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Process Biochemistry 47 (2012) 749–757

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epage: www.elsev ier .com/ locate /procbio

comparative performance evaluation of jute and eggshell matrices tommobilize pancreatic lipase

oham Chattopadhyay, Ramkrishna Sen ∗

epartment of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India

r t i c l e i n f o

rticle history:eceived 29 December 2011eceived in revised form 3 February 2012ccepted 7 February 2012vailable online 15 February 2012

eywords:ipase immobilization

a b s t r a c t

A pancreatic lipase was immobilized on readily available and inexpensive jute and eggshell matrices.The purity of extracted enzyme was confirmed by SDS-PAGE. The maximum protein load for eggshellwas 10.23 mg/g, and for jute, it was 5.7 mg/g. The free enzyme activity retention was greater than 80%for eggshell and 43% for jute. The immobilized lipase was stable over a pH range from 7 to 8 for eggshelland 7.5 to 8.5 for jute with over a temperature range from 25 to 45 ◦C for eggshell and 37 to 40 ◦C forthe jute. FTIR data indicated new bonds on the jute upon immobilization. Although no new bond wasobserved, immobilization data on eggshell fit well with the Langmuir adsorption isotherm model. The

ggshelluteTIREMdsorption isotherm

model constants, � max and Kl , were 13.92 mg/g and 0.382 mL/mg, respectively. Mixed adsorption withboth ionic and hydrophobic interactions was observed. Lipase adsorption was reduced significantly inpresence of Tween 80, whereas the effect was less in case of ionic strength, pH and temperature. Forboth matrices, scanning electron microscopy (SEM) was used to demonstrate the changes in surfacemorphology after immobilization. The performance of eggshell was better than that of jute as a matrixfor immobilizing pancreatic lipase.

. Introduction

Immobilization is the process of attaching or entrapping annzyme to an insoluble material, the matrix, using physical orhemical interactions. The primary objective of immobilizations to reduce the process cost by enhancing enzyme stability andeusability [1]. For further cost minimization, finding a cheap andeadily available matrix that can retain maximum enzyme activitys a major challenge in commercial enzyme immobilization appli-ations. Enzymes can be immobilized by several methods, whichange from carrier-bound to carrier-free techniques [2,3]. Galant al. [4] extensively reviewed different enzyme immobilizationtrategies and indicated that immobilization not only allow us toeuse the enzyme but also to improve its performance by enhanc-ng stability, activity specificity, etc. Various methods have beenxtensively used for enzyme immobilization, including entrapmentsing alginate [5], agarose, polyacrylamide, gelatin, sol–gel and k-arrageenan; adsorption using alumina, macro porous resin, silica,elite and calcium carbonate; as well as covalent binding using chi-

osan [6] and certain biopolymers such as jute [7]. Glutaraldehydes used as a cross-linking agent to stabilize the enzyme–matrixnteraction [8,9]. The selection of matrix and method of

∗ Corresponding author. Tel.: +91 3222283752; fax: +91 3222278707.E-mail address: rksen@hijli.iitkgp.ernet.in (R. Sen).

359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2012.02.003

© 2012 Elsevier Ltd. All rights reserved.

immobilization are critical for retaining maximum enzyme activ-ity and improving enzyme stability. Iyer and Ananthanarayanreviewed the enhancement in enzyme stability after immobiliza-tion in aqueous and non-aqueous environments [10]. Multipointcovalent attachment of an enzyme molecule to a support matrixaids in increasing stability. However, due care must be taken toavoid distorting the enzyme structure [11]. Enzyme active site inac-cessibility or conformation distortion upon immobilization causesmass transfer limitations, which results in reduced enzyme activity[12,13]. On the other hand, it is possible that activity may be lostduring the reaction because the enzyme is leached from the matrix[14]. Modifying the area near enzyme active site during immobi-lization is reportedly beneficial for activation of certain enzymessuch as lipases [11,15,16]. Changing the enzyme orientation on thesupport material is an additional approach employed for effectiveimmobilization [17,18].

Lipases (glycerol ester hydrolase, E.C. 3.1.1.3) are importantenzymes that break down oils and fats into fatty acids and glycerol.Lipases act at the oil–water interface and maintain good activity onhydrophobic substrates for catalysis of reactions such as hydroly-sis, interesterification and transesterification [19]. Lipases have a“lid” in its structure that covers the active site of the enzyme. The

enzyme contacts the oil–water interface, the lid moves away toprovide access for the substrate to enter the active site [20]. Thekinetic mechanism does not depend on the type of reaction. It washypothesized that the mode of action for lipase is similar to a serine

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50 S. Chattopadhyay, R. Sen / Proc

rotease [21]. In a hydrolytic reaction, one mole of oil or fat reactsith three moles of water to produce three moles of fatty acids and

ne mole of glycerol. Recently, lipase demand increased due to itsxtensive use in a transesterification reaction for biodiesel produc-ion [22]. Lipase obtained from various sources (from microbes tohe human and porcine pancreas) was immobilized primarily onydrophobic supports using various strategies. Porcine pancreatic

ipase (PPL) has been widely used for the transesterification due tots low price, easy availability and high stability. The enzyme doesot require any cofactor. It was observed in most of the reports that,he substrate conversion was less than 70% because of the speci-city of PPL toward 1 and 3 positions in the triglyceride. Higheronversion was also reported by some researchers and it was mighte due to the migration of acyl group from 2 positions to 1 or 3osition in the monoglyceride [23]. The materials used for lipase

mmobilization include resins [24], alginate [25], eggshell [26],ol–gel [27,28], sepabead [29] and chitosan [30]. Candida antarcticaipase immobilized on an acrylic resin is currently commerciallyvailable (Novozyme 435) and widely used for biodiesel production24].

To reduce the cost of enzymes and matrix immobilization for aigh-volume, low-value product such as biodiesel and to improvehe stability of enzymes, the study herein is a comparative perfor-

ance evaluation of lipase immobilization on two inexpensive andeadily available matrices such as jute and eggshell.

. Materials and methods

.1. Materials

Steapsin, a crude pancreatic lipase, was purchased from SRL (Mumbai, India).ute and eggshell were collected from a local market and hostel canteens (IIT Kharag-ur, India), respectively. The Bradford protein estimation kit and electrophoresispparatus were purchased from Bangalore Genei Pvt. Ltd. (Bangalore, India). Trib-tyrin was obtained from Himedia (Mumbai, India). Sodium metaperiodate wasurchased from Loba Chemie Pvt. Ltd. (Mumbai, India). An unstained protein molec-lar weight marker was purchased from Fermentas (Germany). All other chemicalsere procured from SRL (Mumbai, India) and were analytical grade.

.2. Methodologies

.2.1. Preparation of the lipase solutionThe crude steapsin powder contained high levels of stabilizing material. To

xtract the protein, the crude powder was dissolved in phosphate buffer (50 mM, pH) and vortexed for 15 min at room temperature. The solution was then centrifugedt 5000 rpm (approximately 3200 × g) for 10 min at 4 ◦C, and the supernatant wassed as the lipase solution in further studies [31].

.2.2. Lipase immobilizationIn this study, the method described by Vemuri et al. [26] was used to immobilize

he lipase on eggshell. In the first step, the eggshells were boiled in a 0.1% SDSolution for 15 min. The shells were washed thrice with distilled water to removeesidual SDS and then washed with acetone three times to remove water. Acetoneas subsequently removed by drying at 60 ◦C for 5 h. The dried eggshells were then

rushed and sieved through a −16, +36 mesh to generate a uniform size. Mixturesf lipase solution containing the requisite amount of lipase and 1 g of eggshell weredded to different conical flasks (50 mL) and incubated at 37 ◦C for specific timeeriods. After incubation, all of the conical flasks containing the mixture were storedt 4 ◦C for 16 h. The resulting enzyme-immobilized matrices were then washed withhosphate buffer (50 mM, pH 7) three times to remove the unbound enzyme.

Jute matrix-based enzyme immobilization, which is reportedly a process ofhemical modification on the jute surface [7], was performed by cutting the rawute into small pieces and boiling them in 0.05% NaOH solution for 30 min. The solu-ion was then neutralized by a wash with water and incubated with 0.5% sodium

etaperiodate (NaIO4) for 3 h under dark conditions to oxidize the OH groups intoCHO groups. The enzyme solution was added to the treated jute and incubated at◦C overnight. The unbound enzyme was washed with phosphate buffer (50 mM,H 7). The immobilized enzyme was suspended and stored in the same buffer untilsed.

.2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)nalysis

To determine the enzyme purity of the extracted lipase solution and ensureffective lipase immobilization on both matrices, an SDS-PAGE gel was generated.he immobilized preparations were boiled in 1% SDS for 15 min to elute the enzyme

chemistry 47 (2012) 749–757

from the matrices. The eluted enzyme and lipase solution (described in Section 2.2.1)were analyzed using SDS-PAGE, and the relative protein positions were comparedwith the standard protein molecular weight marker.

2.2.4. Enzyme loading and activity assayThe enzyme concentration was quantified using the Bradford protein estima-

tion method with bovine serum albumin (BSA) as the standard [32]. Enzyme activitywas determined using the tributyrin method [33]. First, a 2% polyvinyl alcohol (PVA)solution was prepared in distilled water. Next, a PVA solution and tributyrin weremixed at a 9:1 ratio to form an emulsion. A mixture comprising 1 mL emulsion,0.8 mL phosphate buffer (50 mM, pH 7) and 0.2 mL free enzyme solution (contain-ing 10 mg enzyme) was then prepared and incubated at 37 ◦C for 30 min. For theimmobilized enzyme, instead of free lipase, 2 g of lipase-immobilized jute or 1 gof lipase-immobilized eggshell was used. The quantity of immobilized jute andeggshell was determined such that the amount of enzyme was approximately equalin both immobilized systems (using the data for the enzyme loading capacity ofjute and eggshell). The reaction was terminated after 30 min by adding an ace-tone:ethanol (1:1) mixture and then titrated with 0.1 N sodium hydroxide solutionusing phenolphthalein as indicator. The enzyme activity was expressed as butyricacid produced per unit time per mg lipase. The maximum activity was considered100% in calculating the percentage of relative activity for the enzyme.

2.2.5. Variation of pH and temperatureTo determine whether immobilization changed the optimum pH or temperature

for maximum enzyme activity, a series of experiments with varying conditions wasperformed using both matrices and the free enzyme. In the first set of experiments,the pH value of the reaction mixture was varied from 6 to 8.5, but the tempera-ture was maintained at 37 ◦C. Acetate, phosphate and tris–HCl buffer was used tomaintain the desired pH. To determine the effect of temperature, the reactions wereperformed at the temperature range that varied from 25 to 50 ◦C and pH 7. All theexperiments were performed in triplicate, and the data are shown with the standarddeviation.

2.2.6. Immobilization timeThe incubation period is an important parameter for effective immobilization.

For cellulosic fibers, overnight incubation was reported [34]. Thus, for jute, the sam-ples were incubated overnight for efficient immobilization. For the eggshell, lipasedissolved in phosphate buffer (50 mM, pH 7) was mixed with 1 g of eggshell matrixand incubated for different time periods ranging from 30 min to 10 h at 37 ◦C. Themixtures were incubated at 4 ◦C for 16 h and then washed with same buffer thrice toremove unbound enzyme. The enzyme immobilized for different time periods wastested for enzyme activity using the tributyrin method, which was expressed as apercentage of the relative activity.

2.2.7. Fourier transforms infrared spectroscopyThe Fourier transform infrared (FTIR) spectroscopic study was performed in

a Nexus-870 spectrometer (Thermo Nicolet Corporation, Wisconsin, USA). For thejute matrix, three samples were analyzed, including the untreated jute, chemicallytreated jute and enzyme immobilized jute. Similarly, for the eggshell, the matrixsamples were analyzed before and after immobilization. To generate a pellet, 2 mgof each sample was mixed with 100 mg of FTIR grade potassium bromide (KBr). Thespectrometer was used in the transmission mode over a wave number range from4000 to 400/cm. The potassium bromide pellet, which does not absorb in the wavenumber range from 4000 to 400/cm, was used as blank for the study.

2.2.8. Experimental design for adsorption studiesTo study the adsorption of pancreatic lipase on eggshell, 1 g of eggshell was

mixed with different concentrations of the enzyme in 50 mL conical flasks, and themixture was incubated at 37 ◦C for 2 h followed by 4 ◦C for 16 h. The mixtures werethen filtered through Whatman No. 1 filter paper, and the matrix was washed withphosphate buffer (50 mM, pH 7) to remove unbound enzyme. The total protein con-tent in the filtrate was measured using the Bradford method [32]. The differencebetween the initial protein concentration and the concentration after filtration indi-cated the amount of enzyme immobilized onto the matrix. The experimental dataon the initial enzyme concentration and the mass of the enzyme immobilized oneggshell were fit into the Langmuir adsorption isotherm model using MicroCal Ori-gins 8.0 software (MicroCal Software Inc. USA). The linear Langmuir model fromArami et al. [35] is described in Eq. (1).

1�

= 1�max

+ 1�max × Kl

× 1c

(1)

Here, � is the mass of the adsorbed enzyme, � max is the maximum enzymeuptake, c is the initial enzyme concentration and Kl is the Langmuir constant.

To determine the manner of adsorption, eggshell-immobilized lipase was mixed

with a salt solution (NaCl) comprising 0.5 and 1 N and a non-ionic detergent (Tween80) between 1 and 5%. The mixture was then incubated at 37 ◦C for 1 h and washedwith phosphate buffer (50 mM, pH 7) to remove the unbound enzyme from thematrix surface. The activity of the immobilized enzyme was measured and comparedwith that of the untreated immobilized preparation.

S. Chattopadhyay, R. Sen / Process Biochemistry 47 (2012) 749–757 751

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on an eggshell matrix. In this study, the maximum loading capac-ity of the eggshell matrix was 10.23 mg protein/g, which indicatesthat the matrix has a good immobilization capability. In contrast, a

Fig. 1. Photographic images of the jute and eggshell m

To study the influence of different parameters on lipase adsorption, differentets of experiments were conducted with varying buffer concentrations from 25 to00 mM; at a 5.5–8.5 pH range; temperatures from 25 to 50 ◦C and Tween 80 con-entrations from 0.1 to 1% (v/v). The parameters were changed one at a time whileeeping other parameters constant. In the first instance, the pH and temperatureere kept constant at pH 7 and 37 ◦C, respectively, whereas in the pH and temper-

ture experiment, a 50 mM buffer was used. No Tween 80 was added in the firsthree experiments. The last experiment was performed with 50 mM buffer at pH 7nd 37 ◦C.

.2.9. Scanning electron microscopyAll samples (raw jute, chemically treated jute, enzyme-immobilized jute,

ggshell and enzyme-immobilized eggshell) were lyophilized and analyzed in aEOL JSM5800 Scanning Electron Microscope with an Oxford EDS Detector (JEOL,apan). The samples were coated with a thin layer of gold using a spray gun andhen observed at 20 kV with 1500× resolution.

.2.10. Reusing the immobilized lipaseAs stated earlier, enzyme reuse is a major motivation for enzyme immobi-

ization. To study this feature, the immobilized preparations were washed withhosphate buffer (50 mM, pH 7) after a single reaction and reused to detect thenzyme activity. Each reaction was considered a single cycle and the percent relativectivity was determined by taking the activity of the first cycle as 100%.

. Results and discussion

.1. Lipase extraction and immobilization

The protein content in the extracted lipase solution was esti-ated as only 2% (w/w) of the crude powder. The sizes of bothatrices were sufficiently small to maintain a high surface area

atio without adhering to each other. Photographic images of theute and eggshell matrices after lipase immobilization are shownn Fig. 1. As no protein was detected in the flow-through using theradford protein estimation method after the third wash step (dataot shown), it is assured that the unbound enzyme was removed

rom the matrix surfaces, and the detected activity was only fromhe immobilized enzyme.

.2. SDS-PAGE

In the SDS-PAGE gel, two prominent bands with a 32–40 kDaolecular weight range were observed for the extracted lipase

Fig. 2). The same proteins were in the eluted supernatants gener-

ted from boiling the immobilized preparations in SDS. The otherrotein of 25 kDa molecular weight was not immobilized and notresent in eluted fractions. This result indicates that the lipase wasurified and successfully immobilized on both matrices.

after lipase immobilization (with a centimeter scale).

3.3. Enzyme loading and activity

Enzyme loading is one of the most important criteria for matrixselection. A matrix that aids in maximum loading and retains themaximum activity of an enzyme is considered most suitable forimmobilizing an enzyme. Studies on the immobilization capabil-ity of eggshell have primarily been restricted to basic dyes [36],which are reportedly adsorbed poorly (1 mg dye/g of matrix). Therehas been no detailed study on the amount of lipase immobilized

Fig. 2. SDS-PAGE of lipase extracted from a crude sample (E) eluted from theeggshell (ES) and jute (J) matrices. M: molecular weight marker. Molecular weightsare expressed in kiloDaltons (kDa).

752 S. Chattopadhyay, R. Sen / Process Biochemistry 47 (2012) 749–757

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gen bond. Two new peaks were observed at 1250 and 1741/cm forthe chemically modified jute, which corroborates the C O (ester)and C O stretching, respectively. The appearance of new bondmight be from the formation of aldehyde groups upon successful

ig. 3. The effects of pH (a) and temperature (b) on enzyme stability after immobilizxperiments were performed at pH 7.

aximum of 5.7 mg of protein was immobilized/g of jute, which isuch less than with the eggshell matrix.Using the tributyrin assay, the values for the enzyme activities

f lipase on eggshell and jute are shown in Table 1. It is evident thatlmost 83% of the enzyme activity was retained on the eggshellatrix, whereas only 43% activity was retained by the jute matrix

ompared with free enzyme activity at pH 7. A similar result cane inferred from the study by Vemuri et al. [26], which indicatedhat almost 85% of lipase activity was retained after immobiliza-ion on eggshell. Thus, the results indicated that for both enzymeoading and activity, eggshell was superior to jute. The decreasen lipase performance efficiency when immobilized on jute mighte attributed to a blocked enzyme active site from covalent bondormation, whereas lipase immobilized on eggshell had almost theame activity as the free enzyme.

.4. Effects of temperature and pH on the stability of immobilizedipase

In our earlier study, it was reported that the optimum pH andemperature for free lipase were 7 and 37 ◦C, respectively, andhe activity decreased sharply with increasing pH and tempera-ure [31]. For the immobilized lipase, the maximum activity was

aintained between pH 7–8 (for eggshell) and 7–8.5 (for jute),hich resulted in a narrow pH range for similar maximum activ-

ties (Fig. 3a). Though the stability of the immobilized lipase with change in pH was greater for jute, the maximum activity gen-rated was lower compared with eggshell. This reduced activityight result from the method of immobilization on the differentatrices.The optimum temperature for immobilized enzyme is the same

s for free enzyme (Fig. 3b). The literature also confirms that theptimum temperature (40 ◦C) for maximum lipase activity beforend after immobilization remains unchanged [26]. However, it wasnteresting to note that lipase immobilized on eggshell was sta-le through 45 ◦C, and the maximum activity was approximately

◦

onstant from 30 to 40 C. A similar study with tyrosinase showedhat it increased in stability over a temperature range from 20 to0 ◦C when immobilized on eggshell [37]. For jute, the stability was

ower, and the immobilized system was stable from 37 to 40 ◦C. The

able 1nzyme activity (at pH 7 and temperature 37 ◦C) before and after immobilization.

Enzyme Activity (�mol/min/genzyme)

Retention ofactivity (%)

Free 426.6 ± 2.1 100Eggshell immobilized 355.7 ± 1.7 83Jute immobilized 183.4 ± 1.8 43

. All of the pH experiments were performed at 37 ◦C, whereas all of the temperature

experimental data suggested that the enzyme was more stable afterimmobilization on eggshell.

3.5. Standardizing immobilization time

For immobilization of the enzyme on jute, an overnight incuba-tion was performed. For adsorption on eggshell, the percent relativeactivity was highest at 2 h and was the same for the remainder ofthe experimentation time (Fig. 4). Literature reports have suggestedvarious incubation times, such as 15–20 min [38] and 40–60 min[26]. The difference in immobilization time from earlier reportsmay be due to the difference in the characteristics or source of theenzymes immobilized on the eggshell.

3.6. FTIR analysis

To elucidate any changes in the chemical structure of the matrixupon immobilization, an FTIR analysis of raw, chemically modifiedand enzyme-immobilized jute was performed, and the spectra areshown in Fig. 5a. The appearance of a new peak or the disappear-ance of an original peak upon chemical modification is indicatedby an arrow at the respective peak position wave number (/cm).The compound exhibited a moderately strong and broad bandnear 3400/cm, which indicates OH stretching, whereas a strongband at 2921/cm was observed from the intermolecular hydro-

Fig. 4. Incubation time standardization for lipase immobilization on eggshell. Thereaction was performed at 37 ◦C and pH 7.

S. Chattopadhyay, R. Sen / Process Biochemistry 47 (2012) 749–757 753

F rom chemical modification appeared in the spectrum and are indicated with arrows. Noc

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Fig. 6. The Langmuir adsorption isotherm model demonstrates the relationshipbetween the initial protein concentrations, C (mg/mL), and amount of protein

instances, a mixed adsorption can be inferred.

ig. 5. An FTIR spectrum of jute (a) and eggshell (b). The new characteristic peaks fhanges were found for the eggshell.

xidation of certain diols groups present in raw jute by an oxidizinggent, sodium meta-periodate [3,39]. Upon enzyme immobiliza-ion, a new aliphatic amine bond (C N) was identified at 1028/cm,hich indicates a covalent linkage between the aldehyde group on

he jute matrix and the amino group on the lipase, likely throughchiff base formation [3].

For eggshell, there was no change in the three prominent peakst 1417, 712 and 875/cm in the FTIR spectra before and after enzymemmobilization (Fig. 5b). These are the characteristic peaks foralcium carbonate, a major constituent of eggshell [36]. This find-ng indicated no covalent bonds between lipase and the eggshell

atrix, and hence, physical phenomenon such as adsorption mighte responsible for a non-covalent interaction between the enzymend matrix.

.7. Adsorption isotherm

The experimental and calculated values as well as the R2 valuebtained from the Langmuir adsorption isotherm equation arehown in Table 2. The data fit well with the isotherm model, ashown in Fig. 6. The � max value, 13.92 mg protein/g matrix, andl value, 0.382 mL/mg, were obtained for lipase immobilized onhe eggshell matrix. The Kl value was lower where the affinityetween the enzyme and matrix was higher. A similar Kl value0.35 mL/mg) was reported by Tsai et al. [36]. A 10.23 mg maxi-

um of protein was adsorbed/g of eggshell matrix, which is similaro the theoretical maximum value (13.92 mg/g). As the data fromhe adsorption experiment were adequately explained by the Lang-

uir isotherm and the FTIR spectra indicated an absence of covalentonds between the enzyme and matrix, it can be inferred that lipaseas immobilized on eggshell through physical adsorption.

.8. Nature of adsorption

As evident from Fig. 7, the lipase adsorbed on eggshell retainedower activity when it was incubated with NaCl compared withween 80. The effect of salt (NaCl) was more prominent than the

on-ionic detergent (Tween 80), which indicates more ionic thanydrophobic adsorption. The percent relative activity decreased to5 and 21% with the addition of 0.5 and 1 N NaCl, respectively,hereas the activity was reduced by 30 and 60% after treatment

able 2onstants of Langmuir adsorption isotherm.

Constants Values

� max (mg/g) 13.92Kl (mL/mg) 0.382Adjusted R2 0.996

adsorbed/g of support (� , mg/g) for the eggshell matrix.

with 1 and 5% (v/v) Tween 80, respectively, (Fig. 7). The salt andTween compete with the enzyme and replace it on the supportsurface [40]. However, as activity reduction was observed in both

Fig. 7. The effects of NaCl and Tween 80 on lipase desorption from eggshellexpressed as a percentage of relative activity. The conditions included pH 7 and37 ◦C.

754 S. Chattopadhyay, R. Sen / Process Biochemistry 47 (2012) 749–757

Fig. 8. The effects of various parameters on lipase adsorption on eggshell. The conditions are as follows: (i) for the different buffer concentration experiments: pH 7, 37 ◦C andno Tween 80; (ii) for the pH experiments: 37 ◦C, a 50 mM buffer concentration and no Tween 80; (iii) for the temperature experiments: pH 7, a 50 mM buffer concentrationand no Tween 80; (iv) for the variations in Tween 80 concentration: pH 7, 37 ◦C and a 50 mM buffer concentration.

Fe

ig. 9. A scanning electron micrograph of the eggshell matrix (a) and jute fibers (b). ESnzyme immobilized on jute.

: eggshell, E: enzyme immobilized on eggshell, J: chemically treated jute and JE:

ess Biochemistry 47 (2012) 749–757 755

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.9. Effects of various parameter on lipase adsorption

Among the four different parameters, an effect of pH andemperature was not prominent except under certain extreme con-itions, such as pH 8.5 and 50 ◦C (Fig. 8). The maximum enzymectivity was observed over a pH range from 7 to 8 and temperaturerom 30 to 37 ◦C. Below and above the optimum pH values, ionicnteraction involved in adsorption may be responsible for reducednzyme activity. Under extreme temperature conditions, distortionf the enzyme conformation may be responsible for the low activity.or ionic strength, a small decrease in activity was observed, whichecame constant after a certain buffer concentration (100 mM). Asith the previous experiment, adsorption was partially ionic; theecrease in activity was typically from electrostatic interactionsetween the ions, which was saturated after a certain point [41].he effect from Tween 80 was more severe than the other parame-ers studied. With an increasing concentration of detergent, lipasectivity decreased rapidly and was approximately half after addi-ion of only 0.1% Tween 80 (Fig. 7). A similar decrease in adsorptionith increasing detergent concentration was reported in the lit-

rature [42]. This might be from competitive adsorption of theurfactant (through the hydrophobic portions) on eggshell, whichrevents lipase binding.

.10. SEM analysis

Scanning electron microscopy of the eggshell before and afternzyme immobilization is shown in Fig. 9a. SEM of the eggshellndicated a highly porous filamentous structure for the matrix withroteins attached, such as albumin, as was previously reported43]. The SEM image also indicates cavities between the proteinbers. It is difficult to visualize separate protein molecules in anEM image. However, a group of condensed spots was observedn the matrix networks and inside the cavity, which might be fromrotein molecules. The surface morphology changed upon immobi-

ization, and the cavities were occupied by the enzyme molecules,s demonstrated in Fig. 9a. After adsorption of textile dyes [36]nd oxalate oxidase [44] on eggshell, similar changes in surfacerchitecture were observed. A large number of irregular proteinlobules attached to eggshell surface strongly indicated that lipaseas successfully immobilized on the eggshell matrix.

The surface morphology of chemically treated jute fiber visu-lized from SEM before enzyme immobilization was smooth, andhe fibers were clearly visible (Fig. 9b). The chemical treatmentsnvolved in matrix preparation removed the impurities and intra-ellular matter to produce the smooth surface [45]. The changen surface morphology was observed after enzyme immobiliza-ion (Fig. 9b). The increase in surface roughness may be attributedo chemical modification, which was validated by FTIR data thatndicated a covalent interaction between the lipase and the jute.

.11. Reuse of immobilized lipase

Covalent immobilization enhanced the reuse capacity comparedith adsorption because of the strong interaction between the

nzyme and supports. Lipase immobilized on eggshell had reducedctivity after 15–16 cycles, whereas jute-immobilized lipase coulde reused for 20–22 cycles (Fig. 10). As adsorption does not involvehemical bonds between the lipase and eggshell, enzyme leach-ng resulted in lower activity with each reuse. Repeated use ofmmobilized lipase for up to 10–15 cycles has been reported in

he literature [46,47]. Although the jute matrix-immobilized sys-em could be reused for a greater number of cycles, the eggshelletained the initial activity of the immobilized lipase better thanid jute. Ta

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ig. 10. The reuse of lipase after immobilization on eggshell and jute. The data arexpressed as a percentage of relative activity over a number of cycles.

.12. Comparative estimate

The performances of jute and eggshell as matrices for lipasemmobilization are compared in Table 3. The comparative esti-

ate establishes that eggshell is a better matrix than jute for lipasemmobilization.

. Conclusion

The performances of two abundant, readily available and cheapiomaterials, jute and eggshell, were evaluated as matrices for

ipase immobilization. Whereas the enzyme was covalently linkedo the jute matrix, the lipase was immobilized by adsorption onggshell. The adsorption was mixed (both ionic and hydrophobic).he ionic strength of the buffer affected adsorption slightly, but theddition of surfactant severely curtailed lipase adsorption. Morenzyme was loaded on the eggshell, and the activity after immobi-ization was higher compared with jute. Subsequent optimizationtudies demonstrated that enzyme stability was enhanced for themmobilized lipase on both surfaces, and the activity was approxi-

ately constant over the pH range 7–8 for both matrices as well ashe temperature ranges 30–40 ◦C for eggshell and 37–40 ◦C for theute matrix. A 2 h incubation time for immobilization was optimumor maximum enzyme activity on the eggshell matrix. The jute-mmobilized lipase can be reused for 20–22 cycles, whereas theggshell-immobilized lipase could be reused for 15–16 cycles. Fromhe performance evaluation and comparison of jute and eggshell, itan be concluded that eggshell is a more effective matrix for lipasemmobilization than is jute, and these studies may facilitate large-cale use of eggshell immobilized lipase for biodiesel production asell as additional commercially important biotransformations.

cknowledgments

The authors acknowledge Ms. Asmita Mukerji, Ms. Sancharinias and Dr. Ramapati Samanta for their help in the jute studies, andC is thankful to the University Grant Commission (Government ofndia) for the financial support. We would like to acknowledge Dr.soke Deysarkar (PfP Technology LLC., Houston, Texas, USA) andfP (India) Ltd., Mumbai for their financial and moral support. We

re grateful to Prof. Amit Patra, Dean (Alumni affairs & Internationalelations) IIT Kharagpur for his constant encouragement and valu-ble suggestions. The authors are thankful to the central researchacility, IIT Kharagpur, for the instrumentation facilities.

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chemistry 47 (2012) 749–757

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