Protein-Coated Polymer as a Matrix for Enzyme Immobilization: Immobilization

of Trypsin on Bovine Serum Albumin-Coated Allyl Glycidyl Ether–Ethylene

Glycol Dimethacrylate Copolymer

Lakshmi Swarnalatha Jasti, Sandhya Rani Dola, Thenkrishnan Kumaraguru,Sreedhar Bajja, and Nitin W. FadnavisIndian Inst. of Chemical Technology, Uppal Road, Hyderabad, India

Uma AddepallyCentre for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, India

Kishor Rajdeo, Surendra Ponrathnam, and Sarika DeokarPolymer Science & Engineering, Chemical Engineering Division, National Chemical Laboratory, Pashan Road, Pune, India

DOI 10.1002/btpr.1871Published online January 22, 2014 in Wiley Online Library (wileyonlinelibrary.com)

Allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer with 25%crosslink density (AGE-25) shows excellent bovine serum albumin (BSA) adsorption (up to16% (w/w)) at pH 8.0 and the adsorbed BSA is strongly bound. This protein-coated polymerprovides a novel matrix with naturally existing functional groups such as thiol, amino, andcarboxylic acid that are available for covalent immobilization of functional enzymes.Employing appropriate strategies, trypsin as a model protein was covalently bound to BSA-coated matrix both independently, and in a stepwise manner on the same matrix, with lessthan 5% loss of enzyme activity during immobilization. Glutaraldehyde crosslinking afterimmobilization provide stable enzyme preparation with activity of 510 units/g recycled up tosix times without loss of enzyme activity. AFM studies reveal that the polymer surface hasprotein peaks and valleys rather than a uniform monolayer distribution of the protein andthe immobilized enzyme preparation can best be described as polymer supported cross-linked enzyme aggregates (CLEAs). VC 2014 American Institute of Chemical Engineers Bio-technol. Prog., 30:317–323, 2014Keywords: trypsin, bovine serum albumin, immobilization, allyl glycidyl ether, ethyleneglycol dimethacrylate

Introduction

Enzymes immobilized on solid supports have a wide rangeof applications ranging from production of pharmaceuticals1–

3 and diagnostic kits 4 to production of food supplementsand beverages.5 Several strategies are employed for immobi-lizing enzymes, which can vary from simple adsorption orentrapment to covalent bond formation with a polymericsupport bearing functional groups.6–10 A special technique ofcovalent crosslinking of enzymes through bifunctional cross-linker such as glutaraldehyde provides crosslinked enzymecrystals and crosslinked enzyme aggregates (CLEAs), whichare very useful in biotransformations.11 A survey of literatureshows that the most popular reactions for covalent couplingof a protein involve the amino, thiol, and carboxylic acidgroups, and depending on the choice of functional groupsavailable for reaction, well-defined strategies have beendeveloped for formation of the covalent bond between theenzyme and the support.12 In our earlier studies on bindingof a-chymotrypsin, trypsin, alcohol dehydrogenase, etc., on

allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate(EGDM) copolymers, we had observed that the epoxy-activated copolymer was able to bind as much as 25% (w/w)protein, but this was accompanied by a serious loss ofenzyme activity (>80%).13,14 We had to devise a strategy ofusing reverse micelles to overcome the problem ofadsorption-induced denaturation.15 During these studies, wealso observed that the adsorbed proteins were rather stronglybound to the polymer matrix and it was quite difficult towash out the adsorbed protein. This was quite interestingbecause the bound proteins possess all the functional groupsrequired for covalent bonding, especially the thiol, amino,and carboxylic acid, all in the same molecule. Indeed, onecan choose a functional group for binding of another enzymeon the protein-coated polymer surface, without going throughthe complicated synthesis of a specially designed functional-ized polymer matrix. In this article, we successfullydemonstrate this concept. After screening some of the com-mercially available epoxy-activated polymers, we selectedcopolymer of AGE–EGDM with 25% crosslink density(AGE-25) for coating with bovine serum albumin (BSA) bysimple adsorption at pH 8.0. BSA contains 60 Lys, 41 Asp,58 Glu, and 35 Cys residues16 and depending on

Correspondence concerning this article should be addressed to N.W.Fadnavis at fadnavisnw@yahoo.com; fadnavis@iict.res.in

VC 2014 American Institute of Chemical Engineers 317

accessibility functional groups such as ANH2, ASH, andACOOH on the protein-coated polymer surface were usedfor covalent binding of trypsin (EC 3.4.21.4) both independ-ently, and also on the same matrix using a serial couplingprotocol. Binding of trypsin to BSA, on the one hand,presents the worst-case scenario as trypsin is a proteindigester and likely to destroy polymer-bound BSA. On theother hand, immobilized trypsin is interesting from biotech-nological point of view. Production of protein hydrolysatewith low contents of aromatic amino acids from cheesewhey protein has been successfully carried out in the groupof Prof. Guisan using a sequential hydrolysis of proteins cat-alyzed by trypsin, chymotrypsin, and carboxypeptidase Aimmobilized on glyoxyl agarose beads.17,18

Although proteins such as BSA and ovalbumin are rou-tinely used as carrier proteins for coupling with peptides orother haptens,19 to the best of our knowledge, BSA-coatedpolymers have not been used to couple enzymes. We didcome across an interesting concept of BSA supported onaminated sepharose and coated with dextran for selectiveadsorption of small lactoglobulins from dairy whey,20 butthe support was not used for enzyme immobilization.

Materials and Methods

Materials

BSA (fraction V, Cat. No. a-7030), bovine trypsin (Cat.

No. C7762), Na-benzoyl-L-arginine ethyl ester (BAEE), Na-glu-

taryl-L-phenylalanine p-nitroanilide, 1-ethyl-3-[3-dimethylami-

nopropyl]carbodimide hydrochloride (EDC), N-hydroxysuccini

mide, dithiothreitol (DTT), and 4-morpholineethanesulfonic acid

sodium salt (MES) were obtained from Sigma-Aldrich, Banga-

lore, India. N,N0-Disuccinimidyl carbonate (DSC) was obtained

from Alfa Aesar, Hyderabad, India. All other reagents and sol-

vents were analytical grade obtained from Hi Media (Mumbai,

India). Epoxy-activated polymers such as Immobead IB-150,

Sepabeads SP-20SS, and SP-150 were obtained from Sigma.

Immobead IB-350 was obtained from Chiral Vision, Leiden the

Netherlands. DILBEADS TA, VWR, T2, and RG were a gift

from Fermenta Biotech Ltd., Thane, India (http://www.fermen-

tabiotech.com). The macroporous AGE–EGDM copolymer with

relative mole ratio of the cross-linking co-monomer (EGDM) to

epoxy functional monomer (AGE) of 0.25 (AGE-25) was syn-

thesized by suspension polymerization in a jacketed cylindrical

polymerization reactor at a constant volume of cyclohexanol as

porogen with monomer to porogen ratio of 1:1.66 (v/v).13 The

polymer beads with average particle size of 150–250 mm had

specific surface area (BET) of 250 m2/g, pore volume of 0.68

mL/g, and epoxy group content of 5 mmol/g.

UV-visible spectrophotometric measurements were per-

formed on Cary 100 UV-visible spectrophotometer equipped

with temperature control and Cary Win UV software. All

enzyme assays were performed at 30�C. All experiments

were repeated three times and were reproducible within

65%. Atomic force microscopy (AFM) was employed to

characterize the morphology of the polymer particles using

Digital Nanoscope IV (Veeco Instruments, Santa Barbara,

CA, USA). The microscope was vibration-damped. Commer-

cial phosphorous (n) doped silica tips on an I-tape cantilever

with a length of 115–135 mm and resonance frequency of

260 kHz were used. Analysis of experimental data was per-

formed using Graph Pad Prism 5 (www. graphpad.com).

Protein Adsorption Experiments

Prior to protein adsorption experiments, the polymer (500mg) was incubated with tert-BuOH (10 mL) for 2 h in aconical flask. The solvent was decanted and water (10 mL)was added to the polymer, shaken on an orbital shaker at150 rpm for 15 min, and supernatant was discarded. Thepolymer was then left soaking overnight in water (5 mL).The polymer was finally washed with buffer of required pHand freeze dried.

For BSA-binding studies, the buffer-washed and driedpolymer was (500 mg) shaken with BSA solution (5 mL) foron orbital shaker at 150 rpm for 10 h. The supernatant wascollected by centrifugation and the polymer was washedwith buffer (3 3 10 mL) till the supernatant was free of pro-tein. Protein content of the combined washings was deter-mined from its absorbance at 280 nm and also cross-checkedwith Bradford assay method using BSA as a standard. Thedifference in protein content of control and that of the super-natant gave the value for adsorbed protein. The polymer wasstored in refrigerator for further use.

Enzyme Immobilization on BSA-Coated Polymer

Before performing enzyme immobilization experiments,surface saturated BSA-coated polymer was prepared by stir-ring the polymer (500 mg) with BSA solution (5 mL, 25mg/mL, in 0.1 M sodium carbonate buffer pH 8.0) for 12 hin cold. The polymer was washed repeatedly with cold car-bonate buffer till the supernatant was free of protein, andfinally with distilled water. All enzyme-coupling reactionswere performed in a double-walled vessel (volume 25 mL)with cold water circulation (5�C) and a small mechanicalstirrer with Teflon blades.

Trypsin Immobilization via Amino Groups on BSA-CoatedPolymer (Method 1). BSA-coated polymer (500 mg) wasstirred with 4% glutaraldehyde solution in sodium carbonatebuffer (0.1 M, containing 0.1 M NaCl, pH 8.5, 4 mL) in aconical flask on an orbital shaker at 80 rpm for 2 h for acti-vation of the ANH2 groups at room temperature. The poly-mer was separated by centrifugation at 5000 rpm for 10 minand washed with sodium phosphate buffer (0.05 M, pH 8.0,3 3 10 mL). Trypsin solution (5 mg/mL, 5 mL in sodiumphosphate buffer 0.05 M, pH 8.0) was placed in the reactionvessel. BSA-coated polymer activated with glutaraldehydewas added and the contents were stirred slowly at 50 rpmfor 2 h. The polymer was then separated by centrifugationand washed with phosphate buffer (5 3 4 mL). Combinedwashings with supernatant were assayed for protein contentas well as residual enzyme activity. The immobilizedenzyme beads were freeze dried, assayed for enzyme activ-ity, and stored in refrigerator.

Trypsin Immobilization via ASH Groups on BSA-CoatedPolymer (Method 2). These reactions were performed inaqueous solutions, which were degassed and flushed withnitrogen before use and were performed under nitrogenatmosphere. BSA-coated polymer (500 mg) was shaken withDTT (10 mL, 10 mM in 0.1 M sodium acetate buffer con-taining 0.1 M NaCl, pH 4.8) for 2 h. The supernatant wasremoved by centrifugation and the polymer was washed withsodium acetate buffer (pH 7.9) till the washings were free ofthiol as assayed by Ellman’s reagent, 5,50-dithio bis(2-nitro-benzoic acid). The BSA-coated polymer with free thiolgroups was stirred with a solution of DSC (50 mL, 5 mg/mL

318 Biotechnol. Prog., 2014, Vol. 30, No. 2

in dimethylcarbonate) in cold for 2 h. The supernatant wassucked with a pipette, the beads were washed with colddimethylcarbonate (2 3 5 mL), and then with cold phos-phate buffer (sodium phosphate buffer 0.05 M, pH 8.0, 2 3

5 mL). The -SH-activated polymer was stirred with enzymesolution (5 mg/mL, 5 mL in sodium phosphate buffer0.05 M, pH 8.0) for 2 h and then centrifuged at 5000 rpmfor 10 min. The polymer was washed with phosphate buffer(3 3 5 mL), freeze dried, and stored in refrigerator.

Immobilization via ACOOH Groups of BSA-Coated Poly-mer (Method 3). BSA-coated polymer (500 mg) waswashed with MES buffer (0.1 M, pH 5.5, 2 3 5 mL) andstirred with a solution of EDC (1 g) in MES buffer (0.1 M,pH 5.5, 20 mL) for the activation of -COOH groups in coldfor 10 min at 80 rpm. The supernatant was decanted, washedonce with MES buffer (2 3 5 mL), and the activated poly-mer was stirred with enzyme solution (5 mg/mL, 5 mL insodium phosphate buffer 0.05 M, pH 8.0) for 2 h. The poly-mer beads were separated, washed with phosphate buffer(5 3 2 mL), freeze dried, and stored in refrigerator.

Enzyme Activity Measurements

Although trypsin activity is generally measured at 37�C,its stability at this temperature is low (loss of 50% activityin 1 h). In comparison, less than 10% of its activity is lost at30�C in 1 h; hence, all enzyme assays were performed in atemperature-controlled, double-walled vessel at 30�C. Duringimmobilization, the enzyme is primarily located on the sur-face of the polymer and external mass transfer limitations donot appear to influence activity measurements at least at stir-ring speeds above 80 rpm. Thus, the titrimetric assays wereperformed while stirring at 100 rpm with a small mechanicalstirrer. All experiments were repeated three times and werereproducible within 65%. The specific activity of enzymebound to the polymer (units/g) was calculated on the basisof enzyme activity observed for free enzyme (units/mg).

Trypsin Assay. Trypsin activity was measured using Na-benzoyl-L-arginine ethyl ester as substrate. The enzyme solu-tion (10–100 mL) or polymer-bearing immobilized enzyme(25 mg) was added to the substrate solution (10 mL, 10 mM

BAEE in 5 mM Tris-HCl buffer containing 10 mM CaCl2,pH 8.2) and stirred at 100 rpm. Acid produced during hydro-lysis was continuously titrated with 0.1 N NaOH maintainingthe pH at 8.2 for 10 min. Enzyme activity in units isexpressed in terms of mm of NaOH consumed in 1 min. Thenative enzyme showed activity of 60 units/mg.

Results and Discussion

Adsorption of BSA on AGE-25

Screening of Epoxy-Activated Polymers for BSA Binding.Several polymers bearing active epoxy groups are availablein market. To choose the appropriate polymer for our pur-pose, the efficiency of BSA adsorption from aqueous solu-tion was studied for several polymers under comparableconditions (Figure 1). We observed that the Sepabeads SP-20SS and AGE-25 adsorb comparable amounts of BSA, butthe Sepabeads were difficult to handle due to their lightnessand tendency to float in water. In comparison, AGE-25 beadsare heavier, have excellent swelling properties and very easyto handle,13 hence further studies were made with AGE-25copolymer.

Effect of Protein Concentration on Binding. Proteinbinding to the epoxy polymer was studied at pH 8.0 in aque-ous buffer at a fixed amount of polymer and varying BSAconcentration in the solution. It was observed that theamount of BSA bound to the polymer increased withincreasing amount of protein in the solution and reached asaturation level of 160 mg/g (Figure 2).

Such a behavior is routinely observed and is generallyanalyzed using a Langmuir adsorption isotherm or its modi-fied form.21 However, such an analysis has been questioned,as adsorption isotherms imply reversibility of binding and auniform distribution of the protein in a monolayer on thepolymer surface.22 In the present case, we find that BSA isirreversibly bound to the polymer. In addition, the atomicforce microscopic pictures of protein-bound polymer surfacereveal the presence of peaks and valleys indicating that theprotein is bound as small aggregates and not as a monolayer(Figure 3). We thus refrain from analyzing the protein-binding data in terms of adsorption isotherm. It was suffi-cient for our present purpose to observe that at saturation

Figure 1. Adsorption of BSA on various epoxy-functionalizedpolymers in 0.05 M phosphate buffer, pH 8.0, at30s�C. [BSA] 5 30 mg/mL. The polymers are desig-nated as (1) Immobead IB 150, (2) Immobead IB350, (3) Sepabeads SP-20SS, (4) Sepabeads SP-850,(5) DIL BEADS-TA, (6) DIL BEADS-VWR, (7) DILBEADS-T2, (8) DIL BEADS RG, (9) AGE-25.

Figure 2. Adsorption of BSA on AGE-EGDM copolymer AGE-25. [BSA] 5 1–10 mg/mL in 0.05 M phosphate buffer,pH 8.0. [Polymer] 5 25 mg. Residence time 12 h at30� C. Reaction volume 1 mL.

Biotechnol. Prog., 2014, Vol. 30, No. 2 319

level a high loading of BSA (16% w/w) was achieved andthe protein was strongly bound to the polymer.

Covalent Binding of Trypsin to BSA-Coated Polymer

Covalent Binding of Trypsin to BSA-Coated PolymerEmploying Different Methods. BSA contains 60 Lys, 41Asp, 58 Glu, and 35 Cys residues.16 Some of the functionalgroups such as free amino groups of lysine, ACOOH groupsof Asp and Glu, and ASH groups of cysteine may be avail-able for covalent coupling with another protein. To test this

hypothesis, we have carried out immobilization of trypsin onBSA-coated polymer in three different ways as shown inScheme 1.

The following example involving trypsin couplingdescribes our methodology. In our first approach (method 1),the protein-coated polymer (500 mg) was treated with glutar-aldehyde solution. This treatment provided a BSA-coatedpolymer matrix where the free amino groups of BSA wereactivated with an aldehyde function. This was then treatedwith enzyme solution (5 mg/mL, 5 mL, total activity 1500units). The amino groups on enzyme surface were

Figure 3. Atomic force microscopy images of AGE-25 polymer after various stages of BSA and trypsin binding. (a) Plain AGE-25polymer Surface; (b) AGE-25 after coating with BSA at pH 8.0. Peak breadth 180–230 nm and peak height 55 nm. Distancebetween two peaks 240 nm; (c) AGE-25 coated with BSA after crosslinked with glutaraldehyde showing only a small changein morphology; (d) Trypsin bound to AGE-25 coated with BSA at pH 8.0 through ANH2, ACOOH, and ASH functionalgroups of BSA and crosslinked with glutaraldehyde; Average peak breadth 700–900 nm; height 90 nm, peaks separated by700 nm; (e) Trypsin bound to uncoated polymer; (f) Trypsin bound to uncoated polymer after crosslinked withglutaraldehyde.

Scheme 1. A schematic representation of coupling of an enzyme to BSA-coated polymer through amino, carboxyl, and thiol groupspresent in BSA.

320 Biotechnol. Prog., 2014, Vol. 30, No. 2

successfully crosslinked with aldehyde-activated BSA to pro-vide immobilized enzyme preparation with total activity of150 units for 500 mg polymer. The supernatant and com-bined washings showed total activity of 1215 units. Thus,the loss of enzyme activity was less than 10%.

In the second approach (method 2), the enzyme was cova-lently bound to BSA-coated polymer through thiol groupspresent in BSA. Thus, the BSA-coated polymer (500 mg)was treated with a solution of DTT, which reduced the cross-linked thiol residues to free ASH groups. The polymer wasthen treated with a bifunctional reagent DSC to obtain anactivated DSC conjugate, which was treated with trypsinsolution (5 mg/mL, 5 mL, total activity 1500 units) at pH8.0 to obtain immobilized trypsin with activity of 167 unitsfor 500 mg support. The enzyme activity in the supernatantand combined washings was 1266 units. Thus, a total of1433 units (95%) of enzyme activity was recovered.

In the third strategy (method 3), we coupled the enzymeto BSA-coated polymer through the carboxylic acid groupsof BSA. Water-soluble carbodiimide, 1-ethyl-3-(3-dimethyla-minopropyl)carbodiimide (EDC), was used as a coupling rea-gent to obtain immobilized trypsin with total activity of 111units. Again, the combined activity in supernatant and wash-ings was 1250 units giving 91% of total recovery of activity.In contrast, in the case of direct immobilization on nativeuncoated polymer, the enzyme activity in the supernatantand combined washings was 555 units while the polymerexhibited activity of only 16 units. Thus, the total recoveredtrypsin activity was only 62%. It is quite evident that immo-bilization on BSA-coated polymer is far more superior interms of enzyme binding and recovery of enzyme activitythan a direct covalent coupling with epoxy-activatedcopolymer.

Sequential Trypsin Loading and Stabilization. As seenabove, the BSA-coated polymer has three functional groupsavailable for covalent coupling of enzymes. It is possiblethat each method provides some binding sites with one par-ticular functional group, for example, amino, and other bind-ing sites possessing other functional groups, for example,thiol and carboxylate are still free for enzyme coupling. Insuch a scenario, it may be possible to increase enzyme load-ing on the same polymer support. This hypothesis was testedby carrying out sequential loading of trypsin. First, BSA-coated polymer (500 mg) was activated with glutaraldehydeand then coupled with trypsin solution (5 mg/mL, 5 mL) inphosphate buffer of pH 8.0. The recovered polymer had spe-cific activity of 150 units. This preparation of immobilizedtrypsin (500 mg, 150 units) was treated with a solution ofDTT to reduce the crosslinked cysteine thiols and activatedwith DSC in dimethyl carbonate. Here, we would like tomention that DSC has good solubility in dimethyl carbonateand also that the solvent does not cause enzyme denaturationat least for the period it is in contact with immobilized tryp-sin (2 h at 5�C). The succinamidyl conjugate was separatedand treated again with fresh trypsin solution (5 mg/mL, 5mL) in sodium acetate–NaCl buffer of pH 4.8 to obtain afurther trypsin loading so that the polymer now has activityof 210 units. This polymer was then treated with EDC inMES buffer of pH 5.5, separated, and treated with fresh tryp-sin solution (5 mg/mL, 5 mL) in phosphate buffer, pH 8.0.After this step, the total activity of immobilized trypsin was255 units. Thus, the overall procedure provided a BSA-coated polymer matrix with specific activity of 510 units/g.

Based on the activity of native trypsin (60 units/mg), thiscalculates to a loading of 8.5 mg of active trypsin per gramof support.

Based on amino acid composition of BSA (99 carboxylicacid residues of combined Asp and Glu, 60 amino residuesof Lys, and 35 ASH of Cys residues), statistically, onewould expect a binding efficiency to follow the order method3>method 2>method 1. However, in the case of trypsin,all three methods provide rather similar binding efficiencies.As proteins contain several amino groups capable of interact-ing with activated support and can bind at the BSA-coatedpolymer surface with different orientations, it is difficult topredict the binding efficiencies based simply on the basis offunctional group composition of the support. One may needto test all the three methods to arrive at the most appropriatemethod, but it is possible to use all the three different func-tional groups present on the support to improve the enzymeloading.

Atomic Force Microscopy

Changes in surface morphology during sequential bindingof trypsin were observed using AFM (Figure 3). The poly-mer without BSA coating shows a flat plain surface (Figure3a) while the polymer surface coated with BSA at pH 8.0shows protein deposits with average peak breadth of 180–230 nm, peak height 55 nm, and distance between two peaks240 nm (Figure 3b). On treatment with glutaraldehyde foractivation of ANH2 groups, the peak morphology does notchange much (Figure 3c). After sequential binding of trypsinvia the amino, thiol, and carboxylic acid groups of BSA andfinal crosslinking with glutaraldehyde, the surface of BSA-coated polymer shows substantial thickening of peaks withaverage breadth of 700–900 nm at the base and height of 90nm. The peaks are separated by distance of 700 nm (Figure3d). In contrast, the surface of polymer on which trypsin isbound directly (without BSA coat), clumps of protein areobserved (Figure 3e and 3f). Based on recovered enzymeactivities, it appears that binding of trypsin through differentcovalent linkages occurs not only on the BSA but neighbor-ing trypsin molecules also get crosslinked and the peaksthicken. Also, as the polymer matrix goes through variousstages of trypsin contact and washings, some of the boundBSA are digested, loosely adsorbed protein gets slowlywashed off, and only the covalently bound protein remainson the polymer surface.

Possible Location of Trypsin after Sequential Loading andStabilization

In the methodology described above, enzyme immobiliza-tion could occur in two ways: attachment to (functionalized)BSA and binding to polymer surface not covered with BSA.However, activity recovery results indicate (section 3.2.1)that a direct interaction of the enzyme with polymer surface(not covered by BSA) in aqueous medium leads to enzymedenaturation to an appreciable extent. As this does not hap-pen, it seems reasonable to assume that the enzyme immobi-lization occurs through attachment to functionalized BSA.

A second possibility is that, during reduction of SASbonds with DTT, one of the thiol groups of DTT reacts withthe epoxy group of the polymer leaving the second thiolgroup available for activation with DSC and further couplingwith another trypsin molecule. However, experiments under

Biotechnol. Prog., 2014, Vol. 30, No. 2 321

identical conditions with plain polymer discounted thispossibility.

Statistically, the number of reactive functional groups ofBSA available for coupling (about 140 mg of BSA is boundto 1 g polymer) are far more than those available on boundtrypsin (about 5 mg trypsin/g), and it is reasonable to expectthat the incoming trypsin binds to BSA surface rather thantrypsin surface during the course of sequential binding. How-ever, it is still possible that the functional group of alreadybound trypsin may get activated by DSC or EDC and theincoming trypsin molecule may get attached to the existingtrypsin. To check out this possibility, first trypsin was cova-lently bound to the native polymer (without BSA) usingreverse micellar media. This methodology provides immobi-lized trypsin without serious loss of activity.15 This prepara-tion (40 units/g) was subjected to coupling with fresh trypsinusing EDC method (method 3), which resulted in immobi-lized trypsin with increased trypsin activity (620 units/g)confirming that it is indeed possible for two or more trypsinmolecules to get crosslinked. In a similar manner, after glu-taraldehyde treatment, along with covalent links between the

enzyme and BSA, links between neighboring enzyme mole-cules can also form. In such a scenario, it is probably best todescribe our enzyme preparation as polymer-supportedCLEAs. The AFM pictures (Figure 3) do support such anidea.

Properties of Immobilized Trypsin

The protein-coated polymer bears several functionalgroups on the surface, both positively and negativelycharged, which can cause shifts in the basic properties suchas pH-optima and thermal stability of the bound enzyme.For biotechnological applications, these parameters are veryimportant, hence these properties of the immobilizedenzymes were studied and compared with their solublecounterparts.

Effect of pH and Temperature on Activity of ImmobilizedTrypsin. Figure 4a shows the effects of pH on activity offree and immobilized trypsin. The pH versus activity curvefor immobilized trypsin is broad in pH -range from pH 6–9while that for free enzyme is much narrower (pH-optimumpH 8.2–8.5).

The immobilized enzyme also shows much improved ther-mal stability (Figure 4b). When the enzyme is incubated inTris-HCl buffer (0.05 M, 2 mM CaCl2, pH 8.2) for 1 h attemperatures ranging from 30 to 60�C, the soluble enzymeloses its activity rapidly and becomes practically inactiveafter incubation for 1 h at 60�C. In comparison, the immobi-lized enzyme is more stable and retains 60% of its activityunder similar conditions.

Reusability of Immobilized Enzyme. Above experimentsdemonstrated that an enzyme can be bound covalently to BSAmatrix using different strategies. As the location/orientation ofbound enzyme could vary depending on the functional group, itwas interesting to examine whether stability of bound enzymeis dependent on coupling strategy. Figure 5 shows the activityof the immobilized enzymes by different strategies up to 6recycles. It was observed that immobilization by crosslinkingwith glutaraldehyde provides the most stable enzyme prepara-tion followed by that coupled by ACOOH groups. Althoughcoupling by cysteine ASH groups gives more enzyme loading,half of its activity was lost after four recycles. At the momentwe do not have any answer to the question as to why an enzyme

Figure 4. Effect of (a) pH and (b) temperature on activity of (w)soluble and [�] immobilized trypsin. Activity 100% 5 50 units.

Figure 5. Recycle of trypsin immobilized by three differentstrategies. 100% 5 50 units. [�] Crosslinked throughANH2 using glutaraldehyde; (~) Immobilizedthrough ACOOH on BSA using EDC; (•)Immobi-lized through ASH on BSA using DSC. Reactionscarried out in Tris-HCl buffer, pH 8.2 at 30�C.

322 Biotechnol. Prog., 2014, Vol. 30, No. 2

coupled through a ACOOH shows more stability than thatcoupled through an ASH as we have no means of knowing thenumber of ANH2 groups of incoming trypsin that are reactingand the number of activated ACOOH or ASH groups. How-ever, it is well known that glutaraldehyde cross-coupling reac-tion provides multipoint attachments and provides betterstabilization. This was evident from the observation that theimmobilized enzyme preparations obtained after DSC- or EDC-coupling reactions on treatment with 4% glutaraldehyde insodium carbonate buffer (0.1 M, containing 0.1 M NaCl, pH8.5, 4 mL) provided enzyme preparations that were stable andcould be recycled at least seven times without loss of enzymeactivity (Figure 5).

Conclusion

BSA-coated polymers provide an interesting matrix forimmobilization of functional enzymes. The methodology issimple and paves way for immobilization of a wide range ofbiomolecules from peptides to antibodies without the neces-sity of synthesizing specially functionalized supports. Webelieve that this procedure can be quite general if thesequence of chemistry used is carefully chosen. For example,DTT–DSC route cannot be used after a multimeric enzymewith disulfide links is immobilized but a route involvingEDC can be employed with a fair degree of success. Theseaspects are being actively studied.

Acknowledgments

This work was supported by Department of Science andTechnology, New Delhi, India (Grant No. SR/S3/CE/0051/2010), and Council of Scientific and Industrial Research,New Delhi, India.

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