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Enzyme and Microbial Technology 49 (2011) 555– 559

Contents lists available at SciVerse ScienceDirect

Enzyme and Microbial Technology

jou rn al h om epage: www.elsev ier .com/ locate /emt

reparative-scale kinetic resolution of racemic styrene oxide by immobilizedpoxide hydrolase

eniz Yildirim ∗, S. Seyhan Tükel, Dilek Alagöz, Özlem Alptekinniversity of Cukurova, Faculty of Arts and Sciences, Department of Chemistry, 01330 Adana, Turkey

r t i c l e i n f o

rticle history:eceived 13 January 2011eceived in revised form 1 August 2011ccepted 10 August 2011

eywords:poxide hydrolaseupergitinetic resolutionacemic styrene oxide

a b s t r a c t

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L througha Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary aminegroups which were subsequently activated with glutaraldehyde. The amount of introduced primaryamine groups was 220 �mol/g of the support after ethylenediamine treatment, and 90% of these groupswere activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Euper-git C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilizedepoxide hydrolase was 40 ◦C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at40 and 60 ◦C, respectively after 24 h preincubation time whereas the retained activities of immobilized

epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolaseshowed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale(120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in abatch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50%yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initialactivity after 5 reuses.

. Introduction

Enantiopure epoxides and their vicinal diols are versatile inter-ediates in the preparation of biologically active bulk compounds

s well as many fine chemicals and have broad scope of marketemand for their applications [1–4]. A major challenge in modernrganic chemistry is to generate such compounds in high yields,ith high stereo- and regioselectivities [5]. Over the last decades,

hemical (transition-metal-based) and biocatalytic methodologiesave been developed to produce such intermediates in enan-iopure form [6–10]. Due to increased environmental and legalressures, biocatalytic methodologies, which are generally recog-ized as being environmentally safe since they avoid production ofotentially toxic waste in industrial processes, have gained muchttention [11]. Epoxide hydrolases (EHs, EC 3.3.2.3) catalyze theddition of a water molecule to an epoxide resulting in the forma-ion of the corresponding vicinal diol (Fig. 1). EHs are ubiquitous inature, cofactor-independent enzymes [12–14] and they show high

egio, chemo and enantioselectivity on a broad variety of substrates4,15]. However, their limited long-term operational stability andelatively high cost have hampered their commercial application

∗ Corresponding author. Tel.: +90 322 3386081/26; fax: +90 322 3386070.E-mail address: dozyildirim@gmail.com (D. Yildirim).

141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2011.08.003

© 2011 Elsevier Inc. All rights reserved.

to date [16]. EHs have previously been immobilized onto a varietyof different supports to make them cost-effective and long-livedenzymes [11,16–20].

Eupergit supports, copolymers of N,N-methylene-bis-methacrylamide, glycidyl methacrylate, allyl glycidyl etherand methacrylamide were used by many researchers as carriers forimmobilization of various enzymes. These carriers were reportedto be very stable and showed good chemical and mechanical prop-erties (simple immobilization procedure, high binding capacity,low water uptake, high flow rate in column procedures, excellentperformance in stirred batch reactors, etc.) [21–23]. Karbouneet al. [18] determined the immobilization yield for partly purifiedEH onto Eupergit was less than 5% and the immobilized prepara-tion almost showed no activity. Mateo et al. [19] reported 100%immobilization and 30–35% activity yield for Aspergillus niger EHimmobilized onto Eupergit supports whereas 100% immobilizationand ≥95% activity yield were reported for A. niger EH immobilizedonto Eupergit supports modified with ethylenediamine. However,they obtained an immobilization of 5–10% and no activity for A.niger EH immobilized onto Eupergit C modified with iminodiaceticacid. These results indicate that the immobilization and activity

yields strictly depend on the functional groups of Eupergit supportused for A. niger EH immobilization. Therefore, we aimed to furtherexplore the potential of Eupergit C 250 L as support for EH and alsothe potential use of EH immobilized onto Eupergit C 250 L support

556 D. Yildirim et al. / Enzyme and Microbial Technology 49 (2011) 555– 559

-styre

ww

tporsTb

2

2

sn(rwdop

2

2

i(sb(r1al5m3d1

2

(wop1hfectogocBaf–p

Fig. 1. Enantioselective hydrolysis of (R/S)

hich was pretreated with ethylenediamine and further activatedith glutaraldehyde.

In this study, EH from A. niger was covalently immobilized ontohe modified Eupergit C 250 L for the first time and used for areparative-scale hydrolytic kinetic resolution of racemic styrenexide ((R/S)-SO). The optimum conditions for hydrolytic kineticesolution of (R/S)-SO were determined. The thermal and storagetabilities of free and immobilized EH preparations were examined.he operational stability of immobilized EH was investigated for aatch reactor.

. Materials and methods

.1. Materials

Eupergit C 250 L (particle size 250 �m, oxirane content ≥200 �mol/g dryupport), Epoxide hydrolase (1.7 U/mg solid, A. niger sp., recombinant from A.iger), (R/S)-styrene oxide, (S)-styrene oxide ((S)-SO), (R/S)-1-phenyl-1,2-ethanediol(R/S)-PED), (R)-1-phenyl-1,2-ethanediol ((R)-PED), ethylenediamine, ninhydrineagent (2% solution) and glutaraldehyde solution (aqueous solution, 50% (w/w))ere obtained from Sigma–Aldrich (St. Louis, MO, USA). Methanol, acetonitrile andimethyl sulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany). Allther chemicals used in this work were analytical grade and used without furtherurification.

.2. Methods

.2.1. Epoxide hydrolase assayEH activity was assayed by determining the hydrolysis rate of (R/S)-SO accord-

ng to Petri et al. [17]. The reaction mixture containing 350 �L of phosphate buffer100 mM, pH = 7.0) and 50 �L of free enzyme solution (1 mg/mL) prepared in theame buffer or 20 mg immobilized preparation were incubated at 35 ◦C in water-ath for 2 min. The reaction was started by adding 100 �L of (R/S)-SO solution100 mM in DMSO) and allowed to continue for 10 min. During the reaction, theeaction mixture was agitated at 100 rpm. After completion of the reaction time,00 �L of sample taken from the reaction mixture was diluted with 0.5 mL of hex-ne and the quantity of formed PED was determined by using high-performanceiquid chromatography (HPLC) equipped with a C18 column (4.6 mm × 250 mm,

�m) and UV–vis detector at 220 nm. The mobile phase was a methanol–waterixture (50/50%, v/v) at a flow rate of 0.5 mL/min and the column temperature was

5 ◦C. A blank reaction without enzyme was also performed under the same con-itions. One unit EH activity was defined as the amount of enzyme that produced

�mol of PED in 1 min under assay conditions.

.2.2. Modification of Eupergit supportBriefly, 1 g of Eupergit C 250 L was added into 10 mL of ethylenediamine solution

1 M in water, pH = 10) and stirred overnight at room temperature. Then, the supportas exhaustively washed with distilled water and dried at 60 ◦C for 2 h. The amount

f free primary amine (−NH2) groups attached to per gram of ethylenediamine-retreated Eupergit C 250 L was determined as described by Alptekin et al. [24]. To0 mg of ethylenediamine-pretreated Eupergit C 250 L, 100 �L H2O and 200 �L nin-ydrin reagent were added and the mixture was heated in a boiling water bath

or 30 min then cooled at room temperature. 5 mL of a 50/50 (v/v) mixture ofthanol/water was added onto the mixture and mixed well. The absorbance of theolored complex known as Ruhemann’s purple was measured at 570 nm. A calibra-ion curve was plotted using ethylenediamine as standard to determine the ratiof reacted and unreacted –NH2 groups. To activate the support, 25 mL of 2.5% (v/v)lutaraldehyde in potassium phosphate buffer (50 mM, pH = 7.0) was added to 1 gf ethylenediamine-pretreated Eupergit C 250 L and the reaction was allowed toontinue for 2 h. The support was subsequently washed with distilled water on a

uchnel funnel and dried at 60 ◦C for 2 h. The amount of remaining –NH2 groupsfter glutaraldehyde activation was determined as described above. The amount ofunctional aldehyde groups was calculated indirectly by subtracting the amount ofNH2 groups after glutaraldehyde activation from the amount of –NH2 groups afterretreatment with ethylenediamine.

ne oxide catalyzed by Aspergillus niger EH.

2.2.3. Immobilization of epoxide hydrolaseThe immobilization of EH onto the modified Eupergit support was based on the

method described by Knezevic et al. [25]. Briefly, 8 mL of epoxide hydrolase solution(1 mg/mL) prepared in 100 mM phosphate buffer (pH = 7.0) were mixed with 2 g ofthe modified Eupergit supports (Fig. 2). The reaction was allowed to continue ina water-bath at 5 ◦C for 2 h with slow shaking. The resulting immobilized enzymepreparations were washed with the same phosphate buffer until no protein couldbe detected in the filtrate and stored at 5 ◦C until use. The protein content of thesolution was determined using the Lowry method [26]. The amount of protein boundto the support was estimated by subtracting the amount of protein determined inthe filtrate from the total amount of protein used in immobilization procedure. EHimmobilized onto the modified Eupergit C 250 L was mentioned as immobilized EHthroughout the text unless otherwise mentioned.

2.2.4. Characterization of epoxide hydrolaseThe effect of pH on the activities of free and immobilized EH was investigated at

different pHs ranging from 5.0 to 8.0. The optimal temperatures of free and immo-bilized EH preparations were determined in the temperature range of 20–60 ◦C. Theactivation energies (Ea) of free and immobilized EH were estimated by using linearform of the Arrhenius equation:

In k = In A − Ea

RT

where k is reaction rate, A is the constant of integration, R and T are gas constantand absolute temperature, respectively. Ea is determined by plotting k at differenttemperatures versus 1/T.

The thermal stabilities of free and immobilized EH were evaluated by measuringthe residual activity of enzyme exposed to 40 and 60 ◦C. The storage stabilities offree and immobilized EH were investigated at room temperature and 5 ◦C.

2.2.5. Preparative-scale hydrolitic kinetic resolution of racemic styrene oxideThe preparative-scale kinetic resolution of (R/S)-SO was carried out in a 60 mL

capacity batch type reactor. The reaction mixture containing 1 g of immobilizedEH in 40 mL phosphate buffer (100 mM, pH = 6.5) was incubated at 40 ◦C for 5 min.The reaction was started by the addition 10 mL of (R/S)-SO solution (5 M in DMSO).Aliquots (100 �L) withdrawn at different time intervals were mixed with 500 �L ofhexane and analyzed using HPLC equipped with Shodex ORpak CDC-453 HQ chiralcolumn at 220 nm for the quantification of substrate and product concentrationsand their enantiomeric excesses (ee). The mobile phase was an acetonitrile − watermixture (20/80%, v/v) containing 1% acetic acid and 0.1% triethylamine (v/v) at aflow rate of 0.25 mL/min and the column temperature was 20 ◦C. The ee values ofremaining epoxide and formed diol were calculated from the concentration of thetwo enantiomers with the equations given below:

eeepoxide = [S]epoxide − [R]epoxide

[S]epoxide + [R]epoxideand eediol = [R]diol − [S]diol

[R]diol + [S]diol

The enantiomeric ratio (E) was calculated from the enantiomeric excesses ofthe formed diol (eediol) and remaining epoxide (eeepoxide) and conversion degree(c) using the computer program created by Faber and Hönig [27]. E value was alsocalculated using the Sih equation as given below [28]:

E = ln[1 − c(1 + eediol)]ln[1 − c(1 − eediol)]

2.2.6. Purifications of reaction productsAfter completion of reaction time, the immobilized EH was separated by filtra-

tion and the reaction products were extracted by ethyl acetate (3 mL × 40 mL). Thecollected ethyl acetate fractions were dried over Na2SO4 and concentrated undervacuum. Purification of reaction products were accomplished by flash chromatog-raphy (Chromabond Flash RS 25 SiOH). The column was equilibrated with petroleumether/ethyl acetate (80/20, v/v) and the products were eluted with the same mixture

at a flow rate of 1.0 mL/min, using a peristaltic pump.

2.2.7. Operational stability of immobilized epoxide hydrolaseThe operational stability of the immobilized EH was investigated at the con-

ditions described in Section 2.2.5. After 12 h reaction time, the immobilized EH

D. Yildirim et al. / Enzyme and Microbial Technology 49 (2011) 555– 559 557

obiliz

wdfac

3

tdeg2gpta

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htEabhor

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tration (2.5 M) was found as almost constant and the ee of (S)-SOwas determined as 99% after 12 h reaction time. E value for immo-bilized EH was calculated as 246 by the software and as 221 by theSih equation. This value was about 2.5-fold higher than the E value

0

20

40

60

80

100

120

987654pH

Rel

ativ

e A

ctiv

ity (%

)

free EHimmobilized EH

Fig. 2. The modification of Eupergit C 250 L and imm

as separated from the reaction mixture by filtration and washed with 10 mL ofiethylether and 20 mL of the phosphate buffer and stored at 5 ◦C for next cycle. Theormed diol concentration and its ee were determined with chiral HPLC as describedbove. This experiment was repeated with the same immobilized preparation for 5ycles.

. Results and discussion

In this study, the covalent immobilization of A. niger EH ontohe modified Eupergit C 250 L was studied. The amount of intro-uced primary amine groups of Eupergit C 250 L pretreated withthylenediamine was found as 220 �mol/g of support whereas afterlutaraldehyde activation amount of remaining –NH2 groups was2 �mol/g of support. It was concluded that 90% of primary amineroups were activated with glutaraldehyde (Fig. 2). The amount ofrotein bound per gram of modified Eupergit C 250 L was 80% ofhe initial amount of 4 mg protein and EH retained 52% of its initialctivity upon immobilization.

.1. Characterization of epoxide hydrolase

The optimum pH value of free EH was determined as 7.0,owever, the optimum pH value of immobilized EH slightly shiftedo acidic region and found to be 6.5 (Fig. 3). The modification ofupergit support with ethylenediamine gives positively chargedrms to the support that attracts more OH− ions around the immo-ilized enzyme resulting in the pH of enzyme’s microenvironmentigher than the rest of the bulk of the solution. Therefore, theptimal pH of immobilized enzyme shifts a lower pH value withespect to that of free EH.

The free and immobilized EH both showed their maximumctivities at 40 ◦C (Fig. 4). The activities of free and immobilizedH increased with the temperature increasing from 20 to 40 ◦C andhe activity of immobilized EH was almost constant in the tempera-ure range of 50–60 ◦C. However, the activity of free EH dramaticallyecreased above 40 ◦C. The activation energies of free and immobi-

ized EHs were calculated as about 39 and 45 kJ/mol, respectively.he covalent bond formation via amino groups of the immobilizednzyme might have reduced the conformational flexibility, therebyesulting in a higher activation energy for the molecule to reorga-ize and attain the proper conformation for binding to substrate.

The thermal stability of an immobilized enzyme is one of the

ost important criteria of its application. In general, the activity

f the immobilized enzyme, especially in a covalently bound sys-em, is more resistant than that of the soluble form against heatnd denaturing agents [29]. The thermal stabilities of both free

ation of A. niger EH onto modified Eupergit C 250 L.

and immobilized EH preparations were evaluated by incubatingthe respective EH preparations at 40 and 60 ◦C and determiningtheir residual activities at several time points for 24 h (Fig. 5). Thefree EH retained 52 and 33% of its maximum activity at 40 and 60 ◦C,respectively after 24 h, whereas for the immobilized enzyme 90 and75% residual activity was observed for the respective temperatures.

After 30 days storage time, free and immobilized EH showed 29and 77% residual activity at 5 ◦C (Table 1). The free EH completelylost all of its activity after 10 days at room temperature while immo-bilized EH retained 66% of its initial activity after 30 days at roomtemperature.

3.2. Preparative-scale hydrolitic kinetic resolution of racemicstyrene oxide

The immobilized EH catalyzed enantioselective hydrolysis of(R/S)-SO was investigated at pH 6.5 and 40 ◦C. The retention timesof (R)-PED, (S)-SO and (R)-SO were 5.35, 8.63 and 11.05 min, respec-tively. It was determined that the epoxide concentration wasdecreased from 5 M to 2.5 M (50% conversion) and (R)-PED concen-tration increased to 2.5 M after 12 h reaction time. The ee of (R)-PEDwas determined as 99% at that time. The remaining (S)-SO concen-

Fig. 3. The effects of pH on the activities of free and immobilized EH. The activitieswere assayed at 35 ◦C using 100 mM different buffers at pH 5–8 (5.0 and 5.5 acetatebuffers, 6.0 citrate buffer and 6.5, 7.0, 7.5 and 8.0 phosphate buffers). The reactiontime was 10 min and 10 mM (R/S)–SO was used as substrate.

558 D. Yildirim et al. / Enzyme and Microbial Technology 49 (2011) 555– 559

0

20

40

60

80

100

120

706050403020100Temperature (°C)

Rel

ativ

e A

ctiv

ity (%

)

free EHimmobilized EH

Fig. 4. The effects of temperature on the activities of free and immobilized EH. Theafa

(ngcsEatrttr(fwamoE4

3

timfw

Table 1The storage stabilities of free and immobilized EH at room temperature and 5 ◦C.

Storage time (day) Relative activity (%)

Free EH Immobilized EH

Room temp. 5 ◦C Room temp. 5 ◦C

5 55 86 92 9810 0 71 85 92

ctivities were assayed in 100 mM phosphate buffer (pH = 7.0 for free and pH = 6.5or immobilized EH). The reaction time was 10 min and 10 mM (R/S)–SO was useds substrate.

96) of the free EH. This result indicated that enantioselectivity of A.iger EH was enhanced by immobilization onto the modified Euper-it C 250 L. Mateo et al. [19] determined that enantioselectivity ofrude A. niger EH was increased about 2.24-fold against racemictyrene oxide upon immobilization onto ethylenediamine treatedupergit supports. In our study, after flash chromatography (S)-SOnd (R)-PED were obtained with 41 and 48% yield, respectively andhe ee for both (S)-SO and (R)-PED was found as 99%. Yoo et al. [30]eported that (S)-SO was obtained with 41% yield and 98% ee ashe result of kinetic resolution of (R/S)-SO catalyzed by Pichia pas-oris EH. Monfort et al. [31] determined that the hydrolytic kineticesolution of 1-chloro-2-(2,4-difluorophenyl) 2,3-epoxypropane500 g/L) catalyzed by A. niger EH achieved 50% yield and 99% eeor both epoxide and diol forms. They determined yield and eeere as 41.5 and 99.9%, respectively for the epoxide form and

s 43.5 and 94.5%, respectively for the diol form after flash chro-atography. Doumeche et al. [32] achieved the kinetic resolution

f rac-2-(Diethoxymethyl)oxirane (200 g/L) catalyzed by A. nigerH with high ee (>99% for epoxide form, 97% for diol form) after.5 h reaction time and E value was found as >200.

.3. Operational stability of immobilized EH

The operational stability of an immobilized enzyme is one ofhe most important aspects for industrial application. The reusabil-

ties of immobilized enzymes could lower the operational costs and

ake the immobilized enzymes more advantageous than their freeorm. The residual activity of the immobilized EH in the 5th batchas found as 90% of its initial activity. The yield and ee% of (R)-PED

0

20

40

60

80

100

120

24201612840Incubation time (h)

Rel

ativ

e A

ctiv

ity (%

)

free EH at 40 ºC free EH at 60 ºCimmobilized EH at 40 ºCimmobilized EH at 60 ºC

Fig. 5. The thermal stabilities of free and immobilized EH at 40 and 60 ◦C.

[

[

[

20 52 76 8330 29 66 77

were determined as 45 and 99%, respectively after 5 reuses. Mateoet al. [19] reported that A. niger EH immobilized onto Eupergit Cmodified with ethylenediamine was protected its initial activityand ee of remaining epoxide was found to be 98% after 12 cycles.Karboune et al. [20] determined that A. niger EH immobilized ontoDEAE-cellulose retained 90% of its initial activity after 7 reuses inthe preparative-scale (306 g/L) kinetic resolution of (R/S)-p-chlorostyrene oxide. Petri et al. [17] reported that the A. niger EH immo-bilized onto epoxide activated silica gel nearly protected its initialactivity after 9 reuses for kinetic resolution of (R/S)-p-nitro styreneoxide.

4. Conclusion

Our results show that EH was successfully immobilized onto themodified Eupergit C 250 L. The thermal and storage stabilities of EHwere improved upon immobilization onto the modified Eupergit C250 L. The immobilized EH showed about 2.5-fold higher enan-tioselectivity than the free EH towards styrene oxide. The resultsindicate that the immobilized EH is highly effective tool for thepreparations of preparative-scale enantiopure (S)-SO and (R)-PEDwith high yields and enantiomeric excesses.

Acknowledgement

This work was supported by Cukurova University with theproject number of FEF 2007 BAP 22.

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