Bioresource Technology 101 (2010) 2412–2420

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Bioresource Technology

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Purification and characterization of a thermostable intra-cellular b-glucosidasewith transglycosylation properties from filamentous fungus Termitomyces clypeatus

Swagata Pal, Samudra Prosad Banik, Shakuntala Ghorai, Sudeshna Chowdhury, Suman Khowala *

Indian Institute of Chemical Biology, Drug Development and Biotechnology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 August 2009Received in revised form 12 November 2009Accepted 16 November 2009

Keywords:Termitomyces clypeatusIntra-cellular b-glucosidaseCo-aggregationTransglycosylation2-Deoxy-D-glucose

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.11.064

Abbreviations: C, b-glucosidase; S, sucrase; C/S, cratio; IG, intra-cellular b-glucosidase; IFC, intra-csucrase; DG, 2-deoxy-D-glucose; pNPG, p-nitrophesodium dodecyl sulfate; PAGE, polyacrylamide gel emethyl sulfonyl fluoride; GOD–POD, glucose oxidasediethyl amino ethyl; IEC, ion exchange chromatographgel permeation liquid chromatography; HPLC, high peraphy; CD, circular dichroism; pNP, p-nitro phenol; pCAcid; NBT, nitro blue tetrazolium; BCIP, 5-bromo-4DHB, 2,5-dihydroxy benzoic acid; TFA, trifluoroaceassisted laser desorption /ionization-time of flight.

* Corresponding author. Address: Drug DevelopmenIndian Institute of Chemical Biology, (CSIR, Govt. of InKolkata 700 032, West Bengal, India. Tel.: +91 33 22473 5197.

E-mail addresses: sumankhowala@iicb.res.in, s(S. Khowala).

An intra-cellular b-glucosidase was purified to homogeneity by gel filtration, ion exchange chromatogra-phy and HPGPLC from mycelial extract of Termitomyces clypeatus in the presence of the glycosylationinhibitor 2-deoxy-D-glucose. CD spectroscopy demonstrated that the purified enzyme exhibited a-helicalconformation. MALDI-TOF identified the enzyme’s molecular weight as 6688 Daltons, but SDS–PAGE andimmunoblotting indicated that the enzyme formed aggregates. The enzyme also showed unique proper-ties of co-aggregation with sucrase in the fungus. The enzyme showed around 80% stability up to 60 �Cand residual activity was 80–100% between pH ranges 5–8. The enzyme had higher specific activityagainst p-nitrophenyl-D-glucopyranoside than cellobiose and HPLC showed that the enzyme possessestransglycosylation activity and synthesizes cello-oligosaccharides by addition of glucose. The enzymewill be useful in synthetic biology to produce complex bioactive glycosides and to avoid chemical haz-ards. This is the first report of a b-glucosidase enzyme with such a low monomeric unit size.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

b-Glucosidases (EC 3.2.1.21) constitute a group of well character-ized, biologically and industrially important enzymes thatmechanistically catalyze the transfer of glycosyl groups betweenoxygen nucleophiles. The enzyme is regarded as a component ofthe cellulase system in which three categories of enzymes namely,endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) andb-glucosidases (cellobiase, EC 3.2.1.21) act synergistically to cata-lyze the conversion of cellulose into glucose moieties. Cellobiase asa rate limiting regulator plays an important role in degradation ofcello-oligosaccharides influencing the liberation of locked glucose

ll rights reserved.

ellobiase to sucrase activityellular cellobiase free fromnyl-D-glucopyranoside; SDS,lectrophoresis; PMSF, phenyl–peroxidase enzyme; DEAE,y; HPGPLC, high performancerformance liquid chromatog-MB, p-chloromercuribenzoic

-chloro-3-indolyl phosphate;tic acid; MALDI-TOF, Matrix

t and Biotechnology Division,dia), 4, Raja S.C. Mullick Road,499 5813x3491; fax: +91 33

umankhowala@yahoo.com

from the insoluble polysaccharide cellulose that serves as a rawmaterial for production of bioethanol from biomass. The enzymehas high demand in current scenario. The b-glucosidase enzyme cat-alyzes the selective cleavage of glycosidic bonds, which is pivotal inmany crucial biological pathways, such as degradation of structuraland storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnologicalapplications (Bhatia et al., 2002). b-Glucosidases have been sug-gested for various industrial applications such as for flavor enhance-ment and production of biodegradable nonionic surfactants andother compounds (Ducret et al., 2002). The enzyme can be utilizedfor synthesis of diverse oligosaccharides, glycoconjugates, alkyl-and amino-glycosidase. The enzyme has been produced with bacte-ria (Gueguen et al., 1997), yeast (González-Pombo et al., 2008) andfungi (Inglin et al., 1980; Dhake and Patil, 2005). Trichoderma reeseihas been reported to produce extracellular (Chirico and Brown,1987), cell-wall bound (Umile and Kubicek, 1986) and intra-cellular(Inglin et al., 1980) b-glucosidases. Aryl 1,4 b-D glucosidase activitieswas reported in intra-cellular b-glucosidase of cellulolytic fungusSporotrichum chrysosporium (Meyer and Canevascini, 1981) and cel-lobiose-fermenting yeast Candida wickerhamii (Skory et al., 1996).

Finding applications for enzymes in industry is now a greatchallenge and dependent on the development of novel enzymeswith desirable activities and properties. Many glycosidase havesaccharification ability but recent studies created interest inenzymes having the ability to catalyze transglycosylationreactions. Glycosidase-catalyzed transglycosylation is a promising

S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420 2413

alternative to classical chemical glycosylation methods. In compar-ison with chemical methods, enzymatic glycosylation is particu-larly useful for the modification of complex biologically activesubstances, when generally harsh conditions or use of toxic (heavymetals) catalysts are undesirable. These enzymes may be espe-cially useful in the food domain or cosmetics areas where adoptingsynthetic chemistry is sometimes not acceptable. A large numberof well characterized glycosyltransferases catalyze the transfer ofsugars from nucleoside-diphosphate intermediates to mono-, oli-go- and polysaccharide acceptor moieties. There is evidence thatplant endohydrolase–endotransferase can convert xyloglucan olig-omers into cell wall polymers (Thompson et al., 1997). Enzymespossessing transglycosylation properties are in high demand forthe production of bioactive compounds. An example is a glycopep-tide synthesized with the aid of endo-b-N-acetylglucosaminidasefrom Mucor hiemalis (Umekawa et al., 2008). Enhanced transglyco-sylation activity by construction of chimeras between mesophilicand thermophilic b-glucosidase was obtained from highly thermo-stable strain Thermotoga maritime and mesophilic strain Cellvibriogilvus (Goyal et al., 2002). The transferase activity was also studiedin b-glucosidase from Paecilomyces thermophila (Yang et al., 2007),Thermotoga neapolitana (Park et al., 2005), thermophilic fungusMelanocarpus sp. (Kaur et al., 2007). Intra-cellular b-glucosidasewith transferases activity was earlier reported from fungi such asThermoascus aurantiacus (Parry et al., 2001), Botrytis cinerea(Gueguen et al., 1995) and thermophilic fungi Talaromyces thermo-philus (Nakkharat and Haltrich, 2006).

Termitomyces clypeatus, a filamentous fungus belonging to theBasidiomycota is a well known producer of cellulolytic and xylan-olytic enzymes (Khowala et al., 1992; Mukherjee et al., 2006).Enhanced cellobiase activity was reported from the fungus using2-deoxy glucose (DG) as a glycosylation inhibitor in the culturemedium (Mukherjee et al., 2006). Cellobiase of the filamentousfungus formed an aggregate with sucrase in intra- and extracellu-lar preparations and this aggregate was responsible for alterationin the activity, stability and conformations of the enzymes (Muk-herjee et al., 2001). The sucrase of the fungus was purified andcharacterized to be a low-molecular weight protein with chape-ronic properties (Chowdhury et al., 2009). The present study fo-cused on the purification and characterization of a thermostableintra-cellular b-glucosidase, produced in the presence of DG as aglycosylation inhibitor. The enzyme is different from extra- and in-tra-cellular cellobiase preparations from the fungus reported pre-viously (Mukherjee et al., 2001, 2006). The b-glucosidase is anoligomeric enzyme that has transglycosylation activity. Its activity,stability and conformation are affected by its aggregation with su-crase. This is the first report of a b-glucosidase enzyme with trans-glycosylation activity with such low size monomeric units fromany source. The enzyme by virtue of low-molecular size may serveas an interesting model to study protein–protein interaction, pro-tein aggregation and for biomedical as well as biotechnologicalapplications.

2. Methods

Cellobiose, 2-deoxy-D-glucose (DG), Sephacryl S-200, DEAESephadex A-50, p-nitrophenyl-b-D-glucopyranoside (pNPG), SDS,tris, glycine, b-mercaptoethanol, phenyl methyl sulfonyl fluoride(PMSF), coomassie brilliant blue R-250, acrylamide/bis acrylamideand glucose oxidase/peroxidase reagent, were purchased from Sig-ma Aldrich. Prestained proteins marker for SDS–PAGE (Fermentas)was obtained from Pharmacia Fine Chemicals. Dialysis membranes(MW cut off 3 kDa) for desalting purpose were purchased fromMillipore, USA. All other chemicals and salts (analytical grade)were purchased locally.

2.1. Medium preparation and enzyme extraction

Edible fungus T. clypeatus MTCC 5091 was grown in syntheticmedium (500 ml) containing (%, w/v) cellobiose 1; Na-succinate0.5 and other nutrients such as NH4H2PO4 2.5; CaCl2�2H2O 0.037;KH2PO4 0.087; MgSO4�7H2O 0.05; boric acid 0.057; FeSO4�7H2O0.025; MnCl2�4H2O 0.0036; NaMoO4�4H2O 0.0032; ZnSO4�7H2O0.03 as reported earlier (Mukherjee et al., 2006). Sterile solutionof DG was added to the medium before inoculation to a final con-centration of 0.5 mg/ml.

Fungal mycelium after 3 d of growth was filtered throughWhatmann filter paper (24 mm) and the mycelium was washedthoroughly with distilled water to remove the culture medium.The washed mycelium was macerated in 0.1 M sodium acetatebuffer (pH 5.0) with PMSF (50 lM) by using glass beads in the beadbeater (Biospec, Bartlesville, Okla) at 4 �C. The extract was centri-fuged at 10,000 rpm for 30 min; the supernatant was collected asa source of intra-cellular enzyme and stored at �20 �C for furtheruse (Mukherjee and Khowala, 2002).

2.2. Assays of enzyme and protein

b-Glucosidase assay was carried out in the reaction mixture(1 ml) containing 2 mM pNPG in 0.1 M sodium acetate buffer, pH5.0 according to the method described earlier (Mukherjee et al.,2006). Incubation was carried out at 45 �C for 10 min. Reactionwas terminated by the addition of 0.5 ml Na2CO3 (1 M). Intensityof the yellow color developed by liberation of pNP was measuredat 400 nm. A unit of enzyme activity was expressed as the amountof enzyme that produced 1 lmol of pNP per minute under the as-say conditions. Sucrase activity was assayed by measuring the lib-eration of glucose in assay mixture (40 ll) of sucrose (4 mM) in0.1 M sodium acetate buffer, pH 5.0 (Chowdhury et al., 2009) byGOD–POD reagent. The mixture was incubated at 45 �C for 5 min,and reaction was terminated by keeping the solution in a boilingwater bath for 5 min. The solution was cooled to room temperatureand 1 mL of GOD–POD reagent was added to the reaction mixture.Intensity of color was measured at 505 nm after 30 min. The unitsof sucrase activity were expressed in terms of micromoles of glu-cose liberated per minute under the assay condition. Protein wasassayed using Coomassie blue (Bradford) protein assay reagentaccording to the technical instruction manual, with BSA asstandard.

2.3. Transglycosylation assay

Transglycosylation with the intra-cellular enzyme was carriedwith glucose as substrate (Bhiri et al., 2008). Five microgram ofpurified enzyme and 100 mg of anhydrous glucose were incubatedin 200 ll acetate buffer (10 mM, pH 5.0) at 45 �C. Aliquots (50 ll)taken after 3 h and 6 h were centrifuged and loaded on a SepPackcartridge C18 and ion exchange resin to remove salts and proteins.Sugar profile was analyzed in a Rezex RCM carbohydrate column(Phenomenex) by HPLC using double distilled water as mobilephase for detection of the oligosaccharide products. Individual su-gar concentrations were determined by peak area.

2.4. Purification of intra-cellular b-glucosidase

Crude mycelial extract (20 ml) obtained from mycelia of 500 mlmedium was subjected to gel filtration chromatography in batchescontaining 3 mg protein on a Sephacryl S-200 column (100 cm �1.2 cm) equilibrated with 0.1 M acetate buffer at pH 5.0. Fractions(88–126 ml) were collected at a rate of 7 ml/h were pooled andsubjected to anion exchange chromatography in a DEAE SephadexA-50 (21 cm � 3 cm) column equilibrated with 0.01 M acetate

Table 1Purification of intra-cellular cellobiase.

Steps Sample Totalprotein(mg)

Cellobiase (C) Sucrase(S)

C/S

Activity(U)

Specificactivity(U/mg)

Yield(%)

Activity(U)

Step 1 Mycelial extract(20 ml)

30 1300 43.3 100 40 32.50

Step 2 Sephacryl S-200Pool 1 2.50 115.30 46.12 8.83 37 3.11Pool 2 3.60 4.60 1.27 12 0.02 230

Step 3 DEAE SephadexA-50 of pool 2Pool 3 2.70 1.51 0.56 9 – –

Step 4 HPGPLCPeak 1(12.81 min)

0.32 0.17 0.54 1.06 – –

Peak 2(24.97 min)

1.96 – – 6.50 – –

Yield is given in terms of protein.

2414 S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420

buffer at pH 5.0. The unbound enzyme was collected at a rate of18 ml/h and active enzyme fractions were pooled (70–115 ml).The pooled fraction was concentrated by lyophilization and thendialyzed extensively against 10 mM acetate buffer using a 3-kDacut-off dialysis bag. The fraction was then loaded on a HPGPLC col-umn (Ultropac TSK G2000 SW, 0.75 � 60 cm, Pharmacia, Sweden)fitted in series with a pre-column and equilibrated with 0.1 M ace-tate buffer at pH 5.0 and 0.1 M NaCl. Protein peaks eluted at a rateof 1 ml/min and monitored by OD280 were analyzed from the peakarea obtained from the 745B data module (Waters, USA). The frac-tions containing pNPGase activity were identified, dialyzed andlyophilized for further characterization. Molecular weights wereapproximated from HPGPLC by a plot of log of molecular weightversus Kav values of standard proteins. Protein standards (Sigma,USA) glucagon (3.465 kDa) 26.25 min, RNase (13.7 kDa) 23 min,ovalbumin (43 kDa) 17.3 min, BSA (66 kDa) 15.01 min for molecu-lar size determinations were used.

2.5. Electrophoretic analyses

SDS–PAGE (10%, w/v) analysis of purified HPGPLC fraction (peak1) was performed according to the method of Laemmli (1970) andgels were stained with Coomassie brilliant blue R-250. For WesternBlotting, protein samples of enzyme preparations from peak 1(40 lg) and peak 2 (70 lg) of HPGPLC after SDS–PAGE were elec-trotransferred onto a nitrocellulose membrane (Amersham, UK)in a semidry transblot apparatus (Bio-Rad) at a constant currentof 170 mA for 1 h. Prestained protein markers (2 lg) were run inparallel to determine the molecular weight of the protein band.The blotted protein bands were detected by reaction with anti b-glucosidase serum and antirabbit IgG conjugated with alkalinephosphatase as second antibody (Blake et al., 1984). The colorwas developed with NBT and BCIP in 100 mM NaCl and 5 mMMgCl2. Specific antisera against the purified intra-cellular b-gluco-sidase were prepared in rabbit by emulsification of 20 lg of puri-fied enzyme with Freund’s complete adjuvant and injected threetimes at 12-day intervals (Bhiri et al., 2008). Enzyme activity inmycelial extract as well as the purified protein was detectedin situ after native PAGE (Mukherjee et al., 2006) using 10 lgand 50 lg protein, respectively.

2.6. Amino acid composition, N-terminal sequencing and PSI-BLASTanalysis

Amino acid analysis was performed in a PICO.TAG systemaccording to the PICO.TAG operation manual (Waters, USA) using

enzyme protein (30 lg) after extensive dialysis followed by lyoph-ilization and hydrolysis in 6 N HCl with 1% phenol for 22 h at105 �C. N-terminal sequencing of the purified protein (20 lg) fromHPGPLC peak 1 (Table 1, step 4) was done in the Institute ofMicrobial Technology Sector 39-A Chandigarh. The analysiswas done on PROCISE 491 cLC (Applied Biosystems). PSI-BLASTanalysis was performed with the NCBI algorithm using the SWISSPROT fungal protein database. Filter was put on for low complexityregions. All other parameters were kept at default values.Reiterations were carried out until no new significant hits weregenerated.

2.7. Kinetic parameters

Km and Vmax of intra-cellular b-glucosidase were determinedunder standard assay conditions using substrate (pNPG) concen-tration between 0.1 and 10 mM from the Lineweaver–Burk plot.

2.8. pH and temperature optima and stability

The pH optima of intra-cellular b-glucosidase was determinedby performing activity assays at 45 �C in the pH range 3.0–10.0(citrate/phosphate/tris buffer, 0.1 M) using 20 lg enzymes in theassay mixture. Enzyme stability at different pH was determinedby measuring the residual activity at 45 �C, pH 5.0 (50 mM sodiumacetate buffer) after incubating the enzyme (20 lg) for 1 h in thatpH range of 3.0–10.0 (citrate/phosphate/tris buffer, 0.1 M) at roomtemperature. The temperature optimum of the enzyme was deter-mined by assaying the enzyme (20 lg) in pH 5.0 at temperatureranges of 30–80 �C. Thermostability was measured by pre-incubat-ing the enzyme (20 lg) in pH 5.0 for 1 h at the temperature of 30–80 �C and residual activities were measured at 45 �C, pH 5.0.

2.9. Effect of metal ions and reagents

b-Glucosidase activities of crude mycelial extract (2 lg) andpurified enzyme (10 lg) in presence of various metal ions and re-agents were determined by pre-incubating the enzyme with differ-ent concentrations of salts, detergents and reagents in 50 mMsodium acetate buffer (pH 5.0) at room temperature for 1 h andresidual activities were measured.

2.10. Substrate specificity

Substrate specificities of the purified enzyme was determinedby incubating the purified enzyme (0.4 lg) in respective substrates(4 mM) at optimum temperature and pH for 5 min and measuringthe liberated pNP, glucose (Mukherjee and Khowala, 2002) orreducing sugars (Miller, 1959) by the standard procedures.

2.11. Mass spectrophotometry and circular dichroism measurements

Protein peak 1 obtained from HPGPLC was desalted; lyophilizedand 1 ll (20 pmol/ll) was mixed with 3.0 ll super DHB matrixsolution. The ‘‘super-DHB” matrix consisted of a 9:1 (w/w) mixtureof 2,5-DHB and 2-hydroxy-5-methoxybenzoic acid, respectively,since this ratio was found to be the most effective. Stock solutionsof 2,5-dihydroxybenzoic acid (10 mg/ml) was prepared in 50%, v/vaqueous acetonitrile containing 0.1% v/v TFA and that of 2-hydro-xy-5-methoxybenzoic acid (10 mg/ml) was made in high purityabsolute ethanol. The mixture was vortexed and approximately1.5 ll of the matrix–enzyme mixture was spotted onto the MALDItarget plate. The plate was allowed to dry at the room temperatureand mass analysis was performed on a MALDI-TOF/TOF-MS (4700Proteomics Analyzer, Applied Biosystems Inc) in positive ion modeaccumulating 2000 laser shots.

Fig. 2. Purification of intra-cellular b-glucosidase. The enzyme was purifiedthrough (A) Sephacryl S-200 gel chromatography, (B) DEAE Sephadex A-50 ionexchange chromatography and (C) HPGPLC. Fractions were monitored for proteinby measuring OD280 (—j—), cellobiase activity (U/ml) (—h—) and sucrase activity(U/ml) (—4—).

S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420 2415

Circular dichroic (CD) spectra of the protein (30 lg) in the far-UV wavelength range (190–250 nm) were recorded at room tem-perature (22 �C) on a JASCO J-720 spectropolarimeter (calibratedwith d-10-camphorsulfonic acid) using a cylindrical quartz cuvetteof path length 1 mm. The following scan parameters were used:1 nm bandwidth, 2 s. response time, 0.1 nm step resolution and20 nm/min scan speed. Each spectrum was an average of four con-tinuous scans. The acquired spectra were corrected by subtractingblank runs on appropriate protein free buffer solutions (10 mM-acetate buffer, pH 5.0) and subjected to a moderate degree of noisereduction analysis (Mukherjee et al., 2001).

3. Results

3.1. Production of intra-cellular b-glucosidase

Production profile of intra-cellular b-glucosidase showed thatthe specific activity of the enzyme was significantly higher in pres-ence of DG than in absence of the DG until day 3 day (Fig. 1). Maximalenzyme production (40 U/mg) was observed on day 3 in presence ofDG, whereas in absence of DG production was highest (13.6 U/mg)on day 4. It was also observed here that mycelial growth was re-duced by 40–50% in presence of DG and extracellular enzyme pro-duction was maximum on 4th day (Mukherjee et al., 2006).

3.2. Purification of intra-cellular b-glucosidase

The mycelial extract had glucosidase activity 43.3 U/mg associ-ated with sucrase activity 1.33 U/mg with C/S ratio (Cellobiase tosucrase activity ratio) at 32.5 (Table 1). The enzyme was fraction-ated in two pools containing enzyme activity of 46.12 U/mg in pool1 and of 1.27 U/mg in pool 2 in Sephacryl S-200 gel filtration col-umn (step 2) (Fig. 2A). The C/S ratio of pool 1 and pool 2 were3.1 and 230, respectively. At this stage 12% protein was recoveredin pool 2. The pool 2 fraction was next applied to anion exchangechromatography in DEAE Sephadex A-50 (step 3) and b-glucosi-dase (0.56 U/mg) free from sucrase was collected as pool 3(Fig. 2B). In HPGPLC (step 4) the enzyme was resolved in two pro-tein peaks eluted at 12.81 min (peak 1) with enzyme activity(0.54 U/mg) and at 24.97 min (peak 2) with no enzyme activity(Fig. 2C). Recovery of protein in peak 1 and 2 were 0.32 mg and1.96 mg, respectively.

3.3. Characterization of the purified intra-cellular cellobiase

Purified enzyme preparation from HPGPLC (peak 1, step 4) wasused for all the analyses unless otherwise specified.

Fig. 1. Profile of intra-cellular b-glucosidase production in T. clypeatus. Specificactivities of intra-cellular b-glucosidase were ascertained in the mycelial extractsfrom five consecutive days of growth. Data was averaged from three independentculture sets.

3.3.1. Kinetic parametersKm and Vmax of the purified enzyme were, respectively, deter-

mined as 0.148 mM and 0.077 U/mg, though in mycelial extract(containing sucrase) the values were 0.131 mM and 77.51 U/mg,respectively (Table 2). Accordingly the catalytic efficiency of theenzyme decreased from 591.67 U/mg/mM of mycelial preparationto 0.52 U/mg/mM for purified fraction.

3.3.2. Substrate specificityThe purified enzyme showed substrate specificity only towards

substrates with b-1,4 linkages. The enzyme had maximum activitytowards pNPG (0.54 U/mg) and cellobiose (0.15 U/mg) (Table 2),salicin (0.14 U/mg) and methyl b-D-glucopyranoside (0.09 U/mg),but had no hydrolytic activity towards o-nitrophenyl a-D-galacto-pyranoside, methyl a-D-galactopyranoside, melibiose, carboxy-methyl cellulose and starch.

3.3.3. pH and temperature optima and stabilityIntra-cellular b-glucosidase showed optimum activity at pH 5.0

and 45 �C in presence of DG for purified (step 4, Table 1, peak 1) aswell as the enzyme in mycelial extract (step 1, Table 1) as given inTable 2. Stability of the enzyme towards temperature and pH is gi-ven in Table 3A. The purified enzyme had 95% activity at 40 �C

Table 2Comparison of purified intra-cellular b-glucosidase with purified extra and intra-cellular enzymes in presence and absence of DG from T. clypeatus.

Characters IG IFCa EGc EFCa

Molecular weight 6688 Da ND ND 14,000 Dab

pNPG (U/mg) 0.539 2.32 17.9 2.41Cellobiose (U/mg) 0.148 2.06 21.43 2.35pH optima 5.0 5.0 5.4 5.0Temperature optima (�C) 45 47 45 47Thermal stability 70 �C 64% 60% ND NDKm (mM) 0.148 3.448 0.187 4.762

(0.131)d

Vmax (U/mg) 0.077 0.076 0.018 0.076(77.51)d

Kcat (U/mg/mM) 0.52 0.022 0.096 0.016(591.67)d

Conformation Alpha helical Beta sheet ND Random coil

IG, intra-cellular enzyme in presence of DG; EG, extracellular enzyme in presence ofDG; EFC, extracellular enzyme in absence of DG, and IFC, intra-cellular enzyme inabsence of DG.

a Mukherjee et al. (2001).b Saha et al. (2002).c Results not published.d Values in brackets correspond to enzyme in mycelial extract; ND, not

determined.

Table 3Susceptibility of intra-cellular b-glucosidase towards pH, temperature, metal ions andchaotropic agents.

pH Residual activity (%) Temp. (�C) Residual activity (%)

Mycelial extract Purified Mycelial extract Purified

A: Temperature and pH stability4.0 4.30 21.0 30 100 1005.0 10.1 87.5 40 86.5 95.36.0 31.9 100 50 69.5 86.57.0 31.9 95.3 60 60.5 79.58.0 73.2 80.5 70 50.5 64.59.0 100 45.4 80 22.8 46.3

10.0 76.2 32.5Conc. Residual activity (%)

Mycelial extract Purified enzyme

B: Effect of metal ions and chatropic agentsMetal ionsCalcium chloride 20 mM 59.5 53.5Magnesium sulfate 20 mM 62.1 39.2Copper sulfate 2 mM 58.1 44.4Mercuric chloride 2 mM 26.8 1.2Potassium chloride 20 mM 70.9 41.5Zinc sulfate 20 mM 38.6 30.3Manganese chloride 20 mM 42.9 26.3

Chaotropic agentsSDS 0.05% 0.00 50.0Urea 4 M 8.0 66.0EDTA 20 mM 64.3 37.4Sodium azide 20 mM 67.6 42.7pCMB 0.5 mM 66.9 36.9Triton X-100 1% 70.6 100Mercaptoethanol 1% 87.1 152.9DTT 20 mM 75.7 108.4

Notes (A): Protein of mycelial extract (20 lg) and purified enzyme (20 lg) wasincubated at different temperature ranges at pH 5.0 and in different pH at roomtemperature for 1 h followed by measurement of residual enzyme activity usingpNPG as substrate at 45 �C, pH 5.0.(B): Purified enzyme (10 lg) and crude enzyme from mycelial extract (2 lg) werepre-incubated with respective agents for 1 h and followed by measurement ofenzyme activity using pNPG. The activity of the purified enzyme (2 U/ml) as well asmycelial extract (30 U/ml) measured without any additive at 45 �C, pH 5.0 wastaken as 100%.

2416 S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420

which decreased gradually with increase in temperature andshowed about 80% and 46% residual activities even at 60 �C and80 �C, respectively. The purified enzyme showed more than 95%residual activity between pH ranges 6.0–7.0. In comparison myce-lial extract enzyme had 86%, 60% and 22% residual activities at40 �C, 60 �C and 80 �C, respectively. Stability of enzyme in mycelialextract was comparatively low at acidic pH and the enzyme wasmost stable (100%) at pH 9.0.

3.3.4. Susceptibility towards chaotropic agents and metal ionsEffect of metal ions and chaotropic agents on enzyme activity is

presented in Table 3B. In the presence of SDS (0.05%) the enzyme inmycelial extract was inactivated though the purified enzymeshowed about 50% residual activity. In the presence of urea (4 M)the purified enzyme lost about 34% activity whereas the mycelialextract enzyme became almost inactive losing 92% activity. Thepurified enzyme was also less susceptible to the nonionic detergentTriton X-100 and reducing agents mercaptoethanol and DTT thanthe crude enzyme. In presence of Triton X-100 (1%, v/v) the purifiedenzyme was not affected though the enzyme in mycelial extract lost30% activity. Activity of pure enzyme increased by 52% and 8%,respectively, in presence of reducing agents like mercaptoethanol(1%, v/v) and DTT (20 mM), whereas those of crude mycelial extractdecreased by about 13% and 24%, respectively. Purified enzyme wasmore susceptible to sodium azide, pCMB and EDTA and activity de-creased by about 57.3%, 63% and 63%, respectively, though the activ-ity of the mycelial extract enzyme was lost by about 43–46%.

Purified glucosidase was more susceptible to all the metal ionsand activity of the enzyme was inhibited to the extent of 50–60% inthe presence of calcium chloride, magnesium sulfate, copper sul-fate, potassium chloride and zinc sulfate (Table 3B) but the enzymein mycelial extract preparation showed 60–70% residual activity. Inpresence of mercuric chloride purified enzyme became inactiveand in presence of manganese chloride showed 26% activitywhereas in comparison enzyme in mycelial extract had about26% and 42% residual activities, respectively.

3.3.5. Amino acid compositionAmino acid composition of the purified b-glucosidase enzyme

(Table 4) indicated the presence of high amount of polar aminoacids such as cysteine (27%), aspartate and glutamate (22%), serine(6.8%) as well as non polar amino acids isoleucine (17.6%) andglycine (7.2%). The high cysteine content of the enzyme was

supported by the enzyme inactivation with pCMB as a –SH block-ing agents and in the presence of mercuric chloride.

3.3.6. N-terminal sequencing of the purified enzymeN-terminal amino acid sequences of the purified enzyme (peak

1, Table 1, step 4) were done. The first 15 amino acid residues of N-terminal sequence of the enzyme were ‘S–P–P–H–V–L–A–L–L–L–A–V–V–A–A’ PSI-BLAST of the sequence did not reveal any putativeconserved domain. On first iteration, closest hits were obtainedwith RNA polymerase II holoenzyme cyclin like subunit of Neuros-pora crassa and Alpha-glucosiduronase of Aspergillus tubingensis.However, further iterations confined the similarities only to theformer. The lack of sufficient significant hits demonstrated to anextent the novel nature of the b-glucosidase.

3.3.7. Molecular weight and circular dichroism studiesIn HPGPLC the glucosidase was eluted at 12.81 min in activity

peak 1 of approximate size 125.89 kDa (Fig. 2C). Size of proteinfrom the active peak was further estimated as 116 kDa in SDS–PAGE (lane 2, Fig. 3A) and western blot analysis (lane 3, Fig. 3B).Mass of the enzyme from peak 1, however, was observed at6688 Daltons in MALDI-TOF analysis. In HPGPLC another proteinpeak 2, eluted at 24.97 min of size around 6.31 kDa, had no enzymeactivity. However, presence of the glucosidase corresponding tosize 116 kDa (lane 2, Fig. 3B) in peak 2 was confirmed by westernblot analysis of the protein band, which stained positive with theantibody raised with peak 1 (step 4, Table 1) enzyme preparation.

Table 4Amino acids composition of intra-cellular b-glucosidase.

Amino acids % n (mol)

Cys 27Asx-Glx 22Ile 17.6Gly 7.2Ser 6.8Lys 5.4Val 5.08Arg-Thr 5Pro 4.8Leu 4.3Ala 3.3Tyr 2.3His 2.1Phe 1.1Met 0.7Trp N.D.

N.D. – not determined.

Fig. 3. Electrophoretic analyses of purified b-glucosidase. Purified b-glucosidasefrom HPGPLC peak 1 (Lane 2) and protein marker (Lane 1) were subjected to SDS–PAGE (A). For immunoblotting (B) purified intra-cellular b-glucosidase fromHPGPLC peak 1 (Lane 3), peak 2 (Lane 2) and protein marker (Lane 1) were loadedas described in Materials and methods. For activity staining (C) purified intra-cellular b-glucosidase from HPGPLC peak 1 (Lane 2) and mycelial extract (Lane 1)were loaded.

Fig. 4. Circular dichroic spectra of intra-cellular b-glucosidase. The spectra ofpurified enzyme, peak 1, step 4 ( ) and co-aggregated enzyme from Sephacryl S-200 step 2, pool 2 (—) were measured.

S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420 2417

The results confirmed the enzyme to be a homo-oligomeric proteinwith small size monomeric unit. b-glucosidase activity of HPGPLCpeak 1 was also confirmed by enzyme activity staining and thered band was found at the same position of b-glucosidase activitypresent in the mycelial extract (Fig. 3C).

Circular dichroic spectral analysis of the enzyme from peak 1(step 4, Table 1) showed presence of alpha helical conformationas evidenced by double negative bands centered around 223 nmand 250 nm (Fig. 4). Partially purified aggregated enzyme (step 2,Table 1, pool 2) showed weak to moderate negative bands around220 and 280 nm representing no conclusive conformation.

3.3.8. Transglycosylation propertiesDuring analysis of transglycosylation reaction products by the

enzyme showed that at time 0 h incubation period, a single peak(34.5 min) of glucose free of any di, tri or oligosaccharides mixturewas identified (Fig. 5). After 3 h peaks of cello-oligosaccharides at24.945 min and 32.15 min were visible and after 6 h three peaksat 23.5 min, 25.3 min and 28.0 min corresponding, respectively,to cellotetraose, cellotriose and cellobiose were obtained. The max-imum yield of cello-oligosaccharides was estimated at around12.6% in 6 h at experimental conditions as calculated by the peakarea from HPLC chromatogram.

4. Discussion

The intra-cellular b-glucosidase of the fungus T. clypeatusshowed appreciable transglycosylation activities, and the enzymewas characterized as an aryl 1,4 b-D-glucosidase (IG) because ofits narrow substrate specificity only towards substrates withb-1,4 linkages and higher activity (3.6 times) towards pNPG thancellobiose. The enzyme was different from other extra and intra-cellular cellobiase enzyme preparations of the fungus (producedin presence and absence of DG) as observed from the comparisonof the properties towards substrate specificities, temperature opti-ma, thermal stability, kinetic parameters and conformation fromresults given in Table 2. Thermostability, substrate affinity and ki-netic efficiency of IG were highest among all the enzyme prepara-tions. It also became evident that due to restricted glycosylation inpresence of DG the enzyme IG became more thermostable and cat-alytically efficient as compared to the enzyme (IFC) produced andpurified in absence of DG, though IFC showed much better specificactivity on pNPG and cellobiose.

Rigidity of aggregation of proteins leading to their stabilizationis a well known phenomenon (Andya et al., 2003; Cromwell et al.,2006) and it is mediated by disulphide bond formation betweenpreviously unpaired free thiols. The high cysteine content of the in-tra-cellular b-glucosidase reported in this study probably is indic-ative of its necessity for aggregation. This might also account forthe exceptional stability of the enzyme besides the role playedby underglycosylation.

The enzyme (IG) showed higher activity against pNPG as com-pared to cellobiose but other enzyme preparations had somewhatsimilar activities on both the substrates. Conformation of the en-zyme (IG) was also quite different than other enzymes in Table 2.Since the N-terminal sequence did not match with the availabledatabase for the filamentous fungal family, the enzyme appearedto be novel. Intra-cellular b-glucosidase of Trichoderma reesei wasalso similarly reported to be distinctly different from the extra-cellular enzyme preparations of the same fungus (Inglin et al.,1980).

Properties of the enzyme changed significantly after purifica-tion from sucrase in the fungus. The sucrase of the fungus was anovel enzyme and was characterized to have low-molecularweight and possessed chaperonic properties (Chowdhury et al.,2009). Regain of 40% and 53% activity was observed, respectively,

Fig. 5. Transglycosylation assay. Transglycosylation reaction was carried out by incubating the intra-cellular enzyme for 0 h (B), 3 h (C) and 6 h (D). Standards samples ofglucose (34 min), cellobiose (28.3 min), cellotriose (25.8 min) and cellotetraose (24.7 min) were run as control (A).

2418 S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420

for IFC and EFC after in vitro addition of intra and extracellular su-crase in the fungus (Mukherjee et al., 2001). Studies of co-aggrega-tion of intra and extracellular cellobiase (IFC) with sucrase werereported earlier in T. clypeatus produced in absence of DG(Mukherjee et al., 2001) and it was observed that kinetic activityand stability of the enzyme co-aggregates, with different C/S ratio,obtained at different stages of purification were significantly al-tered due to separation from sucrase. However, the enzymes werenot characterized for their substrate specificity and size.

It was observed here that the intra-cellular b-glucosidase (IG)also showed co-aggregation properties with sucrase and kineticactivities and other physicochemical properties changed signifi-cantly after purification (step 3, Table 1), though the magnitudeof the changes in specific activities and stability of the intra-cellu-lar enzyme were quite different from the enzyme IFC (Table 2),earlier produced and purified in absence of DG (Mukherjee et al.,2001). It was interestingly noted that after removal of sucrase, con-formation of the enzyme (IG) was altered to alpha helical formwith better stability towards temperature and pH (Table 2). Theenzyme became less susceptible towards denaturants (urea),detergents (SDS, Triton-X 100) and reducing agents (DTT, b-mercaptoethanol) but susceptibility increased towards metalions in purified enzyme as compared to the crude enzymepreparation. Differences in behaviour between crude and purifiedpreparations were attributed to changed aggregation of the en-zyme by co-aggregation with sucrase. It was also concluded thatco-aggregation of IG with sucrase was different from that of IFC(Mukherjee et al., 2001), due to different conformation of enzymeIG than IFC which existed predominantly in beta sheet form

(Table 2). This change in conformation may be assigned to re-stricted glycosylation of the IG in presence of the glycosylationinhibitor DG used in the study.

Intra-cellular post-translational modification by glycosylation isknown to affect biochemical and biophysical properties of glucosi-dases. In presence of DG production of IG was high from the 1stday and was increased by 4 times on 3rd day as compared to theenzyme produced in absence of DG. It was observed that in DGmedium substrate affinity and thermostability of the enzyme alsoincreased significantly as compared to the other intra and extracel-lular enzymes of the fungus (Table 2). DG was earlier reported toinduce altered glycosylation in the fungus T. clypeatus, and extra-cellular cellobiase activity in cellobiose medium increased by40–50 times due to underglycosylation (Mukherjee et al., 2006).DG is known to affect O-glycosylation in eukaryotes and inhibitsthe incorporation of glucose from UDP-glucose into dolichyl phos-phate glucose and dolichyl pyrophosphate oligosaccharides caus-ing less glycosylation of the glycoproteins (Datema et al., 1983).DG also causes accumulation of DG-6-phosphates and interfereswith carbohydrate metabolism by inhibiting glycolytic enzymesand decreased cell growth (Ralsera et al., 2008). It was reportedthat DG plays dual role such as inhibitory and stimulatory insynthesis of several enzymes. The synthesis of intra-cellulara-glucosidase of mutant Saccharomyces cerevisiae was inhibited(Kuo and Lampen, 1972) though production of thermotolerantb-xylosidase from Kluyveromyces marxianus was stimulated byDG (Rajoka and Khan, 2005).

In T. clypeatus significant increase in regulatory secretion ofextracellular enzyme (cellobiase) and protein was noticed mainly

S. Pal et al. / Bioresource Technology 101 (2010) 2412–2420 2419

in presence of DG, though titer of enzyme and protein were increasedin presence of all the glycosylation inhibitors (tunicamycin, deoxy-nojirimycin and glucono-lactone) used (Mukherjee et al., 2006).

In a separate study to understand the mechanism of excretionof enzymes in the fungus, it was observed that in presence of DGtiters of metabolic enzymes were significantly reduced and wererelated to increased secretion of the enzyme cellobiase (unpub-lished data). It appeared that the intra-cellular b-glucosidase (IG)assisted in maintaining reduced level of intra-cellular glucose bytransglycosylation properties and thereby induced increased secre-tion of the extracellular enzyme in the fungus. This was also sup-ported by the fact that in T. clypeatus titer of the enzymeincreased significantly on 3rd day in presence of DG (Fig. 1). Possi-ble role of induction of extracellular cellobiase was also proposedfor intra-cellular b-glucosidase from T. reesei (Inglin et al., 1980).

The enzyme (IG) was identified to have lowest size monomericunit reported for carbohydrases in the category as confirmed byMALDI-TOF and from size in peak 2 (6.31 kDa) of HPGPLC. The re-sults showed that the monomeric form of the enzyme was avail-able only in HPGPLC but could not show enzyme activity at thisstage. It was concluded that the enzyme was oligomeric or self-aggregated and was stable and active only in aggregated form. Thisaggregation was different from that observed in IFC or EFC enzymepreparations of the fungus as confirmed by protein conformationsand pH and temperature optima and other kinetic properties in Ta-ble 2. The molecular weight (116 kDa) of the purified self-aggre-gated b-glucosidase (IG) obtained was similar in range ofoligomeric intra-cellular b-glucosidases from other sources suchas 98 kDa in Trichoderma reesei (Inglin et al., 1980), 94 kDa and180 kDa in C. wickerhamii (Skory et al., 1996), 178 kDa and106 kDa in Neurospora crassa ** (Yazdi et al., 2003).

5. Conclusion

Recent studies have created the interest in the enzymes catalyz-ing the transglycosylation reactions in food industry. The intra-cel-lular b-glucosidase of T. clypeatus from crude mycelial extract andpurified preparations may be used as suited for specific biotechno-logical applications, respectively for bioconversion and/or in trans-glycosylation reactions. The studies with the enzyme areinteresting with unique observations in regard to self-aggregationand co-aggregation with another glycosidase sucrase of the fungusas well as due to effects of glycosylation affecting the overall prop-erties of the enzyme. The small monomeric unit of the enzyme willbe useful in protein engineering and proteomic studies.

Acknowledgements

Financial support for this research was given in parts by grantsfrom Dept of Biotechnology, Govt. of India and Labonya Prova BoseTrust Kolkata. Acknowledgements must go to Dr. G. Suresh Kumarof IICB, Kolkata for CD analysis and Dr. Debashish Mukherjee (SahaInstitute of Nuclear Physics, Kolkata) for MALDI-TOF analysis of en-zyme and to Director (Institute of Microbial Technology, Chandi-garh) for N-terminal sequencing. Thanks are due to Dr. GouriProsad Datta (Dept. of Physiology, Rammohan College, Kolkata)for his constant encouragement and support.

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