Efficient biosynthesis of glycyrrhetic acid 3-O-mono-β-D-glucuronide(GAMG) in water-miscible ionic liquid by immobilized whole cells ofPenicillium purpurogenum Li-3 in alginate gel

Imdad Kaleem, Huang Shen, Bo Lv, Bin Wei, Aamir Rasool, Chun Li n

School of Life Science, Beijing Institute of Technology, 100081 Beijing, PR China

H I G H L I G H T S

� Various ILs were used to enhance the activity of whole-cell biocatalyst (w-PGUS).� Hydrophilic IL [BMIM]BF4 rectified the catalytic efficiency of immobilized w-PGUS.� Higher GAMG yield was achieved by the combination of immobilized w-PGUS and IL.� High operational stability of immobilized w-PGUS was found in IL medium.

a r t i c l e i n f o

Article history:Received 28 April 2013Received in revised form11 September 2013Accepted 15 October 2013Available online 23 November 2013

Keywords:Alginate gelIonic liquidOperational stabilityCatalytic activityBiotransformationPencillium purpurogenum Li-3

a b s t r a c t

Five immobilizing materials were investigated for the catalytic efficiency of whole cells of Penicilliumpurpurogenum Li-3 (w-PGUS) and alginate gel was found the best polymer for the biotransformation ofglycyrrhizin (GL) into glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG). The activity of composi-tionally optimized w-PGUS alginate beads was examined in different ionic liquids (ILs). The 30% (v/v)hydrophilic IL [BMIM]BF4 in reaction mixture exhibited an explicit biocompatibility with alginatesupport; consequently aggrandized the catalytic efficiency of the immobilized w-PGUS compared withpure buffer and ILs media. The optimal biotransformation conditions of the immobilized w-PGUS wereevaluated and compared with buffer and IL co-solvent media: including w-PGUS beads concentration(70 g/l vs. 80 g/l), substrate concentration (5 mM vs. 4 mM), temperature (45 1C vs. 45 1C), pH (5.4 vs.5.6), and shaking speed (180 rpm vs. 160 rpm). Under the optimized conditions; the yield of GAMG was2.62 g/l after 62 h in IL co-solvent medium compared to 2.34 g/l after 72 h in buffer medium. Theimmobilized w-PGUS also retained 45.80% and 18.13% of their original activity in IL co-solvent and buffermedia respectively; after ten repeated batches displaying high operational stability for GL biotransfor-mation.

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1. Introduction

Glycyrrhizin (GL), a primary active ingredient of licorice root, iscomposed of one molecule of glycyrrhetic acid (GA) as aglycon andtwo molecules of glucuronic acid attached to the C-3 atom of theaglycon moiety (Muro et al., 1986). GL can be hydrolyzed intoglycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG), glycyrrheticacid (GA) and glucuronide by the cells expressing β-glucuronidase(Fig. 1). The commercial importance of GAMG is evident from itsenormous use in food and pharmaceutical industry (Fenwick et al.,1990; Park et al., 2004).

Biosynthesis of GAMG is generally preferred over chemicalsynthesis due to high specificity of β-glucuronidase resulting inthe increase of product yield. There are many biological sources ofβ-glucuronidase including prokaryotes and eukaryotes, but itsfungal sources are quite limited. β-glucuronidase from fungalsource could be very advantageous due to its high selectivityand stability over a wide range of pH and temperature making ithighly suitable for practical/industrial applications (Shuping et al.,2013). Our research group previously screened a wild-type Peni-cillium purpurogenum Li-3 (w-PGUS) strain having a specific β-glucuronidase (PGUS, EC 3.2.1.31), which could directly hydrolyzeGL into GAMG (Feng et al., 2006). The lower expression of theenzyme and rapid in vitro loss of enzymatic activity were the mainrestraints for the higher yield of GAMG.

Immobilizing strategies are usually devised to retain theoriginal enzyme activity for a longer time period and minimize

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n Corresponding author. Tel./fax: þ86 10 68913171.E-mail address: lichun@bit.edu.cn (C. Li).

Chemical Engineering Science 106 (2014) 136–143

the perturbations in the physical and chemical parametersrequired for biotransformation. Entrapment in gel is the mostcommon method for the whole cell immobilization which exhibitsseveral advantages such as non-toxicity, mechanical stability, andporous controllability, and it also has enormous applications inindustrial biocatalysts for production of useful compounds (Gilland Ballesteros, 2000; Jian et al., 2009; Park and Chang, 2000).There are few studies on the immobilization of β-glucuronidasefrom other sources (Salam et al., 2011), but the whole-cellimmobilization of P. purpurogenum Li-3 could enhance the opera-tional stability of intracellular β-glucuronidase for the efficientbiosynthesis of GAMG yet to be explored.

The low enzymatic activity could be improved by mediumengineering. In recent years, ionic liquids (ILs) have been used aspure solvents, as co-solvents in the aqueous phase, or as biphasicsystems together with other solvents and even with combinationof polymer for the enhancement of both chemical and enzymaticreactions (Virtanena et al., 2007; Zhao 2010). ILs-containing mediaaffect the activity, enantioselectivity and stability of the biocata-lysts; hence increase the catalytic efficiency of an enzyme ormicrobial cells (Choi et al., 2012; Illanes et al., 2012). Modernapproaches have enhanced the efficiency of many importantenzymatic reactions by utilizing ILs (Arai et al., 2010; Wonget al., 2006). ILs, such as 1-butyl-3-methylimidazolium hexafluor-ophosphate [Bmim]PF6 and 1-butyl-3-methylimidazolium tetra-fluoroborate [BMIM]BF4, are well known as positive markers inmany biological reactions (Dong-Mei et al., 2010; Xiao et al., 2012).Previously, our research group reported the biosynthesis ofGAMG by whole cells of P. purpurogenum Li-3 (w-PGUS) in anionic liquid/buffer biphasic system which significantly improvedthe yield of GAMG but the operational stability was unsustainable(Jin-Yan et al., 2012).

High enzymatic activity and operational stability are thebasic criteria for all biotransformation reactions to be applied onlarge scale. To achieve these dual goals, many researchers havebeen employing the activity enhancer (IL) and activity stabilizer(polymer support) together in the same biocatalytic system. Manyenzymes have been immobilized with ILs on a certain biocompa-tible support to attain a high efficiency and reusability of thebiocatalyst (Clousier et al., 2011; Hu et al., 2012; Kim et al., 2012).Keeping in view the lower activity, expression and operationalstability of β-glucuronidase from P.purpurogenum (w-PGUS) Li-3;firstly we entrapped the whole cells in alginate gel beads theninvestigated the biocompatibility and catalytic efficiency in bufferand ILs media. The hydrophilic IL [BMIM]BF4 showed a remarkablebiocompatibility with immobilized w-PGUS and also displayed animproved activity and operational stability in this monophasicsystem. The prime focus of the study was to stabilize and enhancethe intracellular activity of the β-glucuronidase for the higherGAMG yield.

2. Material and methods

2.1. Chemicals and solvents

The ILs, 1-butyl-3-methylimidazolium hexafluorophosphate[Bmim]PF6, 1-butyl-3-methylimidazolium tetrafluoroborate [BM-IM]BF4, 1-ethyl-3-methylimidazolium tetrafluoroborate [EMIM]BF4, N-butyl-pyridinium tetrafluoroborate [BPy]BF4 and N-ethyl-pyridinium tetrafluoroborate [EPy]BF4 (purity 99%) were pur-chased from Henan Lihua Pharmaceutical Co. Ltd. (Henan, China).Glycyrrhizin (GL) and glycyrrhetic acid (GA) were purchased fromSigma Chemical Co. (USA). Glycyrrhetic acid 3-O mono-β-D-glu-curonide (GAMG) was generously donated by Nanjing Universityof Technology (China). Methanol was of chromatographic grade.All other chemicals were of analytical grade.

2.2. Whole-cell biocatalyst (w-PGUS)

The whole cells of P.purpurogenum (w-PGUS), were incubatedin flasks containing 100 mL of basal medium (5.0 g/L glucose,3.0 g/L NaNO3, 1.0 g/L K2HPO4, 0.5 g/L KCl, 0.5 g/L MgSO4 �7H2O,0.01 g/L FeSO4 �7H2O) at pH 5.0. The cells were grown withshaking at 170 rpm at 30 1C for 48 h. After 48 h, 10% of the culturewas transferred to the inducement medium (200 mL, pH 5.0) thatcontained 3.0 g/L glycyrrhizic acid ammonium dissolved in wholecell biocatalyst (w-PGUS) basal medium (described above), and thecells were grown at 30 1C, and 170 rpm for 96 h. The cells wereharvested by centrifugation (10,000 rpm, 5 min, 4 1C), washedtwice with distilled water, separated from the aqueous mediumby centrifugation (10,000 rpm, 5 min) and stored at 4 1C.

2.3. Immobilization of w-PGUS in different materials

The w-PGUS immobilizing efficiency of the different immobi-lizing materials was determined as follows:

Immobilization efficiencyð%Þ¼w� PGUS activity in immobilizing material

Free w� PGUS activity� 100 ð1Þ

2.3.1. Sodium alginate gel (w-PGUS/NaAlg)The entrapment of w-PGUS was conducted as described by Fraser

and Bickerstaff (1997), with some modifications. The w-PGUS (3 g)cell suspension was added to 4% sterilized NaAlg solution (w/v) to afinal concentration of 2% alginate. Beads were prepared by droppingthe cell suspension from a sterilized syringe into constantly stirred2% CaCl2 solution (w/v). The beads were kept in the stirring gelationmedium for 30–40 min, then twice washed with distilled waterand stored at 4 1C. The biotransformation efficiency of these beads

Fig. 1. Biotransformation of GL into GAMG, GA and glucuronide catalyzed by β-glucuronidase of whole cells of Penicillium purpurogenum Li-3.

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(100 g/l) was performed in 3 mM GL solution at 40 1C, pH 5.5 andshaking speed of 150 rpm for 96 h.

2.3.2. DiatomiteThe sterilized diatomite or kieselguhr, (5 g) was gradually

added to the cell suspension (20 ml H2O, 3.0 g w-PGUS) with aconstant stirring speed 30 rpm for 30 min. The saline washedbeads were dried and used for the GL biotransformation under theabove mentioned reaction conditions.

2.3.3. Agarose gelThe w-PGUS (3 g) cell suspension was added into the agarose

solution and made up 6% (w/v), then after its settlement, the driedbulk was cut into the uniform pieces (1.5 cm�1 cm�0.2 cm). Thewashed and dried pieces were put into the reaction mixture forthe activity analysis under the same set of conditions.

2.3.4. ChitosanThe weighed amounts of chitosan (0.5 g) and w-PGUS (3 g)

were added in 2% dilute acetic acid to formulate a colloidalsolution of the chitosan (20 ml), and it was drop wise poured into25 ml solution mixture of NaOH and ethanol (composition; 7.5%NaOH solution and 95% ethanol into 4:1 ratio) by a syringe. Theresulting beads were twice washed and examined for thebiotransformation of GL.

2.3.5. Polyvinyl alcohol (PVA)The suspension of the w-PGUS was added in 20% PVA (100 ml)

sterilized solution and this mixture was filled in a glass column fordrop by drop discharge into a sterile saturated boric acid solution.The beads were washed with saline and investigated for thecatalytic hydrolysis of GL.

2.4. Selection of immobilizing material

The best immobilizing material was selected primarily on thebasis of its biotransformation activity and reaction mediumbiocompatibility. All reactions were performed in 10 ml GL solu-tion, pre-incubated in 20 ml glass vials and then specific amountsof immobilized w-PGUS were added to these reaction flasks toinitiate the biotransformation processes and the product wasanalyzed by HPLC.

2.5. Determination of optimized compositional parametersof whole cells alginate beads (w-PGUS/Alg)

The compositional efficiency of w-PGUS alginate beads wasevaluated by determining the optimal concentrations of CaCl2 andNaAlg. The reactions were performed with various compositionalconcentrations of CaCl2 and NaAlg in 20 ml glass vials containing100 g/l w-PGUS beads in 10 ml reaction mixture having 3 mM GLconcentration at pH 5.5, 40 1C, and shaking speed of 150 rpm for60 h.

2.6. SEM analysis

The internal and external morphology of w-PGUS microcap-sules was observed by scanning electron microscope (SEM)(Philips XL30 ESEM). The alginate beads samples were dried inair and overnight lyophilized in a freeze drier. The samples weretreated by gold sputtering before SEM observation. The densityand distributional ratio of w-PGUS mycelia inside the core ofmicrocapsules was observed using a TriStar 3000 surface areaanalyzer at an adsorption temperature of 77 K.

2.7. Relative activity of the immobilized w-PGUS in buffer and ILs

The activity analysis of immobilized w-PGUS in ionic liquids[Bmim]PF6, [BMIM]BF4, [EMIM]BF4, [BPy]BF4, and [EPy]BF4 wasconducted by preparing different dilutions of these ionic liquids inacetate buffer (pH 5) containing 4 mM GL, placing the reactionmedia at 40 1C and shaking speed of 160 rpm. These reactionparameters were applied after the initial experimentation for thecomparative activity analysis of the immobilized w-PGUS indifferent ILs. The immobilized w-PGUS (whole-cell beads) cata-lyzed in pure buffer (acetate buffer pH 5.0) was considered as acontrol. The immobilized biocatalyst (100 g/l) was taken in 20 mlglass vials containing 10 ml of each reaction medium and incu-bated at 40 1C and 160 rpm for 10 h. Aliquots were withdrawn forHPLC analysis to determine the relative enzyme activity of theimmobilized w-PGUS.

2.8. Optimization of biocatalytic conditions for immobilized w-PGUSin buffer and IL co-solvent media

All reaction parameters of the immobilized w-PGUS in thebuffer and IL/buffer media were optimized and compared on thebasis of actual GAMG yield determined by HPLC. The parametersinvestigated were the substrate (GL) concentration, the immobi-lized w-PGUS beads concentration, pH, temperature, shakingspeed and time course of the reaction.

2.9. Reusability of immobilized w-PGUS

The immobilized w-PGUS beads were filtered after each reac-tion batch, rinsed with buffer and then introduced into the freshlyprepared substrate solution for the next reaction cycle under thesimilar conditions. The reusability of the immobilized w-PGUS wasdetermined as follows:

Reusable efficiencyð%Þ ¼w � PGUS beads activity in the nth cyclew � PGUS beads activity in the 1st cycle

� 100

ð2Þ

2.10. HPLC analysis

Quantitative analysis of the substrate and products in thereaction systems was performed by HPLC. GL, GAMG and GA wereseparated on an octadecylsilane (ODS) column (Shim-pack,VP-ODS, 4.6�250 mm2, Shimadzu Corporation, Kyoto, Japan)under the following chromatographic conditions: UV detectionwavelength 254 nm; flow rate 1.0 mL/min; mobile phase, water(pH 2.85 with 0.6% (v/v) acetic acid) and methanol at 19:81 (v/v);and injection volume 10 mL. The retention times of GL, GAMG andGA were 7.5, 13.5 and 22.7 min, respectively.

The initial reaction rate (V0), conversion rate of GL and theproduction rate of GAMG were determined as follows:

V0 ¼ ðGL0�GLeÞ=t ð3Þ

GL conversion rateð%Þ ¼ ðGL0�GLe=GL0Þ � 100 ð4Þ

GAMG productionrateð%Þ ¼ ðPGAMG=GL0Þ � 100 ð5Þ

Where GL0 and GLe are the initial and unreacted concentrations ofsubstrate (GL) at time 0 and time t, and PGAMG is the concentrationof GAMG at time t.

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3. Results and discussion

3.1. Immobilizing efficiency of different materials

The catalytic activity of the immobilized w-PGUS in fivedifferent materials such as chitosan, kieselghur, agrose, sodiumalginate and polyvinyl alcohol was investigated and comparedwith (1.17 U/g) free w-PGUS (Fig. 2).Their relative activities were;chitosan 51.75%, kieselghur 73.93%, agarose 81.3%, PVA 92.42% andNaAlg 97.96% compared to free w-PGUS after 96 h.

Many researchers reported that alginate gel has high immobi-lizing efficiency for whole-cell biocatalyst/enzyme compared toother immobilizing materials. They also demonstrated that thehigh efficiency of β-glucuronidase (GUS) in microcapsules was dueto strong mechanical strength and low swelling degree during therepeated use therefore rendered the storage stability to theenzyme in the alginate gel (Jian et al., 2009).

3.2. Optimization of compositional parameters of alginate beads

Six different concentrations of CaCl2 and NaAlg were investi-gated as shown in Table 1. The results demonstrated that 2% CaCl2and 2.5% NaAlg were optimal concentration for beads constructionand consequently efficient for the GL biotransformation. Theexternal and internal surface morphology of the w-PGUS-alginate beads could be observed in the SEM images (Fig. 3). Thepolymers and cross linkers concentration play an important role inthe immobilization of the whole cells by regulating the porosity ofthe core and gel carrier matrix (Salam et al., 2011).

3.3. Selection of IL and its volumetric ratio

The ILs were selected on the basis of relative enzymatic activityof the immobilized w-PGUS in pure buffer and ILs. The immobi-lized w-PGUS activity in pure buffer medium was considered as100%. The immobilized w-PGUS showed high initial reaction rate

in [BMIM]BF4 co-solvent medium compared to the pure buffer andfour ILs (Fig. 4). There was 1.35 mMn-1 initial reaction rate of theimmobilized w-PGUS in [BMIM]BF4 co-solvent medium comparedto the pure buffer medium (1.26 mMn-1). All other ILs showed lowactivity compared to the pure buffer solution at all dilutions. Thedifference in the activities could be attributed to the chemicalcomposition, hydrophobic and hydrophilic nature of the ILs media;therefore it influenced the course of catalysis. The water miscible[BMIM]BF4 displayed more compatibility with alginate core andlead the rapid mass transfer subsequently induced the enzymewhich consequently increased the biotransformation rate com-pared to other ILs (Liu et al., 2006; Lozano et al., 2007). Clousieret al., (2011) also reported the alginate-supported ionic liquid[BMIM]BF4, which was successfully applied to the palladiumcatalyzed allylation and amination reactions.

Among the various volumetric ratios of [Bmim]BF4, the 30% IL/buffer ratio was best for the efficient GL biotransformation. The[Bmim]BF4 had an ascending effect on the GL catalysis at the lowvolumetric ratio and exhibited the positive impact of IL on thebiotransformation rate; while above 30% IL/buffer ratio, the rate ofreaction decreased dramatically and demonstrated the negativeeffect of the high ionic strength on the active site of the enzyme.[Bmim]BF4 is a moderately polar ionic liquid containing coordinat-ing anions (BF4) and could be helpful in the activation of theenzymes. High concentration of the ions in the reaction systemcould retard the enzymatic activity by inflicting an inhibitoryeffect on the enzyme's active site, and it also limits the masstransfer of substrate by increasing the viscosity of the reactionmedium. Therefore, most of the reaction media contain ILs as co-solvent to control ionic strength (Hernandez-Fernandez et al.,2007; Xiao et al., 2009).

3.4. Optimization of biotransformation parameters for immobilizedw-PGUS in buffer and IL/buffer media

The GL solution of different concentrations ranging from 2 to7 mM were prepared in the acetate buffer and IL/buffer; andanalyzed for the constant alginate beads concentration undersimilar reaction conditions (Fig. 5a). The yield of GAMG steadilyincreased with the increase of GL concentration and reachedmaximum at 4 mM (2.08 g/l GAMG yield) in pure buffer GLmedium, on the other hand, in case of IL co-solvent medium5 mM (2.40 g/l GAMG) was the maximum yield but furtherincrease in GL concentration rapidly reduced the GAMG yield.

The initial increase of GL concentration enhanced the produc-tion of GAMG while after optimal GL concentration; furtherincrease had reductive effect on the yield of GAMG which couldbe due to the increased viscosity of the reaction mixture, limiteddiffusion of GL into the beads core, consequently into the cells.Moreover, excessive substrate concentration also inflicts an inhi-bitory effect on the active site of enzyme. The IL co-solvent buffermedium exhibited more compatibility with GL and it could be dueto the hydrophilic nature of [BMIM]BF4 which facilitated thetransport of substrate across the beads as well as the cell

Fig. 2. Immobilizing efficiency of whole-cell biocatalyst (w-PGUS) in variousmaterials.

Table 1Compositional efficiency of immobilized w-PGUS in different concentrations of CaCl2 and NaAlg.

Reagent Percent GAMG yield Process constants

1% 1.5% 2% 2.5% 3%

CaCl2 44.8572.76 46.7073.47 53.4074.23 48.5373.19 45.4374.87 NaAlg 2%, 5 g/l w-PGUSNaAlg 41.6073.13 44.8074.23 51.1372.98 60.6073.94 37.6073.18 CaCl2 2%, 5 g/l w- PGUS

All reactions were performed at pH 5.5, temperature 40 1C, GL concentration 3 mM, and shaking speed of 150 rpm for 60 h.

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membrane hence biotransformed the high concentration of GL(Constantinescu et al., 2007; Nakashima et al., 2006).

The effect of immobilized w-PGUS beads quantity on the yieldin buffer and IL/buffer reaction mixtures has been shown in Fig. 5(b). The optimal w-PGUS alginate beads concentrations for max-imum GAMG production in IL/buffer and buffer reaction mediawere 70 g/l and 80 g/l, respectively. The GAMG yield in IL/buffermedium was 68.01% compared to 65.30% in buffer medium. Theincrease in w-PGUS alginate beads enhanced the GAMG yield byproviding more active sites for the diffusion of the GL into the cellswhile further increase in the alginate beads quantity engendered anegative impact on the hydrolytic activity of w-PGUS because toomuch concentration of the beads hindered the mass transfer(Fatima et al., 2007). IL co-solvent medium provided a favorableenvironment for the higher hydrolytic activity of the immobilizedw-PGUS, therefore, IL co-solvent medium recruited 12.5% lessimmobilized biocatalyst (w-PGUS) than the pure buffer mediumat the similar GL concentration.

The pH profile of the immobilized w-PGUS was investigated inthe 5–6 pH range. The maximum immobilized w-PGUS activity inpure buffer reaction medium was recorded at pH 5.6 (72.62%GAMG), while for IL/buffer system the pH optima was 5.4 (74.14%

GAMG). On contrary to this, at upper and lower of the pH optima,the GAMG yield was comparatively low (Fig. 5 c).

The pH plays a substantial role for the ionization of thesubstrate, ionic liquid and the binding of substrate at the activesites of the enzymes by modulating its polarity (Wang et al., 2009).GL is a weak tribasic acid; the pH variability can change theionization degree of the carboxyl group and accordingly affectsbinding to the enzyme thus affects biotransformation rate. The pHof the IL co-solvent reaction medium influenced the biotransfor-mation of the GL by efficiently mobilizing the substrate within thepH range 5.0 to 5.4; in contrast to this, the pure buffer reactionmedium showed a slightly high acidic 5.6 pH. This slight differencemight be due to the anionic nature of the alginate, which recruitsthe cations, and change the microenvironment around the immo-bilized enzyme. The IL co-solvent medium slightly activated theenzyme under acidic condition due to the presence of anions byshifting the pH towards slightly acidic (Lou et al., 2009).

Temperature is a key factor in all biotransformation processes;increase in temperature lowers the activation energy and conse-quently accelerate the reaction rate until optimal temperatureachieved as shown in Fig. 5(d). The temperature's effect on theGAMG yield was investigated by incubating the reaction mixturesat various temperatures and under the constant conditions. Theimmobilized w-PGUS demonstrated similar 45 1C optimum tem-perature in both reaction media. However, the reaction mediumwith IL as co-solvent proved to be a more productive GAMG(80.73%) than buffer medium (74.13%). The initial increase of thereaction rate in the IL co-solvent medium could be attributed tothe hydrophilic nature of [BMIM]BF4, which accelerated the reac-tion rate by increasing the mass transfer and also activated theactive sites of the enzymes by furnishing the ionic strength(Katsoura et al., 2007). Both systems demonstrated bell shapedprofiles and at higher temperature decreased the catalytic activity,which could be the consequence of denaturation/unfolding of theenzyme (Katsoura et al., 2007).

Shaking speed regulate the diffusion of the substrate into thetarget site in a reaction system, hence changes the reaction rate.High viscosities of the ILs restrict the substrates and products in/out diffusion from active site of enzyme (Kaar et al., 2003).Therefore, the effect of different shaking speeds (120–220 rpm)on GAMG yield by immobilized w-PGUS was evaluated in bufferand IL co-solvent media. By increasing the shaking speed gradually

Fig. 3. SEM images of internal morphology w-PGUS alginate beads (a,b) external morphology, (c,d) internal morphology.

Fig. 4. Effect of various ILs volumetric ratios (v/v) on the relative enzyme activity ofimmobilized w-PGUS. Data are representative of three independent experiments;*Po0.05.

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increased GAMG yield till the optimum values for both reactionmedia, suggesting the mass transfer limitation. The maximumGAMG yield of 78.40% and 81.30% was achieved at 160 rpm and180 rpm in buffer and IL co-solvent media, respectively (Fig. 5e).After the optimal values, the raise in the shaking speed inflicted anegative impact on the GAMG yield; as higher agitation could have

damaged the cells or inactivated the enzyme by altering theconformation of the active site (Manolov 1992). The difference inshaking speed for maximum yield of GAMG could be attributed tothe viscosity variation of two media. IL co-solvent medium wasrelatively viscous than aqueous medium which restrained themass transfer and therefore necessarily required more agitation

Fig. 5. (a) Effect of substrate (GL) concentration on the GAMG yield catalyzed by immobilized w-PGUS in buffer and IL/buffer media. The reactions were performed in purebuffer and 30% IL/buffer media containing different GL (2–7 mM) concentrations at pH 5.0, 40 1C temperature, 100 g/l immobilized w-PGUS, and shaking speed of 160 rpmfor 60 h. (b) Effect of beads concentration of w-PGUS on the GAMG yield catalyzed by immobilized w-PGUS in buffer and IL/buffer media. The reactions were performed withdifferent concentrations of immobilized w-PGUS beads in pure buffer and 30% IL/buffer media containing 4 mM and 5 mM concentrations of GL respectively, at pH 5.0,temperature 40 1C, and shaking speed of 160 rpm for 60 h. (c) Effect of pH on the GAMG yield catalyzed by immobilized w-PGUS in buffer and IL/buffer media. The reactionsin pure buffer media were performed at different pH levels containing 4 mM concentrations of GL, 80 g/l concentration of immobilized w-PGUS, 40 1C temperature, andshaking speed of 160 rpm for 60 h. The reactions in 30% IL/buffer media were performed at different pH levels containing 5 mM concentration of GL, 70 g/l concentration ofimmobilized w-PGUS, 40 1C temperature, and shaking speed of 160 rpm for 60 h. (d) Effect of temperature on the GAMG yield catalyzed by immobilized w-PGUS in bufferand IL/buffer media. The reactions in pure buffer media were performed in 4 mM concentrations of GL at pH 5.6, 80 g/l concentration of immobilized w-PGUS, and 160 rpmshaking speed for 60 h under different temperatures. The reactions in 30% IL/buffer media were performed in 5 mM concentration of GL at pH 5.4, 70 g/l concentration ofimmobilized w-PGUS, and shaking speed of 160 rpm for 60 h under different temperatures. (e) Effect of shaking speed on the GAMG yield catalyzed by immobilized w-PGUSin buffer and IL/buffer media. The reactions in buffer media were performed in 4 mM concentrations of GL at pH 5.6, 80 g/l concentration of immobilized w-PGUS, andincubated at 45 1C temperature for 60 h at different shaking speeds. The reactions in 30% IL/buffer media were performed in 5 mM concentration of GL at pH 5.4, 70 g/lconcentration of immobilized w-PGUS, and incubated at 45 1C temperature for 60 h at different shaking speeds. (f) Time course of biotransformation of GL into GAMGcatalyzed by immobilized w-PGUS in buffer and IL/buffer media under the determined optimized conditions. The reactions in pure buffer were performed with 4 mM GLconcentration at pH 5.6, 80 g/l immobilized w-PGUS, 5.6 pH, 45 1C temperature and 160 rpm shaking speed. The reactions in 30% IL/buffer media were performed in 5 mMconcentration of GL at pH 5.4, 70 g/l concentration of immobilized w-PGUS, 45 1C temperature and 180 rpm shaking speed. Data are representative of three independentexperiments; nPo0.05.

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(Katsoura et al., 2007). Above optimum shaking speed, GAMGyield was decreased in both reaction media but it was steady incase of IL co-solvent medium compared to the buffer medium,which illustrated a rapid fall of GAMG yield.

The GL biotransformation efficiency of immobilized w-PGUSwas evaluated against the time course in buffer and IL co-solventreaction mixtures as shown in Fig. 5(f). The immobilized w-PGUSexhibited a faster reaction rate in IL co-solvent medium comparedto the buffer medium. The maximum GAMG yield (85.33%, 2.62g/l) was recorded after 62 h in IL co-solvent medium; however onthe other hand the highest GAMG yield (84.76%, 2.34 g/l) wasachieved in buffer medium after 72 h. The hydrophilic IL acceler-ated the reaction rate and overall 1.16% increased the reaction ratecompared to the buffer medium. The enhancement of biotrans-formation rate of the GL in IL co-solvent medium could be due tothe increase of the mass transfer and activation of the enzymaticactivity. There are several researches on the enhancement ofcatalytic efficiency of enzymes due to the presence of ionic liquidsin the reaction systems (Bose et al., 2012; He et al., 2009).

3.5. Operational stability of immobilized w-PGUS

The major advantage of the immobilized whole-cell biocata-lysts or enzymes in the polymer support is to attain the opera-tional stability for repeated use. The w-PGUS could be easilyremoved from both reaction mixtures by simple filtration orcentrifugation. The repeated use of alginate entrapped w-PGUSin IL co-solvent and buffer media was evaluated for 12 repeatedcycles. It was found that the immobilized whole-cell biocatalyst(w-PGUS) retained higher activity in IL co-solvent than the buffermedia after each successive batch reactions (Fig. 6).

After ten repeated batches, the operational stability of immo-bilized w-PGUS catalyzed in IL co-solvent was significantly higher(45.80%) than the buffer medium (18.13%). After 12 reuses, theimmobilized w-PGUS retained 27.20% of its original activity in ILco-solvent mediumwhile almost no activity in the buffer medium.The alginate gel matrix engendered an environment where immo-bilized w-PGUS act like enzyme containers suspended in the innercore of capsules (Kim et al., 2012). The ILs could improve theoperational stability and catalytic efficiency of these enzymecontainers. Arai et al. (2010), also reported the improved stabilityof the immobilized filamentous fungus on the biomass supportparticles (BSPs) in hydrophilic [BMIM]BF4 which subsequentlyenhanced the fatty acid methyl ester (ME) yield. The continuousloss or decay in the immobilized w-PGUS activity could beascribed to the inactivation of enzyme by protein denaturation,

adsorption or accumulation of the reaction products in theimmobilized cells (Moniruzzaman et al., 2012).

4. Conclusions

In this study, efficiency and operational stability of the alginateentrapped w-PGUS biocatalyst was investigated in buffer and ILsco-solvent media. The immobilized w-PGUS displayed signifi-cantly higher catalytic efficiency and yield in [BMIM]BF4 co-solvent medium compared to the buffer medium. The reactionrate in IL co-solvent system enhanced up to 1.16% compared to thebuffer system. The immobilized w-PGUS retained 45.80% and18.13% of its original activity in the IL co-solvent and buffer mediarespectively, after ten batches successive reuse and demonstratedthe excellent operational stability. The combination of immobi-lized w-PGUS and [BMIM]BF4 could have an excellent potential forhigher and efficient GAMG production.

Acknowledgments

This work was financially supported by National Science Founda-tion of China (No. 21176028, 21276025, 21376028), Doctoral Fund ofMinistry of Education of China (No. 20091101110036, 20121101110050),National Science Foundation of Beijing (No. 2112035), NationalHigh Technology Research and Development Program of China (863Program No. 2012AA02A704), and the Major State Basic ResearchDevelopment Program of China (973 Program No. 2013CB733900).

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