Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on...

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 80:198–205 (2005)DOI: 10.1002/jctb.1179

Simultaneous purification and immobilizationof bitter gourd (Momordica charantia)peroxidases on bioaffinity supportSuhail Akhtar, Amjad Ali Khan and Qayyum Husain∗Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh-202002, UP, India

Abstract: This paper demonstrates the construction of an inexpensive bioaffinity adsorbent by simplyincubating Sephadex G 50 matrix with jack bean meal extract at room temperature. Sephadex G 50adsorbed 17 mg Con A (concanavalin A) per g of the matrix. Con A-adsorbed Sephadex was employedfor the immobilization of glycoenzymes directly from ammonium sulfate-fractionated proteins of bittergourd. The obtained bioaffinity support was very efficient for high yield immobilization of peroxidasesfrom bitter gourd and it bound nearly 425 enzyme units per g of the matrix. Bitter gourd peroxidaseimmobilized on lectin–Sephadex support showed a very high effectiveness factor, ‘η,’ of 1.25. ImmobilizedBGP preparation was quite stable against the denaturation induced by pH, heat, urea, Triton X 100, Tween20, SDS, Surf Excel and water-miscible organic solvents: dimethyl sulfoxide and dimethyl formamide.Low concentration of detergents like SDS, Tween 20, and Triton X 100 enhanced the activity of solubleand immobilized bitter gourd peroxidase. Peroxidase bound to the bioaffinity support exhibited very highresistance to proteolysis caused by the trypsin treatment. Con A–Sephadex-bound bitter gourd peroxidaseretained 85% of its initial activity after treatment with 2.5 mg trypsin per cm3 of incubation mixture for 1 hat 37 ◦C while the soluble enzyme lost nearly 40% of the initial activity under similar incubation conditions.Immobilized bitter gourd peroxidase preparation appeared to be more rigid to proteolysis mediated bytrypsin compared with soluble bitter gourd peroxidase. 2004 Society of Chemical Industry

Keywords: bioaffinity support; bitter gourd; immobilization; peroxidase; phenol removal; remediation;stabilization; wastewater

NOTATIONBGP bitter gourd peroxidaseCon A concanavalin ADMSO dimethyl sulfoxideDMF dimethyl formamideSDS sodium dodecyl sulfate

INTRODUCTIONPeroxidases (EC 1.11.1.7) are hemeproteins whichcatalyze the oxidation of a large number of aro-matic compounds at the expense of H2O2.1 Per-oxidases have a number of applications in clin-ical/chemical/environmental analysis, bio-bleachingprocesses, fuel and chemical production from woodpulp or in the production of dimeric alkaloids, oxi-dation/biotransformation of organic compounds andsynthesis of phenolic resins.2–5 It has been reportedthat peroxidases can be used in the detoxification

of various phenols and aromatic amines and thedecolorization and detoxification of dyes present inpolluted wastewater/industrial effluents from severaltextile dyes, and the paper and pulp and otherindustries.6–9 This great diversity in applications is dueto a wide substrate specificity of peroxidase catalysis.10

The use of soluble enzyme has some inherent demer-its whereas the immobilized form of the enzyme hasseveral advantages, such as enhanced stability, eas-ier product recovery and purification, protection ofenzymes against denaturants, proteolysis and reducedsusceptibility to contamination. A large number ofmethods have been employed for the immobilizationof peroxidases from various sources but most of theimmobilized enzyme preparations either use commer-cially available enzyme or expensive supports whichadds more cost to the system.7–11 Such immobilizedsystems cannot fulfil the requirement for the treat-ment of huge volumes of industrial effluents containing

∗ Correspondence to: Qayyum Husain, Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh-202002,UP, IndiaE-mail: [email protected]/grant sponsor: Council of Scientific and Industrial Research, New Delhi, IndiaContract/grant sponsor: University Grants CommissionContract/grant sponsor: DST, New Delhi, India(Received 13 April 2004; revised version received 24 August 2004; accepted 26 August 2004)Published online 8 December 2004

2004 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2004/$30.00 198

Bioaffinity-based immobilization/stabilization of bitter gourd peroxidases

various hazardous compounds. However, among thetechniques used for the immobilization of enzymes,bioaffinity-based supports have several merits overthe other known method. There has been remark-able interest in the bioaffinity-based procedures ofenzyme immobilization.12–14 Ease of immobilization,lack of chemical modification and usually accompany-ing enhancement in stability are some of the advan-tages offered by the bioaffinity-based procedures,which also offer the additional benefit of orienting theenzyme favorably on the support.15,16 These supportshave been used for the high yield and stable immo-bilization of glycoenzymes/enzymes. Bioaffinity-basedprocedures can be employed for the immobilization ofenzymes directly from the crude homogenate or par-tially purified enzyme preparation.12,14 Horseradishroot is the only traditional source for the commercialproduction of peroxidases but it is cultivated in rel-atively cool climates and not in most parts of India.Therefore efforts are being made to search for newsources of peroxidases. Several new sources have beendiscovered but none of them has been exploited forthe large-scale purification of the enzyme.6,7

In this study an attempt has been made toinvestigate a new source of peroxidase, vegetable bittergourd. Our preliminary studies demonstrated veryhigh peroxidase activity in the ammonium sulfate-fractionated bitter gourd proteins. The purpose ofthis study was to find a cheaper and easily availablealternative for the commercially available enzymesand its immobilization and utilization on a largescale. Peroxidases from bitter gourd are glycosylatedand this was observed by precipitating bitter gourdproteins with Con A. Con A-precipitated proteins ofbitter gourd exhibited very high peroxidase activity.Con A-adsorbed Sephadex support was obtainedby simply incubating the Sephadex with jack beanextract at room temperature and this Con A-adsorbedSephadex support was used for the immobilization ofperoxidases from the ammonium sulfate-fractionatedproteins of bitter gourd. Bioaffinity-based immobilizedenzyme preparation was compared with its solublecounterpart for its stability against various physical andchemical parameters. Immobilized BGP preparationwas significantly stable against pH, heat, urea,detergents, proteolysis and water-miscible organicsolvents.

MATERIALS AND METHODSMaterialsSephadex G 50 and methyl α-D-mannopyranosidewere the products of Amersham Biosciences Uppsala,Sweden and Sigma Chem Co, USA, respectively.Jack bean meal was procured from DIFCO, Detroit,USA. o-Dianisidine–HCl was obtained from theCenter for Biochemical Technology, New Delhi,India. Sodium dodecyl sulfate, dimethyl sulfoxideand dimethyl formamide were obtained from SRLChemicals, Mumbai, India. Surf Excel was purchased

from the local market. Other chemicals and reagentsemployed were of analytical grade and were usedwithout any further purification.

Ammonium sulfate fractionation of bitter gourdproteinsBitter gourd (50 g) was homogenized in 100 cm3 of0.1 M sodium acetate buffer, pH 5.6. Homogenatewas filtered through four layers of cheesecloth. Thefiltrate was then centrifuged at 10 000 × g on a RemiR-24 cooling centrifuge. The clear solution thusobtained was subjected to salt fractionation by adding20–80% (w/v) (NH4)2SO4. It was stirred overnight at4 ◦C to obtain the maximum amount of precipitate.The precipitate was collected by centrifugation at10 000 × g on a Remi R-24 cooling centrifuge. Theobtained precipitate was redissolved in 0.1 M sodiumacetate buffer, pH 5.6, and dialyzed against the samebuffer.17 Specific activity of the ammonium sulfate-fractionated BGP was 10.29-fold higher than the crudepreparation.

Preparation of bioaffinity supportSephadex (5.0 g) was incubated and stirred with100 cm3 clear solution of jack bean meal extractprepared in 0.1 M sodium phosphate buffer, pH 6.2,overnight at 4 ◦C. Unbound proteins were removed byextensive washing with the assay buffer.18 The specificbinding of Con A with Sephadex was confirmed byeluting the bound lectin using 1.0 M methyl α-D-mannopyranoside.

Immobilization of BGP on Con A–SephadexsupportBGP (5733 enzyme units) was added to 5.0 g of ConA–Sephadex support and stirred in sodium phosphatebuffer, pH 6.2, at 4 ◦C overnight. Unbound BGP wasremoved by extensive washing with the assay buffer.18

Effect of trypsin-mediated proteolysis on theactivity of soluble and immobilized BGPSoluble and immobilized BGP preparations (1.33EU) were incubated with 0.25–2.5 mg trypsin cm−3

of incubation mixture at 37 ◦C for 1 h. Both BGPpreparations were also incubated with 0.5 mg trypsincm−3 solution at 37 ◦C for up to 4 h to monitor theactivation of BGP mediated by trypsin.

Treatment of soluble and immobilized BGP withdetergentsSDS (0.1–1.0% w/v) and 0.5–5.0% (v/v) of TritonX 100 and Tween 20 were used as final assayconcentration to observe the effect of detergentson the activity of BGP. Soluble and immobilizedenzyme preparations (1.33 EU) were incubated withthe detergents in 50 mM sodium acetate buffer, pH5.6, at 37 ◦C for 1 h.

J Chem Technol Biotechnol 80:198–205 (2005) 199

S Akhtar, AA Khan, Q Husain

Incubation of soluble and immobilized BGP withwater-miscible organic solventsSoluble and immobilized BGP preparations (1.33EU) were incubated with 10–60% (v/v) of water-miscible organic solvents, DMSO/DMF, prepared in0.1 M sodium acetate buffer, pH 5.6, at 37 ◦C for 1 h.Peroxidase activity was determined at all the indicatedorganic solvent concentrations after the incubationperiod.19 Other assay conditions were the same asdescribed in the text.

Measurement of peroxidase activityPeroxidase activity was estimated from the change inthe optical density (A460 nm) at 37 ◦C by measuringthe initial rate of oxidation of o-dianisidine–HCl byhydrogen peroxide using the two substrates in saturat-ing concentrations. The immobilized preparation wascontinuously agitated for the entire duration of assay.The assay was highly reproducible with immobilizedpreparations.20

One unit of peroxidase activity was defined as theamount of enzyme protein that catalyzes the oxidationof 1 µmol of o-dianisidine–HCl per min at 37 ◦C.

Determination of protein concentrationThe protein concentration was determined by theprocedure of Lowry et al.21 Bovine serum albuminwas used as standard.

RESULTSIt is well recognized that Sephadex is used for thepurification of Con A and other similar lectins. Inview of its property to adsorb Con A specifically,this property has been exploited for preparing ConA–Sephadex adsorbent directly from jack bean meal.Con A–Sephadex was obtained by simply incubatingSephadex G 50 with 10% jack bean meal extractovernight at 4 ◦C. After binding of Con A onSephadex, the complex was washed with assaybuffer till the traces of unbound proteins wereremoved. Sephadex adsorbed nearly 17 mg proteinper g of gel from the solution. Con A-boundSephadex was selected as a bioaffinity medium for theimmobilization of glycoenzymes. The precipitation ofbitter gourd glycoproteins was achieved by incubatingthe ammonium sulfate-fractionated proteins with ConA extract. The obtained precipitate expressed veryhigh peroxidase activity. These results suggestedthat peroxidases from bitter gourd are glycosylated.Moreover, our group has also purified peroxidasesfrom bitter gourd by using Con A–Sepharosesupport to 100-fold purity as compared with crudehomogenate (data not shown). It further confirmsthe glycosylated nature of bitter gourd peroxidases.In view of the glycoproteinic nature of bitter gourdperoxidases, these could be directly immobilized onCon A–Sephadex support from ammonium sulfate-fractionated proteins or from the crude homogenateof bitter gourd. Unbound proteins were removed by

extensive washing with assay buffer. Con A–Sephadexadsorbed 425 enzyme units of peroxidase per gof the matrix. The effectiveness factor ‘η’ of theimmobilized enzyme preparation was 1.25. Thisvery high effectiveness factor of immobilized bittergourd peroxidases suggested that the immobilizedpreparation was quite porous and effective in catalysis.

The stability of soluble and Con A–Sephadex-bound BGP preparations was monitored against var-ious physical and chemical parameters because theseparameters can affect the activity of the enzymesused for the treatment of organic pollutants presentin the wastewater. Con A–Sephadex-bound BGPshowed broadening in the pH–activity profile as com-pared with the native enzyme (Fig 1). Immobilizedenzyme retained significantly higher enzyme activ-ity on both sides of the pH-optimum in comparisonto free enzyme. The pH-optimum of immobilizedenzyme remained unchanged from pH 5.0 to 6.0,although soluble enzyme showed a pH-optimum atpH 5.0. In an earlier study immobilized preparationsof glycoenzymes on lectin supports have also showna similar type of broadening in pH–activity profiles.12

Bioaffinity-bound BGP exhibited a marginal broaden-ing in temperature–activity profile. Soluble and ConA–Sephadex-bound BGP preparations exhibited thesame temperature optima of 40 ◦C. However, ConA–Sephadex-bound BGP retained greater fractionsof activity on both sides of the temperature-optimacompared with soluble enzyme (Fig 2).

As shown in Fig 3, soluble BGP incubated at 60 ◦Cfor 2 h retained half of its initial enzyme activitywhile the immobilized enzyme incubated under similarconditions was significantly more stable to heatinactivation. The immobilized BGP exhibited 85%of the original activity after 2 h of heat treatment.Con A–Sephadex-bound BGP was more resistant toinactivation induced by 4.0 M urea compared with its

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Figure 1. pH–activity profile of the soluble and immobilized BGP.The appropriate amount of soluble and immobilized BGP was takenfor the preparation of pH–activity profile. The reaction mixture wasincubated at 37 ◦C for 15 min in buffers of the pH range from 3.0 to9.0. The buffers used were 100 mM glycine–HCl for pH 3.0, sodiumacetate for pH 4.0, 5.0, sodium phosphate for pH 6.0, 7.0, 8.0 andTris–HCl for pH 9.0. All the results are the mean of triplicate samples(average standard deviation <5%).

200 J Chem Technol Biotechnol 80:198–205 (2005)


Temperature (°C)

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Figure 2. Temperature–activity profiles of soluble and immobilizedBGP. The activity of appropriate amounts of soluble and immobilizedBGP was monitored at various indicated temperatures. Activityexpressed at 40 ◦C was taken as control for calculating percentactivity. All the results are the mean of triplicate samples (averagestandard deviation <5%).

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Figure 3. Thermal denaturation of soluble and ConA–Sephadex-bound BGP. Soluble and immobilized BGP (1.33 units)were incubated at 60 ◦C for varying time intervals in 100 mM sodiumacetate buffer, pH 5.6. Aliquots of each preparation were taken out atindicated time intervals and enzyme activity was determined.Un-incubated samples were taken as 100% for the calculation ofremaining percent activity. All the results are the mean of triplicatesamples (average standard deviation <5%).

soluble counterpart. Exposure of soluble enzyme with4.0 M urea for 2 h resulted in the loss of 52% activitywhereas the immobilized enzyme retained more than80% of the initial enzyme activity (Fig 4).

Figure 5 shows the stability of soluble and immobi-lized BGP in the presence of 0.25–2.5 mg trypsin cm−3

of incubation mixture. Soluble BGP was rapidly inac-tivated in the presence of increasing concentrations oftrypsin and retained 60% of the initial activity after 1 hincubation with 2.5 mg trypsin cm−3 at 37 ◦C whilethe immobilized BGP was remarkably more stableagainst proteolysis mediated by trypsin. ImmobilizedBGP expressed over 85% of the initial activity withsimilar treatment. The soluble and immobilized BGPwere activated at low concentrations of trypsin as com-pared with the trypsin-untreated preparations of BGP

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Figure 4. Effect of 4.0 M urea on soluble and ConA–Sephadex-bound BGP. Soluble and immobilized BGPpreparations (1.33 units) were incubated in 4.0 M urea in 100 mM

sodium acetate buffer, pH 5.6. Enzyme activity was determined atdifferent time intervals. For calculating the percent activity untreatedsamples were considered as 100%. All the results are the mean oftriplicate samples (average standard deviation <5%).

Trypsin (mg cm-3)

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

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Figure 5. Effect of trypsin concentration on the activity of soluble andimmobilized BGP. Soluble and Con A–Sephadex-bound BGPpreparations (1.33 units) were independently incubated withincreasing concentration of trypsin (0.25–2.5 mg) in a total volume of1.0 cm3 of 100 mM sodium acetate buffer, pH 5.6, at 37 ◦C for 1 h. Allthe results are the mean of triplicate samples (average standarddeviation <5%).

(Fig 5). The highest activation of soluble and immo-bilized BGP was observed at 0.5 mg trypsin cm−3 and0.75 mg trypsin cm−3 of incubation mixture, respec-tively. In order to investigate the time-dependentactivation of soluble and immobilized BGP, boththe preparations were incubated with 0.5 mg trypsincm−3 of incubation mixture for different times. Theactivity of soluble enzyme was enhanced up to 120%after 90 min incubation at 37 ◦C while the activity ofimmobilized BGP was increased up to 140% after 2 hincubation at 37 ◦C with trypsin (Fig 6).

Wastewater coming out from various eliminationsites contains several types of denaturants includingdetergents, which can strongly denature the enzymesused for the treatment of polluted wastewater. Inorder to use such enzymes for the removal of



Time (min)

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Figure 6. Effect of fixed concentration of trypsin on the activity ofsoluble and immobilized BGP for different time intervals. Soluble andCon A–Sephadex-bound BGP preparations (1.33 units) wereindependently incubated with 0.5 mg trypsin in a total volume of1.0 cm3 of 100 mM sodium acetate buffer, pH 5.6, at 37 ◦C for differenttime intervals. All the results are the mean of triplicate samples(average standard deviation <5%).

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Figure 7. Activation of soluble and immobilized BGP activity by SDS.Soluble and immobilized BGP preparations (1.33 units) wereincubated with increasing concentration of SDS (25–500 µM)prepared in 100 mM sodium acetate buffer, pH 5.6, at 37 ◦C for 1 h. Allthe results are the mean of triplicate samples (average standarddeviation <5%).

aromatic pollutants from wastewater it becomesnecessary to monitor the stability of enzymes in thepresence of denaturants. In this study four differentdetergents have been selected for comparative stabilityof soluble and immobilized BGP. Both soluble andimmobilized enzyme preparations were activated bylow concentrations of SDS, however, the immobilizedBGP achieved further activation at higher levels ofdetergent (Fig 7). This experiment indicated thatthe presence of lower concentrations of SDS isnot harmful to the enzyme structure and enzymescan work more efficiently on the industrial effluentscontaining compounds such as soap and detergents.Lower concentrations of Triton X 100 and Tween20 also activated soluble and immobilized BGP andin this case the extent of activation was much higher(data not shown). In order to investigate the effectof higher concentrations of detergents on the activityof BGP, soluble and immobilized BGP preparations

were incubated with 0.1–1.0% (w/v) SDS and 0.5–5%(v/v) of Triton X 100 and Tween 20 for 1 h at 37 ◦C.Pre-incubation of soluble and immobilized enzymepreparations with 1% (w/v) SDS for 1 h resulted in theloss of 58% and 19% of the original enzyme activity,respectively (Fig 8). Soluble and Con A–Sephadex-bound BGP preparations were treated with increasingconcentration of Triton X 100 and Tween 20 for 1 h at37 ◦C. Immobilized BGP retained very high activity inthe presence of 5.0% (v/v) Triton X 100 and Tween 20while soluble enzyme rapidly lost its activity (Table 1).

Surf Excel is a very common detergent used inhouseholds and laundry. Unused detergent is normallypresent in the wastewater coming out of municipal

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Figure 8. Inactivation effect of SDS on the activity of soluble andimmobilized BGP. Soluble and immobilized BGP preparations(1.33 units) were incubated with (0.1–1.0% w/v) SDS prepared in100 mM sodium acetate buffer, pH 5.6, at 37 ◦C for 1 h. Enzymeactivity was determined after the incubation period. All the results arethe mean of triplicate samples (average standard deviation <5%).

Table 1. Effect of Tween 20 and Triton X 100 on soluble and

immobilized BGP

Percent remaining BGP activity

Detergent

concentrationTween 20 Triton X 100

(%, v/v) Soluble Immobilized Soluble Immobilized

0.5 40 98 61 1001.0 38 98 58 991.5 37 96 50 992.0 36 93 51 942.5 35 90 49 933.0 33 88 48 903.5 31 85 46 884.0 29 79 45 864.5 27 77 44 855.0 28 75 43 81

Soluble and immobilized BGP preparations (1.33 units) were incubatedwith Tween 20/Triton X 100 [0.5–5.0% (v/v) prepared in 50 mM sodiumacetate buffer, pH 5.6 at 37 ◦C for 1 h].Peroxidase activity was assayed at all the indicated detergentsconcentrations and other assay conditions were the same asmentioned in the text.The activities of soluble and immobilized BGP in assay buffer withoutany detergent were taken as control (100%) for the calculation ofpercent activity.Each value represents the mean for three-independent experimentsperformed in duplicate, with average standard deviation <5%.



% Surf Excel (w/v)

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Figure 9. Effect of Surf Excel on the activity of soluble and ConA–Sephadex-bound BGP. Soluble and immobilized BGPpreparations (1.33 units) were incubated with varying concentrationsof Surf Excel (0.1–1.0% w/v) in 100 mM sodium acetate buffer, pH5.6, at 37 ◦C for 1 h. All the results are the mean of triplicate samples(average standard deviation <5%).

waste. This wastewater is somewhere mixed with theeffluents released by industries. In order to make theimmobilized preparation more efficient for wastewatertreatment, we have investigated the effect of Surf Excelon the activity of immobilized BGP. Soluble BGP wasmore sensitive to the Surf Excel exposure and lostnearly 90% enzyme activity after 1 h incubation with1% (w/v) detergent. However, the immobilized BGPwas markedly more resistant to inactivation inducedby Surf Excel and retained over 76% of the initialactivity (Fig 9).

Industrial effluents and municipal wastewatercontain organic solvents together with other aromaticpollutants. Therefore it is of utmost importance toinvestigate the role of some water-miscible organicsolvents on the activity of immobilized enzymes.The exposure of soluble enzyme with varyingconcentrations of DMSO (10–60% v/v) resulted in theloss of a greater fraction of enzyme activity while theimmobilized enzyme was quite resistant to inactivationinduced by DMSO. Exposure of immobilized enzymepreparation with 50% (v/v) DMSO for 1 h hadnegligible effect on its activity, although the solubleenzyme lost nearly 33% of its original activity withsimilar treatment (Table 2). The incubation of solubleand immobilized BGP with increasing concentrationof DMF resulted in continuous loss of enzymeactivity. However, the immobilized enzyme was moreresistant to inactivation mediated by DMF. Thetreatment of soluble BGP with 60% (v/v) DMF for1 h resulted in the loss of 45% of the initial activitywhile the Con A–Sephadex-bound BGP exhibitedpronouncedly very high stabilization against similartreatment and retained more than 85% of its originalactivity (Table 2).

DISCUSSIONA large number of methods are employed for theimmobilization/stabilization of enzymes but very few

Table 2. Effect of DMF and DMSO on the activity of soluble and

immobilized BGP

Percent remaining BGP activity

DMF DMSOOrganic solvent

(% v/v) Soluble Immobilized Soluble Immobilized

10 96 98 95 9820 87 95 90 9530 78 91 86 9440 72 88 77 9250 57 87 67 9060 55 86 58 90

Soluble and immobilized BGP preparations (1.33 units) were incubatedwith increasing concentration of DMSO/DMF (10–60% v/v) in 50 mM

sodium acetate buffer, pH 5.6 at 37 ◦C for 1 h.Peroxidase activity was assayed at all the indicated organic solventconcentrations and other assay conditions were same as mentionedin the text.The activities of soluble and immobilized BGP in assay buffer withoutany organic solvent were taken as control (100%) for the calculationof percent activity.Each value represents the mean for three-independent experimentsperformed in duplicate, with average standard deviation <5%.

can meet the requirement of enzyme immobilizationdirectly from the crude homogenate. In this work aneffort has been made to immobilize the BGP directlyfrom the ammonium sulfate-fractionated proteinsof bitter gourd on Con A–Sephadex. It is welldocumented that Sephadex can be used for the affinity-based purification of Con A from jack bean mealextract.12 Therefore, this property has been exploitedfor the preparation of bioaffinity media for theimmobilization of BGP directly from the ammoniumsulfate-fractionated bitter gourd proteins. BGP wasimmobilized in very high yield on Con A–Sephadexand it bound 425 EU of BGP g−1 of the adsorbent.The immobilization yield was quite superior over othermethods used for the immobilization of peroxidases.22

BGP bound to Con A–Sephadex support exhibitedvery high stabilization against pH, heat and ureadenaturation (Figs 1–4). Several earlier reports arealso available on the use of Con A support for highyield immobilization of glycoenzymes.12,14 Bioaffinity-bound BGP was remarkably stable against proteolysismediated by trypsin (Fig 5). In another study, ourlaboratory has explained the stabilization of ConA–Sephadex-bound glucose oxidase against trypsintreatment.18

Lower concentrations of SDS stimulated the solubleand immobilized BGP. This experiment indicatedthat the presence of lower concentrations of SDS isnot harmful to the enzyme function. Such enzymescan work more efficiently on industrial effluentscontaining compounds such as soap and detergents.Con A–Sephadex-bound BGP was quite resistantagainst denaturation induced by detergents such asTriton X 100, Tween 20 (Table 1), SDS and SurfExcel (Figs 8 and 9). Numerous detergents normallyflow in the municipal wastewater and these can affect



the activity of enzymes. Therefore, it is necessaryto know about the effect of detergents on theenzyme activity prior to their use in the removalof organic compounds present in the wastewater.These observations suggest that Con A–Sephadex-bound BGP preparation was remarkably more stableagainst the exposure caused by very high concentrationof several detergents. Potential applications of thisenzyme can be made for the treatment of wastewatercontaining hazardous aromatic pollutants.

Organic solvents are also very common pollutantsalong with aromatic compounds and their presencecan influence the structure of enzymes. Enzymesemployed for the treatment of wastewater containingpollutants would be affected by the presence of suchsolvents. In view of their presence in wastewaterit becomes necessary to investigate the stabilityof enzymes in the presence of these solvents.The Con A–Sephadex-bound BGP was markedlymore stable against the exposure to DMSO andDMF (Table 2). There have been reports thatimmobilization of enzymes by multipoint attachmentprotects them from denaturation by organic solventsin cosolvent mixtures.23–25 These workers havedescribed that potato polyphenol oxidase adsorbedon chitin behaved differently compared with solubleenzyme in aqueous–organic cosolvent mixtures.Moreover, they have evaluated that the enzymespolyphenol oxidase, peroxidase, trypsin and acidphosphatase showed stimulation of enzyme activitywithin a specific concentration range of water-miscibleorganic solvent present in the medium.26 Enzymeimmobilized by adsorption on Eudragit S-100, chitinand chitosan exhibited enhanced activity in organiccosolvent mixtures when the concentration of theorganic solvent was around 10–20% (v/v).27 Morerecently in our laboratory it has been shown thatenzymes immobilized on protein supports were alsoquite resistant to denaturation induced by variouswater-miscible organic solvents.19,28

The Con A–Sephadex-bound BGP preparation haspronounced stability against pH, heat, urea, proteol-ysis, detergents and water-miscible organic solvents.Several earlier reports described that the immobiliza-tion of glycoenzymes on Con A support resulted inthe stabilization of enzymes against various forms ofdenaturation.12,16 Protease resistance is an additionalattribute of the bioaffinity-based immobilization ofBGP. It indicated that Con A–Sephadex-bound BGPpreparation has great potential in the treatment oforganic pollutants present in industrial effluents. ConA–Sephadex-adsorbed enzyme has only non-covalentforces between the support and enzyme molecules andsometimes it leads to the desorption of enzyme orCon A or both from the support. Cross-linking ofbioaffinity-adsorbed enzyme could be done by usingbifunctional or multifunctional reagents to prevent thedissociation/desorption of enzyme or Con A–enzymecomplex from the Sephadex G 50 support.29

CONCLUSIONBioaffinity supports are significantly useful inthe immobilization of enzymes directly from thecrude homogenate, thus avoiding the high cost ofenzyme purification.12,14 Peroxidase-based removal/detoxification of phenols and aromatic amines hasbeen a subject of considerable interest during thepast two decades. However, practical applications oflarge-scale enzymatic removal have always been lim-ited due to high cost and lower operational stabilityof peroxidases.1,4 The method of enzyme immobiliza-tion developed in this work has an advantage becausethere is no need for the use of commercially availablelectin for the preparation of the bioaffinity support.Moreover, this procedure emphasized the immobiliza-tion of BGP directly from the crude homogenate orammonium sulfate-precipitated proteins. It has furtherreduced the cost of the immobilized enzyme prepara-tion. BGP adsorbed on Con A support showed highyield of immobilization and markedly high stabilizationagainst several types of denaturants. In the near future,enzyme reactors containing such inexpensive immobi-lized preparations can be exploited for the treatmentof wastewater containing toxic and hazardous com-pounds.

ACKNOWLEDGEMENTSThe Council of Scientific and Industrial Research,New Delhi, India is gratefully acknowledged for assis-tance in the form of project entitled ‘Decolorizationand detoxification of textile and some other indus-trially important dyes from the wastewater by usingimmobilized polyphenoloxidases’. The authors arealso thankful to the University Grants Commissionand DST, New Delhi, India for providing specialgrants to the Department in the form of DRS andFIST, respectively, for developing infrastructure facil-ities.

REFERENCES1 Ghioureliotis M and Nicell JA, Assessment of soluble products

of peroxidase-catalyzed polymerization of aqueous phenol.Enzyme Microb Technol 25:185–193 (1999).

2 Ryu K, McEldon JM, Pokora AR, Cyrus W and Dordick JS,Numerical and Monte-Carlo simulation of phenolic polymer-ization catalyzed by peroxidase. Biotechnol Bioeng 42:807–814(1993).

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