Journal of Biotechnology 122 (2006) 274–284

Hydrolyses and transglycosylations performed by purified�-d-glucosidase of the marine molluscAplysia fasciata

Giuseppina Andreotti, Assunta Giordano, Annabella Tramice,Ernesto Mollo, Antonio Trincone∗

Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy

Received 30 May 2005; received in revised form 20 September 2005; accepted 6 October 2005

Abstract

The purification and characterisation of the�-glucosidase from the marine molluscAplysia fasciata are reported. Overallsubstrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable charac-teristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxineglucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last twoacceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl trans-ferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptorwhich is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reactionb©

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atch, etc.).2005 Elsevier B.V. All rights reserved.

eywords: �-Glucosidase; Transglycosylation; Oligosaccharide synthesis; Marine organism;Aplysia fasciata; Biocatalysis

. Introduction

The investigation of transglycosylation potential oflycosyl hydrolases can provide enzymes with new

nteresting catalytic activities. These enzymes usu-lly hydrolyse glycosidic bonds but are also able toatalyse the stereospecific formation of such linkagesScigelova et al., 1999), finding application in the enzy-

∗ Corresponding author. Tel.: +39 081 8675095;ax: +39 081 8041770.

E-mail address: antonio.trincone@icb.cnr.it (A. Trincone).

matic synthesis of oligosaccharides. The great intfor them has been also recently demonstrated bycessful genetic manipulation of some of their repretatives (glycosynthases) (for a review seePerugino eal., 2004).

The marine environment furnished different souof glycosyl hydrolases (Kusaykin et al., 2003) andthe effort for the identification of new enzymes, eforming any desired glycosidic bond, is of currsignificance. We are actively involved in the seafor enzymes from marine organisms thus recentlyfocused our attention on the sea hareAplysia fasciata

168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2005.10.002

G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284 275

Poiret 1789, a large mollusc easily collectable andvery common in Mediterranean habitats belonging tothe order Anaspidea (Carefoot, 1987). Glycosyl hydro-lases interesting for biotechnological applications suchas�-mannosidase,�-glucosidase and�-galactosidasewere abundant both in the hepatopancreas and in vis-ceral mass extracts of this organism. Both�-enzymespossess interesting catalytic properties for the syn-thesis of�-mannosides (Andreotti et al., 2005) and�-galactosides (Giordano et al., 2005). The reactionwith maltose (producing good yield of panose, iso-maltose and higher tetra- and pentasaccharides) andother few transglucosylation reactions performed by�-glucosidase activity were already studied (Giordano etal., 2004) using the crude homogenate; the use of a widevariety of acceptors was hampered by the presence ofother hydrolytic activities in the total visceral extract.Transglycosylation activity of the�-glucosidases is abiotechnologically relevant issue in that these enzymesare applied both in food industry to produce oligosac-charides (Voragen, 1998) and to conjugate sugars tobiologically relevant materials improving their chem-ical properties and physiological functions (Kren andMartinkova, 2001).

In this paper we report on the purification and char-acterisation of the�-glucosidase fromA. fasciata as wewere prompted by the interesting preliminary resultsobtained using the crude homogenate. Overall substratespecificity of the pure enzyme for both hydrolytic andtransglycosylation reactions was studied in order toa m ab lu-c sticso ando is ofd arer

2

2

Flow,P pur-c itro-p romS and

TLC silica gel plates were from E. Merck (Darm-stadt, Germany). All other chemicals were of analyticalgrade.

HPLC was performed on a Mlton Roy appara-tus equipped with UV detector (Waters Ass., USA)(254 nm).

A Dionex 500 chromatographic system, controlledby PeakNetTM software (Sunnyvale, CA), was used toseparate and detect carbohydrates.

AKTA-Prime system (Amersham PharmaciaBiotech) was used for protein purification.

Agilent UV–vis Spectroscopy System was used foranalytical UV measurements.

NMR spectra were recorded on Bruker instrumentsat 600, 400 and/or 300 MHz. Samples for NMR analy-sis were dissolved in the appropriate solvent and boththe downfield shift of the signal of the solvent oracetone-d6 (31.07 ppm for13C NMR spectra in D2O)were used as internal standard.

Acetylation of compounds was performed withpyridine/Ac2O at room temperature. The solvents wereremoved by a N2 stream and the reaction mixture waspurified by silica gel chromatography or preparativeTLC.

Protein concentrations were routinely esti-mated using the Bio-Rad Protein System, with thebovine serum albumin as the standard (Bradford,1976).

TLC solvents: (A) (EtOAc:CH3COOH:2-propanol:HCOOH:H2O, 25:10:5:1:15, v/v/v/v/v), (B) (EtOAc:CH edo ).

2

spc r thep se-w ed

2α

m-p he

ssess specific features which are of interest froiotechnological point of view (i.e. production of gosides of different nature); remarkable characterif this enzyme for the bioconversion of maltosether malto-oligosaccharides and for the synthesifferent trisaccharides of cellobiose and sucroseeported.

. Material and methods

.1. Materials and instrumental

Q-Sepharose Fast Flow, SP-Sepharose Fasthenyl-Sepharose Fast Flow, Superdex-200 werehased from Amersham Pharmacia Biotech. Nhenyl glycosides and maltose were obtained figma (St. Louis, MO). Reverse-phase silica gel

H3OH:H2O, 70:20:10, v/v/v), (C) (CHC13:CH3OH:2O, 65:25:4, v/v/v). ESI-MS spectra were obtainn a Q-T of mass spectrometer, Micro (Mcromass

.2. Enzyme source

Visceral mass extract ofA. fasciata was prepared areviously described (Giordano et al., 2004). Half ofrude material (49 g) from 20 animals was used fourification either of�-mannosidase described elhere (Andreotti et al., 2005) and�-d-glucosidase herescribed.

.3. Isolation and characterisation of-d-glucosidase

All purification steps were carried out at room teerature in K-acetate 50 mM pH 5.5 (buffer A). T

276 G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284

Table 1Purification of�-glucosidase fromAplysia fasciata

Purification stepa Volume (ml) Activity (U) Protein (mg) Specific activity (U mg−1) Yield (%) Purification-fold

Homogenate 12 345 226.8 1.52 100 1.00Superdex-200 185 294 138.7 2.12 85 1.39SP-Sepharose 195 351 82.1 4.28 100 2.79Q-Sepharose 20 281 20.8 13.5 80b 8.83Phenyl-Sepharose 1.85 253 6.6 38.4 74 25.05

a The enzyme was purified contemporary with a�-mannosidase (Andreotti et al., 2005) and the activities were separated after the thirdchromatography step. The procedure described here is related only to the�-glucosidase activity.

b An additional minor�-glucosidase activity was separated at this stage.

purification protocol for�-glucosidase is the same asthe one reported for�-mannosidase activity (Andreottiet al., 2005). The two enzymes were separated after theanionic exchange chromatography on a Q-Sepharosecolumn. An hydrophobic interaction chromatography(Phenyl-Sepharose Fast Flow) was necessary to obtainpure �-glucosidase (Table 1). The pooled sample of�-glucosidase (20 ml) from Q-Sepharose was treatedwith solid ammonium sulphate to give 20% satura-tion. After stirring for 1 h at room temperature, thesample was loaded at flow rate of 1 ml min−1 onto aPhenyl-Sepharose Fast Flow column (1 cm× 9.5 cm)which had been equilibrated with buffer A containingthe same amount of ammonium sulphate. The col-umn was washed with the equilibration buffer until theabsorbance at 280 nm returned to baseline, and retainedproteins were eluted with a 60-ml linear gradient of20–0% ammonium sulphate. The�-d-glucosidase waseluted when ammonium sulphate was 7.2%. The frac-tions were pooled, dialysed against 50 mM K-acetatepH 5.5, concentrated by ultrafiltration and stored at−20◦C.

The molecular mass of native�-d-glucosidase wasestimated by gel filtration on a Superdex-200 columnequilibrated with 50 mM K-acetate pH 5.5, which hadpreviously been calibrated with myoglobin (18 kDa),ovalbumin (43 kDa), bovine serum albumin (67 kDa),catalase (232 kDa) and ferritin (440 kDa). Blue dextranand pyridoxal phosphate were used to determine thevoid and the total volume, respectively. The flow ratew

ionsw ning1e ingC

2.4. Assay for p-nitrophenyl glycosides and othersubstrates

�-d-Glucosidase activity was assayed under stan-dard conditions at 34◦C in 60 mM Na-phosphate/30 mM citrate at pH 5.8 in the presence of 5 mM pNP-�-d-Glc (0.5 ml); aliquots (0.05 ml) were withdrawn atintervals and the reaction was stopped with the additionof Na2CO3 1 M (0.45 ml). One unit of�-d-glucosidaseactivity was defined as that amount of enzyme requiredto catalyse the release of 1.0�mol of p-nitrophenolper min. One millimolar substrate concentration wasused for determination of temperature (between 25 and50◦C at pH 5.8) and pH optimum in the range 4.5–7.5(at 34◦C); while 0.15–17 mM substrate were usedfor determination of kinetic parameters. The activitytowardsp-nitrophenyl�-d-galactopyranoside (pNP-�-d-Gal) andp-nitrophenyl�-d-mannopyranoside (pNP-�-d-Man) was determined under standard conditions asdescribed above in presence of 20 mM substrate.

A qualitative analysis of substrate specificity wastested on three groups of substrates.p-Nitrophenyl glu-cosides (pNP-�-d-Glc, pNP-�-d-Glc-(1-4)-�-d-Glc,pNP-�-d-Glc-(1-4)-�-d-Glc) were tested at 33 mMconcentration. Disaccharides (maltose, trehalose, iso-maltose, saccharose) and maltotriose were tested at100 mM while maltoheptaose and carbohydrate poly-mers were all reacted at 10 mg/ml.

All these reactions were performed using 72�g ofpure protein per ml of reaction mixture. The prod-u rds,o gra-p ex)( w).

wass

as 1 ml min−1.The molecular mass under denaturing condit

as estimated by SDS-PAGE on slab gel contai0% acrylamide using standard procedures (Sambrookt al., 1989). Proteins were located on the gels usomassie Brillant Blue staining.

cts were identified by TLC using authentic standar High Performance Anion Exchange Chromatohy with Pulsed Amperometric Detection (DionHPAEC-PAD) or by NMR spectroscopy (see belo

The time course for bioconversion of maltosetudied using 93 mM maltose and 18�g of pure protein

G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284 277

per ml. Aliquots were withdrawn at different timeintervals and the enzyme was inactivated by heat-ing for 2 min. The denatured proteins were removedby centrifugation. Chromatographic analysis of reac-tion mixtures was achieved by HPAEC-PAD usinga CarboPacTM PA1 analytical column (Dionex) witha linear gradient from 100 mM sodium hydroxide to100 mM sodium hydroxide/200 mM sodium acetateover 20 min at 0.25 ml min−1. Authentic standardswere used for calibration. Chemical identity of prod-ucts was established as previously reported (Giordanoet al., 2004).

The enzymatic activity towards maltose, isomaltoseand pNP-�-d-Glc-(1-4)-�-d-Glc was measured by theamount of glucose released quantified with glucoseenzymatic kit (Sigma). The reaction mixture (0.3 ml)containing 60 mM Na-phosphate/30 mM citrate at pH5.8 and an appropriate amount of substrate was incu-bated at 34◦C. The reaction was initiated by additionof the enzyme, aliquots (0.05 ml) were withdrawn atintervals and the reaction stopped by heating. Kineticparameters were determined using standard reactionmixtures containing 0.6–20 mM maltose or 0.8–25 mMpNP-�-d-Glc-(1-4)-�-d-Glc. Activity towards isomal-tose was tested at 22 mM.

One unit of enzyme activity was defined as thatamount of enzyme required to catalyse the hydrolysisof 1.0�mol of substrate per min.

2

ves-ti le thenm

f .5,5

K-p

2

ingp -

tion, monitoring the progress by TLC (solvents A, B orC, see Section2.1).

Analytical scale transglycosylation reactions usingpNP-�-d-Glc (33 mM) as donor were conducted at fivemolar excess of hetero-acceptors indicated inTable 2;reactions using maltose (29 mM) as donor were con-ducted at 1:1 molar ratio of acceptors; in both systems9�g of pure enzyme per ml (TLC solvent B) were used.

The products of pyridoxine glycosylation were sep-arated by reverse-phase chromatography and prepara-tive TLC (solvent C); purified bands were analysed by1H NMR spectroscopy using DMSO-d6 as solvent.

The reaction mixture for semipreparative�-glucosylation of pNP-�-d-Glc was purified by reversephase RP-18 (eluting firstly with water and then withmethanol) and silica gel chromatography (eluting withgradient of methanol in ethyl acetate).

Semi-preparative scale reactions for�-glucosyl cel-lobiose and�-glucosyl sucrose formation were con-ducted using 25 mg (83�mol) of pNP-�-d-Glc andfive molar excess of cellobiose or sucrose, dissolved in2.5 ml of phosphate buffer 50 mM pH 5.8; 18�g of pureenzyme were added to start the reaction at 34◦C underagitation. After the total consumption of donor (TLCsolvent B, ca. 6 h) the reaction mixtures were placedat 100◦C for 5 min, cooled and the products purifiedon Bio gel P2 column chromatography by eluting withwater. The fractions were analysed by TLC (solventA); those containing the reaction products were pooledand lyophilised and the products subjected to NMRse ted toa ty-l singC

3

co-s ech-n ofg st undi nw ridep tetra-a lation

.5. Stability under different conditions

Both the pH and temperature stabilities were inigated by incubating the pure enzyme at 96�g ml−1

n the presence of 90�g ml−1 of BSA. Residuanzymatic activity under standard conditions waseasured after 19 h incubation.

The pH stability was investigated at 34◦C at dif-erent pH values: 50 mM K-acetate buffer at pH 50 mM K-phosphate buffer at pH 5.8 and 6.5.

Thermal stability was measured in 50 mMhosphate pH 5.8 at 30, 34 and 38◦C.

.6. Transglycosylation reactions

All reactions were conducted in sealed vials ushosphate buffer 50 mM pH 5.8 at 34◦C under agita

pectroscopy using D2O as solvent and acetone-d6 forxternal reference. The products were also subjeccetylation (Section2.1) overnight and the perace

ated derivatives used for NMR measurements uDC13 as solvent and external reference.

. Results and discussion

The ability of�-glucosidases to perform transglyylation reactions is a relevant issue from the biotological point of view (food industry, productionlycoconjugates, etc.). An�-glucosidase activity wa

he most abundant glycosyl hydrolase activity fon the visceral mass ofA. fasciata and the reactioith maltose (producing good yield of the trisacchaanose, the disaccharide isomaltose and highernd pentasaccharides) and other few transglucosy

278 G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284

Table 2Qualitative assessment of bioconversion of different substrate by�-glucosidase fromAplysia fasciata

Substrates Reactivity Notes

Donor Acceptor

Starch – −Amylopectin (�-1,4/�-1,6) – −Amylose (�-1,4) – −Isomaltose (�-1,6) – −Pullulan (�-1,4/�-1,6) – −Saccharose (�-1,2-�-fru) – −Trehalose (�-1,1) – +/− Glucose formationGlucal – −Maltose (�-1,4) – + Production of panose, isomaltose and lesser amount of tetra-

and pentasaccharidesPanose – –Maltotriose – + Oligomers up to M6 present in early stage then panose and

isomaltose formationMaltoheptaose – + Formation of product from M7 to glucose then panose and

isomaltose formationpNP-�-d-Glc – + Interconversion of regioisomeric disaccharides and hydrolysispNP-�-d-Glc-(1-4)-�-d-Glc – + Trisaccharide formation and hydrolysispNP-�-d-Glc-(1-4)-�-d-Glc – + Trisaccharide formation and hydrolysispNP-�-d-Glc Cellobiose + Trisaccharide formation (seeFig. 3)pNP-�-d-Glc Saccharose + Trisaccharide formation (seeFig. 3)pNP-�-d-Glc Trehafose +/− Trisaccharide formationpNP-�-d-Glc Isomaltose + Trisaccharide formationpNP-�-d-Glc �-Cyclodextrin −pNP-�-d-Glc Ascorbic acid −pNP-�-d-Glc l-Menthol −pNP-�-d-Glc Glycerol +/− Glucosyl glycerolpNP-�-d-Glc Melibiose +/− Trisaccharide formationpNP-�-d-Glc Lactose −pNP-�-d-Glc Phenyl thiocellobioside −Maltose pNP-�-d-Glc + pNP-�-d-Glc-(1-4)-�-d-Glc then pNP-�-d-Glc-(1-6)-�-d-Glc

(15–20% yield)Maltose pNP-�-d-Glc-(1-4)-�-d-Glc + Formation of chromophoric trisaccharideMaltose pNP-�-d-Gal − Trace amount of pNP-disaccharideMaltose pNP-�-d-Gal − Trace amount of pNP-disaccharideMaltose Pyridoxine + Glucosyl pyridoxines (seeFig. 3)

reactions performed by using the crude homogenatewere already studied (Giordano et al., 2004); the use ofa wide variety of acceptors was hampered by the pres-ence of other hydrolytic activities in the total visceralextract.

3.1. Purification of α-glucosidase and structuralcharacterisation

The results of chromatographic purification of�-glucosidase from the visceral mass ofA. fasciata

are shown inTable 1. After the last step on aPhenyl-Sepharose column the sample was obtainedin a homogeneous form as determined by SDS-PAGE (Fig. 1). The purified enzyme gave onlyone band of 69 kDa on SDS-PAGE, while themolecular mass of the native�-glucosidase deter-mined by analytical Superdex-200 gel filtration was255 kDa, indicating that this enzyme is a homote-tramer. The enzyme was purified 25-fold with a spe-cific activity of 38.4 U/mg and the overall yield was74%.

G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284 279

Fig. 1. SDS-PAGE (10% acrylamide) of the�-glucosidase fromAplysia fasciata (A) and standard mixture of proteins (M) pre-stained broad-range markers (Bio-Rad). Proteins were detected withComassie staining.

3.2. Thermal and pH properties

The effects of acidity and heat on the activity andstability of�-glucosidase were examined. The enzymeshowed a pH optimum at 5.8 and the optimum temper-ature at this pH was in the range 36–44◦C. When theenzyme was incubated for 19 h at 34◦C in the presenceof 0.09 mg ml−1 BSA, at pH 5.5 (in acetate buffer),5.8 and 6.5 (in phosphate buffer), it retained 67, 64and 69% of its hydrolytic activity, respectively. Theextrapolated half-life times were approximately 30 hin all these cases. When the experiment was performedat the same pHs but in the presence of 60 mM Na-phosphate/30 mM citrate buffer, a stronger decrease ofthe activity was monitored. When the enzyme was incu-

bated for 19 h at pH 5.8 at 30 and 34◦C it retained 70and 64% of its activity, respectively. The half-life timeswere ca. 30 h. Only 34% of the activity was detectedafter incubation at 38◦C, and the half-life was 12 h.

In agreement with these observations pH 5.8 and34◦C were chosen as standard conditions for perform-ing hydrolytic and transglycosylation experiments.

3.3. Catalytic properties: hydrolysis andtransglycosylation

3.3.1. HydrolysisThe �-glucosidase fromA. fasciata was not reac-

tive using starch, amylopectin, amylose, isomaltose,panose, pullulan and saccharose. A very feeble reactionwas detected using trehalose. No enzymatic hydra-tion of glucal (formation of 2-deoxyglucose) wasdetectable. A qualitative assessment of reactivity of theenzyme toward each substrate is reported inTable 2.

The less reactive among thep-nitrophenyl substrateswas pNP-�-d-Glc-(1-4)-�-d-Glc while the reactionsusing pNP-�-d-Glc-(1-4)-�-d-Glc and pNP-�-d-Glcwere complete in few minutes in the conditions adoptedas judged by TLC.

Kinetic parameters for the hydrolysis of maltose,isomaltose, pNP-�-d-Glc and pNP-�-d-Glc-(1-4)-�-d-Glc are reported inTable 3. The hydrolytic activityfor �-1-4 glucosidic linkage was higher than that of�-1-6, in fact specific activity for maltose was 40 timeshigher than that for isomaltose.

3m

b-s er-f aridep e( tra-a sultsp nate( ds withm d int andi alsoi per-f tionw ion

.3.2. Transglycosylations and maltose and otheralto-oligosaccharides bioconversionAs indicated inTable 3, maltose was a good su

trate for the�-glucosidase and its bioconversion, pormed by the pure enzyme, produced the trisacchanose (�-Glc-(1-6)-�-Glc-(1-4)-Glc) and isomaltos�-Glc-(1-6)-Glc) together with lesser amount of tend pentasaccharides, in agreement with the rereviously obtained by using the crude homogeGiordano et al., 2004). Maltotriose was also a gooubstrate with a pattern similar to the one observedaltose: maltotetra- up to maltohexaose were foun

he early stages of reaction (Dionex) but panosesomaltose were later identified as end productsn this reaction. Using maltoheptaose the enzymeormed a reaction which is in accord to the reacith maltose. In the first few minutes the format

280 G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284

Table 3Kinetic parameters for hydrolysis of various substrates by�-glucosidase fromA. fasciata

Substrate Km (mM) kcat (s−1) kcat/Km (mM−1 s−1) VMAX (U/mg)

Maltose 5.70 489 86.0 115Isomaltose N.D.a N.D.a N.D.a 2.9b

pNP-�-d-Glc 0.26 163 627 38.4pNP-�-d-Glc-(1-4)-�-Glc 2.06 75.9 36.8 17.9

a Not determined because of the slight activity.b Activity measured by using 22 mM substrate.

of products from maltohexaose down to glucose wasclearly assessed by TLC and HPAEC-PAD analysis; assoon as maltose accumulated the formation of panoseand isomaltose also started. After 20 min panose wasthe most abundant among products of uppermost MW(Table 2).

In Fig. 2 the time course for the bioconversion ofmaltose is reported. Within the first hour of reactionin the conditions adopted ca. 60% of maltose wasconsumed forming maltotriose and panose; the con-centration of maltotriose decreased to a very low valueafter 360 min as well as the concentration of the regioi-somer panose increased to a plateau (after ca. 180 min),reaching a value of ca. 8 g/1 in the reaction mixture.

F pure� 3,m es area

The �-glucosidase fromA. fasciata when reactedwith pNP-�-d-Glc (Table 2) initially produced dif-ferent products at Rfs corresponding to disaccharidesand trisaccharides of the substrate as assessed by TLCand previously reported by using crude homogenate.The interconversion of different regioisomeric disac-charides was noticed by TLC (solvent B) and thesecompounds were also hydrolysed back to form againthe substrate pNP-�-d-Glc (Giordano et al., 2004).Interconversion reactions were also observed usingdirectly disaccharides such as pNP-�-d-Glc-(1-4)-�-Glc and pNP-�-d-Glc-(1-4)-�-d-Glc; using the latter,the monomer pNP-�-d-Glc, pNP-�-d-Glc-(1-6)-�-d-Glc and glucose were the products after 24 h whiletrisaccharidic compounds were noticed in the earlystages of reaction.

In Table 2reactions using pNP-�-d-Glc as donorand different substrates as hetero-acceptors are alsoreported; the survey of these transglycosylation reac-tions indicated that cellobiose, saccharose and isoma-ltose were all subjected to�-glycosylation forminghigher MW products. Trehalose, melibiose and glyc-erol also formed products to a lesser extent. The disac-charide lactose as well as other compounds indicatedin Table 2were not glycosylated; dimers of donor how-ever were observed in all reactions but in the ones usingascorbic acid and phenyl thio-cellobioside indicatingstrong enzyme inhibition.

Conveniently, cheap maltose can be also used asdonor and formation of transglycosylation productsw dif-f toseb on(G rst,tG LCa d in

ig. 2. Time course for the bioconversion of maltose using-glucosidase fromAplysia fasciata. M2, maltose; P, panose; Maltotriose; IM, isomaltose. Glucose, tetra- and pentasaccharidlso present in the reaction mixture.

as observed even using it at 1:1 molar ratio witherent acceptors along with products due to malioconversion (Table 2). In the early stages of reacti30 min) using pNP-�-d-Glc as acceptor, pNP-�-d-lc-(1-4)-�-d-Glc was the sole product appearing fi

hen isomaltoside derivative, pNP-�-d-Glc-(1-6)-�-d-lc, was also identified as indicated by TLC and HPnalyses with authentic standards; it accumulate

G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284 281

the reaction mixture up to the end when this prod-uct became the most abundant isomer (yield ca.15–20%). Usingp-nitrophenyl�-cellobioside (pNP-�-d-Glc-(1-4)-�-d-Glc), trisaccharidic product forma-tion was observed (see below) while both�- and �-anomers ofp-nitrophenyl galactopyranoside were notglycosylated in significative amount.

Pyridoxine acted as acceptor furnishing mono�-glucosyl derivatives at both –CH2OH of the moleculeas established by1H and13C NMR spectra for 1 and 2(Fig. 3) as reported (Suzuki et al., 1997).

Results obtained for hydrolysis and transglyco-sylation experiments on different substrates suggestthe production of panose by intramolecular arrange-ment from maltotriose firstly formed, instead of anintermolecular direct�-1-6 glucosylation of maltose,namely (i) the decrease of maltotriose while increas-ing panose concentration during maltose bioconver-sion (Fig. 2), (ii) the disappearing of pNP-�-d-Glc-(1-4)-�-d-Glc formed, and the accumulation of pNP-�-d-Glc-(1-6)-�-d-Glc in the glucosylation of pNP-�-d-Glc by maltose, (iii) the poor results for lac-tose and other galactopyranosides possessing 4-axialhydroxyl group (Table 2) showing the poor capabil-ity for direct �-1-6 glucosylation. The overall results

obtained with malto-oligomers indicated the prefer-ential enzymatic formation of�-1-4 linkages in theearly stages of the reaction and accumulation of�-1-6 products (isomaltose and panose). Furthermorea possible molecular limit in the acceptor site ofthe enzyme is conceivable as it is indicated by theabsence of higher MW products in the maltoheptaosereaction.

It is of interest the finding that higher tetra- and pen-tasaccharides are among reaction products also start-ing from high maltose concentration; a similar featureobtaining high MW products has also been recentlyreported in the synthesis of L-fucose oligosaccharidesfor two eukaryotic wild type exo-glycosidases (Berteauet al., 2004) one of which belongs to a marine source;nevertheless, any conclusion should strictly considerthe nature and function of the enzyme and the substrateaffinity in the acceptor site.

According to the Michaelis constants for pNP-�-d-Glc (0.26 mM) and maltose (5.7 mM) (Table 3) it couldbe suggested that the subsite + 1 of this enzyme has anaffinity for the aryl group higher than for a glucosylresidue. Although thekcat value for maltose (489 s−1)was three times higher than that for pNP-�-d-Glc(163 s−1), the catalytic efficiency showed that, in vitro,

tained

Fig. 3. �-Glycosylated products ob by�-glucosidase fromAplysia fasciata.

282 G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284

pNP-�-d-Glc is the preferred substrate for this enzyme(kca/Km = 627 mM−1 s−1). However, a still good cat-alytic efficiency was observed in the hydrolysis of pNP-�-d-Glc-(1-4)-�-Glc (kcat/Km 36.8 mM−1 s−1), whileno hydrolytic activity was detected when the pureenzyme was incubated in the presence of pNP-�-d-Gal or pNP-�-d-Man (data not shown). The reportedsubstrate specificity, together with the fact that thisenzyme is not able to hydrate glucal, suggested that theenzyme fromAplysia fasciata may belong to family Itype of�-glucosidases (Kimura, 2000) correspondingto family 13 of glycosyl hydrolases GH (Kimura etal., 2004) however sequence similarity study is neces-sary to establish it. Isomers of glycosylated pyridoxineobtained (Table 2) are important molecules from the

nutritionally point of view and are more stable thanpyridoxine against light and heat (Asano and Wada,2003). Traces of disaccharidic derivatives of the accep-tor were also noticed by TLC.

3.4. α-Glucosyl cellobiose and relatedchromophoric derivative and α-glucosyl sucroseformation

Encouraged by the positive preliminary resultsof cellobiose and saccharose shown inTable 2, weperformed both reactions with these acceptors on asemi-preparative scale. The chromophoric derivativeof cellobiose, pNP-�-d-Glc-(1-4)-�-d-Glc, was alsotested. Interest in oligoglucosides possessing mixed

F tylatedG ucosida stic1 cture d olumn,c eric si te1 etylate )a ppm both correlating with anomeric13C NMR signals at 100.0 and 100.3 ppm.Srm

ig. 4. NMR data of glucosyl cellobiose (A in D2O) and of its acelc-(1-4)-p-d-Glc-(1-4)-�-d-Glc (C in CDCl3) synthesised by�-gl

H–13C multiple bond correlations found in HMBC spectra. Struontaining the most abundant trisaccharide, showed four anom3C NMR signal. (B) Diagnostically in the1H NMR spectrum of acnomeric�-signals (J1–2= 7.6 Hz) were clearly visible at ca. 4.56

tarting from these anomeric doublets, in the COSY spectrum of this

ing finding H-4 signal of this unit at 3.94/75.2 ppm, typical values for aultiple bond correlations found confirmed such assignments. Mass

derivative (B in CDC13) and of peracetylated derivative of pNP-�-d-se fromAplysia fasciata; double headed arrows indicate diagno

rawing not complete for simplicity. (A) The fraction from Biogel cgnals in the1H NMR spectrum (in D2O) each correlating with appropriad derivative, two slightly shifted (for�- and�-forms at reducing end

derivative, it was possible to follow the correlations of the Glc2 pyranoseglycosylated position in a peracetylated compound. Finally the1H–13C

spectra are in agreement with trisaccharidic structure.

G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284 283

�- and�-linkages recently arisen (Ning et al., 2003);in fact �-glucosyl cellobioses have been recently pro-duced from sucrose donor and cellobiose acceptor byalternansucrase (Morales et al., 2001) while glucosylsucroses were also synthesised enzymatically by�-glucosidase from spinach (Sugimoto et al., 2003) andother enzymes.

The trisaccharides obtained with cellobiose andsucrose (ca. 25% yield, ca. 4 g/1) were purified bychromatography on Biogel P2 and analysed by NMRspectroscopy for structural determination. In the reac-tion using cellobiose the�-1-4 glucosyl derivative wasproduced as the most abundant (ca. 90%) product (3,Fig. 3); other minor isomers were also present. The�-1-4 interglycosidic linkage of the major compound wasassigned by (i) negative comparison with previouslyreported NMR data for�-1-2 and�-1-6 isomers andDEPT experiments (Morales et al., 2001), (ii) rulingout of �-1-3 linkage for the absence of signals highlyshifted (downfield O-glycosylation�-shift, >80 ppmfor �-1-3 linkage) in13C NMR spectrum and (iii) directproof by two-dimensional NMR spectroscopy studyof acetylated derivative (Fig. 4). The chromophoricderivative of cellobiose, pNP-�-d-Glc-(1-4)-�-d-Glc,formed the corresponding elongated product pNP-�-d-Glc-(1-4)-�-d-Glc-(1-4)-�-d-Glc (3a, Fig. 3) as estab-lished by1H NMR spectroscopy study on acetylatedderivative (Fig. 4C).

The sucrose was also�-glucosylated at position4 of glucose unit forming erlose (4G-�-d-glucosyls bycp ati-c ns-f lsi ndN thep yla-t turew rtu-n l-l ingf ruc-t byt osta isacc lm ure

was shown to be a tetrasaccharide by the presence oftwo glucose anomeric signal (5.41/95.5 and 5.30/95.6)other than the one due to the fructose linked glucosemoiety (5.58/89.8) and by mass spectrum (1277.6,M+ + Na).

3.5. Conclusion

The main physiological role of the exo-type gly-cosidases is to produce monosaccharides then utilisedas carbon and energy source (Kato et al., 2002).The �-glucosidase from molluscA. fasciata heredescribed is probably involved in this function. Theresults reported here indicated that the enzyme actson relatively short maltooligosaccharides; in eachexample end products, which may have importantroles in gene regulation (Miller and Reznikoff, 1978;Mandels et al., 1962) are panose and isomaltose reflect-ing the very poor hydrolytic activity of our enzymeon �-1-6 linkage. Finally the interesting survey oftransglycosylation features of this enzyme promptedfor the optimisation of single important reactionssuch as pyridoxine glucosylation, synthesis of chro-mophoric (pNP) di- and trisaccharides, glucosylationof cellobiose and sucrose. Remarkably, while for cel-lobiose and sucrose reaction yields and concentra-tion of products are comparable to those obtainedusing glycosyl transferases (Morales et al., 2001), theothers reactions were realised at a 1:1 molar ratiomaltose:acceptor, a very interesting attribute from a

o-.,

R.V.

B-for

Theioneject

nkonimi

ucrose,4, Fig. 3) as could be easily establishedomparison of the13C NMR signals (in D2O) of ourroduct with those reported for the erlose enzymally prepared by using cyclodextrin glucosyl traerase (Martin et al., 2004). However, minor signan the 13C NMR spectrum of native material aMR analysis of peracetylated mixture indicatedresence of two other minor products. After acet

ion the peracetylated erlose was obtained in mixith an uncharacterised minor trisaccharide; foately in the1H NMR spectrum it was easy to fo

ow 1H–1H correlations of the pyranose ring startrom anomeric signal of the glucose attached to fose (HI 5.57/89.8) securing the erlose structurehe presence of H-4 signal at 3.98 ppm for the mbundant. Mass spectra are in agreement with trharidic structure (989.4, M+ + Na+). An additionainor compound purified from the acetylated mixt

-

synthetic point of view (effortless purification, prductivity of each reaction batch, etc.) (Scigelova et al1999).

Acknowledgments

The authors wish to thank E. Pagnotta andD’Ambrosio for technical assistance. D. Melck,Mirra, S. Zambardino of the NMR service of ICNaples, and M. Zampa are also acknowledgedrunning NMR and mass spectra, respectively.present research was partially supported by RegCampania, L.R. N. 5 28.03.2002 Research Pro(A. Giordano). Dr. A. Tramice wishes to thaCentro Regionale di Competenza in ApplicaziTecnologico-Industriali di Biomolecole e Biosistefor fellowship.

284 G. Andreotti et al. / Journal of Biotechnology 122 (2006) 274–284

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