of H2O2, which can destroy PB. This makesmultienzyme systems consisting of ascomycetousCDHs and cellobiohydrolases the promising candi-dates for preparation of the PB-based molecular

sieves based on highly porous (ligno)cellulose ma-terials for, e.g., selective absorption of 137Cs cation.

This research was supported by the European Proj-ect NMP4-SL-2009-229255 “3D-Nanobiodevices”and the Program “Molecular and Cell Biology” of thePresidium of Russian Academy of Sciences. Dr.Roland Ludwig is the recipient of the APART fellow-ship 11322 by the Austrian Academy of Sciences. Theauthors are indebted to Prof. Raisa Mikhailova, Dr.Tatyana Semashko, and Dr. Magdalena Kujawa forproviding purified samples of glucose oxidases andpyranose dehydrogenase and to Dr. Karen Kara-petyan for kind assistance by purification of CDHfrom Chaetomium sp.

The authors declare no conflict of interest.

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