Hydrolyses and transglycosylations performed by purified α-d -glucosidase of the marine mollusc Aplysia fasciata

Journal of Biotechnology 122 (2006) 274–284

Hydrolyses and transglycosylations performed by purified ␣-d-glucosidase of the marine mollusc Aplysia 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 mollusc Aplysia fasciata are reported. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable characteristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last two acceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl transferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptor which is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.). © 2005 Elsevier B.V. All rights reserved. Keywords: ␣-Glucosidase; Transglycosylation; Oligosaccharide synthesis; Marine organism; Aplysia fasciata; Biocatalysis

1. Introduction The investigation of transglycosylation potential of glycosyl hydrolases can provide enzymes with new interesting catalytic activities. These enzymes usually hydrolyse glycosidic bonds but are also able to catalyse the stereospecific formation of such linkages (Scigelova et al., 1999), finding application in the enzy∗ Corresponding author. Tel.: +39 081 8675095; fax: +39 081 8041770. E-mail address: [email protected] (A. Trincone).

0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.10.002

matic synthesis of oligosaccharides. The great interest for them has been also recently demonstrated by successful genetic manipulation of some of their representatives (glycosynthases) (for a review see Perugino et al., 2004). The marine environment furnished different sources of glycosyl hydrolases (Kusaykin et al., 2003) and the effort for the identification of new enzymes, each forming any desired glycosidic bond, is of current significance. We are actively involved in the search for enzymes from marine organisms thus recently we focused our attention on the sea hare Aplysia fasciata

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Poiret 1789, a large mollusc easily collectable and very common in Mediterranean habitats belonging to the order Anaspidea (Carefoot, 1987). Glycosyl hydrolases interesting for biotechnological applications such as ␤-mannosidase, ␣-glucosidase and ␤-galactosidase were abundant both in the hepatopancreas and in visceral mass extracts of this organism. Both ␤-enzymes possess interesting catalytic properties for the synthesis of ␤-mannosides (Andreotti et al., 2005) and ␤-galactosides (Giordano et al., 2005). The reaction with maltose (producing good yield of panose, isomaltose and higher tetra- and pentasaccharides) and other few transglucosylation reactions performed by ␣glucosidase activity were already studied (Giordano et al., 2004) using the crude homogenate; the use of a wide variety of acceptors was hampered by the presence of other hydrolytic activities in the total visceral extract. Transglycosylation activity of the ␣-glucosidases is a biotechnologically relevant issue in that these enzymes are applied both in food industry to produce oligosaccharides (Voragen, 1998) and to conjugate sugars to biologically relevant materials improving their chemical properties and physiological functions (Kren and Martinkova, 2001). In this paper we report on the purification and characterisation of the ␣-glucosidase from A. fasciata as we were prompted by the interesting preliminary results obtained using the crude homogenate. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied in order to assess specific features which are of interest from a biotechnological point of view (i.e. production of glucosides of different nature); remarkable characteristics of this enzyme for the bioconversion of maltose and other malto-oligosaccharides and for the synthesis of different trisaccharides of cellobiose and sucrose are reported.

2. Material and methods 2.1. Materials and instrumental Q-Sepharose Fast Flow, SP-Sepharose Fast Flow, Phenyl-Sepharose Fast Flow, Superdex-200 were purchased from Amersham Pharmacia Biotech. Nitrophenyl glycosides and maltose were obtained from Sigma (St. Louis, MO). Reverse-phase silica gel and

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TLC silica gel plates were from E. Merck (Darmstadt, Germany). All other chemicals were of analytical grade. HPLC was performed on a Mlton Roy apparatus equipped with UV detector (Waters Ass., USA) (254 nm). A Dionex 500 chromatographic system, controlled by PeakNetTM software (Sunnyvale, CA), was used to separate and detect carbohydrates. AKTA-Prime system (Amersham Pharmacia Biotech) was used for protein purification. Agilent UV–vis Spectroscopy System was used for analytical UV measurements. NMR spectra were recorded on Bruker instruments at 600, 400 and/or 300 MHz. Samples for NMR analysis were dissolved in the appropriate solvent and both the downfield shift of the signal of the solvent or acetone-d6 (31.07 ppm for 13 C NMR spectra in D2 O) were used as internal standard. Acetylation of compounds was performed with pyridine/Ac2 O at room temperature. The solvents were removed by a N2 stream and the reaction mixture was purified by silica gel chromatography or preparative TLC. Protein concentrations were routinely estimated using the Bio-Rad Protein System, with the bovine serum albumin as the standard (Bradford, 1976). TLC solvents: (A) (EtOAc:CH3 COOH:2-propanol: HCOOH:H2 O, 25:10:5:1:15, v/v/v/v/v), (B) (EtOAc: CH3 OH:H2 O, 70:20:10, v/v/v), (C) (CHC13 :CH3 OH: H2 O, 65:25:4, v/v/v). ESI-MS spectra were obtained on a Q-T of mass spectrometer, Micro (Mcromass). 2.2. Enzyme source Visceral mass extract of A. fasciata was prepared as previously described (Giordano et al., 2004). Half of crude material (49 g) from 20 animals was used for the purification either of ␤-mannosidase described elsewhere (Andreotti et al., 2005) and ␣-d-glucosidase here described. 2.3. Isolation and characterisation of α-d-glucosidase All purification steps were carried out at room temperature in K-acetate 50 mM pH 5.5 (buffer A). The

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Table 1 Purification of ␣-glucosidase from Aplysia fasciata Purification stepa

Volume (ml)

Activity (U)

Protein (mg)

Specific activity (U mg−1 )

Yield (%)

Purification-fold

Homogenate Superdex-200 SP-Sepharose Q-Sepharose Phenyl-Sepharose

12 185 195 20 1.85

345 294 351 281 253

226.8 138.7 82.1 20.8 6.6

1.52 2.12 4.28 13.5 38.4

100 85 100 80b 74

1.00 1.39 2.79 8.83 25.05

a The enzyme was purified contemporary with a ␤-mannosidase (Andreotti et al., 2005) and the activities were separated after the third chromatography 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 as the one reported for ␤-mannosidase activity (Andreotti et al., 2005). The two enzymes were separated after the anionic exchange chromatography on a Q-Sepharose column. An hydrophobic interaction chromatography (Phenyl-Sepharose Fast Flow) was necessary to obtain pure ␣-glucosidase (Table 1). The pooled sample of ␣-glucosidase (20 ml) from Q-Sepharose was treated with solid ammonium sulphate to give 20% saturation. After stirring for 1 h at room temperature, the sample was loaded at flow rate of 1 ml min−1 onto a Phenyl-Sepharose Fast Flow column (1 cm × 9.5 cm) which had been equilibrated with buffer A containing the same amount of ammonium sulphate. The column was washed with the equilibration buffer until the absorbance at 280 nm returned to baseline, and retained proteins were eluted with a 60-ml linear gradient of 20–0% ammonium sulphate. The ␣-d-glucosidase was eluted when ammonium sulphate was 7.2%. The fractions were pooled, dialysed against 50 mM K-acetate pH 5.5, concentrated by ultrafiltration and stored at −20 ◦ C. The molecular mass of native ␣-d-glucosidase was estimated by gel filtration on a Superdex-200 column equilibrated with 50 mM K-acetate pH 5.5, which had previously been calibrated with myoglobin (18 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), catalase (232 kDa) and ferritin (440 kDa). Blue dextran and pyridoxal phosphate were used to determine the void and the total volume, respectively. The flow rate was 1 ml min−1 . The molecular mass under denaturing conditions was estimated by SDS-PAGE on slab gel containing 10% acrylamide using standard procedures (Sambrook et al., 1989). Proteins were located on the gels using Comassie Brillant Blue staining.

2.4. Assay for p-nitrophenyl glycosides and other substrates ␣-d-Glucosidase activity was assayed under standard 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 at intervals and the reaction was stopped with the addition of Na2 CO3 1 M (0.45 ml). One unit of ␣-d-glucosidase activity was defined as that amount of enzyme required to catalyse the release of 1.0 ␮mol of p-nitrophenol per min. One millimolar substrate concentration was used for determination of temperature (between 25 and 50 ◦ 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 used for determination of kinetic parameters. The activity towards p-nitrophenyl ␣-d-galactopyranoside (pNP-␣d-Gal) and p-nitrophenyl ␣-d-mannopyranoside (pNP␣-d-Man) was determined under standard conditions as described above in presence of 20 mM substrate. A qualitative analysis of substrate specificity was tested on three groups of substrates. p-Nitrophenyl glucosides (pNP-␣-d-Glc, pNP-␣-d-Glc-(1-4)-␣-d-Glc, pNP-␤-d-Glc-(1-4)-␣-d-Glc) were tested at 33 mM concentration. Disaccharides (maltose, trehalose, isomaltose, saccharose) and maltotriose were tested at 100 mM while maltoheptaose and carbohydrate polymers were all reacted at 10 mg/ml. All these reactions were performed using 72 ␮g of pure protein per ml of reaction mixture. The products were identified by TLC using authentic standards, or High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (Dionex) (HPAEC-PAD) or by NMR spectroscopy (see below). The time course for bioconversion of maltose was studied using 93 mM maltose and 18 ␮g of pure protein

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per ml. Aliquots were withdrawn at different time intervals and the enzyme was inactivated by heating for 2 min. The denatured proteins were removed by centrifugation. Chromatographic analysis of reaction mixtures was achieved by HPAEC-PAD using a CarboPacTM PA1 analytical column (Dionex) with a linear gradient from 100 mM sodium hydroxide to 100 mM sodium hydroxide/200 mM sodium acetate over 20 min at 0.25 ml min−1 . Authentic standards were used for calibration. Chemical identity of products was established as previously reported (Giordano et al., 2004). The enzymatic activity towards maltose, isomaltose and pNP-␤-d-Glc-(1-4)-␣-d-Glc was measured by the amount of glucose released quantified with glucose enzymatic kit (Sigma). The reaction mixture (0.3 ml) containing 60 mM Na-phosphate/30 mM citrate at pH 5.8 and an appropriate amount of substrate was incubated at 34 ◦ C. The reaction was initiated by addition of the enzyme, aliquots (0.05 ml) were withdrawn at intervals and the reaction stopped by heating. Kinetic parameters were determined using standard reaction mixtures containing 0.6–20 mM maltose or 0.8–25 mM pNP-␤-d-Glc-(1-4)-␣-d-Glc. Activity towards isomaltose was tested at 22 mM. One unit of enzyme activity was defined as that amount of enzyme required to catalyse the hydrolysis of 1.0 ␮mol of substrate per min. 2.5. Stability under different conditions Both the pH and temperature stabilities were investigated by incubating the pure enzyme at 96 ␮g ml−1 in the presence of 90 ␮g ml−1 of BSA. Residual enzymatic activity under standard conditions was then measured after 19 h incubation. The pH stability was investigated at 34 ◦ C at different pH values: 50 mM K-acetate buffer at pH 5.5, 50 mM K-phosphate buffer at pH 5.8 and 6.5. Thermal stability was measured in 50 mM Kphosphate pH 5.8 at 30, 34 and 38 ◦ C. 2.6. Transglycosylation reactions All reactions were conducted in sealed vials using phosphate buffer 50 mM pH 5.8 at 34 ◦ C under agita-

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tion, monitoring the progress by TLC (solvents A, B or C, see Section 2.1). Analytical scale transglycosylation reactions using pNP-␣-d-Glc (33 mM) as donor were conducted at five molar excess of hetero-acceptors indicated in Table 2; reactions using maltose (29 mM) as donor were conducted at 1:1 molar ratio of acceptors; in both systems 9 ␮g of pure enzyme per ml (TLC solvent B) were used. The products of pyridoxine glycosylation were separated by reverse-phase chromatography and preparative TLC (solvent C); purified bands were analysed by 1 H NMR spectroscopy using DMSO-d as solvent. 6 The reaction mixture for semipreparative ␣glucosylation of pNP-␤-d-Glc was purified by reverse phase RP-18 (eluting firstly with water and then with methanol) and silica gel chromatography (eluting with gradient of methanol in ethyl acetate). Semi-preparative scale reactions for ␣-glucosyl cellobiose and ␣-glucosyl sucrose formation were conducted using 25 mg (83 ␮mol) of pNP-␣-d-Glc and five molar excess of cellobiose or sucrose, dissolved in 2.5 ml of phosphate buffer 50 mM pH 5.8; 18 ␮g of pure enzyme were added to start the reaction at 34 ◦ C under agitation. After the total consumption of donor (TLC solvent B, ca. 6 h) the reaction mixtures were placed at 100 ◦ C for 5 min, cooled and the products purified on Bio gel P2 column chromatography by eluting with water. The fractions were analysed by TLC (solvent A); those containing the reaction products were pooled and lyophilised and the products subjected to NMR spectroscopy using D2 O as solvent and acetone-d6 for external reference. The products were also subjected to acetylation (Section 2.1) overnight and the peracetylated derivatives used for NMR measurements using CDC13 as solvent and external reference.

3. Results and discussion The ability of ␣-glucosidases to perform transglycosylation reactions is a relevant issue from the biotechnological point of view (food industry, production of glycoconjugates, etc.). An ␣-glucosidase activity was the most abundant glycosyl hydrolase activity found in the visceral mass of A. fasciata and the reaction with maltose (producing good yield of the trisaccharide panose, the disaccharide isomaltose and higher tetraand pentasaccharides) and other few transglucosylation

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Table 2 Qualitative assessment of bioconversion of different substrate by ␣-glucosidase from Aplysia fasciata Substrates

Reactivity

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) Glucal Maltose (␣-1,4)

– – – – – – – – –

− − − − − − +/− − +

Panose Maltotriose

– –

+

Maltoheptaose

–

+

pNP-␣-d-Glc pNP-␤-d-Glc-(1-4)-␣-d-Glc pNP-␣-d-Glc-(1-4)-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc pNP-␣-d-Glc Maltose

– – – Cellobiose Saccharose Trehafose Isomaltose ␣-Cyclodextrin Ascorbic acid l-Menthol Glycerol Melibiose Lactose Phenyl thiocellobioside pNP-␤-d-Glc

+ + + + + +/− + − − − +/− +/− − − +

Maltose Maltose Maltose Maltose

pNP-␤-d-Glc-(1-4)-␤-d-Glc pNP-␤-d-Gal pNP-␣-d-Gal Pyridoxine

+ − − +

reactions performed by using the crude homogenate were already studied (Giordano et al., 2004); the use of a wide variety of acceptors was hampered by the presence of other hydrolytic activities in the total visceral extract. 3.1. Purification of α-glucosidase and structural characterisation The results of chromatographic purification of ␣glucosidase from the visceral mass of A. fasciata

Notes

Glucose formation Production of panose, isomaltose and lesser amount of tetraand pentasaccharides – Oligomers up to M6 present in early stage then panose and isomaltose formation Formation of product from M7 to glucose then panose and isomaltose formation Interconversion of regioisomeric disaccharides and hydrolysis Trisaccharide formation and hydrolysis Trisaccharide formation and hydrolysis Trisaccharide formation (see Fig. 3) Trisaccharide formation (see Fig. 3) Trisaccharide formation Trisaccharide formation

Glucosyl glycerol Trisaccharide formation

pNP-␤-d-Glc-(1-4)-␣-d-Glc then pNP-␤-d-Glc-(1-6)-␣-d-Glc (15–20% yield) Formation of chromophoric trisaccharide Trace amount of pNP-disaccharide Trace amount of pNP-disaccharide Glucosyl pyridoxines (see Fig. 3)

are shown in Table 1. After the last step on a Phenyl-Sepharose column the sample was obtained in a homogeneous form as determined by SDSPAGE (Fig. 1). The purified enzyme gave only one band of 69 kDa on SDS-PAGE, while the molecular mass of the native ␣-glucosidase determined by analytical Superdex-200 gel filtration was 255 kDa, indicating that this enzyme is a homotetramer. The enzyme was purified 25-fold with a specific activity of 38.4 U/mg and the overall yield was 74%.

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bated for 19 h at pH 5.8 at 30 and 34 ◦ C it retained 70 and 64% of its activity, respectively. The half-life times were ca. 30 h. Only 34% of the activity was detected after incubation at 38 ◦ C, and the half-life was 12 h. In agreement with these observations pH 5.8 and 34 ◦ C were chosen as standard conditions for performing hydrolytic and transglycosylation experiments. 3.3. Catalytic properties: hydrolysis and transglycosylation

Fig. 1. SDS-PAGE (10% acrylamide) of the ␣-glucosidase from Aplysia fasciata (A) and standard mixture of proteins (M) prestained broad-range markers (Bio-Rad). Proteins were detected with Comassie staining.

3.2. Thermal and pH properties The effects of acidity and heat on the activity and stability of ␣-glucosidase were examined. The enzyme showed a pH optimum at 5.8 and the optimum temperature at this pH was in the range 36–44 ◦ C. When the enzyme was incubated for 19 h at 34 ◦ C in the presence of 0.09 mg ml−1 BSA, at pH 5.5 (in acetate buffer), 5.8 and 6.5 (in phosphate buffer), it retained 67, 64 and 69% of its hydrolytic activity, respectively. The extrapolated half-life times were approximately 30 h in all these cases. When the experiment was performed at the same pHs but in the presence of 60 mM Naphosphate/30 mM citrate buffer, a stronger decrease of the activity was monitored. When the enzyme was incu-

3.3.1. Hydrolysis The ␣-glucosidase from A. fasciata was not reactive using starch, amylopectin, amylose, isomaltose, panose, pullulan and saccharose. A very feeble reaction was detected using trehalose. No enzymatic hydration of glucal (formation of 2-deoxyglucose) was detectable. A qualitative assessment of reactivity of the enzyme toward each substrate is reported in Table 2. The less reactive among the p-nitrophenyl substrates was pNP-␤-d-Glc-(1-4)-␣-d-Glc while the reactions using pNP-␣-d-Glc-(1-4)-␣-d-Glc and pNP-␣-d-Glc were complete in few minutes in the conditions adopted as judged by TLC. Kinetic parameters for the hydrolysis of maltose, isomaltose, pNP-␣-d-Glc and pNP-␤-d-Glc-(1-4)-␣d-Glc are reported in Table 3. The hydrolytic activity for ␣-1-4 glucosidic linkage was higher than that of ␣1-6, in fact specific activity for maltose was 40 times higher than that for isomaltose. 3.3.2. Transglycosylations and maltose and other malto-oligosaccharides bioconversion As indicated in Table 3, maltose was a good substrate for the ␣-glucosidase and its bioconversion, performed by the pure enzyme, produced the trisaccharide panose (␣-Glc-(1-6)-␣-Glc-(1-4)-Glc) and isomaltose (␣-Glc-(1-6)-Glc) together with lesser amount of tetraand pentasaccharides, in agreement with the results previously obtained by using the crude homogenate (Giordano et al., 2004). Maltotriose was also a good substrate with a pattern similar to the one observed with maltose: maltotetra- up to maltohexaose were found in the early stages of reaction (Dionex) but panose and isomaltose were later identified as end products also in this reaction. Using maltoheptaose the enzyme performed a reaction which is in accord to the reaction with maltose. In the first few minutes the formation

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Table 3 Kinetic parameters for hydrolysis of various substrates by ␣-glucosidase from A. fasciata Substrate

Km (mM)

kcat (s−1 )

kcat /Km (mM−1 s−1 )

VMAX (U/mg)

Maltose Isomaltose pNP-␣-d-Glc pNP-␤-d-Glc-(1-4)-␣-Glc

5.70 N.D.a 0.26 2.06

489 N.D.a 163 75.9

86.0 N.D.a 627 36.8

115 2.9b 38.4 17.9

a b

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

of products from maltohexaose down to glucose was clearly assessed by TLC and HPAEC-PAD analysis; as soon as maltose accumulated the formation of panose and isomaltose also started. After 20 min panose was the most abundant among products of uppermost MW (Table 2). In Fig. 2 the time course for the bioconversion of maltose is reported. Within the first hour of reaction in the conditions adopted ca. 60% of maltose was consumed forming maltotriose and panose; the concentration of maltotriose decreased to a very low value after 360 min as well as the concentration of the regioisomer panose increased to a plateau (after ca. 180 min), reaching a value of ca. 8 g/1 in the reaction mixture.

Fig. 2. Time course for the bioconversion of maltose using pure ␣-glucosidase from Aplysia fasciata. M2, maltose; P, panose; M3, maltotriose; IM, isomaltose. Glucose, tetra- and pentasaccharides are also present in the reaction mixture.

The ␣-glucosidase from A. fasciata when reacted with pNP-␣-d-Glc (Table 2) initially produced different products at Rfs corresponding to disaccharides and trisaccharides of the substrate as assessed by TLC and previously reported by using crude homogenate. The interconversion of different regioisomeric disaccharides was noticed by TLC (solvent B) and these compounds were also hydrolysed back to form again the substrate pNP-␣-d-Glc (Giordano et al., 2004). Interconversion reactions were also observed using directly 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)-␣-dGlc and glucose were the products after 24 h while trisaccharidic compounds were noticed in the early stages of reaction. In Table 2 reactions using pNP-␣-d-Glc as donor and different substrates as hetero-acceptors are also reported; the survey of these transglycosylation reactions indicated that cellobiose, saccharose and isomaltose were all subjected to ␣-glycosylation forming higher MW products. Trehalose, melibiose and glycerol also formed products to a lesser extent. The disaccharide lactose as well as other compounds indicated in Table 2 were not glycosylated; dimers of donor however were observed in all reactions but in the ones using ascorbic acid and phenyl thio-cellobioside indicating strong enzyme inhibition. Conveniently, cheap maltose can be also used as donor and formation of transglycosylation products was observed even using it at 1:1 molar ratio with different acceptors along with products due to maltose bioconversion (Table 2). In the early stages of reaction (30 min) using pNP-␤-d-Glc as acceptor, pNP-␤-dGlc-(1-4)-␣-d-Glc was the sole product appearing first, then isomaltoside derivative, pNP-␤-d-Glc-(1-6)-␣-dGlc, was also identified as indicated by TLC and HPLC analyses with authentic standards; it accumulated in

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the reaction mixture up to the end when this product became the most abundant isomer (yield ca. 15–20%). Using p-nitrophenyl ␤-cellobioside (pNP␤-d-Glc-(1-4)-␤-d-Glc), trisaccharidic product formation was observed (see below) while both ␣- and ␤anomers of p-nitrophenyl galactopyranoside were not glycosylated in significative amount. Pyridoxine acted as acceptor furnishing mono ␣glucosyl derivatives at both –CH2 OH of the molecule as established by 1 H and 13 C NMR spectra for 1 and 2 (Fig. 3) as reported (Suzuki et al., 1997). Results obtained for hydrolysis and transglycosylation experiments on different substrates suggest the production of panose by intramolecular arrangement from maltotriose firstly formed, instead of an intermolecular direct ␣-1-6 glucosylation of maltose, namely (i) the decrease of maltotriose while increasing panose concentration during maltose bioconversion (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 lactose and other galactopyranosides possessing 4-axial hydroxyl group (Table 2) showing the poor capability for direct ␣-1-6 glucosylation. The overall results

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obtained with malto-oligomers indicated the preferential enzymatic formation of ␣-1-4 linkages in the early stages of the reaction and accumulation of ␣1-6 products (isomaltose and panose). Furthermore a possible molecular limit in the acceptor site of the enzyme is conceivable as it is indicated by the absence of higher MW products in the maltoheptaose reaction. It is of interest the finding that higher tetra- and pentasaccharides are among reaction products also starting from high maltose concentration; a similar feature obtaining high MW products has also been recently reported in the synthesis of L-fucose oligosaccharides for two eukaryotic wild type exo-glycosidases (Berteau et al., 2004) one of which belongs to a marine source; nevertheless, any conclusion should strictly consider the nature and function of the enzyme and the substrate affinity in the acceptor site. According to the Michaelis constants for pNP-␣-dGlc (0.26 mM) and maltose (5.7 mM) (Table 3) it could be suggested that the subsite + 1 of this enzyme has an affinity for the aryl group higher than for a glucosyl residue. Although the kcat 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,

Fig. 3. ␣-Glycosylated products obtained by ␣-glucosidase from Aplysia fasciata.

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pNP-␣-d-Glc is the preferred substrate for this enzyme (kca /Km = 627 mM−1 s−1 ). However, a still good catalytic efficiency was observed in the hydrolysis of pNP␤-d-Glc-(1-4)-␣-Glc (kcat /Km 36.8 mM−1 s−1 ), while no hydrolytic activity was detected when the pure enzyme was incubated in the presence of pNP-␣-dGal or pNP-␣-d-Man (data not shown). The reported substrate specificity, together with the fact that this enzyme is not able to hydrate glucal, suggested that the enzyme from Aplysia fasciata may belong to family I type of ␣-glucosidases (Kimura, 2000) corresponding to family 13 of glycosyl hydrolases GH (Kimura et al., 2004) however sequence similarity study is necessary to establish it. Isomers of glycosylated pyridoxine obtained (Table 2) are important molecules from the

nutritionally point of view and are more stable than pyridoxine against light and heat (Asano and Wada, 2003). Traces of disaccharidic derivatives of the acceptor were also noticed by TLC. 3.4. α-Glucosyl cellobiose and related chromophoric derivative and α-glucosyl sucrose formation Encouraged by the positive preliminary results of cellobiose and saccharose shown in Table 2, we performed both reactions with these acceptors on a semi-preparative scale. The chromophoric derivative of cellobiose, pNP-␤-d-Glc-(1-4)-␤-d-Glc, was also tested. Interest in oligoglucosides possessing mixed

Fig. 4. NMR data of glucosyl cellobiose (A in D2 O) and of its acetylated derivative (B in CDC13 ) and of peracetylated derivative of pNP-␤-dGlc-(1-4)-p-d-Glc-(1-4)-␣-d-Glc (C in CDCl3 ) synthesised by ␣-glucosidase from Aplysia fasciata; double headed arrows indicate diagnostic 1 H–13 C multiple bond correlations found in HMBC spectra. Structure drawing not complete for simplicity. (A) The fraction from Biogel column, containing the most abundant trisaccharide, showed four anomeric signals in the 1 H NMR spectrum (in D2 O) each correlating with appropriate 13 C NMR signal. (B) Diagnostically in the 1 H NMR spectrum of acetylated derivative, two slightly shifted (for ␣- and ␤-forms at reducing end) anomeric ␤-signals (J1–2 = 7.6 Hz) were clearly visible at ca. 4.56 ppm both correlating with anomeric 13 C NMR signals at 100.0 and 100.3 ppm. Starting from these anomeric doublets, in the COSY spectrum of this derivative, it was possible to follow the correlations of the Glc2 pyranose ring finding H-4 signal of this unit at 3.94/75.2 ppm, typical values for a glycosylated position in a peracetylated compound. Finally the 1 H–13 C multiple bond correlations found confirmed such assignments. Mass spectra are in agreement with trisaccharidic structure.

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␣- and ␤-linkages recently arisen (Ning et al., 2003); in fact ␣-glucosyl cellobioses have been recently produced from sucrose donor and cellobiose acceptor by alternansucrase (Morales et al., 2001) while glucosyl sucroses were also synthesised enzymatically by ␣glucosidase from spinach (Sugimoto et al., 2003) and other enzymes. The trisaccharides obtained with cellobiose and sucrose (ca. 25% yield, ca. 4 g/1) were purified by chromatography on Biogel P2 and analysed by NMR spectroscopy for structural determination. In the reaction using cellobiose the ␣-1-4 glucosyl derivative was produced as the most abundant (ca. 90%) product (3, Fig. 3); other minor isomers were also present. The ␣-14 interglycosidic linkage of the major compound was assigned by (i) negative comparison with previously reported NMR data for ␣-1-2 and ␣-1-6 isomers and DEPT experiments (Morales et al., 2001), (ii) ruling out of ␣-1-3 linkage for the absence of signals highly shifted (downfield O-glycosylation ␣-shift, >80 ppm for ␣-1-3 linkage) in 13 C NMR spectrum and (iii) direct proof by two-dimensional NMR spectroscopy study of acetylated derivative (Fig. 4). The chromophoric derivative of cellobiose, pNP-␤-d-Glc-(1-4)-␤-d-Glc, formed the corresponding elongated product pNP-␤-dGlc-(1-4)-␤-d-Glc-(1-4)-␣-d-Glc (3a, Fig. 3) as established by 1 H NMR spectroscopy study on acetylated derivative (Fig. 4C). The sucrose was also ␣-glucosylated at position 4 of glucose unit forming erlose (4G -␣-d-glucosyl sucrose, 4, Fig. 3) as could be easily established by comparison of the 13 C NMR signals (in D2 O) of our product with those reported for the erlose enzymatically prepared by using cyclodextrin glucosyl transferase (Martin et al., 2004). However, minor signals in the 13 C NMR spectrum of native material and NMR analysis of peracetylated mixture indicated the presence of two other minor products. After acetylation the peracetylated erlose was obtained in mixture with an uncharacterised minor trisaccharide; fortunately in the 1 H NMR spectrum it was easy to follow 1 H–1 H correlations of the pyranose ring starting from anomeric signal of the glucose attached to fructose (HI 5.57/89.8) securing the erlose structure by the presence of H-4 signal at 3.98 ppm for the most abundant. Mass spectra are in agreement with trisaccharidic structure (989.4, M+ + Na+ ). An additional minor compound purified from the acetylated mixture

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was shown to be a tetrasaccharide by the presence of two glucose anomeric signal (5.41/95.5 and 5.30/95.6) other than the one due to the fructose linked glucose moiety (5.58/89.8) and by mass spectrum (1277.6, M+ + Na). 3.5. Conclusion The main physiological role of the exo-type glycosidases is to produce monosaccharides then utilised as carbon and energy source (Kato et al., 2002). The ␣-glucosidase from mollusc A. fasciata here described is probably involved in this function. The results reported here indicated that the enzyme acts on relatively short maltooligosaccharides; in each example end products, which may have important roles in gene regulation (Miller and Reznikoff, 1978; Mandels et al., 1962) are panose and isomaltose reflecting the very poor hydrolytic activity of our enzyme on ␣-1-6 linkage. Finally the interesting survey of transglycosylation features of this enzyme prompted for the optimisation of single important reactions such as pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. Remarkably, while for cellobiose and sucrose reaction yields and concentration of products are comparable to those obtained using glycosyl transferases (Morales et al., 2001), the others reactions were realised at a 1:1 molar ratio maltose:acceptor, a very interesting attribute from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.) (Scigelova et al., 1999). Acknowledgments The authors wish to thank E. Pagnotta and R. D’Ambrosio for technical assistance. D. Melck, V. Mirra, S. Zambardino of the NMR service of ICBNaples, and M. Zampa are also acknowledged for running NMR and mass spectra, respectively. The present research was partially supported by Regione Campania, L.R. N. 5 28.03.2002 Research Project (A. Giordano). Dr. A. Tramice wishes to thank Centro Regionale di Competenza in Applicazioni Tecnologico-Industriali di Biomolecole e Biosistemi for fellowship.

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