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Journal of Biotechnology 139 (2009) 229–235

Contents lists available at ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

eoxynojirimycin enhanced the transglycosylation activityf a glycosidase from the China white jade snail

ing Hua,b, Hongwei Luana,b, Guangbo Gea,b, Huixin Liua,b, Yanyan Zhanga,b,un Zhoua,b, Yong Liua, Ling Yanga,∗

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhong-shan Road, Dalian 116023, ChinaGraduate University of the Chinese Academy of Sciences, Beijing, China

r t i c l e i n f o

rticle history:eceived 8 July 2008eceived in revised form 31 October 2008ccepted 9 December 2008

a b s t r a c t

A �-d-glycosidase (G I) from the China white jade snail showed non-Michaelis–Menten mode in catalyzingthe reaction using pNPGlu and pNPFuc as the substrate and monitoring the released pNP. We determinedquantitatively both the transglycosidic and hydrolytic products of pNPGlu and pNPFuc solvolysis for thedetailed kinetic analysis on G I-catalyzed hydrolysis and transglycosylation reaction. The inhibition kinetic

eywords:-d-Glycosidaseransglycosylation activityineticsllosteric effect

studies using deoxynojirimycin (DNJ) and butanol as inhibitors were preceded. DNJ only inhibited com-petitively the hydrolysis of cellobiose and pNPGlu while “activated” the transglycosylation of pNPGlu andpNPFuc. This was evident from the increased Vmaxtr value with no change of the apparent Kmtr. In contrast,butanol exhibited a competitive inhibition to the transglycosylation reaction and non-competitive inhibi-tion to the hydrolysis. The results indicated that the non-Michaelis–Menten kinetic behavior was caused

ubstrlation

he China white jade snail by the co-occurrence of sincrease the transglycosy

. Introduction

Glycosidases (EC 3.2.1.) are a widespread group of enzymeshat hydrolyze the glycosidic bond between two or more carbohy-rates or between a carbohydrate and a non-carbohydrate moietyHenrissat, 1991). Recently interest in these enzymes has gained

omentum owing to their biosynthetic abilities (Thiem, 1995; Criytnd Vic, 1998). Transglycosylation is a common feature of most gly-osidases (Tramice et al., 2007; Park et al., 2007, 2005; Fourage etl., 2000; Fourage and Colas, 2001; Andreotti et al., 2006; Ferrer etl., 2005; Kurakake et al., 2005; Eneyskaya et al., 2007).

The possibility of using transglycosidic reactions to produceommercially important glycosides, such as alkyl glucosides,ructo- and galacto-oligosaccharides, is economically enticing sincet would occur in one-step procedure (van Rantwijk et al., 1999;irayama, 2002; Yazaki et al., 1997). However, the transglycosyla-

ion efficiency is low due to the co-occurrence of hydrolysis. Hereto-ore, the hydrolytic and transglycosidic reaction are considered toccur at the same site and transglycosylation happens when an

cceptor (an alcohol or a sugar) replaces water in the final step of theeaction. Scheme 1 shows the mode of transglycosylation reactionith the substrate itself as the acceptor (Faijes and Planas, 2007;ommalai et al., 2005). Accordingly, the unique strategy to enhance

∗ Corresponding author. Tel.: +86 411 84379317; fax: +86 411 84676961.E-mail address: yling@dicp.ac.cn (L. Yang).

168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2008.12.006

ate transglycosylation reaction. This study provided a simple method toyield by using DNJ to inhibit hydrolysis.

© 2008 Elsevier B.V. All rights reserved.

the transglycosylation efficiency is to modify the exiting glycosi-dases by site-directed mutagenesis (Perugino et al., 2004). However,the method might lead to the alteration of the enzyme propertyincluding stability and substrate specificity. It is necessary to furtherstudy the glycosidase-catalyzed hydrolysis and transglycosylationreaction for discovering the pathway to enhance the transgly-cosylation efficiency. However, the complicated reaction processmakes the detailed analysis and comparison of the transglycosidicand hydrolytic reaction difficult. In most studies, the substrateswith p-nitrophenyl or methylumbelliferyl moiety were employedto detect the aglycone formation rate by simple colorimetric assay.The kinetic parameters for transglycosylation were derived bynon-linear regression fitting to the modified Michaelis–Mentenequation (Eneyskaya et al., 2007; Seidle and Huber, 2005; Kawaiet al., 2004). Alternatively, the kinetic parameters were obtainedindirectly by the difference between the overall rate (hydrolysisand transglycosylation) and hydrolysis rate (Bonnin et al., 1997;Gopalan et al., 1992; Hays et al., 1998). Only few studies determinedthe transglycosidic kinetic parameters by directly detecting the rateof transglycosylation products (Bruins et al., 2003).

Previously we reported three glucosidases (G I, G II and G IV)(Hu et al., 2007, 2008; Luan et al., 2006) from the China white jade

snail. Among them, G I exhibited the transglycosylation activityin the presence of various alcohols (Hu et al., 2008). For detailedkinetic analysis of hydrolysis and substrate transglycosylation reac-tion, here we further monitored quantitatively the hydrolysis andtransglycosylation products of the reaction with pNPGlu or pNPFuc

230 Y. Hu et al. / Journal of Biotechnology 139 (2009) 229–235

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atb

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f(wawoAMg

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hydrolytic Vmax (Vmaxhy) values for pNPGlu and pNPFuc were cal-culated by Hill equation fitting with the data of glucose and fucoseformation. The transglycosidic Km (Kmtr) and transglycosidic Vmax

(Vmaxtr) values were obtained by Hill equation fitting with the dataof pNPGluGlu and pNPFucFuc formation.

Table 1The linear equation, coefficient and the linear range of various products.

Products Equation Coefficient (R2) Linear range

pNPa y = 10.013x + 0.004 0.9998 0.01–0.07 �molGlucosea y = 3.7923x + 0.0033 0.9999 0.01–0.1667 �molpNPGluGlub y = 886,283x + 2830.7 0.9999 0.0125–8 nmol

cheme 1. The scheme for hydrolysis (1) and substrate transglycosylation (2), whehe Michaelis complex, the putative common glucosyl-enzyme intermediate and trf the glycolytic cleavage step; k3, rate of the hydrolytic step; k4, rate of the transgly

s both acceptor and donor by UFLC. Based on the analysis method,he inhibition study was performed on G I to compare the differenceetween the hydrolysis and transglycosylation reaction.

. Materials and methods

.1. Special chemicals

p-Nitrophenyl-�-d-glucopyranoside (pNPGlu), pNP-�-d-ucopyranoside (pNPFuc), p-nitrophenyl-�-d-galactopyranosidepNPGal) and p-nitrophenyl-�-d-glucopyranoside (pNP-�-d-Glu)ere purchased from Sigma–Aldrich. Cellobiose as well as glucose

nd fucose were obtained from J&K Chemical Ltd., China. DNJas a product of Wako Pure Chemical Co., Japan. Glucose Kit wasbtained from Shanghai Kehua Bio-engineering Co. Ltd., China.cetonitrile (HPLC grade) was obtained from Tedia Co. Ltd., USA.illi-Q water was used. The general chemicals used were of AR

rade.

.2. Enzyme source

The acetone powder of the viscera of the China white jade snail,hich was used as the source of glycosidase, was purchased from

ingKeHongDa Biotechnology Co. Ltd., China. The enzyme, G I, wasurified from the acetone powder, according to a previous methodHu et al., 2008). The final product was homogeneous by SDS-PAGE.

.3. Colorimetric assay of glycosidase activity

�-d-Glycosidase activity was assayed as follows. A mixture (totalolume 0.3 ml) containing 0.02 ml of 10 mM pNP-glycoside men-ioned above, 50 mM citrate–phosphate buffer (pH 5.5), and 0.005 Uf the enzyme was incubated for 30 min at 40 ◦C. The reactionas stopped by the addition of 0.25 M NaOH (1.5 ml), and then

he amount of liberated p-nitrophenol (pNP) was determined byeasuring absorbance at 405 nm with UV–vis spectrophotometer

JASCO V-530, Japan). The reference cuvette contained all reac-ants except the enzyme. Enzyme activities against cellobioseere determined by measuring the amount of released glucoseith the glucose oxidase–peroxidase method (GOD–POD) (Leary

t al., 1992). The amount of glucose released was divided by twoecause two glucose units were generated for each unit of substrateydrolyzed.

The kinetic parameters were evaluated by Hill equationv = Vmax × [S]n/(Km + [S]n)) fitting (Segel, 1975). The substrateoncentration ranges for cellobiose, pNPGlu, pNPFuc, pNPGal,nd pNP-�-d-Glu were 0.107–8, 0.06–8, 0.0267–2, 0.107–8, and.333–16 mM.

.4. UFLC/MS condition

Transglycosylation products and sugar product (fucose) werenalyzed by ultra-fast liquid chromatography (UFLC) system cou-

Enz.Gly-X, Enz-Gly, and Gly-Gly-X represent the aglycone moiety in the substrate,ycosylation product itself, respectively. k1, substrate dissociation constant; k2, rateic step.

pled with a binary solvent pump, an autosampler injector, a columnoven and a PDA detector (SHIMADZU, Japan). A Shim-pack XR-ODS column (2.2 �m, 100 mm × 2.0 mm i.d.) was used and themobile phase consisted of water (A) and CH3CN (B). For transg-lycosylation products, gradient elution was carried out with thefollowing profile: 0–5 min, 92% A; 5–10 min, 92–5% A; 10–12 min,5% A; 12–16 min, balance to 92% A. The flow rate was 0.4 ml min−1;for fucose, the gradient elution was as follows: 0–3 min, 100% A;3–10 min, 100–5% A; 10–14 min, balance to 100% A. The flow ratewas 0.3 ml min−1. The injection volume was 10 �l and scan wave-length was set at 200 nm. Mass detection was performed on aShimadzu LCMS-2010EV instrument with an electrospray ioniza-tion (ESI) interface in negative ion mode from m/z 100 to 600. Thedetector voltage was set at 1.55 kV. Curved desolvation line (CDL)and heat block temperatures were both set at 250 ◦C. The nebulizergas flow was set at 1.5 l min−1 and the drying gas (N2) pressure wasset at 0.06 MPa. Data acquisition was carried out by LCMS solutionversion 3.41 software.

2.5. Kinetic analysis of hydrolysis and transglycosylation

pNPGlu (0.06–8 mM) or pNPFuc (0.0267–2 mM) was incubatedwith 0.005 U G I for 30 min at 40 ◦C in a 300-�l citrate–phosphatebuffer (0.1 M, pH 5.5), then the reaction was terminated by boilingfor 5 min. The transglycosylation products (pNPFucFuc/pNPGluGlu)were quantitatively analyzed by UFLC method described above. Thestandard curves were prepared by pNPGlu and pNPFuc standardas no pNPFucFuc or pNPGluGlu standards were available; it wasassumed that the pNPFucFuc or pNPGluGlu had the same molarabsorbance as the respective parent glycosides. Hydrolysis wasmonitored by measuring the rate of produced glucose and fucose,which were quantitatively analyzed by colorimetric method andUFLC/MS method, respectively. The ion [M+2H2O]− (199.0) was sta-ble and selected for the quantitative analysis of the produced fucose.The calibration curves for various products are listed in Table 1.The accuracy and precision of the back-calculated values for eachconcentration were less than 15%. The hydrolytic Km (Kmhy) and

Fucosec y = 255,132x + 4,41,921 0.9961 0.25–20 nmolpNPFucFucb y = 953,608x − 48,637 0.9953 0.0125–8 nmol

a The amount was determined by colorimetric method.b The amount was determined by UFLC/PDA method.c The amount was determined by UFLC/MS method.

technology 139 (2009) 229–235 231

2r

m(oogpw

3

3

c

Table 2Apparent kinetic parameters for the solvolysis of various substrates by G I.

Km (mM) Vmax (�mol mg−1 min−1) Vmax/Km n

pNP�-d-Fuca 0.12 ± 0.01 10.7 ± 0.20 89.2 1.54 ± 0.12pNP�-d-Glua 3.34 ± 0.40 24.7 ± 1.02 7.4 0.76 ± 0.02Cellobioseb 0.57 ± 0.04 3.11 ± 0.08 5.5 1.04 ± 0.06pNP�-d-Gala 3.04 ± 0.32 20.32 ± 0.90 6.7 0.98 ± 0.04pNP�-d-Glua 0.83 ± 0.11 0.58 ± 0.03 0.7 1.04 ± 0.15

a

Fes((5w

Y. Hu et al. / Journal of Bio

.6. Inhibition kinetic study of hydrolysis and transglycosylationeaction

Effect of DNJ on the hydrolysis of cellobiose and pNPGlu wereeasured in the presence of inhibitor at various concentrations

0.2–1.2 mM). Inhibitor constants (Ki) were determined by replotsf data from Lineweaver–Burk plots (Segel, 1975). Effect of DNJn the transglycosidic reaction of pNPGlu or pNPFuc was investi-ated by adding 10 mM DNJ. The kinetics behavior of cellobiose andNPGlu solvolysis including the hydrolysis and transglycosylationere also analyzed in the presence of 0.54 M butanol.

. Results

.1. Hydrolysis substrate specificity

The enzyme has a broad substrate specificity and hydrolyze glu-osides, fucosides and galactosides. Table 2 shows that pNPFuc is

ig. 1. Profile of transglycosylation and hydrolysis products generated from pNPGlu and pach substrate (pNPGlu or pNPFuc) for 30 min at 40 ◦C in a 300-�l citrate–phosphate bufubstrate; (B) MS spectrum for peak 1, m/z = 462.1 [M−H]− , transglycosylation product (pND) MS spectrum for peak 3, m/z = [M−H]− , product (pNP); (E) MS spectrum for hydrolysisF) chromatography profile for the reaction with pNPFuc as the substrate; (G) MS spectrum, m/z = 466.1 [M+2H2O]− , transglycosylation product (pNPFucFuc); (I) MS spectrum for pehich is not presented in UFLC profile, m/z = 198.9 [M+2H2O]− .

Activity determined by measuring the rate of release of pNP.b Activity determined by measuring the glucose released from sugar hydrolysis

using a glucose oxidase kit.

the best substrate for G I in terms of catalytic efficiencies, whichis given by the Vmax/Km ratio. Moreover, G I follows a typical

Michaelis–Menten mode in hydrolyzing cellobiose, pNPGal andpNP-�-d-Glu, but behaves in a non-Michaelis–Menten mode in thehydrolysis of pNPGlu and pNPFuc with a negative (n = 0.76) andpositive cooperativity (n = 1.54), respectively.

NPFuc by G I. The activity was assayed by incubating enzyme (0.005 U) with 10 mMfer (0.1 M, pH 5.5). (A) Chromatography profile for the reaction with pNPGlu as thePGluGlu); (C) MS spectrum for peak 2, m/z = 336.0 [M+2H2O]− , substrate (pNPGlu);product (glucose), which is not presented in UFLC profile, m/z = 214.9 [M+2H2O]−;for peak 4, m/z = 319.9 [M+2H2O]− , substrate (pNPFuc); (H) MS spectrum for peak

ak 6, m/z = [M−H]− , product (pNP); (J) MS spectrum for hydrolysis product (fucose),

232 Y. Hu et al. / Journal of Biotechnology 139 (2009) 229–235

Fig. 2. The hydrolysis and transglycosylation kinetics of G I using pNPGlu (A) and pNPFuc (B) as the substrate. pNPGlu (0.06–8 mM) or pNPFuc (0.0267–2 mM) was incubatedwith G I (0.005 U) for 30 min at 40 ◦C in a 300-�l citrate–phosphate buffer (0.1 M, pH 5.5). (A) The amounts of pNP (�) was measured by colorimetric assay. The amounts ofglucose (�) and pNPGluGlu (�) were quantitatively analyzed by colorimetric and UFLC/PDA method, respectively. (B) The amounts of pNP (�) was measured by colorimetrica by UFa ed on

3

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3

tryepodwTda1tph

ssay. The amounts of fucose (©) and pNPFucFuc (�) were quantitatively measurednd the data points represent the means ± S.D. Dashed curve: results calculated bas

.2. Transglycosylation reaction

We previously had reported the transglycosylation activity ofI in the presence of alcohols (Hu et al., 2008). TLC and MS

nalysis of the products indicated that G I exhibited a significantransglycosylation activity when using various alcohols as accep-ors in producing corresponding alkyl-glycosides. In the presenttudy, we investigated the transglycosylation activity using pNPGlur pNPFuc as both donor and acceptor. As the results shown inig. 1, the MS spectrum revealed the molecular weight of an ionM−H]−: m/z = 462.1, corresponding to pNPGluGlu (Fig. 1B) and the

olecular weight of the ion [M−H]−: m/z = 430.3, correspondingo pNPFucFuc (Fig. 1H). However, the expected transglycosyla-ion product (cellotriose) was not detected with cellobiose as theubstrate (data not shown). The results indicated that G I cat-lyzed the transglycosylation reaction with pNPGlu and pNPFucs both donor and acceptor to format disaccharide glycosidespNPGluGlu and pNPFucFuc). Therefore, it is possible that theransglycosylation activity led to the atypical kinetics for pNP for-

ation.

.3. The kinetic analysis of hydrolysis and transglycosylation

To investigate the hydrolysis and transglycosylation reaction,he detailed kinetic study on transglycosylation and hydrolysiseaction of G I was performed. As shown in Fig. 2A, both hydrol-sis and transglycosylation kinetics with pNPGlu as a substratexhibited non-Michaelis–Menten kinetics. In detail, the glucoseroduction increased in responds to the increased concentrationf [pNPGlu] for up to 2 mM. However, the glucose productionecreased when the [pNPGlu] concentration was above 2 mM,hile pNPGluGlu was produced when [pNPGlu] was up to 1 mM.

he data for pNPFuc showed the similar trends. The hydrolysis rateecreased when [pNPFuc] concentration was between 0.667 mM

nd 1 mM and the transfer products presented at [pNPFuc] up tomM (Fig. 2B). Thus, the transglycosylation is positively related to

he initial substrate concentration and the formation of transferroduct (pNP-disaccharide) is obviously relative to the decreasedydrolysis rate. Furthermore, it was noteworthy that there was a

LC/MS and UFLC/PDA method, respectively. Measurements were made in triplicateHill equation fitting of the data of the combination of v(hy) and v(tr) data.

good agreement between the profile (dashed line) derived fromthe combination of v(tr) and v(hy) data and the kinetic curvesobtained by non-linear regression of v(pNP) data. Therefore, it isevident that non-Michaelis–Menten kinetics was due to simultane-ous G I-catalyzed transglycosylation (bimolecular pathway) as wellas hydrolysis reaction (unimolecular) (Scheme 1). The apparentKmhy and Vmaxhy for pNPGlu (0.37 mM and 7.93 �mol mg−1min−1)and those for pNPFuc (0.12 mM and 8.99 �mol mg−1 min−1) wereobtained by Hill equation fitting of the glucose or fucose formationdata. The values of apparent Kmtr and Vmaxtr for pNPGlu (4.01 mMand 11.92 �mol mg−1 min−1) and those for pNPFuc (1.38 mM and5.57 �mol mg−1 min−1) were obtained by Hill equation fitting ofpNP-disaccharide formation data.

3.4. Enhancement of the transglycosylation activity by DNJ

We investigated the transglycosylation ratio at different con-centration [pNPGlu] or [pNPFuc] in the absence and presence ofDNJ. The transglycosylation ratios were increased with the increaseof the initial substrate concentration (about 54.3% and 36.7%,respectively, at 8 mM [pNPGlu] and 2 mM [pNPFuc]) (Fig. 3). Thetransglycosylation reactions were favored over the hydrolysis reac-tion after addition of DNJ. At the high substrate concentration, thetransfer yields were very high and reached almost 100%. The trans-glycosylation ratio for pNPGlu or pNPFuc was increased 2.1- or2.7-fold by DNJ.

3.5. Inhibition kinetic study

3.5.1. Effect of DNJ to cellobiose, pNPGlu and pNPFuc solvolysisLineweaver–Burk plots indicated that DNJ inhibited compet-

itively the hydrolysis of cellobiose (Ki = 0.479 mM) and pNPGlu(Ki = 0.219 mM). Using pNPGlu as the substrate, DNJ enhanced theapparent Vmaxtr value for 1.3-fold without affecting the apparent

Kmtr value, which led to increased formation of pNPGluGlu. WhenpNPFuc served as the substrate, the apparent Vmaxtr increased 1.9-fold and apparent Kmtr remained unchanged. Additionally, whenadding enough amount of DNJ, the apparent Km values for pNPGlu(4.42 mM) and pNPFuc (1.32 mM) were in good agreement with the

Y. Hu et al. / Journal of Biotechnology 139 (2009) 229–235 233

Fig. 3. Effect of DNJ to transglycosylation kinetics of G I with pNPGlu (A) and pNPFuc (B) as the substrate. pNPGlu (0.06–8 mM) or pNPFuc (0.0267–2 mM) was incubated withG I (0.005 U) for 30 min at 40 ◦C in the presence of DNJ. The activity was measured using UFLC procedure that provided estimates for the amounts of the transglycosylationproduct pNPGluGlu in absence (�) or presence of DNJ (�) and pNPFucFuc in absence (�) or presence of DNJ (©). Measurements were made in triplicate and the data pointsrepresent the means ± S.D.

Table 3Effect of DNJ on the hydrolysis and transglycosylation reaction of pNPGlu and pNPFuc.

Product pNPGlu pNPFuc Inhibition type

Km (mM) Vmax (�mol/(mg min)) Km (mM) Vmax (�mol/(mg min))

pNPa/−DNJ 3.34 ± 0.40 24.7 ± 1.02 0.12 ± 0.01 10.7 ± 0.20pNPa/+DNJ 4.42 ± 0.40 14.02 ± 0.54 1.32 ± 0.09 12.9 ± 0.42

Sugar product/−DNJ 0.37 ± 0.06b 7.93 ± 0.67b 0.12 ± 0.01b 8.99 ± 1.1bCompetitive

Sugar product/+DNJ NDc NDc NDc NDc

Transfer productd/−DNJ 4.01 ± 0.24 11.92 ± 0.46 1.38 ± 0.25 5.57 ± 1.21Non-Competitive

Transfer productd/+DNJ 4.33 ± 0.44 16.07 ± 1.19 1.35 ± 0.02 10.36 ± 0.11

a Activity determined by measuring the rate of release of pNP by colorimetric assay.b assay

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Activity determined by measuring the rate of release of glucose by colorimetricc Not detected.d Activity determined by measuring the rate of release of products by UFLC assay

mtr values (4.01 mM for pNPGlu and 1.38 mM for pNPFuc) (Table 3).he result showed that DNJ was a competitive inhibitor for hydroly-is and a non-competitive activator for transglycosylation, resultingn an enhanced transglycosylation rate. The kinetic data suggestedhat the hydrolysis and transglycosylation might be occurring atifferent site.

.5.2. Effect of butanol on cellobiose and pNPGlu solvolysis

New transfer products, alkyl-glycosides, were produced instead

f the auto-condensation products (pNPGluGlu/pNPFucFuc) inhe present of 2.2 M alcohols as acceptors (data not shown).his suggested that the enzyme-catalyzed substrate transglyco-ylation (pNPGlu/pNPFuc) was competing simultaneously with

able 4ffect of butanol on pNPGlu and cellobiose solvolysis including the hydrolysis and transgl

roduct pNPGlu

Km (mM) Vmax (�mol/(mg min))

lucosea/−butanol 0.37 ± 0.06 7.93 ± 0.67lucosea/+butanol 0.32 ± 0.12 4.98 ± 0.83

NPGluGlub/−butanol 4.01 ± 0.24 11.92 ± 0.46NPGluGlub/+butanol 5.54 ± 0.63 13.51 ± 1.30

a Activity determined by measuring the rate of release of glucose by colorimetric assayb Activity determined by measuring the rate of release of products by UFLC assay.c Not determined.

.

the alcoholysis. We next investigated the inhibition type ofbutanol to the solvolysis of cellobiose and pNPGlu. Using cel-lobiose as the substrate, the value of apparent Vmaxhy decreasedfrom 3.11 �mol mg−1 min−1 to 2.23 �mol mg−1 min−1 while theapparent Kmhy did not alter with addition of 0.54 M butanol,which indicated that butanol exhibited a non-competitive inhi-bition to cellobiose (Table 4). With pNPGlu as the substrate,the effect of butanol on transglycosylation reaction was to

decrease the apparent Vmaxhy value from 7.93 �mol mg−1 min−1

to 4.98 �mol mg−1 min−1 without altering the apparent Kmhy.Inversely, butanol was exclusively to increase the apparent Vmaxtrwithout changing the apparent Kmtr (Table 4). Obviously, the datasuggested that butanol could only compete with the substrate for

ycosylation reaction.

Cellobiose Inhibition type

Km (mM) Vmax (�mol/(mg min))

0.57 ± 0.04 3.11 ± 0.08Non-Competitive

0.60 ± 0.18 2.23 ± 0.27

–c –cCompetitive

–c –c

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34 Y. Hu et al. / Journal of Bio

inding to the transfer site but not interact with hydrolysis site.oreover, the transfer site was relative hydrophobic and preferred

o accept alcohols as acceptors.

. Discussion

Many glycosidases can be useful transferases via transglyco-ylation reaction in spite of their natural function as hydrolyasesTramice et al., 2007; Park et al., 2007, 2005; Andreotti et al., 2006;errer et al., 2005; Kurakake et al., 2005). When considering theynthesis capability, it is vital to compare the rate of transgly-osylation and hydrolysis because they occurred simultaneously.owever, few studies detailing the properties of the transglycosidic

eactions have been reported due to the complicated kinetic proce-ure (Eneyskaya et al., 2007; Seidle and Huber, 2005; Kawai et al.,004; Bonnin et al., 1997; Gopalan et al., 1992; Bruins et al., 2003). Aualitative study on the transglycosylation activity of G I had beenreviously proceeded using various alcohols as acceptors (Hu et al.,008). In this paper, we employed highly sensitive UFLC/PAD andFLC/MS method to quantify the transglycosylation product andydrolysis product for detailed kinetic studies on transglycosylationnd hydrolysis reactions.

In the inhibition kinetic studies, the hydrophobic butanol is aompetitive inhibitor for the transglycosylation reaction and a non-ompetitive inhibitor for the hydrolysis reaction. DNJ competitivelynhibited the hydrolysis but activated the transglycosylation reac-ion. Based on the kinetic data, we speculate that the hydrolysisite and transfer site of G I might be independent of each other. Ineneral, DNJ, which is an analog ofd-glucose, could cause a compet-tive inhibition of the glucosidases (Dale et al., 1985). Nevertheless,n our study, DNJ is a competitive inhibitor for hydrolysis but anctivator for transglycosylation reaction, leading to the enhancedransfer efficiency. We reported, for the first time, that the transg-ycosylation rate could be increased by DNJ. This uncommon featureertainly is worth more attention and further study.

It has been reported that the hydrophobicity of the environmentn active center is critical for the transglycosylation activity of gly-osidases (Hong et al., 2006; Cha et al., 1998; Tang et al., 2006).he transglycosylation activity of a neopullulanase was increasedhen one residue located on the entrance of the water moleculeas replaced with more hydrophobic amino acid residue. Inversely,

he transglycosylation activity was decreased when the residue waseplaced by hydrophilic amino acid residue (Kuriki et al., 1996).ommalai et al. (2005) confirmed that the hydrophobicity of theglycone sites of glucosidases from cassava and Thai rosewoodere possibly the main reason allowing the enzymes to catalyze

n efficient transglycosylation reaction. Similarly, the transfer sitef G I could accept hydrophobic alcohols and some substratespNPFuc and pNPGlu) as acceptors, while not recognize hydrophilicellobiose as the acceptor. Thus the transfer site is likely moreydrophobic compared to the hydrolysis site. This implies that thenvironment of the active center is important in determining theeaction mode: the active center of the transfer site of G I withydrophobic residues cause the water molecules to be excluded andubstrates and/or hydrophobic alcohols to predominate, therebyainly catalyzing the transglycosylation activity. In contrast, the

ctive center of hydrolysis site with hydrophilic residues makes theater molecule mainly attacking the active center to allow for theydrolysis reaction.

In conclusion, the hydrolysis and transglycosylation sites of G I

ight be distinctive. DNJ only inhibited the hydrolysis activity but

ncreased the transglycosylation rate of G I. This provides a conve-ient pathway to enhance the transglycosylation rate via inhibitingydrolysis with DNJ. Certainly, the kinetic results may still note sufficient to fully explain the distribution of catalytic activi-

logy 139 (2009) 229–235

ties between the hydrolysis and transglycosylation. The structuralanalysis is needed to confirm these conclusions.

Acknowledgements

This work was funded by the “973 Program” from Ministry ofScience and Technology of the China (2003CB716005), DICP Innova-tion Fund of Chinese Academy of Sciences and the National NaturalScience Foundation of China (30772608).

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