Journal of Microbiological Methods 76 (2009) 295–300

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Journal of Microbiological Methods

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Novel precipitated fluorescent substrates for the screening ofcellulolytic microorganisms

Dina R. Ivanen, Natalia L. Rongjina, Sergey M. Shishlyannikov, Galina I. Litviakova,Luidmila S. Isaeva-Ivanova, Konstantin A. Shabalin, Anna A. Kulminskaya ⁎Petersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation Biophysics Division, 188300, Orlova roscha 1, Gatchina, Leningrad District, Russia

⁎ Corresponding author. Molecular and Radiation BNuclear Physics Institute of RAS, 188300, Orlova roschaRussia. Tel./fax: +7 81371 32014.

E-mail address: kulm@omrb.pnpi.spb.ru (A.A. Kulmi

0167-7012/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.mimet.2008.12.008

a b s t r a c t

a r t i c l e i n f o

Article history:

New substrates, 2-(2′-benzo Received 6 July 2008Received in revised form 11 December 2008Accepted 15 December 2008Available online 25 December 2008

Keywords:2-(2′-Benzothiazolyl)-phenylcellooligosaccharidesCellulasesFluorescent precipitated substrates

thiazolyl)-phenyl (BTP) cellooligosaccharides with degree of polymerization (d.p.)2–4 (BTPG2–4) were synthesized for the screening of microbial cellulolytic activity in plate assays. Thesubstrateswere veryefficient thatwas shown for several cellulolytic bacteria, including yeast-like isolates fromKamchatka hot springs. Three tested bacterial strains and eighteen of 30 of the yeast isolates showed ability todegrade cellulose with cellobiohydrolase, β-glucosidase and endo-cellulase activities measuredwith standardsubstrates. The structures of 2-(2′-benzothiazolyl)-phenyl oligosaccharides were solved by NMR- and mass-spectrometry. The usefulness of the 2-(2′-benzothiazolyl)-phenyl substrates were also shown duringpurification of the B. polymyxa cellulolytic complex, which consists of at least three types of the enzymes:cellobiohydrolase, endo-β-D-glucanase and β-glucosidase.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

A considerable interest in enzymes capable of degrading celluloseand hemicelluloses exists due to their increasing use in bio-bleachingand conversion of biomass to chemical feedstocks and fuels (Prade,1996; Demain et al., 2005; Lynd et al., 2005). Many microorganismshave been described to be producers of such enzymes. Well-characterized and generally used producers of cellulolytic complexesare such microbial cultures as fungal strains like Trichoderma andAspergillus. However, new cellulose-degrading microorganisms stillare required for the specific biotechnology needs.

For the screening of a large number of polysaccharide-degradingmicroorganisms, efficient plate screening methods are a prerequisite.Traditionally, carboxymethyl cellulose or microcrystalline celluloseare used as substrates for detection of cellulolytic enzymes. Cellulaseactivity is detected visually upon appearance of a halo aroundmicroorganisms-producers after dying with, for example, Congo redand washing (Zhang et al., 2006). Such approach is not quantitativeand not sensitive enough due to a poor correlation between enzymeactivity and halo size (Sharrock, 1988). Short cellooligosaccharidespossessing modified reducing terminal with chromogenic/fluorogenicgroup could serve as a good alternative. Thus, cellobiosides carryingnitrophenyl- or umbelliferyl groups at the reducing end are substratestraditionally used for cellulases. Fluorogenic substrates are generally

iophysics Division, Petersburg, Gatchina, Leningrad District,

nskaya).

l rights reserved.

preferred due to the higher sensitivity, and several examples such asfluorescein, resorufin and 4-methylumbelliferone arewell-established(Huang, 1991; Xu and Ewing, 2004; Wittrup and Bailey, 1988;Eggertson and Craig, 1999; Fia et al., 2005; Irwin et al., 2004). Note,some difficulties still exist in use of such substrate.

A major limitation of most of the fluorescent substrates traditionallyused in plate screening is that an aglycone, released by hydrolysis, has atendency to diffusewidely, and therefore this kind of compounds are notsuitable for an incorporation into agar plates. These limitationsstimulated the development of chromogenic substrates, which producepractically non-diffusible precipitating products. Most commonly andwidely used substrate includes galactosides of indoxyl and its haloge-nated derivatives, such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyr-anoside, so-called X-gal (Itahana et al., 2007). Similar substratewas synthesized for cellulases (Chernoglazov et al., 1989). This com-pound, 5-bromo-indoxyl-β-cellobioside was hydrolyzed by the enzymereleasing free chromophoric group, which is subsequently oxidized toindigo-derivative with simultaneous H2O2 formation. Hydrogen per-oxide is further detected with isoluminol-peroxidase. Such two-stepprocedure leads to incorrect localization of enzymatic activity due topossible movement of the primary reaction products. At the best of ourknowledge, there are no any adequate substrates have been synthesizedfor cellulase detection. On one hand, such compounds should containmore than one saccharide residue along with chromogenic aglyconemoiety. On the other hand, liberated insoluble aglycone should provide aconsequent accurate localization of the microbial colony producing thecellulase. We took advantages from modification of different glycosideswith benzothiazoles, which form highly fluorescent precipitates aftertheir release (US Patent 5316906). As a result, 2-(2′-benzothiazolyl)-

296 D.R. Ivanen et al. / Journal of Microbiological Methods 76 (2009) 295–300

phenyl cellooligosaccharides with degree of polymerization (d.p.) 2–4(BTPG2–4) were produced. In the present paper, we demonstrate thepotential of new compounds for glucanase/cellulase detection inagarized medium during the growth of microorganisms.

2. Materials and methods

2.1. Chemicals

All chemicals were obtained from Sigma Chemical (St. Louis,MO, USA) or Acros Organics (Geel, Belgium) unless otherwise noted.4-Methylumbelliferyl β-D-cellotrioside (MUG3) was synthesized ac-cording to standard procedures described for BTP cellooligosacchar-ides (see below). All reagents were of analytical or research grade.

2.2. Synthesis of the BTP-substrates

Synthesis of the BTP-cellooligosaccharides with d.p. 2–4 was carriedout following four-step procedure. Common structure of the substratesproduced is shown in Fig. 1. Initial set of peracetylated cellooligosacchar-

Fig. 1. General formulae of cellooligosaccharide-derivatives produced during the synthesi

ides (1a–c) was obtained by the chromatographic separation of celluloseacetolizate according to Dickey and Wolfrom (1949). Then individualacetates were activated by treatment with 33% HBr in acetic acid at roomtemperature during 30–60 min. Each of the corresponding acetobromo-derivatives of cellooligosaccharides with d.p. 2–4 (2a–c) was further usedin the standard Koenigs–Knorr type glycosidation, which involvestreatment of fluorescent dye with a brominated sugar, soft acid catalyst(silver carbonate) and a nonnucleophilic base (2,4,6-collidine) underanhydrous conditions (Nalewayet al.,1994). The desired substrates (4a–c)were prepared from the corresponding peracetates (3a–c) by de-acetylationwith sodiummethoxide inmethanol. All resultant compoundswere identified by 1H and 13CNMRandMS-analysis. Due to low solubility,the BTP substrates were dissolved in 10% DMF in the 50 mM sodiumacetate buffer solution (pH 4.5) for further application. After hydrolysiswith 1,4-β-glucanase or cellobiohydrolase, released aglyconewas found tobe insoluble and yellow-fluorescent under UV-light.

2.2.1. 2-(2′-benzothiazolyl) phenyl-β-cellobiose1H NMR (400 MHz, DMSOd6), δ ppm: 1H NMR (500 MHz, ) 5.36

(1H, d, J1,2 7.6 Hz, H1′), 4.36 (1H, d, J1,2 7.8 Hz, H1), 3.76 (1H, dd, J5,6a

s of BTP-glycoside substrates (A). (B) Formulae of 2-(2′-benzothiazolyl)phenyl (BTP).

297D.R. Ivanen et al. / Journal of Microbiological Methods 76 (2009) 295–300

9.3, J6a,6b 11.2 Hz, H6a′), 3.71 (1H, dd, J5,6a 6.7, J6a,6b 11.4 Hz, H6a), 3.70(1H, ddd, J4,5 8.5, J5,6b 3.0 Hz, H5′), 3.67 (1H, dd, J2,3 9.5 Hz, H2′), 3.66(1H, dd, H6b′), 3.55 (1H, dd, J3,4 8.5 Hz, H3′), 3.52 (1H, dd, Hz, H4′),3.44 (1H, dd, J5,6 2.3 Hz, H6b), 3.23 (1H, ddd, J4,5 9.6 Hz, H5), 3.18 (1H,dd, J1,2 9.3 Hz, J2,3 9.1 Hz, H3), 3.08 (1H, dd, H4), 3.04 (1H, dd, H2).

13C NMR (126 MHz, DMSOd6) d ppm 131.43 (1C, s, C3 ), 128.06 (1C,s, C5 ), 125.43 (1C, s, C10 ), 124.22 (1C, s, C11 ), 121.68 (1C, s, C9), 121.35(1C, s, C4), 120.99 (1C, s, C12), 114.14 (1C, s, C2), 102.33 (1C, s, C1),99.01 (1C, s, C1′), 78.82 (1C, s, C4′), 75.97 (1C, s, C5), 75.97 (1C, s, C3),74.43 (1C, s, C5′ ), 74.41 (1C, s, C3′), 72.58 (1C, s, C2), 72.30 (1C, s, C2′),69.36 (1C, s, C4), 60.33 (1C, s, C6), 59.16 (1C, s, C6′).

ESI+ MS [M+Na]+ m/z 574.554 calcd. for C25H29NO11S, observed:574.561.

2.2.2. 2-(2′-benzothiazolyl) phenyl-β-cellotriose1H NMR (400 MHz, DMSOd6), δ ppm: 5.37 (1H, d, J1,2 8.0 Hz, H1″),

3.71 (1H, dd, J2,3 10.0 Hz, H2″), 3.77 (1H, dd, J3,4 9.9 Hz, H3″), 3.83 (1H,dd, J4,5 9.8 Hz, H4″), 3.61 (1H, m, H5″), 3.72 (1H, m, H6a″), 3.42 (1H, m,H6b″), 4.42 (1H, d, J1,2 8 Hz, H1′), 3.11 (1H, dd, J2,3 10.0 Hz, H2′), 3.36(1H, dd, J3,4 9.9 Hz, H3′), 3.41 (1H, dd, J4,5 9.8 Hz, H4′), 3.70 (1H,m, H5′),3.68 (1H, m, H6a′), 3.56 (1H, m, H6b′), 4.26 (1H, d, J1,2 8 Hz, H1), 3.00(1H, dd, J2,3 10.0 Hz, H2), 3.16 (1H, dd, J3,4 9.9 Hz, H3), 3.05 (1H, dd, J4,59.8 Hz, H4), 3.20 (1H, m, H5), 3.71 (1H, m, H6a), 3.41 (1H, m, H6b).

13C NMR (126MHz, DMSOd6) d ppm 162.40 (1C, s,C8),154.52 (1C, s,C1),151.45 (1C, s, C7),135.72 (1C, s,C3),132.08 (1C, s, C10),128.72 (1C, s,C11),126.09 (1C, s, C13),124.87 (1C, s, C6),122.40 (1C, s, C9),122.04 (1C,s, C12),121.67 (2C, s, C4),114.80 (1C, s, C2),103.17 (1C, s, C1),102.56 (1C,s, C1′), 99.64 (1C, s, C1″), 80.31 (1C, s, C4″), 79.34 (1C, s, C4′), 76.74 (1C,s, C3), 76.40 (1C, s, C3′), 75.07 (1C, s, C2), 75.01 (1C, s, C2′), 74.83 (1C, s,C2′), 74.79 (1C, s,C2″), 73.20 (2C, s, C3′, C3″), 72.96 (2C, s, C2″), 69.97(1C, s, C4), 60.97 (1C, s, C6), 60.30 (1C, s, C6′), 59.76 (1C, s, C6″).

ESI+ MS [M+H]+ m/z 714.712 calcd. for C31H39NO16S, observed:714.707.

2.2.3. 2-(2′-benzothiazolyl) phenyl-β-cellotetraose1H NMR (400 MHz, DMSOd6), δ ppm: 5.37 (1H, d, J 7.78 Hz, H1′″),

4.41 (1H, d, J 7.87 Hz, H1″), 4.25 (1H, d, J 7.83 Hz, H1′), 4.34 (1H, d, J7.95 Hz, H1). ESI+ MS [M+H]+ m/z 876.853 calcd. for C37H49NO21S,observed: 876.859.

2.3. Microorganisms and culture conditions

The strains of Bacillus halodurans LMG 21649, Bacillus polymyxaLMG 13294 and Pseudomonas aeruginosa LMG 12229 were obtainedfrom BCCM/LMG collection and were maintained as recommended bythe supplier (http://bccm.belspo.be/). The strains were chosen asbacterial producers of cellulose-degrading enzymes.

Yeast collection (30 individual strains collected from hot springs ofKamchatka) was a gift of Dr. Yarowoi (PNPI). Isolates were maintainedon a potato-dextrose agar (ATCC medium # 336). Liquid medium usedfor characterization of cellulase complex in the yeast and bacterialisolates contained 1% of yeast extract, 0.5% of peptone, and 0.6% offilter paper strips. The cultures were grown in 100 ml shaking flaskscontaining the above medium for 3 days at 30 °C.

2.4. Agar-plate assays

For the testing of enzyme activities at agar-plates, either MUG3 orBTPG2–4 were used in a medium consisted of (g per l of distilledwater): carboxymethyl-cellulose, 10; asparagine, 0.5; yeast extract,0.5; Mg2SO4 7H2O; (NH4)2SO4, 0.5; KH2PO4, 1; KCl, 0.5; CaCl2, 1; agar,15. Substrates (MUG3 or BTPG2–4) were stored as 10 mM stocksolutions in DMSO and immediately before using were diluted 10times with 50 mM sodium acetate buffer, pH 4.5. The solutionsobtained were filter-sterilized (0.2-µm pore size), added to liquidagarized medium up to a final concentration of 0.1 mM and poured

into petri dishes. Plates with yeast isolates were incubated at 30 °C.Plates with B. polymyxa LMG 13294, Pseudomonas aeruginosa LMG12229 and B. halodurans LMG 21649 were incubated at 30 °C and 37 °Cas recommended by BCCM. All agar plates with fluorescent substrateswere examined daily under UV light (366 nm) for 3 days.

2.5. Partial enzymes purification

Culture of B. polymyxa LMG 13294 was grown in 8 L of mediumdescribed above andwith 1% of filter paper for 60 h at 30 °C using 20-Lfermenter with aeration of 1 L of air per 1 L of medium.

After the growth, cells were removed by centrifugation (3000 ×g,40 min) and cell-free extract was concentrated by precipitation in 75%-saturated (NH4)2SO4. The resulting precipitate was collected bycentrifugation and re-suspended in 250 ml of 100 mM sodiumphosphate buffer, pH 7.0, mixed with 20% of insoluble cellulose(Sigmacell, cat. # S-6790) and kept at 4 °C with periodic shaking for30min. The suspensionwaswashed two timeswith the same phosphatebuffer and gently re-suspended for removal of unbound proteins. A finalwash was performed with 10 mM phosphate buffer, pH 7.0. Elution ofbound proteins was achieved by addition of equal volume of 1%triethylamine inwater, followedby vigorous agitation and centrifugation(3000 ×g, 50 min) to remove Sigmacel. The resulting supernatant waslyophilized and stored for further assays. Unbound fractions werecollected, concentrated using an Amicon PM-30membrane to 34ml anduploaded onto Sephacryl S-200 column(15×1000mm) for gel-filtration.Fractions containing β-glucosidase activity and endo-β-glucanaseactivity were collected, concentrated on the Amicon PM-30 membraneand used for further assays. All purification stepswere carried out at 4 °C.

2.6. Enzyme assays

Glucanase activities were determined by measuring the amount ofreducing sugar released from β-glucan by DNS method after 30-minincubation at 37 °C in 50 mM sodium acetate buffer, pH 6.0. Endo-cellulase activities were determined following standard assay condi-tions after 1 h incubation of the reaction mixture at 37 °C with0.05 mM sodium acetate buffer (pH 4.7) using a strip of filter paper(5 mg) and appropriate enzyme solution (Ghose, 1987). Cellobiohy-drolase activity was assayed with p-nitrophenyl (PNP) β-D-cellobio-side as a substrate in 10 mM sodium acetate buffer, pH 5.0, at 30 °C(Tuohy et al., 2002). β-Glucosidase activity was measured using PNPβ-D-glucopyranoside as a substrate in 50 mM sodium phosphatebuffer, pH 6.0, at 37 °C (Arrizubieta and Polaina, 2000). Liberation ofp-nitrophenol from the substrates was measured spectrophotome-trically at 400 nm in alkaline solution.

2.7. General methods

Protein concentrationwasmeasured by Lowrymethodwith BSA asa standard (Lowry et al., 1951). SDS-PAGE of the partially purifiedenzymes was done in 10% gel according to Laemmli (1970) usingmolecular weight calibration kit MW SDS 200 (29–205 kDa) fromSigma. The enzyme bands were revealed by silver staining.

All 1H and 13C NMR spectra were recorded with an AV-400 andAV-500 Bruker spectrometers in DMSO at ambient temperature usinga signal of DMSO as an internal standard (δH 2.5 and δC 39). Massspectrometric analysis of novel substrates was performed with aQ-Tof™ 2 mass spectrometer fitted with a nanoflow ion source(Waters Corporation, Micromass MS Technologies, Manchester, U.K.)as described (Eneyskaya et al., 2005).

3. Results and discussion

Three bacterial strains (B. halodurans LMG 21649, B. polymyxa LMG13294 and Pseudomonas aeruginosa LMG 12229) were tested with

Fig. 2. Agar-plate assays for B. polymyxa LMG 13294 with fluorescent substrates (A: BTPG3; B:MUG3): Filter-sterilized (0.2-µm pore size) 2-(2′-benzothiazolyl) phenyl-β-cellotriosideand 4-methylumbelliferyl β-cellotrioside (final concentration 0.1 mM) were added to the CM-cellulose-medium. Plates with B. polymyxa LMG 13294 were incubated at 30 °C andwere visually examined daily under UV light (366 nm) for 3 days.

Table 1Enzymatic activities in liquid supernatants of bacterial microorganisms

Microorganism name, collectionnumber

Enzymatic activitya, U/mL

FP-aseb Cellobiohydrolasec β-Glucosidased

B. halodurans LMG 21649 0.21 0.07 0.08B. polymyxa LMG 13294 0.77 0.3 0.06Pseudomonas aeruginosa LMG 12229 0.08 0.02 0.05

a Experimental error is ±5% values for at least 3 determinations.b Measured using the standard FP method (Ghose, 1987).c Assayed with PNP cellobioside (Tuohy et al., 2002).d Measured using PNP β-D-glucopyranoside (Arrizubieta and Polaina, 2000).

298 D.R. Ivanen et al. / Journal of Microbiological Methods 76 (2009) 295–300

new substrates. These cultures were chosen as bacterial producers ofmulti-enzyme cellulose-degrading complexes. The bacteria weregrown on the agar containing BTP cellobioside (BTPG2), BTP cellotrio-side (BTPG3) or BTP cellotetraoside (BTPG4) at 30 °C for three days. Allthree cultures most efficiently degraded only BTPG3 releasing yellow-fluoresced precipitate. B. polymyxa LMG 13294 showed the sameefficiency towards both BTP cellobioside and cellotetraoside whileweak fluorescence was observed with B. halodurans LMG 21649 andPseudomonas aeruginosa LMG 12229 (data not shown).

To reveal advantages of 2-(2′-benzothiazolyl)phenol in compar-ison with traditionally used fluorophore, 4-methylumbelliferol, insolid medium, we used B. polymyxa LMG 13294, the most effectivebacterial producer of cellulose-degrading complex. The microorgan-ism was grown on agarized medium containing BTPG3 and MUcellotrioside (MUG3) as fluorescent markers of the cellulase activities.A release of 4-methylumbelliferol was observed overnight. Furtherkeeping plates at the same conditions led to noticeable diffusion of thefluorophore into the medium in contrast to the case when BTPcellotrioside was used. Hydrolysis of BTPG3 by the B. polymyxa LMG13294 cellulolytic complex resulted in non-diffused yellow colorpointing to the enzymatic activity (Fig. 2). It is interesting, that thefluorescence remained stable for two weeks after the plates wereplaced at 4 °C.

The appearance of yellow-fluoresced precipitate in the solidmedium containing BTP cellooligosaccharides after the growth ofbacteria indicated the presence of cellulose-degrading enzymes. Threedifferent enzymes are well known to be involved into the cellulosedegradation: 1,4-β-D-glucan cellobiohydrolase (CBHs, E.C. 3.2.1.91,exoglucanase), which specifically liberates cellobiose dimers fromthe non-reducing ends of cellulose chains; endo-1,4-β-D-glucan4-glucanohydrolase (E.C. 3.2.1.4, endoglucananse), which cleavesinternal cellulosic linkages; and β-D-glucoside glucohydrolase(E.C. 3.2.1.21, exo-β-D-glucosidase), which specifically cleaves offglucose from the nonreducing ends of cellooligosaccharides. Allthree bacterial cultures were grown in liquidmedium containing 1%of filter paper as cellulase inducer. After centrifugation of culturebroths, supernatants were examined using PNP β-D-cellobioside/PNP β-glucopyranoside for measurements of cellobiohydrolase/β-glucosidase activities and by standard FP test for measurementof endo-cellulase activity. Observed differences in action towardsBTP cellooligosaccharides with various lengths in solid medium,which selected bacterial cultures exhibited, might be explained bydiverse distribution of exo- and endo-acting cellulolytic enzymes.B. polymyxa LMG 13294 displayed higher level for all tested acti-vities while B. halodurans LMG 21649 and Pseudomonas aeruginosaLMG 12229 were more active toward PNP β-glucopyranoside and

less active toward PNP cellobioside (Table 1). These results mayindicate that both cultures posses mainly β-glucosidases and atsome extent endo-cellulase, while the level of cellobiohydrolases isessentially lower. Comparable values for cellulolytic activitiesmight be explained also by the function of exo-acting enzymespresented in both cultures. By contrast, B. polymyxa LMG13294possess all three types of glycoside hydrolases at detectable level.

To evaluate the effectiveness of the novel substrates for thescreening of new cellulolytic microorganisms, all synthesized com-poundswere tested during the search among 30 yeast strains from ourown collection. Of these, 18 individual cultures showed their ability tohydrolyze BTP cellooligosaccharides that expressed in appearance ofyellow-colored fluorescent precipitates in agarized medium (Fig. 3).During this screening, we observed for the yeast isolates noticeablepreference in degrading longer BTP cellooligosaccharides, BTPG3 andBTPG4. It is appeared that BTPncellobioside is not convenient substratefor all types of cellulolytic enzymes presented in microbial cells. Thismight be explained by reasons mentioned above for bacteria. Anotherexplanation of such preference is specific tunnel architecture of theactive site of cellulases, which allows binding of long oligosaccharidesto facilitate their hydrolysis. This is in consistence with literature dataon mapping of active sites of various glycanases, where values forhydrolytic efficiency, kcat/KM, reported to be lower for short oligo-saccharides than for longer (Fontaine et al., 1997; Stubbs et al., 1999).

All 18 microorganisms were grown in liquid medium and threeenzymatic activities of cellulolytic complex were assayed. It was fond,that all strains demonstrate detectable level of cellulase activities. Thecultures Y038, Y019, Y004, Y035 and Y042 exhibited the highestintensities on the agar plates with all substrates. For these strains, acorrelation was observed in measurements of increased cellulolyticactivities using new and traditional substrates (Table 2).

In spite of poor solubility of the novel substrates in water, whichlimits their application in continuous assay, we aimed to evaluate theirusefulness in isolation procedures of cellulases. The strain B. polymyxa

Fig. 3. Screening of yeast microorganisms using BTPG2, BTPG3, BTPG4 as cellulase markers: Filter-sterilized (0.2-µm pore size) 2-(2′-benzothiazolyl) phenyl-β-cellotrioside (finalconcentration 0.1 mM) were added to the CM-cellulose-medium. Plates were incubated at 25 °C and were visually examined daily under UV light (366 nm) for 3 days.

299D.R. Ivanen et al. / Journal of Microbiological Methods 76 (2009) 295–300

LMG 13294 was used to show the effectiveness of the new substratesduring purification and characterization of cellulolytic complex withlow activity. Culture supernatant of B. polymyxa LMG 13294 wasconcentrated by ammonium sulfate precipitation, and then exo- andendo-active cellulolytic enzymes were fractionated using theirdifference in affinity toward microcrystalline cellulose. Fractionscontained exo-cellulase activities (mainly cellobiohydrolase andtraces of β-glucosidase) were bound to cellulose and eluted withwater solution of 1% of triethylamine. Unbound fractions containedendo-1,4-β-D-glucanase and 1,4-β-D-glucosidase activities and wereseparated by gel filtration on the Sephacryl S-200 column (Fig. 4). Thefractions with cellobiohydrolase, endo-cellulase and β-glucosidasebefore loading on the column and after the separation were testedwith BTPG3. Fraction with bacterial cellobiohydrolase (tube 2 at theFig. 4) showed no release of yellow-colored fluorescence, whilefractions with endo-cellulase (open circles, tube 4) and β-glucosidase

Table 2Detection of yeast producers of the enzymes of cellulose complex onto agar-plates usingBTP cellooligosaccharides with d.p. 2–4 and their enzymatic activities in liquidsupernatants

Microorganism name,collection number

Agar-plate screeningwith

Enzymatic activitya, U/mL

BTPG2 BTPG3 BTPG4 FP-aseb CBHc β-Glucosidased

Trichosporon sp., Y073 + ++ ++ 0.27 0.05 0.01Debaryomyces hansenii, Y038 ++ ++ +++ 0.65 0.09 0.02Cryptococcus albidus, Y001 + + + 0.08 0.06 0.005Torulaspora delbrueckii, Y007 + + + 0.02 0.05 b0.001Rhodotorula mucilaginosa,Y018

+ ++ ++ 0.15 0.07 0.03

Sporichiobolus salmonicolusi,Y051

+ ++ ++ 0.47 0.04 0.01

Rhodotorula minuta, Y052 + ++ ++ 0.32 0.02 0.01Candida sace, Y019 ++ +++ +++ 0.88 0.18 0.05Candida albidus, Y004 +++ ++ +++ 1.05 0.25 0.08Crtyptococcus sp., Y006 + + + 0.05 0.03 b0.001Bullera variabilis, Y022 + + + 0.06 0.02 b0.001Cryptococcus laurentii, Y023 + + + 0.03 0.01 b0.001Candida haemulonii, Y033 ++ ++ ++ 0.57 0.04 0.02Rhodotorula aurentia, Y035 ++ ++ +++ 0.28 0.12 0.08Sporobolomyces roseus, Y028 + ++ ++ 0.51 0.07 0.06Metschnikowia pulherina, Y042 + +++ +++ 0.83 0.19 0.03Tremella foliana, Y016 + ++ ++ 0.29 0.05 0.01Cryptococcus hongericus, Y009 + ++ + 0.08 0.09 0.02

Designations +, ++, +++ indicate visual intensity of the yellow-fluoresced precipitatespot in UV-light.

a Experimental error is ±5% values for at least 3 determinations.b Measured using the standard FP method (Ghose, 1987).c Assayed with PNP cellobioside (Tuohy et al., 2002).d Measured using PNP β-D-glucopyranoside (Arrizubieta and Polaina, 2000).

(open squares, tube 3) correlated well with their level of activitiesmeasured using standard substrates. Specific activity for the cellobio-hydrolase was calculated to be 6.5 U/mg, for the β-glucosidase−0.95 U/mg and for the endo-cellulase −7.5 U/mg. The absence of thecolor release in the hydrolysis of BTP cellotrioside by the bacterialcellobiohydrolase might be explained by the specific architecture ofthe active site of the enzyme enabling effectively cleave off cellobio-sides but not cellotrioside or glucose from cellooligosaccharidesubstrate.

In conclusion, due to relatively expensive cost of the large-scaleproduction of cellulase itself and its products of hydrolysis, the searchfor new microbial producers of the enzymes becomes a vital task forbiotechnology. Application of new strains and an optimization oftechnological processes would result in reduction of final costs. One ofthe important tools for adequate solution for screening of suchproducers is the use of highly sensitive and relatively simple

Fig. 4. Gel-filtration of the enzymes with low affinity to microcrystalline cellulose onSephacryl S-200. The column (30 mm×800 mm) was equilibrated with 50 mM Na-phosphate buffer, pH 7.0, volume of fractions collected was 5 ml. ●, Protein (A280);□, β-glucosidase activity; ○, β-glucanase activity. Inside: Detection of cellulose acti-vities with BTPG3. Tube 1 contained endo-1,4-β-D-glucanase and 1,4-β-D-glucosidaseactivities before loading to Sephacryl S-200; tube 2: fraction with cellobiohydrolaseeluted from microcrystalline cellulose; tube 3: β-glucosidase eluted from SephacrylS-200, fractions 25–33; tube 4: endo-cellulase, fractions 37–43.

300 D.R. Ivanen et al. / Journal of Microbiological Methods 76 (2009) 295–300

compounds suitable for primary characterization of cellulose-degrad-ing complex. We presented here new fluorescent substrates, whichrelease yellow-colored slow-diffusible precipitate upon hydrolysis bycellulases, β-glucanases and β-glucosidases, which are produced bymicroorganisms even inminor quantities. Moreover, many cellulolyticbacteria are strict anaerobes (e.g. Clostridia) for which a screeningwith compounds described is the only possibility, because usuallyused 5-bromo-4-chloro-indolyl aglycone requires an oxidation step.Therefore, it could make BTP compounds the “X-gal” of investigators,who are looking for cellulolytic microorganisms.

Use of the new BTPGn substrates for agar-incorporated assay ofcellulases derived from additional species and in live cell or live tissueformats are underway and should facilitate the analysis of cellulaseactivity in a variety of important biological and bioengineeringsystems.

Acknowledgments

We are grateful to Prof. Harry Brumer and Mr. Gustav Sundqvist(AlbaNova University Centre, Stockholm, Sweden) for providing MS-spectra of the synthesized compounds. This work was supported inpart by grants from the Program for Basic Research in Molecular andCell Biology from the Presidium of Russian Academy of Sciences(PRAS), from a bilateral research program between Flanders (Belgium)and Russian Foundation for Basic Research (RFBR, grant number 05-04-50825-MF_a).

References

Arrizubieta, M.J., Polaina, J., 2000. Increased thermal resistance and modification of thecatalytic properties of a beta-glucosidase by random mutagenesis and in vitrorecombination. J. Biol. Chem. 275, 28843–28848.

Chernoglazov, V.M., Ermolova, O.V., Vozny, Ya.V., Klyosov, A.A., 1989. A method fordetection of cellulases in polyacrylamide gels using 5-bromoindoxyl-beta-D-cellobioside: high sensitivity and resolution. Anal. Biochem. 182, 250–252.

Demain, A.L., Newcomb, M., Wu, J.H., 2005. Cellulase, clostridia, and ethanol. Microbiol.Mol. Biol. Rev. 69, 124–154.

Dickey, E.E., Wolfrom, M.L., 1949. A polymer-homologous series of sugar acetates fromthe acetolysis of cellulose. J. Am. Chem. Soc. 71, 825–828.

Eggertson, M.J., Craig, D.B., 1999. β-Galactosidase assay using capillary electrophoresislaser-induced fluorescence detection and resorufin-β-D-galactopyranoside assubstrate. Biomed. Chromatogr. 13, 516–519.

Eneyskaya, E.V., Ivanen, D.R., Shabalin, K.A., Kulminskaya, A.A., Backinowsky, L.V.,Brumer III, H., Neustroev, K.N., 2005. Chemo-enzymatic synthesis of 4-methylum-belliferyl β-(1→4)-D-xylooligosides: new substrates for β-D-xylanase assays. Org.Biomol. Chem. 3, 146–151.

Fia, G., Giovani, G., Rosi, I., 2005. Study of β-glucosidase production by wine-relatedyeasts during alcoholic fermentation. A new rapid fluorimetric method todetermine enzymatic activity. J. Appl. Microbiol. 99, 509–517.

Fontaine, T., Hartland, R.P., Beauvais, A., Diaquin, M., Latge, J.P., 1997. Purification andcharacterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus. Eur. J.Biochem. 243, 315–321.

Ghose, T.K., 1987. Measurement of cellulose activities. Pure Appl. Chem. 59, 257–268.Huang, Z.J., 1991. Kinetic assay of fluorescein mono-β-D-galactoside hydrolysis by β-

galactosidase: a front-face measurement for strongly absorbing fluorogenicsubstrates. Biochemistry 30, 8530–8534.

Irwin, J.A., Morrissey, P.E., Ryan, J.P., Walshe, A., O'Neill, S.M., Carrington, S.D., Matthews,E., Fitzpatrick, E., Mulcahy, G., Corfield, A.P., Dalton, J.P., 2004. Glycosidase activity inthe excretory–secretory products of the liver fluke Fasciola hepatica. Parasitology129, 465–472.

Itahana, K., Campisi, J., Dimri, G.P., 2007. Methods to detect biomarkers of cellularsenescence: the senescence-associated beta-galactosidase assay. Methods Mol.Biol. 371, 21–31.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature 227, 680–685.

Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements withthe Folin phenol reagent. J. Biol. Chem. 193, 265–275.

Lynd, L.R., van Zyl, W.H., McBride, J.E., Laser, M., 2005. Consolidated bioprocessing ofcellulosic biomass: an update. Curr. Opin. Biotechnol. 16, 577–583.

Naleway, J.J., Fox, C.M.J., Robinhold, D., Terpetsching, E., Olson, N.A., Haugland, R.P., 1994.Synthesis and use of new fluorogenic precipitating substrates. Tetrahedron Lett. 35,8569–8572.

Prade, R.A., 1996. Xylanases: from biology to biotechnology. Biotechnol. Genet. Eng. Rev.13, 101–131.

Sharrock, K.R., 1988. Cellulase assay methods: a review. J. Biochem. Biophys. Methods17, 81–105.

Stubbs, H.J., Brasch, D.J., Emerson, G.W., Sullivan, P.A., 1999. Hydrolase and transferaseactivities of the beta-1,3-exoglucanase of Candida albicans. Eur. J. Biochem. 263,889–895.

Tuohy, M.G., Walsh, D.J., Murray, P.G., Claeyssens, M., Cuffe, M.M., Savage, A.V.,Coughlan, M.P., 2002. Kinetic parameters and mode of action of the cellobiohy-drolases produced by Talaromyces emersonii. Biochim. Biophys. Acta 1596, 366–380.

US Patent 5316906. 1994. Enzymatic analysis using substrates that yield fluorescentprecipitates.

Wittrup, K.D., Bailey, J.E., 1988. A single-cell assay of β-galactosidase activity in Sac-charomyces cerevisiae. Cytometry 9, 394–404.

Xu, H., Ewing, A.G., 2004. A rapid enzyme assay for β-galactosidase using optically gatedsample introduction on a microfabricated chip. Anal. Bioanal. Chem. 378,1710–1715.

Zhang, Y.-H.P., Himmel, M.E., Mielenz, J.R., 2006. Outlook for cellulose improvement:Screening and selection strategies. Biotechnol. Adv. 24, 452–481.