Journal Identification = PRBI Article Identification = 9240 Date: June 27, 2011 Time: 8:20 am

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Process Biochemistry 46 (2011) 1698–1702

Contents lists available at ScienceDirect

Process Biochemistry

jo u rn al hom epa ge: www .e lsev ier .com/ locate /procbio

hort communication

highly efficient diastereoselective synthesis of �-isosalicin by maltase fromaccharomyces cerevisiae

usan Velickovic a, Aleksandra Dimitrijevic a, Filip Bihelovic a, Dejan Bezbradicab, Ratko Jankova,enad Milosavic a,∗

Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, SerbiaFaculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 15 February 2011eceived in revised form 10 May 2011ccepted 11 May 2011

a b s t r a c t

In this report, �-isosalicin, a potent anticoagulant and skin whitening agent, was synthesized by a highlyefficient chemoselective and diastereoselective reaction, catalyzed by maltase from bakers’ yeast (Sac-charomyces cerevisiae). The highest yield of this one-step transglucosylation reaction was achieved with50 mM of salicyl alcohol as a glucose acceptor. The key reaction factors were optimized using response

eywords:-Isosalicinlucosidasealicyl alcoholransglucosylation

surface methodology (RSM) with an enzyme concentration of 10 U/mL. The optimum temperature of thereaction was determined as 36.5 ◦C, the optimal maltose concentration was 40% (w/v), the optimal pHwas 6.5, and the optimal reaction time was 16 h. Under these conditions 75% of �-isosalicin was obtained,with a yield of 10 g/L, and no by product formation was observed.

© 2011 Elsevier Ltd. All rights reserved.

akers’ yeast

. Introduction

Development of stereoselective methods for the synthesis oflycosidic linkages presents a considerable challenge to synthetichemists [1–3]. Chemical syntheses of glycosidic moieties areainly based on time-consuming protection and deprotection

trategies, activation or metal catalysis, but are often accompaniedy the formation of unwanted diastereomers and low yields [4,5].owever, these difficulties can be overcome by the applicationf enzymatic syntheses [6]. Transglycosylation reactions areell known and widely used methods for glucoside syntheses.lycosidases, responsible for catalytic hydrolysis of the glycosidic

inkage, are increasingly being used in carbohydrate synthesis7]. �-Glucosidase (maltase) is one of the most abundant glucosylydrolases present in baker’s yeast and has been used for theynthesis of various glucosides [7–9].

Glucosides of o-hydroxybenzyl alcohol (salicyl alcohol) con-inue attracting increasing attention due to a variety of biologicalctivities such as anti-inflammatory and analgesic [10], anti-ancer [11], antipyretic [12] and allergy preventive activity [13].

-Isosalicin is particularly important due to its effect on blood coag-lation, since �-isosalicin is an even more efficient anticoagulant

∗ Corresponding author. Tel.: +381 11 333 6656; fax: +381 11 2184 330.E-mail address: nenadmil@chem.bg.ac.rs (N. Milosavic).

359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2011.05.007

than heparin [14]. Furthermore, �-isosalicin is a potential skinwhitening agent, due to its tyrosinase inhibitory activity [15].

Transglucosylation reactions require a narrow range of condi-tions (temperature, pH, concentration of reactants and durationof reaction) for maximum utilization of the biocatalytic activityof an enzyme. Response surface methodology (RSM) has beenwidely employed for the optimization of enzymatic processesas well as other catalytic studies and it is also useful in simul-taneous analysis of the effects of several independent variables[6]. In this study, the synthesis of �-isosalicin [2-hydroxybenzyl-�-d-glucopyranoside], based on chemo- and diastereoselectiveglucosylation of 2-hydroxybenzyl alcohol (salicyl alcohol) with �-1,4-glucosidase from Saccharomyces cerevisiae was investigated.The optimal condition for the synthesis of �-isosalicin by maltasefrom baker’s yeast, with maltose as the glucose donor and salicylalcohol as the glucose acceptor, was determined. During optimiza-tion of conditions, the reaction was monitored by both, TLC andHPLC. The product was isolated, and its structure was confirmedby spectroscopic methods (1H and 13C NMR, HRMS and opticalrotation).

2. Materials and methods

2.1. Chemicals and enzyme

All commercially available reagents and solvents were used as obtained withoutfurther purification. �-1,4-Glucosidase (3.2.1.20) was isolated from baker’s yeastby a previously published procedure [16], and it showed a molecular weight of63 kDa on SDS PAGE. Its specific activity against 4-nitrophenyl-�-d-glucopyranoside

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D. Velickovic et al. / Process Biochem

Table 1Experimental design with experimental yields of transglucosylation. Includes alsocoded values (−2, −1, 0, 1, 2) of the variables and their corresponding actual values(values in brackets) used in the design of the experiments. In all experiments themaltase concentration was 10 U/mL.

Expt. no. Maltose(X1)

pH (X2) Temperature(X3)

Time (X4) Expt.yield

1 −1 (30%) −1 (4.5) −1 (35 ◦C) −1 (10 h) 02 1 (50%) −1 (4.5) −1 (35 ◦C) −1 (10 h) 25.63 −1 (30%) 1 (6.5) −1 (35 ◦C) −1 (10 h) 63.74 1 (50%) 1 (6.5) −1 (35 ◦C) −1 (10 h) 65.675 −1 (30%) −1 (4.5) 1 (45 ◦C) −1 (10 h) 1.36 1 (50%) −1 (4.5) 1 (45 ◦C) −1 (10 h) 18.67 −1 (30%) 1 (6.5) 1 (45 ◦C) −1 (10 h) 60.618 1 (50%) 1 (6.5) 1 (45 ◦C) −1 (10 h) 619 −1 (30%) −1 (4.5) −1 (35 ◦C) 1 (26 h) 2.18

10 1 (50%) −1 (4.5) −1 (35 ◦C) 1 (26 h) 43.911 −1 (30%) 1 (6.5) −1 (35 ◦C) 1 (26 h) 57.812 1 (50%) 1 (6.5) −1 (35 ◦C) 1 (26 h) 6013 −1 (30%) −1 (4.5) 1 (45 ◦C) 1 (26 h) 1.3914 1 (50%) −1 (4.5) 1 (45 ◦C) 1 (26 h) 20.3315 −1 (30%) 1 (6.5) 1 (45 ◦C) 1 (26 h) 53.616 1 (50%) 1 (6.5) 1 (45 ◦C) 1 (26 h) 5417 −2 (20%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 56.918 2 (60%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 63.1419 0 (40%) −2 (3.5) 0 (40 ◦C) 0 (18 h) 2.2520 0 (40%) 2 (7.5) 0 (40 ◦C) 0 (18 h) 56.621 0 (40%) 0 (5.5) −2 (30 ◦C) 0 (18 h) 69.1522 0 (40%) 0 (5.5) 2 (50 ◦C) 0 (18 h) 53.423 0 (40%) 0 (5.5) 0 (40 ◦C) −2 (2 h) 52.724 0 (40%) 0 (5.5) 0 (40 ◦C) 2 (34 h) 6025 0 (40%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 6526 0 (40%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 65.427 0 (40%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 61.3428 0 (40%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 65.81

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3.2. The effect of salicyl alcohol concentration

In order to maximize the utilization of the catalytic potential ofmaltase it is necessary to achieve high product yields and avoid the

29 0 (40%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 68.0530 0 (40%) 0 (5.5) 0 (40 ◦C) 0 (18 h) 66.24

as 100 U/mg protein and it showed no activity against isomaltose. Km for 4-itrophenyl-�-d-glucopyranoside was 0.27 mM. 1 U of enzyme activity was defineds the amount of enzyme that liberates 1 �mol of glucose at 25 ◦C for 1 min from-nitrophenyl-�-d-glucopyranoside.

.2. Transglucosylation reaction, identification and quantification of products

In all experiments, the reaction mixture contained 10 U/mL �-glucosidase from. cerevisiae and 50 mM salicyl alcohol in a 0.1 M sodium citrate/phosphate buffer pH.5, unless otherwise indicated. The maltose concentration, reaction temperature,nd reaction time were varied in accordance to the experimental plan. The reactionas quenched by acidification with 0.1 M HCl to pH 3.

The reaction mixture was then centrifuged, and analyzed by an Akta PurifierPLC (column: Waters Spherisorb 5 �m ODS 2, 4.6 mm × 250 mm; gradient from0% AN (v/v) with 1 mM HCl to 20% AN (v/v) with 1 mM HCl. The length of theradient was two column volumes, at a flow of 1.0 mL/min at 268 nm.

Thin-layer chromatography separations were performed on silica gel (60 F254,erck) using ethyl acetate/methanol/water (10:1.7:1.4, v/v/v) as the solvent, and

he spots were visualized by immersion in a ceric ammonium molybdate (CAM)ip.

.3. Optimization of reaction conditions

Experiments were conducted using a central composite design with 30 exper-mental points, that helps in investigating linear, quadric and cross product effectsf four factors, each varied at five levels [17]. The assay conditions for reactionarameters were taken at zero level (center point), level one (+1 and −1) and levelwo (+2 and −2). The design of the experiments employed is presented in Table 1,ith both, actual and coded values of factors. For the data evaluation response sur-

ace methodology (RSM) was used, and the second order polynomial equation wasested:

= b0 +k∑

i=1

bixi +k∑

i=1

biix2i

+i<j∑

i

∑

j

bijxixj

here Y is the dependent variable (response variable), xi and xj are the independentariables (factors), b0, bi , bii , bij are regression coefficients of the applied model. Theisher test was used to determine model adequacy, and the Student distributionas applied to evaluate the significance of the coefficients.

istry 46 (2011) 1698–1702 1699

2.4. Purification and structural analysis of the transglycosylated product

The reaction mixture (100 mL), quenched by acidification with 0.1 M HCl to pH 3,was loaded on a pre-equilibrated (1 mM HCl) column packed with 50 mL of PuroliteMN102 (a synthetic macroporous polystyrene resin, commercialized by Purolite,Wales, UK). The column was washed with 1 mM HCl to remove all non-phenoliccompounds (glucose and maltose). Retained phenolic compounds were eluted with96% (v/v) ethanol, the sample was concentrated under vacuum and redissolved in20% of ethanol. The sample was applied on a gel filtration column (Sephadex G-10)in order to separate �-isosalicin from unreacted salicyl alcohol.

NMR spectra were obtained on a Varian Gemini 200 (1H NMR at 200 MHz, 13CNMR at 50 MHz); chemical shifts are expressed in ppm (ı) using 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt as an internal standard. Optical rotation wasmeasured on an Autopol IV, Rudolph Research Analytical automatic polarimeter.Mass spectra were recorded on an Agilent Technologies 6210 TOF LC/MS instrument(LC: series 1200).

3. Results

3.1. Transglycolysation, product purification and its structuralanalysis

Since maltase from S. cerevisiae, obtained and purified by thesame procedure, has been previously reported as an efficient andselective catalyst in transglucosylation reactions [9,16], a prelimi-nary experiment was performed at the previously reported optimalconditions: at 30 ◦C, pH 5.5, and 40% maltose. The reaction mixturewas analyzed using TLC in order to determine the number of reac-tion products, and only one product (Rf 0.45) was detected. Then,the product was purified using the described method (Section 2.4.)and its structure was analyzed by spectroscopic methods (1H and13C NMR, HRMS, optical rotation). Obtained results confirmed that�-isosalicin is the obtained product.

1H NMR (DMSO-d6): 7.36 (d, J = 7.2, 1H), 7.04–7.13 (m, 1H), 6.81(s, 1H), 6.77 (s, 1H), 4.20–6.40 (bs, 5H), 4.77 (d, J = 4, 1H), 4.65 and4.43 (AB quartet, J = 13, 2H), 3.35–3.65 (m, 5H), 3,27 (dd, J1 = 3.7,J2 = 9.3, 1H), 3.10 (t, J = 8.7, 1H).

13C NMR (DMSO-d6): 155.0 (C), 128.8 (CH), 128.4 (CH), 124.7(C), 119.0 (CH), 115.1 (CH), 98.5 (CH), 73.6 (CH), 73.2 (CH), 72.3(CH), 70.6 (CH), 63.8 (CH2), 61.2 (CH2). HRMS (ESI) calcd. forC13H18O7Na+ [M+Na]+: 309.0944, found: 309.0941.

[�]D = 129.9 (c 1.67, methanol).

Fig. 1. Influence of salicyl alcohol concentration on the yield of conversion of salicylalcohol to �-isosalicin, and on the concentration of �-isosalicin in the reaction mix-ture after 2 h of the transglucosylation reaction. Reaction mixture: 10 U/mL maltase,40% maltose, pH 5.5, 40 ◦C.

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1700 D. Velickovic et al. / Process Biochemistry 46 (2011) 1698–1702

Fig. 2. Contour plots showing the effects of different variables on the conversion of salicyl alcohol in the synthesis of �-isosalicin. (a) The effect of maltose concentration andpH; (b) the effect of temperature and pH; (c) the effect of pH and reaction time.

Journal Identification = PRBI Article Identification = 9240 Date: June 27, 2011 Time: 8:20 am

D. Velickovic et al. / Process Biochemistry 46 (2011) 1698–1702 1701

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ig. 3. Reaction between maltose and salicyl alcohol catalyzed by maltase (�-1,4-glroduct yield) and optimal conditions for the synthesis of �-isosalicin are presente

se of excessive concentrations of salicyl alcohol because it com-licates downstream processing and increases production costs.herefore, the initial concentration of salicyl alcohol was optimizedith respect to both, the substrate conversion degree and the prod-ct yield. In all experiments the maltose concentration was 40%w/v), while the salicyl alcohol concentration was varied in theange 10–100 mM. It can be seen (Fig. 1) that the highest con-ersion (92%) was achieved with 10 mM of salicyl alcohol. Furtherncrease of the concentration of salicyl alcohol resulted in a con-inuous decrease of the conversion degree. This decrease becameery steep with the increase of acceptor concentrations above0 mM leading to conversions as low as 40%. On the other hand,he yield (expressed in g of product per L) increased very steeplyith the increase of salicyl alcohol concentration until 60 mM. Fur-

her increase resulted in only a slight increase of the yield from 65o 73 g/L. Since changes in the salicyl alcohol concentration exhib-ted opposite effects on the conversion degree and product yield,

e used 50 mM salicyl alcohol in further experiments, becauset enables achievement of high yields and satisfying conversionegrees.

.3. Optimization of the reaction conditions using RSM

The transglucosylation reaction of salicyl alcohol catalyzed by �-lucosidase from baker’s yeast was optimized with respect to thealtose concentration, pH, temperature and duration of the reac-

ion. Response surface methodology was used because it allowshe determination of optimum values of key experimental fac-ors, as well as understanding of possible relationships betweenactors. Experiments were performed in accordance with experi-

ental design and obtained results are given in Table 1. Statisticalnalysis (Fisher’s test) has shown that the proposed second orderolynomial model is adequate for fitting experimental results at thepplied significance value (p < 0.05). After further analysis by thetudent test and exclusion of insignificant coefficients the followingodel was obtained:

= 66.2 + 5.05x1 + 19.7x2 − 3.32x3 + 0.47x4 − 3.37x21

− 11.0x22 − 3.05x2

3 − 4.28x24 − 6.19x1x2 − 3.04x2x4 (1)

The obtained model indicates that all examined experimen-al factors had an influence on the product formation. Also, all

uadratic coefficients are significant and have a negative value,

ndicating that the effects of all examined factors can be describedy a quadratic function with a maximum, which allows easy deter-ination of their optimum values. Additionally, it is noticeable that

ase) from Saccharomyces cerevisiae. The optimal concentration of the reactant (andw each molecule and arrow, respectively.

two pairs of interactions between factors have been detected: anegative interaction between the maltose concentration and pH(−6.19) and a negative interaction between the pH and reactiontime (−3.04). Generally, the pH seems to be the most influentialreaction factor, since the corresponding linear and quadratic coeffi-cients have the highest values. For illustration, the effects of severalpairs of experimental factors have been depicted in contour plots(Fig. 2). It can be seen that any change of pH value from opti-mum leads to a steep decrease of conversion, while other factorsexhibited a more moderate decrease. The optimum values are: tem-perature 36.5 ◦C, maltose concentration 40%, pH 6.5, and reactiontime 16 h.

In order to perform an experimental evaluation of the obtainedregression model, an additional experiment has been performed (intriplicate) in the obtained optimal conditions. The obtained yieldwas 71.6 ± 2.1%, which confirms that the model has good adequacy(deviation from the predicted value was 3.4%) and it enables anincrease in the enzyme’s productivity.

4. Discussion

The preliminary experiment has shown that maltase from S.cerevisiae has a good prospect for application in transglycosylationof salicyl alcohol, since it catalyzed formation of only one product,and no other product was detected by TLC or HPLC. 1H and 13C NMRanalyses proved that the obtained product is �-isosalicin indicatingthat the reaction occurs as illustrated in Fig. 3. From the couplingconstant of the anomeric proton NMR signal at 4.77 ppm (J = 4 Hz)it was concluded that glucopyranose was attached to aglycone by�-linkage, which was additionally confirmed by a rapid hydroly-sis of the product by amyloglucosidases. The chemical shift of thecharacteristic benzylic carbon in 13C NMR spectrum (60.1 ppm) isidentical as in a previously published spectrum of �-isosalicin [15].

Further experiments have been focused on the increase of prod-uct yield by the optimization of key factors. Optimization of theinitial concentration of salicyl alcohol was performed with a highmolar excess of maltose (1:12–1:120), since it is a significantlycheaper substrate so it is economically justified to base a novelprocess on such a strategy for increasing the product yield. Theopposite trends of the acceptor concentration effect on the con-version degree and product yield were observed, so the optimumconcentration was determined as 50 mM (molar ratio 1:60), sinceit enables almost a maximum product yield at high conversion

degrees. Further optimization of other key reaction factors usingRSM enabled attaining the conversion degree of the substrate above70%. As far as we know, this is the first report that maltase cancatalyze the production of �-isosalicin with such high yields.

Journal Identification = PRBI Article Identification = 9240 Date: June 27, 2011 Time: 8:20 am

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All previously published papers on �-isosalicin enzyme syn-hesis have shown some drawbacks: in the amyloglucosidaseransglycosylation reaction the yield is relatively low, 2.3 mg-isosalicin per 50 mg starch [1] while dextransucrase and cyclo-altodextrin glucanyltransferase produce a complex mixture of

roducts [10]. Although maltose phosphorylase gives the highestield (mol/mol 86%, 25 g L−1, 240 h), 1 U produces only 0.12 mg ofhe products [18]. In our present work, 1 U of �-glucosidase pro-uces as much as eight folds (1.06 mg) of �-isosalicin. Even more,he reaction rate of synthesis of �-isosalicin with �-glucosidase is5 times higher than with maltose phosphorylase. Therefore, it cane concluded that the obtained high yields and perfect diastere-selectivity imply that �-glucosidase from S. cerevisiae has a goodrospect for application in the synthesis of �-isosalicin.

cknowledgements

The authors are grateful for the financial support of the Min-stry of Science of the Republic of Serbia (Project Nos. 172049 and46010).

eferences

[1] Jung JH, Seo DH, Ha SJ, Song MC, Cha JC, Yoo SH, et al. Enzymatic synthesisof salicin glycosides through transglycosylation catalyzed by amylosucrasesfrom Deinococcus geothermalis and Neisseria polysaccharea. Carbohyd Res2009;344:1612–9.

[2] Nimpiboon P, Nakapong S, Pichyangkura R, Ito K, Pongsawasdi P. Sunthesis ofa novel prebiotic trisaccharide by type I �-glucosidase from B. licheniformisstrain TH4-2. Process Biochem 2011;46:448–57.

[3] Gonzalez-Pombo P, Farina L, Carrau F, Batista-Viera F, Brena BM. A novelextracellular �-glucosidase from Issatchenkia tericcola: isolation, immobiliza-

tion and application for aroma enhancement of white Muscat wine. ProcessBiochem 2011;46:385–9.

[4] Bridiau N, Taboubi S, Marzouki Z, Legoy MD, Maugard T. Beta-galactosidasecatalyzed selective galactosylation of aromatic compounds. Biotechnol Progr2006;22:326–30.

[

istry 46 (2011) 1698–1702

[5] Bojarova P, Petraskova L, Ferrandi EE, Monti D, Pelantova H, Kuzma M,et al. Glycosyl azides—an alternative way to disaccarides. Adv Synth Catal2007;349:1514–20.

[6] Thangavel P, Balaraman M, Soundar D. A response surface methodologicalstudy on prediction of glucosylation yields of thiamin using immobilized �-glucosidase. Process Biochem 2009;44:251–5.

[7] Arrojo L, Dolores M, De Segura AG, Dolores L, Alcalde M, Patricia GA,et al. Transformation of maltose into prebiotic isomaltooligosaccharides by anovel �-glucosidase from Xantophyllomyces dendrorhous. Process Biochem2007;42:1530–6.

[8] Milosavic N, Prodanovic R, Jankov R. A simple and efficient one-step, regiose-lective, enzymatic glucosylation of arbutin by alpha-glucosidase. TetrahedronLett 2007;48:7222–4.

[9] Prodanovic R, Milosavic N, Sladic D, Velickovic T, Vujcic Z. Synthesisof hydroquinone-alpha-glucoside by alpha-glucosidase from baker’s yeast.Biotechnol Lett 2005;27:551–4.

10] Yoon SH, Fulton B, Robyt JF. Enzymatic synthesis of two salicin analoguesby reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glu-canyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase.Carbohydr Res 2004;339:1517–29.

11] El-Shemy HA, Aboul-Enein A, Aboul-Enein M, Issa S, Fujita K. The effect ofwillow leaf extracts on human leukemic cells in vitro. J Biochem Mol Biol2003;36:387–9.

12] Bonaterra GA, Heinrich EU, Kelber O, Weiser D, Metz J, Kinscherf R.Anti-inflammatory effects of the willow bark extract STW 33-I (Proactiv) in LPS-activated human monocytes and differentiated macrophages. Phytomedicine2010;17:1106–13.

13] Ogawa Y, Oku H, Iwaoka E, Iinuma M, Ishiguro K. Allergy-preventive phenolicglycosides from Populus sieboldii. J Nat Prod 2006;69:1215–7.

14] Seo ES, Lee JH, Park JY, Kim D, Han HJ, Robyt JF. Enzymatic synthesis and anti-coagulant effect of salicin analogs by using the Leuconostoc mesenteroidesglucansucrase acceptor reaction. J Biotechnol 2005;117:31–8.

15] Shin HK, Kong JY, Lee JD, Lee TH. Syntheses of hydroxybenzyl-�-glucosides by amyloglucosidase-catalyzed transglycosylation. Biotechnol Lett2000;22:321–5.

16] Prodanovic R, Milosavic N, Sladic D, Zlatovic M, Bozic B, Velickovic T, et al. Trans-glucosylation of hydroquinone catalysed by alpha-glucosidase from baker’syeast. J Mol Catal B: Enzym 2005;35:142–6.

17] Rodriguez-Nogales JM, Roura E, Contreras E. Biosynthesis of ethyl

butyrate using immobilized lipase: a statistical approach. Process Biochem2005;40:63–8.

18] Kino K, Shimizu Y, Kuratsu S, Kirimura K. Enzymatic synthesis of alpha-anomer-selective d-glucosides using maltose phosphorylase. Biosci Biotechnol Biochem2007;71:1598–600.