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Enzyme and Microbial Technology 43 (2008) 517–522

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

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nvertase embedded-PVC tubing as a flow-through reactor aimed atonversion of sucrose into inverted sugar

aroj Kumara,b, Vinay Singh Chauhanb, Pradip Nahara,∗

Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi 110007, IndiaDepartment of Biotechnology, Bundelkhand University, Jhansi 284 128, UP, India

r t i c l e i n f o

rticle history:eceived 24 April 2008eceived in revised form 7 August 2008ccepted 11 August 2008

eywords:

a b s t r a c t

A simple flow-through reactor system is prepared by covalent linking of a biomolecule on the innersurface of a polyvinyl chloride (PVC) tube. This is achieved by introducing an active functional group onthe surface of an inert PVC tube through 1-fluoro-2-nitro-4-azidobenzene (FNAB), a precursor of highlyreactive nitrene, which can insert to any C–H bond. CCl4 lacking C–H bond is taken as a solvent for loadingFNAB solution into the tube. FNAB loaded tube is then allowed to expose to sunlight for 20 min during

olyvinyl chloride-Fluoro-2-nitro-4-azidobenzeneunlightlow-through reactornvertaseovalent immobilization

which azido group of FNAB generates nitrene and attaches itself to PVC tube through insertion reaction.Invertase is immobilized in the activated PVC tube at 50 ◦C in 45 min. Invertase embedded-PVC tube isused as a flow-through reactor to convert sucrose to invert sugar. The flow-through reactor convertedsucrose into invert sugar with 97% yield as shown by HPLC analysis. The reactor is reused for eight cycleswith 17% loss of its initial activity. The reactor system is cheap as PVC tube is working both as a carrier ofbiomolecule and a reaction vessel. This reactor system overcomes the problem of back pressure and can

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. Introduction

The invert sugar has important applications in beverage andood industries for making jams, noncrystallizable cream, arti-cial honey, liquid sugar, etc. [1,2]. The invert sugar can berepared from sucrose either chemically by acid hydrolysis ornzymatically. Invertase is a highly efficient enzyme for convert-ng sucrose to glucose and fructose [3]. The enzymatic method is

ore preferable as product is colourless compared to acid hydrol-sis method where drastic reaction conditions (temperature andH) make the products coloured [4–8]. Besides, enzyme-basedransformation is cost efficient and environmental friendly. Innzymatic method, usually invertase is immobilized on a solidupport for conversion of sucrose to glucose and fructose by dif-erent immobilization techniques such as physical entrapment [9],

icroencapsulation [10], adsorption [11,12] or covalent attachment13–15]. Covalent method is preferable as it restricts the leach-

ng of the immobilized enzyme by making a stable covalent bond

ith the support. Covalent immobilization of invertase was car-ied out on different polymers, e.g. nylon-6 beads [16], magneticolyvinyl alcohol [17], poly(p-chloromethylstyrene) [18], poly(2-

Abbreviations: PVC, polyvinyl chloride; FNAB, 1-fluoro-2-nitro-4-azidobenzene.∗ Corresponding author. Tel.: +91 11 27666156/7; fax: +91 11 27667471.

E-mail address: pnahar@igib.res.in (P. Nahar).

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141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2008.08.002

ersion in a flow-through system.© 2008 Elsevier Inc. All rights reserved.

ydroxymethyl methaacrylate-co-glycidyl methaacrylate) [3] foraking bioreactors. There are several types of bioreactors avail-

ble for enzyme-based transformations such as batch stirred-tankeactors, packed-bed reactors or membrane reactors. Batch stirred-ank reactor operates batch-wise and consists of a vessel in whichhe reactant fluid mixture is stirred. The immobilized enzyme iseparated from the reaction medium at the end of the reaction byltration or centrifugation. Main drawback of such reactor is timeonsuming procedure for emptying, cleaning and filling the tanks well as separating the enzyme. Membrane or diaphragm reactorrovides very attractive alternative to batch reactor technology. Itan be operated one or two liquid phases. In this reactor, enzyme ismmobilized either onto the flat-sheet or hollow fiber membrane.ue to ability of such membrane in the secretion of two immisci-le fluids, membrane reactors are usually employed for biphasic

iquid system. In such system flow of the reacting fluid play anmportant role for proper mixing and reaction. However, one ofhe major disadvantages of such reactor is back pressure that mayccur due to clogging of the membrane. In packed-bed reactors orxed-bed reactors immobilized enzyme are usually packed in theolumn or jacketed thermostated pipe. Packed-bed reactors have

een traditionally used for large scale catalytic reactions due toheir efficiency and ease of operation and maintenance [19]. Onef the disadvantages of packed-bed reactors is the changed flowharacteristics due to alterations in the bed porosity during oper-tion. Several modifications such as tapered beds to reduce the

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ressure drop across the length of the reactor, inclined bed, hor-zontal bed, rotary horizontal reactor have been tried with limiteduccess. Besides, preparation of such reactor itself is cumbersomend costly affair.

In this communication, we report a simple flow-through reac-or system by making dual use of PVC tube, one as carrier ofiomolecule and other as a reaction vessel. As a carrier, invertaseas immobilized onto the activated PVC tube. PVC tube was chosenecause of its tolerance to pH and temperature [20–23]. Photoacti-ation of a PVC tube was carried out by a simple single step methody a photolinker in CCl4 (a solvent lacking C–H bond) after modifi-ation of the reported protocol [24,25].

. Materials and methods

.1. Materials

Invertase, horseradish peroxidase (HRP), o-dianisidine, glucose, fructose anducrose were purchased from Sigma, USA. Glucose oxidase was purchased fromoehringer Mannheim Gmbh, Germany. Transparent PVC tube was purchased

ocally. 1-Fluoro-2-nitro-4-azidobenzene (FNAB) was prepared from 4-fluoro-3-itroaniline through the diazotization reaction as reported earlier [26]. Intensityf sunlight was measured by a digital luxmeter. Phosphate-buffered saline (PBS)as prepared by mixing 0.85% NaCl with 0.01 M phosphate buffer (pH 7.2). All the

xperiments were carried out in triplicates.

.2. Activation of polyvinyl chloride tube at different photolinker concentrationsnd sunlight exposure time

PVC tube (diameter 6 mm) was cut into 15 cm and placed it into 100 ml of glasseaker by bending it in a U-shaped size. The open portions remained upward. Dif-erent concentrations (25, 50, 100, 200 and 400 �mol) of FNAB solutions were maden 500 �l of CCl4. FNAB solution (500 �l) of each concentration was poured into eachVC tube in such a way that the solution occupied the middle portion of the tube.he occupied portion of the tube was marked by a marker pen. It was then exposedo sunlight (intensity: 48,315 lx, temperature: 21 ◦C) for 20 min.

Photoactivation time was optimized by exposing the PVC tubes loaded withNAB solution (100 �mol/500 �l of CCl4/tube) to sunlight for 5, 10, 15, 20, 30 and0 min, respectively. Control experiments were carried out in dark. After the photo-hemical reaction the tubes were washed with methanol to remove unbound FNABnd its degraded products. Activated PVC tubes were then dried either by vacuumr passing hot air and placed in a beaker. Invertase solution 350 �l (1 mg/ml solu-ion in sodium acetate buffer, 0.05 M, pH 5) was added to each tube in such a wayhat the solution remained in the activated portion. The tube was then kept in anncubator at 50 ◦C for 45 min and washed four times with washing buffer (0.05% ofween-20 in 0.01 M PBS) to remove unbound invertase. Immobilized invertase washen assayed as described in the following section.

.3. Assay of invertase activity

The enzymatic activity of immobilized invertase was checked by adding 350 �lf sucrose solution (0.5 M) into PVC tube and incubating at 55 ◦C for 60 min. Theydrolyzed products transferred in a micro centrifuge tube. Glucose was assayedy adding 800 �l of assay buffer (0.5 mg of glucose oxidase, 0.12 mg of HRP and.24 mg of o-dianisidine in 2 ml of PB) to 200 �l of hydrolyzed sugar. After 30 minolour development was stopped by adding 200 �l of 5% H2SO4. Absorbance wasecorded at 490 nm.

.4. HPAE-PAD analysis of hydrolyzed products

High performance anion exchange chromatography-pulse amperometric detec-or analyser (Dionex DX 500 Bio LC) equipped with a gradient pump (GP 40), annion exchange column (carbopac PA-1, 4 mm × 250 mm) and an eluant degas mod-le (EDM-2) was used for the analysis of carbohydrates. Analysis of glucose andructose was carried out using an isocratic 18 mM NaOH eluant. A 30 min columnash with 200 mM NaOH followed by a 30 min equilibrium with the starting elu-

nt was used to yield reproducible retention time. The flow rate was maintained at.8 ml/min at ambient temperature. The detection of the hydrolyzed products wasarried out by PAD using gold electrode and an Ag/AgCl reference electrode. Triple

ulsed amperometry was used and the following pulse potential duration was given:1 = +0.10 V for 50 s, E2 = +0.60 V for 100 s, and E3 = −0.60 V for 50 s. Integration wasone from 300 to 500 m s. The response time of the PAD was set to 5 s. Glucose,ructose and sucrose were used as standard sugars. The standard sugar solutions50 �M) were prepared in deionized water. The standard mixture of sugars was runefore and after analysis of each sample. The identification of sugar peaks was basedn comparison of standards using a detector sensitivity of 50 nC full scale.

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Technology 43 (2008) 517–522

.5. Optimization of incubation temperature and time for immobilization ofnvertase

Invertase (350 �l from 1 mg/ml of sodium acetate buffer, pH 5) was pourednto the activated tubes and incubated for 45 min at 40, 45, 50, 55, 60, and 65 ◦C,espectively.

To optimize time, invertase was loaded into the activated PVC tubes and incu-ated at 50 ◦C for 25, 35, 45, 60, 90, and 120 min, respectively. Control experimentsere carried out with untreated PVC tubes. After washing immobilized invertaseas assayed.

.6. Kinetics of free and immobilized invertase

The kinetic parameters were determined by measuring the initial reaction ratesith varying concentration of sucrose (20–400 mM in acetate buffer, pH 5) at 55 ◦C.

m and Vmax values were determined from the Lineweaver–Burk plots.

.7. Thermal stability of immobilized invertase

Thermal stability of immobilized and free invertase was checked by incubatinghem in buffer, pH 5 for periods ranging from 30 min to 180 min at 55, 65, and 75 ◦C.fter each incubations assay was carried out as described in Section 2.3. Activity of

nvertase without incubation (initial activity) was treated as control.

.8. Continuous flow-through reactor

100 cm of 105 cm long PVC tube was loaded with 5 mmol of FNAB/25 ml ofCl4 and exposed to sunlight (intensity was 47,258 lx) for 30 min after which itas washed. Invertase solution (25 mg/25 ml of sodium acetate buffer) was loaded

nto the activated area of PVC tube and incubated for 45 min at 50 ◦C. The invertasemmobilized-PVC tube was coiled and put in a water bath at 50 ◦C. One end of tubeas attached to a small funnel through which 25 ml of 0.5 M of sucrose solution was

dded at a flow rate of 22 ml/h. Hydrolyzed sugar was collected from other end ofhe tube. Aliquots of hydrolyzed sugar were collected in stipulated time and assayed.

.9. Storage stability and reusability of immobilized invertase

Activity of immobilized and free invertase was determined after storage inodium acetated buffer, pH 5 at 30 ◦C for 0, 40, 80, 120, 160, 200 and 240 days.

The flow-through reactor system comprising immobilized invertase was reusedor eight times as described in Section 2.8. In each time the system operated for 8 h.he activity of covalently immobilized enzyme was expressed as a percentage of itsesidual activity with respect to its initial activity.

. Results and discussion

.1. Photochemical activation of PVC tube

We have made a simple flow-through reactor system by allowingVC tube to work as a carrier as well as reaction vessel. Preparationf such system involves two simple steps, namely activation of PVCube and immobilization of enzyme which are schematically rep-esented in Fig. 1. Activation is carried out by a heterobifunctionalhotolinker FNAB, a precursor of nitrene which has a property of

nserting into C–H bond. Therefore, to avoid undesirable reaction,Cl4 is chosen as a solvent as it has no C–H bond.

The determination of FNAB concentration was importantor optimum activation of PVC tube. Maximum activation wasbserved when 200 �mol of FNAB, dissolved in 500 �l of CCl4 wassed to activate 2 cm of PVC tube. Further increase in FNAB did notnhance its activation. However, 100 �mol of FNAB also showedomparable result; hence used as optimum (Fig. 2A).

Light is another prerequisite for photoactivation. The rate limit-ng step for activation reaction was the formation of intermediateitrene which increased with increase in light exposure time.aximum activation was obtained in 30 min of sunlight exposure

Fig. 2B). However, activation time for inner surface of PVC tube is

light higher than the activation of open surface which usually takes0–20 min [24,25]. One reason for higher activation time might beue to obstruction of light to reach the photolinker present insidehe PVC tube which was 80% transparent. Intensity of sunlight was

easured as 48,513 lx which was above the minimum intensity

S. Kumar et al. / Enzyme and Microbial Technology 43 (2008) 517–522 519

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ig. 1. Schematic representation of photochemical activation of polyvinyl chlorideitrene which insert into C–H bond of the PVC (i) producing an activated support,ctivated surface to produce an immobilized protein (iv).

evel (26,300 lx) required for optimum activation [27]. When acti-ation of PVC tube was carried out in the presence of artificial lighthat is UV light at 365 nm in an UV Stratalinker (Stratagene, USA)o significant difference in activation was observed compared to

ig. 2. Optimization of (A) FNAB concentration and (B) light exposure time for acti-ation of PVC tube. (A) Different concentrations of FNAB solutions were poured intoVC tubes and exposed to sunlight (intensity of 48,315 lx) for 20 min. (B) PVC tubesoaded with 100 �mol of FNAB were exposed to sunlight (�) for 5, 10, 15, 20, 30,nd 40 min, respectively. Control experiment was carried out in dark (�). Efficacyf the activation was checked by immobilizing invertase into the tubes followed byt’s assay.

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ce. 1-Fluoro-2-nitro-4-azidobenzene (ii), under sunlight generates highly reactivea labile fluoro group (iii). Amino group of the protein replaces fluoro group of the

unlight activation. Hence, in absence of sunlight PVC tube can bectivated by artificial light. The advantage of using sunlight is thatt can activate PVC tube of any size or shape for commercial oresearch use without investing money as sunlight is accessible inost part of the world in most of the time.

.2. Covalent immobilization of invertase onto PVC surface

The activated PVC tube did not require any additional reagentr catalyst for immobilization. However, temperature enhancedhe process of immobilization. Invertase was best immobilized inhe activated PVC tube at 50 ◦C (Fig. 3A). Time depended immo-ilization study showed that 45 min was enough to immobilize

nvertase at the optimum temperature (Fig. 3B). Control experimentarried out onto an untreated PVC surface showed five times lessbsorbance than that of activated PVC. Efficacy of immobilizationnto the activated PVC was checked by enzymatic analysis of glu-ose, which was one of the sucrose hydrolyzed products, catalyzedy invertase.

Invertase-catalyzed reaction products (glucose and fructose)ere further confirmed by an HPAE-PAD method with anion

xchange column. Analysis of reaction mixture showed three peaksorresponding to glucose, fructose and sucrose which were con-rmed by the peaks of standard sugar solutions. A small sucroseeak is due to unreacted sugar (Fig. 4).

.3. Kinetic constants

When an enzyme is immobilized, microenvironment aroundhe bound enzyme is altered leading to variations in the kineticarameters Km and Vmax with respect to free enzyme. Microen-ironments of the immobilized invertase depend on the type ofmmobilization and the solid matrix. In view of this we have stud-ed the kinetic parameters of the immobilized and free invertase byineweaver–Burk plot. The plot gave two straight lines which con-orm to the Michaelis–Menten equation for the enzyme-catalyzedeaction (Fig. 5). The Km and Vmax value of covalently immobi-ized enzyme as obtained from the Lineweaver–Burk plot wereound to be 33.5 mM and 281 U mg−1 protein, whereas for free

nzyme they were 22.8 mM and 437 U mg−1 protein, respectively.he Km value of immobilized invertase was 1.4 times greater thanhe free enzyme. Similar results were obtained by Prodanovic etl., who reported 1.5 times greater Km value for invertase cova-ently immobilized onto glycidyl methaacrylate membrane than

520 S. Kumar et al. / Enzyme and Microbial Technology 43 (2008) 517–522

Fig. 3. Optimization of incubation (A) temperature and (B) time for immobilizationoo2a

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f invertase into activated (�) and untreated (♦) PVC tube. Incubation was carriedut (A) for 45 min at 40, 45, 50, 55, 60, and 65 ◦C, respectively and (B) at 50 ◦C for5, 35, 45, 60, 90, and 120 min, respectively. Absorbance was recorded after enzymessay.

hat of the free invertase [28]. The change in the affinity of inver-ase to its substrate was probably due to lower accessibility of

he substrate to the active site of the immobilized invertase [29].ecrease in Vmax value of immobilized invertase than that of the

ree invertase was due to restriction in mobility of the immobilizednzyme. Similar results were also observed by other researchers14,17].

ig. 4. Analysis of reaction mixture comprising glucose (1), fructose (2) and unre-cted sucrose (3) obtained by enzymatic hydrolysis of sucrose by HPAE-PAD.

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ig. 5. Kinetics of immobilized (�) and free (♦) invertase using Lineweaver–Burklot. The inverse values of substrate concentrations are plotted against the inversealues of initial rates.

.4. Thermal stability

Thermal stability of immobilized and free invertase was deter-ined by incubating them in buffer at 55, 65, and 75 ◦C in

ifferent times. Residual activity of invertase was calculatedith respect to its initial activity. Activity of invertase either

mmobilized or free decreased with increase in temperature.owever, immobilized invertase due to restricted conforma-

ional mobility showed better thermal stability than the freenzyme [14,17]. At 55 ◦C, immobilized invertase preserved 97%f its initial activity after 90 min which decreased to 94% afterh. At higher temperature sharp fall of activity was observed

Fig. 6).The activation energy of immobilized and free enzyme was

alculated from Arrhenius plot at different temperature rangesrom 25 ◦C to optimum temperature (55 ◦C). The plots of immo-ilized and free enzyme were linear and the activation energy of

mmobilized and free enzyme was found to be 5.1 kcal/mol and.6 kcal/mol, respectively. Lower activation energy for immobilized

nzyme compared to free enzyme was previously reported by oth-rs [11,17].

ig. 6. Thermal stability of immobilized (dark) and free (hollow) invertase at 55 ◦C�); 65 ◦C (�) and 75 ◦C (�).

S. Kumar et al. / Enzyme and Microbial

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ig. 7. Operational stability of immobilized invertase in a continuous flow reactor.

.5. Operational parameters of continuous flow system

The reactor system was operated for 40 h at 50 ◦C at a flow ratef 22 ml/h for invertase-catalyzed hydrolysis of sucrose. The flow-hrough reactor converted sucrose into invert sugar with 97% yields shown by HPLC analysis (Fig. 4). Hence, during 40 h, 880 ml of.5 M sucrose solution was passed through the reactor amountingo 150.48 g of sucrose of which 146 g of sucrose was converted tonvert sugar. Thus, 3.65 g of sucrose converted to 1.92 g of glucosend similar amount of fructose per hour. During this period inver-ase lost 10% of its initial activity (Fig. 7). The loss in activity may beue to higher operational temperature during a prolong run. Such

oss of activity at higher temperature was also observed by otheresearcher [3,16,17,30].

.6. Storage stability and reusability

The storage stability of the immobilized invertase was alsonhanced over that of the free enzyme. Immobilized invertasehen stored at room temperature (30 ◦C) for 240 days, it lost about

8% of its initial activity. The improved storage stability of immo-ilized enzymes can be attributed to a reduction in the rate of

enaturation of the enzyme (Fig. 8). The flow-through reactor waseused at least for eight cycles with 83% of residual activity (dataot shown). Loss of activity of immobilized invertase either during

ong storage or repeated use of reactor may be due to deactivationf invertase as well as leaching of physically adsorbed invertase

Fig. 8. Storage stability of immobilized (�) and free (♦) invertase.

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Technology 43 (2008) 517–522 521

hich may occur onto the unactivated spots of the matrix alongith covalent immobilization of invertase.

. Conclusions

A simple flow-through reactor system comprising PVC-tubembedded with invertase is prepared for conversion of sucrose intonvert sugar. In this reactor system PVC is used as a carrier of enzymes well as a reaction vessel. Operation and maintenance cost of sucheactor is also low. Besides, there is no need for specially designedhermal jacket for maintaining the reactor temperature, instead,he tube can be coiled and put in a water tank operating at an opti-

al temperature. Further, there is no need of stirring in the reactionixture as the products formed can continuously removed.This simple flow-through reactor system could be custom made

ither by increasing the length of the reactor or incorporating dif-erent enzymes at different length for single step enzyme-basedransformations, online diagnostics or affinity chromatography.

cknowledgements

The authors thank Dr. H.R. Das, Head, Lectin Research Labora-ory, IGIB and Mrs. Hemlata Gautam, Technical Officer, IGIB for theirelp in HPLC analysis.

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