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Process Biochemistry 49 (2014) 604–616

Contents lists available at ScienceDirect

Process Biochemistry

jo u r n al homep age: www.elsev ier .com/ locate /procbio

uning of Lecitase features via solid-phase chemical modification:ffect of the immobilization protocol

ristina Garcia-Galana,1, José C.S. dos Santosa,b,1, Oveimar Barbosac, Rodrigo Torresc,rnandes B. Pereirad, Vicente Cortes Corberana, Luciana R.B. Gonc alvesb,oberto Fernandez-Lafuentea,∗

Instituto de Catálisis-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, SpainDepartamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760 Fortaleza, CE, BrazilEscuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander,ucaramanga, ColombiaUniversidade Federal de Alfenas, 37130-000 Alfenas, MG, Brazil

r t i c l e i n f o

rticle history:eceived 26 November 2013eceived in revised form 20 January 2014ccepted 23 January 2014vailable online 30 January 2014

eywords:nzyme immobilizationnzyme chemical modificationnzyme stabilizationnzyme hyperactivationlutaraldehyde,4,6-Trinitrobenzensulfonic acid

a b s t r a c t

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two dif-ferent strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activationon octyl-agarose beads. Both immobilized preparations have been submitted to different individual orcascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS)modification) in order to check the effect of these modifications on the catalytic features of the immobi-lized enzymes (including stability and substrate specificity under different conditions). The first point tobe remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase wasmore active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalentpreparations. Moreover, the effects of the chemical modifications strongly depend on the immobilizationstrategy used. For example, using one immobilization protocol a modification improves activity, whilefor the other immobiled enzyme is even negative. Most of the modifications presented a positive effect onsome enzyme properties under certain conditions, although in certain cases that modification presenteda negative effect under other conditions. For example, glutaraldehyde modification of immobilized or

modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymesat pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could bedetected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stabil-ity of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent

10-fo

preparation at pH 9 (by a

. Introduction

Phospholipases A1 hydrolyze the 1-acyl group of a phospho-ipid to a lysophospholipid and a free fatty acid. They are ofarticular interest for industrial applications, to produce 2-acyl-

ysophospholipids (good emulsifiers), glycerides with interestingatty acid composition (eicosapentaenoic acid, conjugated linoleiccid and docosahexaenoic acid) and degumming process of oils

∗ Corresponding author at: Departamento de Biocatálisis, ICP-CSIC, C/Marie Curie, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. Tel.: +34 91 585 48 09;ax: +34 585 47 60.

E-mail addresses: rfl@icp.csic.es,fernandezlafuente@hotmail.com (R. Fernandez-Lafuente).

1 Both authors have evenly contributed to the paper.

359-5113/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2014.01.028

ld factor).© 2014 Elsevier Ltd. All rights reserved.

[1–5]. Due to their interesting applications, new phospholipaseshave been screened and discovered in the recent years [6–8].In this context, a new enzyme preparation with phospholipaseA1 activity, namely Lecitase® Ultra, has been patented and madecommercially available [9]. Following the supplier information,this is a preparation obtained from the fusion of the genes ofthe lipase from Thermomyces lanuginosus and the phospholipasefrom Fusarium oxysporum and developed principally for degum-ming processes [9]. This new enzyme presented the stability of thelipase from T. lanuginosus and the activity of the enzyme from F.oxysporum [9]. However, few manuscripts may be found in the liter-ature on the uses and biocatalyst design of this interesting enzyme

[10–18].

In this paper, the effects of different chemical modificationson the diverse catalytic features of Lecitase have been studied.This enzyme, as lipases, has a catalytic mechanism the so-called

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nterfacial activation, with a lid covering the active center inomogenous medium (closed form) in equilibrium with a formhere the active center is exposed to the medium (open form)

19,20]. In the presence of hydrophobic surfaces, such as oil drops,he open form of the lipase becomes adsorbed via the largeydrophobic pocket formed by the internal face of the lid andhe surroundings of the active center [19]. As these conforma-ional changes give the lipases a large flexibility to its active center,t is relatively simple to modulate the lipase properties (activ-ty, selectivity or specificity) via different approaches, like genetic

odifications [21,22], medium engineering [23], but also physi-ochemical tools like immobilization or chemical immobilization24,25]. Lecitase properties have been already altered via immo-ilization [26–28], but this is the first report on the effects of thehemical modification on its catalytic features.

Chemical modification has been performed on already immo-ilized Lecitase. This simplifies the process, avoids proteinrecipitation during or after modification, and may even permito pre-stabilize the enzyme [29].

Moreover, if the hypothesis is correct and immobilization is aay to tailor the exact conformation of the enzyme, it may beossible that the chemical modification of different enzyme prepa-ations may give very different results. Some reports using lipase

from Candida antarctica seem to confirm this possibility [30].onsidering the former possibility, we have selected two very dif-erent immobilization strategies. The first one is the immobilizationf the enzyme via interfacial activation on octyl-agarose [31], aay to fix the open form of the Lecitase, leaving the active cen-

er and the groups around it in a quite hydrophobic environment32].

The second one is the covalent immobilization on agarose acti-ated with bromide cyanogen groups [33]. This immobilizationethod will be performed at pH 7 and at a temperature of 4 ◦C.nder these conditions, the possibilities of multipoint covalentttachment are reduced [34], and the enzyme will keep the pos-ibility of conformational equilibrium between open and closedorm [19,20]. At first glance, all the external groups except thosenvolved in the immobilization will be susceptible to the chemical

odification.In this paper, three different chemical modifications have been

ttempted. The first one was the modification of the exposed car-oxylic acids of the enzyme with ethylenediamine (EDA) afterctivation with carbodiimide (DEC, N-3-dimethylaminopropyl-′-ethylcarbodiimide hydrochloride) [35,36]. This modificationaintains the possibility of ionization of the enzyme surface but

roduces a great alteration of the ionic interactions (breaking ionicridges). This is a well described modification of proteins, and itas been used to modulate the properties of lipases (e.g., speci-city [29,30,37]). The pK of the new amino group is around 9.2,hat makes it more reactive than the �-amino group of Lys (pK 10.7),

oreover the addition of all external carboxylic groups is in mostrotein 2- to 3-fold higher than the amount of external Lys groups.owever, the modification is not fully specific because it has beenescribed that the carbodiimide may transform some carboxylicroups to urea groups (if amine attack to the carboxyl activatedroups is not rapid enough) and can also modify Tyr residues. Thisast chemical modification may be reversed by incubation in theresence of hydroxylamine, re-obtaining free Tyr [38].

The second modification has been the modification ofhe primary amino groups of the protein surface by 2,3,4-rinitrobenzenesulfonate (TNBS). This highly specific modificationf primary amino groups is traditionally employed to quantify

hese groups in proteins and supports [39,40]. The modifica-ion of the amino groups of the enzyme surface will produce aydrophobization of the enzyme. It will leave a secondary amineith a hydrophobic group pendant on it. This reagent has been

hemistry 49 (2014) 604–616 605

successfully employed to detect the implication of Lys residues inthe function of enzymes and proteins [41]. However, very few stud-ies have considered this reagent as a way to modulate the enzymeproperties, we have been able to only find in the literature themodification of invertase (with moderately good results in termsof stabilization) [42], porcine pancreatic �-amylase (changing theenzyme specificity) [43] and lipase B from C. antarctica (alteringmost of the catalytic features) [30].

The third modification consisted in the treatment with glu-taraldehyde. Glutaraldehyde is a reagent widely used to modifyproteins; mainly involving primary amino groups of proteins,although it may eventually react with other groups (e.g., thiols,phenols, and imidazols) [44–47]. Moreover, it is a very effectivecrosslinker [47,48]. An intramolecular crosslinking is by far moredifficult to achieve than the one-point chemical modification or aninter-molecular crosslinking. In an intramolecular crosslinking, thedistance between the groups involved needs to match the lengthof the crosslinker and even if the distance is adequate, it requiresthe correct alignment of the two involved chemical groups, whichare located in a rigid protein surface [29]. The degree of modi-fication of an aminated surface by glutaraldehyde may be quiteeasily controlled by the use of different glutaraldehyde concen-trations, reaction pH values and reaction times [49]. It has beenshown that amino-glutaraldehyde groups are quite reactive withother amino-glutaraldehyde groups or with free glutaraldehyde,thus being the best way to get a glutaraldehyde crosslinking [47,50].Inter or intra-crosslinking bonds should always cause a certainincrease of enzyme rigidity in that area of the protein [51] that mayproduce or not a certain stabilization of the enzyme. On the otherhand, one-point chemical modification with glutaraldehyde meansto add a relatively hydrophobic group [52] hung from the enzymeamino groups, and that may greatly affect the global physical prop-erties of the enzyme surface (e.g., hydrophobicity), also altering theenzyme catalytic properties.

In this paper, as the modifications with glutaraldehyde and TNBSare based on the reaction with primary amino groups, they havebeen performed also in previously aminated enzymes, to maximizetheir possible effects (e.g., inter or intramolecular crosslinkings).

The hypothesis is that these chemical modifications can producegeneral conformational changes in the enzyme structure or alterthe movements of the lid, in both cases tuning the final enzymefeatures. These changes may be different depending on the immo-bilization strategy used.

2. Materials and methods

Lecitase was obtained from Novozymes (Spain). Octyl-agarose and cyanogenbromide crosslinked 4% agarose (CNBr) beads were from GE Healthcare.Ethylenediamine (EDA), N-(3-dimetylaminopropyl)-N-ethylcarbodiimide (DEC),2,4,6-trinitrobenzensulfonic acid (TNBS), glutaraldehyde (50% aqueous solution),p-nitrophenylbutyrate (p-NPB), Triton X-100, sodium dodecyl sulphate (SDS), cetyltrimethyl ammonium bromide (CTAB) R and S methyl mandelate and methyl pheny-lacetate were from Sigma Chemical Co. (St. Louis, MO, USA). Glutaraldehyde (25%(v/v) stabilized in ethanol) was from Fluka. All reagents and solvents were of ana-lytical grade.

2.1. Standard determination of enzyme activity

This assay was performed by measuring the increase in absorbance at348 nm produced by the released p-nitrophenol in the hydrolysis of 0.4 mM p-nitrophenylbutyrate (p-NPB) in 100 mM sodium phosphate at pH 7.0 and 25 ◦C (εunder these conditions is 5150 M−1 cm−1). To start the reaction, 50–100 �l of lipasesolution or suspension was added to 2.55 ml of substrate solution. One internationalunit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 �mol ofp-NPB per minute under the conditions described previously. Protein concentrationwas determined using Bradford’s method [53] and bovine serum albumin was used

as the reference.

In the determination of the effects of the pH value on the enzyme activity, thefollowed protocol was similar but the buffer in the measurements was changedaccording to the pH value: sodium acetate at pH 5, sodium phosphate at pH 6–8and sodium borate at pH 9 and pH 10. At 25 ◦C, all the preparations remained fully

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table after incubation for several hours at any of these pH values. In some instances,ifferent concentrations of some detergents were used.

.2. Immobilization of Lecitase on octyl-agarose beads

Lecitase was immobilized on octyl-agarose beads at low ionic strength [31,32]..8 ml of commercial extract (16 mg protein/ml having a pNPB activity of 5.6 U/mgrotein) was diluted in 67.5 ml of 5 mM sodium phosphate at pH 7. Then, 15 g ofet octyl-agarose beads was added. Activity of supernatant and suspension was

ollowed using pNPB. After, immobilization the suspension was filtered and theupported lipase was washed several times with distilled water.

.3. Immobilization of Lecitase on CNBr-agarose beads

A volume of 2.8 ml of commercial Lecitase was diluted in 67.5 ml of 5 mM sodiumhosphate containing 0.05% (w/v) sodium dodecyl sulfate at pH 7 at 4 ◦C. Then,5 g of wet CNBr-support was added. Activity of supernatant and suspension wasollowed using pNPB. The enzyme-support immobilization was ended by incubat-ng the support with 1 M ethanolamine at pH 8 for 12 h. Finally, the immobilizedreparation was washed with abundant distilled water.

.4. Chemical modification of immobilized Lecitase

Fig. 1 shows the preparation of the different Lecitase preparations and the abbre-iations used in the paper.

.4.1. Solid phase amination of LecitaseTen grams of immobilized Lecitase, immobilized Lecitase-TNBS (see below) or

mmobilized Lecitase-GLU (see below) was added to 100 mL of 1 M EDA at pH 4.75nder continuous stirring [37]. Modification started with the addition of solid DECto a final concentration of 10 mM). After 2 h of gentle stirring at 25 ◦C, the aminatederivates had more than 95% of the exposed carboxylic groups modified [35,36]. Thisas confirmed by comparing the developed color after titration with TNBS, that didot increase using 100 mM DEC or 2 M EDA. These preparations were washed withistilled water and incubated in 1 M hydroxyl amine at pH 8 for 12 h to recover theyr residues that could have been modified by DEC [38]. Finally, the immobilizednzyme preparations were washed with an excess of distilled water and stored at◦C.

.4.2. Modification of the primary amino groups of immobilized Lecitase withNBS

12 g of immobilized Lecitase or Lecitase-EDA (see above) was added to 100 mlf a solution of 0.1% (w/v) TNBS in sodium phosphate at pH 8.0 and the mixture wasncubated for 60 min at room temperature. Then, the modified enzyme preparation

as washed with distilled water [30]. The developed color did not increase when theNBS concentration and volume were doubled, suggesting that 100% of the exposedrimary amino groups in the protein had been modified.

When this method was used to determine the amount of primary amino groupsn the protein, 200 mg were re-suspended in a cuvette containing 2.2 ml of 100 mMorate at pH 9 and the absorbance at 430 nm was determined [30]. As a reference,ctyl-agarose or blocked CNBr free of enzyme was treated in a similar way.

.4.3. Glutaraldehyde modification of immobilized LecitaseBoth immobilized Lecitase prepared as previously described, as well as both

ecitase-EDA preparations, were incubated with 0.1% (v/v) glutaraldehyde solutionn 25 mM sodium phosphate buffer at pH 7 and 25 ◦C for 1 h under mild stirring.his range of conditions permitted to fully modify the primary amino groups ofhe enzyme with just one glutaraldehyde molecule [54]. The suspension was thenltered and washed with water to remove the excess of glutaraldehyde.

Five grams of immobilized and modified Lecitase was added to 200 mL of 0.1 Modium borate containing 1 mg/mL of NaBH4 at pH 8.5 and 4 ◦C; the reaction mix-ure was continuously stirred for 30 min. The reduced biocatalysts were filterednd washed several times with distilled water. This treatment produced a 10%ecrease in CNBr-Lecitase activity, while it presented no effect on the activity ofctyl preparations.

.5. Thermal inactivation of different Lecitase immobilized preparations

To check the stability of enzyme derivatives, 1 g of immobilized enzyme wasuspended in 5 ml of 10 mM of sodium acetate at pH 5, sodium phosphate at pH 7r sodium carbonate at pH 9 at different temperatures. Periodically, samples wereithdrawn and the activity was measured using pNPB. Half-lives were calculated

rom the observed inactivation courses.

.6. Hydrolysis of methyl phenylacetate

Enzyme activity was determined by using methyl phenylacetate; 200 mg of themmobilized preparations were added to 0.2 mL of 5 mM substrate in 100 mM bufferontaining 50% CH3CN. The buffer was sodium acetate at pH 5, sodium phosphate atH 7 and sodium carbonate at pH 8.5. All experiments were carried on at 25 ◦C under

hemistry 49 (2014) 604–616

continuous stirring. The conversion degree was analyzed by RP-HPLC (Spectra PhysicSP 100 coupled with an UV detector Spectra Physic SP 8450) using a Kromasil C18(15 cm × 0.46 cm) column. Samples (20 �L) were injected and eluted at a flow rate of1.0 mL/min using acetonitrile, 10 mM ammonium acetate aqueous solution (35:65,v/v) and pH 2.8, as mobile phase and UV detection was performed at 230 nm. Acid hasa retention volume of 3 mL while ester has a retention volume of 12 mL. One unit ofenzyme activity was defined as the amount of enzyme necessary to produce 1 �molof phenyl acetic acid per minute under the conditions described above. Activity wasdetermined by triplicate with a maximum conversion of 20–30%, and data are givenas average values.

2.7. Hydrolysis of R and S methyl mandelate

Enzyme activity was also determined by using R or S methyl mandelate. 400 mgof the immobilized preparations was added to 2 mL of 50 mM substrate in 100 mMsodium acetate at pH 5, 100 mM sodium phosphate at pH 7 or 100 mM sodiumcarbonate at pH 9 and 25 ◦C under continuous stirring. The conversion degree wasanalyzed by RP-HPLC (Spectra Physic SP 100 coupled with an UV detector SpectraPhysic SP 8450) using a Kromasil C18 (15 cm × 0.46 cm) column. Samples (20 �L)were injected and eluted at a flow rate of 1.0 mL/min using acetonitrile/10 mMammonium acetate (35:65, v/v) at pH 2.8 as mobile phase and UV detection wasperformed at 230 nm. Acid has a retention volume of 2.5 mL while ester has a reten-tion volume of 10 mL One unit of enzyme activity was defined as the amount ofenzyme necessary to produce 1 �mol of mandelic acid per minute under the con-ditions described above. Activity was determined by triplicate with a maximumconversion of 20–30%, and data are given as average values. As the reaction is not offirst order at 10 mM, E cannot be calculated from reaction rate observed using the Rand the S isomers (VR/VS), and this direct ratio is the one used in the paper.

2.8. SDS-PAGE experiments

SDS-polyacrylamide gel electrophoresis was performed according to Laemmli[55] using a Miniprotean tetra-cell (Biorad), 12% running gel in a separation zoneof 9 cm × 6 cm, and a concentration zone of 5% polyacrylamide. One hundred mil-ligrams of the immobilized enzyme samples was re-suspended in 1 mL of rupturebuffer (2% SDS and 10% mercaptoethanol), boiled for 5 min and a 20 �L aliquot of thesupernatant was used in the experiments. Gels were stained with Coomassie bril-liant blue. Low molecular weight markers from Fermentas were used (10–200 kDa).

3. Results

3.1. Immobilization of Lecitase on octyl- and CNBr-agarose

Fig. 2a shows the immobilization course of Lecitase on octyl-agarose and CNBr-agarose.

In octyl-agarose, most of the enzyme activity is already immo-bilized only after 0.5 h, and the activity increase by more than a2-fold factor.

When immobilizing the enzyme in CNBr, the enzyme is alsorapidly incorporated to the support (more than 90%), but theactivity suffers a drastic drop (less than 5% of the initial activityis observed in the support). Enzyme inactivation closely followsenzyme immobilization. This is surprising, as the immobilizationconditions are very mild and it an intense enzyme/support reactionshould not be expected.

Trying to improve the enzyme activity recovery in CNBr, weassayed several detergents, trying to find one that can keep theopen form of the Lecitase and keep the enzyme in monomeric form[56,57] while having a low impact on its stability. It was found thatSDS was the one with a lower effect on Lecitase stability (resultsnot shown). Fig. 2b shows the effect of the presence of differentconcentrations of SDS during immobilization on enzyme activity.Immobilization rates remained very high, and using 0.05% SDS dur-ing the immobilization around 70% of the initial enzyme activityafter 2.5 h of immobilization were recovered. The effects of thedetergent could be several. On the one hand, some areas of theenzyme could be blocked by this detergent avoiding the immobi-lization by that area, even preventing the reaction of some group.

On the other hand, a detergent may stabilize the open form of thelipase, if the problem after immobilization in CNBr is the increasingdifficulty to the opening of the enzyme form, the immobilization inthe presence of detergent may solve it [58]. Moreover, if Lecitase

C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616 607

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ends to form dimers (like many lipases do [56,57]), the detergentill break them and will permit to immobilize the monomeric form

f the enzyme.These immobilization conditions were used as standard ones for

he preparation of the biocatalysts employed in this paper.

.2. Effect of the chemical modifications on enzyme activity

Both enzyme preparations were submitted to the chemicalodifications showed in Fig. 1. The effect on the enzyme activity

nder standard conditions may be found in Table 1.Using octyl-Lecitase, the amination produced an increase on

nzyme activity (by around a 35%), while the TNBS (around 50%ctivity recovery) and glutaraldehyde (around 70%) produced aecrease in enzyme activity.

The modification of the aminated enzyme with TNBS decreasedhe activity by 40%, slightly less than modifying the unchangedctyl-Lecitase, even though now the number of modified groups

ill be much higher. The glutaraldehyde treatment of this biocat-

lyst reduced the activity by around 50%, in fact this modificationeversed the positive effect of the amination and it was less activehan the just glutaraldehyde modified enzyme.

aldehyde; TNBS: 2,4,6-trinitrobenzensulfonic acid; EDA: ethylendiamine.

On the other hand, the amination of the enzyme modified withTNBS or glutaraldehyde caused a slight decrease in the enzymeactivity.

The situation was different using the covalently immobilizedenzyme. First, the initial activity is much lower than using octyl-agarose, almost a 30-fold factor (measures are now performed inabsence of detergent).

Amination produced a slight improvement on enzyme activity,while glutaraldehyde modification decreased the activity by 40%.However, TNBS modification increased the activity by more than a2.5-fold factor. The glutaraldehyde or TNBS treatment of the ami-nated enzyme produced a severe decrease in enzyme activity (20and 40% activity recovery respectively), much more significant thanusing the unmodified enzyme. Amination of TNBS modified enzymeproduced the loss of all the positive effect of the TNBS modification,and even promoted a further decrease on enzyme activity (activitydropped from 2.5 to 0.32 U/mg). This is surprising considering thatboth independent modifications have positive effects on enzyme

activity. Amination of the enzyme modified with glutaraldehydealso have a negative effect on activity, but not so negative.

Thus, the effects of the chemical modifications on the enzymefeatures strongly depend on the immobilization strategy, and they

608 C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616

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ig. 2. Immobilization courses of Lecitase on octyl- and CNBr-agarose. Experimenctyl-agarose; circles: CNBr-agarose. Solid lines: suspension, dashed lines: supernat.05% SDS (v/v); Rhombus: 0.1% SDS (v/v).

re not necessarily additive ones, in some cases the accumulativeodification has a lower impact on the enzyme activity that each

f the individual modifications or even the opposite. Consideringhat we expect to alter the delicate balance of forces that stabi-ize the open form of the lipase, a specific explanation for each ofhe individual results may be almost impossible. However, differ-nces on the immobilized preparations may be at least a partialxplanation to the different effect of the chemical modification ofovalent and interfacially activated immobilized Lecitase prepa-ations. Lecitase immobilized on octyl-agarose has stabilized thepen form. That means that any effect of the chemical modifica-ion founded on changes in the open/closed equilibrium will be

inimized now. Moreover, the area of this immobilized enzymeolecules is in close contact with the hydrophobic surface of the

upport, and this may alter its reactivity (in some instances, it maye that the group is not accessible to the modification, in others,ome partition may increase or decrease its apparent reactivity).

To increase the information on the effect of the modification onhe enzyme activity, we have studied the activity from pH 5 to pH0.

.3. Effect of pH value on the activity of the different Lecitasereparations

Fig. 3a shows the pH/activity profile of the octyl- and CNBr-ecitase preparations. The profiles are very different. While theovalently immobilized enzyme has a clear optimum of activityt pH 6 and only retains around 40% of activity at pH 10, the octylreparation does not have a clear optimal pH value in the rangetudied, activity in a flat from pH 8–10, with a certain tendency toncrease the activity even at the highest pH value analyzed (higherH values presented a too rapid chemical hydrolysis of the pNPB toe studied). Considering that octyl-Lecitase has already stabilizedhe open form of the enzyme, the importance of the pH value on

he movement of the using the CNBr preparation could be a likelyxplanation for these very different results.

Regarding the effect of the chemical modifications on this pro-le, it is very diverse.

able 1ffect of the chemical modifications on the pNPB activities of the different preparationlutaraldehyde. Activity is given as U/mg.

Biocatalyst Chemical modification

None EDA TNBS GLUTA

Octyl-Lecitase 26 ± 1 35 ± 1 12.5 ± 0.5 18. ± 1

CNBr-Lecitase 0.9 ± 0.05 1.1 ± 0.05 2.4 ± 0.1 0.54 ± 0.04

nditions are detailed in Section 2. (A) Immobilization in absence of SDS. Squares:B) Effect of SDS on the Lecitase immobilization courses on CNBr-agarose. Triangles:

First, we will focus on the octyl preparations (Fig. 3b). Amina-tion almost left the profile unaltered. This was quite surprisingconsidering the radical change of the surface ionic interactionscomparing the modified and unmodified enzyme, and the dra-matic change that this chemical modification should produce onhow the pH may affect these interactions. Modification with TNBSdecreased the activity at pH values under 8 and the effect is moresignificant at acidic pH values (at pH 5 the TNBS octyl deriva-tive maintained only 15% of the activity while the unmodified oneretained 61%). Modification with glutaraldehyde produced a nar-rower activity/pH profile, appearing a clear activity maximum atpH 8, with a more significant decrease of activity at both acid andbasic pH values.

Amination of the TNBS-Lecitase preparation promoted a certainrecovery of activity at acid pH value (retaining around 50% of theactivity ay pH 5). However, the effect of the amination is clearlynegative for the activity of the glutaraldehyde modified preparationat acidic pH value (the activity decrease from 50% to 15% at pH 5),while shifting the optimal pH of the glutaraldehyde preparationfrom 8 to 9.

Modification with TNBS of the aminated enzyme has almost noeffect on the activity/pH profile. And the modification with glu-taraldehyde of the aminated enzyme has an effect on the pH/profilesimilar to the modification of the unmodified octyl preparation, butleaving a clear optimal pH value at pH 9

Fig. 3c shows the effect of the modifications on the pH/activitycurve of Lecitase immobilized on CNBr. In this case, the changes arefar more evident than when using octyl-Lecitase.

Amination produced that the maximum activity detected maybe found at the highest studied pH value, pH 10. Under these con-ditions the absolute activity of the modified enzyme was 6-foldhigher than that of the unmodified enzyme (0.5 versus 3 U/mgenzyme). However, at pH 5 the modification reduced the activityof the enzyme by almost a 2-fold factor.

Modification with TNBS moved the optimal pH value from 6 topH 9 (at this pH value the modification increased by 8 the enzymeactivity, from 0.5 to 4 U/mg), at acid pH this difference is shorter(e.g., at pH 5 went from 0.8 to 1.3).

s. Activity was determined at pH 7 and 25 ◦C as indicated in Section 2. GLUTA,

EDA-TNBS EDA-GLUTA TNBS-EDA GLUTA-EDA

20.5 ± 1 16.5 ± 1 11.5 ± 0.6 15.2 ± 10.45 ± 0.03 0.26 ± 0.02 0.32 ± 0.02 0.4 ± 0.03

C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616 609

0153045607590

105

1098765pH

Rel

ativ

e A

ctiv

ity, (

%)

0153045607590

105

1098765

pH

Rel

ativ

e A

ctiv

ity, (

%)

0153045607590

105

1098765

pH

Rel

ativ

e A

ctiv

ity, (

%)

A

CB

Fig. 3. Effect of the pH on the activity versus pNPB of the different Lecitase preparations. Experiments have been performed as described in Section 2. (A) Effect of theimmobilization protocol: octyl-Lecitase: squares; CNBr-Lecitase: triangles. (B) (octyl-Lecitase) and (C) (CNBr-Lecitase). Effect of the chemical modification. One modification:S s: GLUG Cros

oim

adiHw(hfa

oaa

sapet

3

tLm

olid black lines. Squares: No modification; Triangles: EDA, Circles: TNBS, RhombuLUTA. Amination of modified Lecitase: Dashed gray lines. Gray Rhombus: TNBS, Gray

Modification with glutaraldehyde produced a slight shift of theptimal pH value (to pH 7), with a very significant decrease of activ-ty at alkaline pH values (at pH 10 the activity decreased after the

odification from 0.5 to 0.08).Amination of the TNBS modified enzyme caused the maximal

ctivity to be detected at pH 10. At pH 10, the amination pro-uced an increment on enzyme activity (from 2.1 to 2.6 U/mg), and

s 5-fold higher than that of the no modified enzyme (0.5 U/mg).owever, at acid pH the effect is very negative, at pH 5 the activityent from 1.3 to 0.06, lower than that of the just aminated enzyme

0.8 U/mg). The amination of the glutaraldehyde modified Lecitaseave not a clear effect on the pH/activity profile, just a better per-

ormance at alkaline pH value (in fact, activities become identicalt pH 10).

The treatment of aminated enzyme with TNBS maintained theptimal pH value; but the activity decreased at more alkaline orcid pH values, giving a narrower curve. Using glutaraldehyde, theminated enzyme left the activity/pH profile almost unaltered.

The results show how the effect of a chemical modification istrongly dependent on the pH value, some positive modificationst a pH value, may become very negative at other. The effect of theH on the enzyme activity depends on the immobilization strategymployed; the likely explanations may be some of the detailed inhe previous points.

.4. Effect of the presence of detergents on enzyme activity

Figs. 4–6 show the effect of the presence of different concen-rations of detergents on the activity of the different immobilizedecitase preparation. The effect of the detergent on enzyme activityay be due to different reasons. First, it can stabilize the open form

TA. Modification of aminated Lecitase: Solid gray lines. Gray Circles: TNBS, Circles:ses: GLUTA.

of the lipase [58]. Second, the detergent may alter the enzyme con-formation, producing enzyme inactivation or hyperactivation, butalso enzyme destabilization [59,60]. Moreover, the detergent mayact as a competitive inhibitor [58,59]. Thus, it is not unlikely thatfor each enzyme preparation (that we expect to have an alteredconformation) the effect of a detergent may be fully different, forexample, the octyl preparations cannot suffer enzyme activation bystabilization of the open form, because they already has this formstabilized [31,32].

Using SDS (Fig. 4a), octyl-Lecitase increased the activity at0.001 and 0.01% (v/v) to 120%, but at 0.1% the activity decreaseto 60% (no enzyme desorption was detected using these detergentconcentrations). Using CNBr-Lecitase, the increment in activity ismore significant and reaches a maximum at 0.01% (over 215%),decreasing to 75% at 0.1% detergent. The differences between bothimmobilized preparations are in the expected trend, a higher effecton the CNBr-Lecitase than in the octyl-Lecitase was found.

Next, we analyze if the chemical modification may alter thiseffect of the detergent, considering its anionic nature.

Starting with the octyl preparations (Fig. 5a), amination seemsto avoid the positive effect of the SDS on enzyme activity, and evenproduces a decrease in enzyme activity at lower concentrations(e.g., at 0.01% (v/v) SDS, the activity is under 90%, while beforethe activity was 120%). TNBS modification provoked that a posi-tive effect of SDS was detected at the highest concentration used(0.1%) while glutaraldehyde treatment had almost no effect on theSDS influence on enzyme activity. Amination of the glutaralde-

hyde modified enzyme eliminates the positive effects of SDS onthe glutaraldehyde modified enzyme, while amination of the TNBSmodified enzyme increases the negative effects of SDS on the TNBSmodified enzyme. Treatment with glutaraldehyde of the aminated

610 C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616

0

50

100

150

200

250

0.10.010.001

Detergent Concentration, (%)R

elat

ive

Act

ivity

, (%

)

A

CB

0

200

400

600

800

1000

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

0

50

100

150

200

250

300

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

Fig. 4. Effect of different detergents (A: SDS, B: CTAB, C: Triton X-100) on the pNPB activity of different immobilized Lecitase preparations. Experiments have been performedas described in Section 2. Octyl-Lecitase: squares; CNBr-Lecitase: triangles.

A

CB

0

40

80

120

160

200

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

0

100

200

300

400

500

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

0

25

50

75

100

125

150

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

Fig. 5. Effect of different detergents (A: SDS, B: CTAB, C: Triton X-100) on the pNPB activity of differently modified octyl-Lecitase preparations. Experiments have beenperformed as described in Section 2. One modification: Solid black lines. Squares: No modification; Triangles: EDA, Circles: TNBS, Rhombus: GLUTA. Modification of aminatedLecitase: Solid gray lines. Gray Circles: TNBS, Circles: GLUTA. Amination of modified Lecitase: Dashed gray lines. Gray Rhombus: TNBS, Gray Crosses: GLUTA.

C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616 611

A

CB

0

40

80

120

160

200

0.10.010.001

Detergent Concentration, (%)R

elat

ive

Act

ivity

, (%

)

0

300

600

900

1200

1500

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

0

50

100

150

200

250

300

0.10.010.001

Detergent Concentration, (%)

Rel

ativ

e A

ctiv

ity, (

%)

Fig. 6. Effect of different detergents (A: SDS, B: CTAB, C: Triton X-100) on the pNPB activity of differently modified CNBr-Lecitase preparations. Experiments have beenp modiL ecitase

e0w

pctmetmopaietaa

ubttt

4eamb

erformed as described in Section 2. One modification: Solid black lines. Squares: Noecitase: Solid gray lines. Gray Circles: TNBS, Circles: GLUTA. Amination of modified L

nzyme permitted to keep around 100% using SDS from 0.001 to.1% (v/v), while treatment with TNBS gave as result an enzymeith a behavior very similar to the unmodified enzyme.

Using CNBr-Lecitase results are different (Fig. 6a). Aminationromoted that the enzyme activity decreased at lower SDS con-entrations; while TNBS modification caused the positive SDS effecto be more progressive, doubling the enzyme activity at the maxi-

um SDS utilized in this study (0.1%, v/v). Glutaraldehyde modifiednzyme progressively decreased the activity in all the SDS concen-ration analyzed. Amination of the TNBS modified enzyme almost

ade that the enzyme activity is not influenced by the presencef SDS, using the glutaraldehyde modified enzyme the aminationrevented the decrease of activity at 0.001 and 0.01% SDS but nott 0.1%. TNBS modification of aminated enzyme had a effect sim-lar to that of the same modification on unmodified immobilizednzyme (the maximum activity was detected at 0.1% SDS) whilehe glutaraldehyde modification permitted to improve the activityt high SDS concentration (from 44% the activity increase to 190%t 01% SDS).

CTAB was also utilized. This cationic detergent may help tonderstand the effects of the other detergent, even though it haseen found that long term incubation of Lecitase on solutions ofhis detergent decreased its activity. However, in the few minuteshat the activity assay takes; this inactivation is fully negligible inhe range of studied concentrations.

Using octyl-Lecitase (Fig. 4b), the activity increased to more than40% at 0.1% CTAB, the maximum utilized concentration (where

nzyme was not found in solution). Using CNBr, the increment onctivity reached an 8-fold factor. Thus, CTAB presented an evenore positive effect on enzyme activity compared to SDS using

oth immobilized preparations. Next, we have analyzed the effect

fication; Triangles: EDA, Circles: TNBS, Rhombus: GLUTA. Modification of aminated: Dashed gray lines. Gray Rhombus: TNBS, Gray Crosses: GLUTA.

of the chemical modification on the enzyme activity on the pres-ence of CTAB (Figs. 5b and 6b). Most modifications reduced thepositive effects of CTAB on enzyme activity for both immobilizedpreparations.

Using aminated octyl-Lecitase (Fig. 5b), the increase in activitywas also observed in all the CTAB concentrations studied, but witha maximum at 0.1% (v/v) CTAB of only 290%. However, after themodification with TNBS, the enzyme progressively decreased itsactivity with the increase in the CTAB concentration. Modificationwith glutaraldehyde also reduced the positive effects of CTAB, only85% of the initial activity was observed at 0.1%. The amination of thispreparation has almost no influence on the detergent effect, while ifthe TNBS preparations was aminated the activity decrease with theCTAB concentration (to less than 45% at 0.1% CTAB, almost 7 timeslower than modifying the native immobilized enzyme). Both mod-ifications of aminated enzyme also eliminated the positive effect ofthe CTAB (Fig. 5b).

Using the covalently immobilized preparation (Fig. 6b), ami-nation strongly decreased the positive effect of CTAB on enzymeactivity, in fact the maximum activation (only 160%) is observedat 0.01%, with a decrease on enzyme activity at 0.1%. Glutaralde-hyde modification reduced the effect, but still the hyperactivationwas significant and the maximum activity is obtained at 0.1%. Themost favorable result was using TNBS, where the activity increasedprogressively till reaching a 1500% increase, considering that thispreparation was already 2.5 more active than the native enzyme,the final activity in 0.1% CTAB is almost 5-fold higher using this

modified enzyme than using the native enzyme (in absolute val-ues). The TNBS modification of the aminated enzyme also greatlyimproved the performance of the enzyme in CTAB, with a hyperac-tivation of 8.5-folds at 0.1%. The modification with glutaraldehyde

6 s Bioc

on(aigii

t(

eHpa

aacdtwoitegamTa0eeir

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12 C. Garcia-Galan et al. / Proces

f the aminated enzyme produced that CTAB presented a slight andegative effect on enzyme activity (more than 70% at 0.1% CTAB)Fig. 6b). Amination of the enzymne modified with TNBS produced

decrease on the hyperactivation caused by the CTAB, but the max-mum value was still observed at 0.1% CTAB. The amination of thelutaraldehyde modified enzyme produced a moderate insensitiv-ty of the enzyme to the effects of CTAB (a mild progressive decreasen activity, till reaching 80% at 0.1% CTAB).

Finally, we analyzed the effect of a nonionic detergent onhe enzyme activity of the different preparations, Triton X-100Figs. 4c, 5c, and 6c).

Octyl-Lecitase progressively decreased the activity in the pres-nce of Triton (activity is only a 30% in 0.1% detergent (v/v)) (Fig. 4c).owever, CNBr-Lecitase significantly increased the activity in arogressive way when Triton was added to the medium (hyper-ctivation become almost 3 times at 0.1% detergent) (Fig. 4c).

Using octyl-Lecitase (Fig. 5c), the amination reduced the neg-tive effects of this detergent on enzyme activity (70% of residualctivity at 0.1%), glutaraldehyde modification provoked that lowoncentrations had no effect on enzyme activity and only 0.1% pro-uced a clear decrease on enzyme activity, while TNBS made thathe enzyme activity rapidly decreased in presence of this detergent,ith a 25% of residual activity at 0.1% Triton. Curiously, amination

f this TNBS modified preparation permitted to observe a certainncrease in enzyme activity in the presence of low Triton concen-ration (140% using 0.001% detergent), and reduced the negativeffects of higher detergent concentrations (50% using 0.1% deter-ent) (Fig. 5c). Amination of glutaraldehyde modified enzyme hadlmost no influence on the effect of Triton X-100 on glutaraldehydeodified enzyme activity. On the other hand, the modification with

NBS of the aminated enzyme permitted to have a certain hyper-ctivation at 0.001 and 0.01% detergent (around 1.4-folds), but at.1% the relative activity was very similar to the solely aminatednzyme. The modification with glutaraldehyde of this aminatednzyme, however, permitted to keep unaltered the enzyme activ-ty in all the range of [Trinton X-100] studied, becoming the oneetaining more activity at 0.1% (Fig. 5c).

Using the covalent preparation (Fig. 6c), amination permittedo obtain a similar enzyme performance at low detergent concen-ration, while hyperactivation at higher concentrations is lower.lutaraldehyde modification caused a drastic decrease on enzymectivity in the presence of this detergent (15% at 0.1% detergent),hile TNBS modification almost left the effect of this detergent on

nzyme activity unaltered (Fig. 6c). Amination of glutaraldehydeodified enzyme partially reverted the very negative effect of the

lutaraldehyde modification, while the amination of TNBS modifiednzyme produced a lack of effect of Triton on all the range of studiedoncentrations. TNBS modification of aminated enzyme promotedhat Triton recovered the effect on enzyme activity found for thenmodified enzyme or just TNBS modified enzyme, reverting the

ack of effect of the amination. However, glutaraldehyde modifica-ion of the aminated enzyme increased the negative effect of Triton-100 on enzyme activity (Fig. 6c).

The results suggest that the positive effects of the detergent maye not only based on the stabilization of the open form of lipase,ut also on some “global” conformational changes, as in some caseshe octyl preparation showed hyperactivation in the presence ofetergents, lower than that of CNBr but quite significant still.

The use of enzymes enriched in cationic groups or with moreydrophobic groups pending on its structure greatly modifies theffect of the detergents. However, rules cannot be observed. Ami-ation of enzymes should increase the number of SDS molecules

hat interacts with the enzyme, making the interaction of theationic detergent more difficult. This could produce effects sim-lar to those of the SDS using much lower concentrations whileequiring higher amounts of CTAB. However that trend is not

hemistry 49 (2014) 604–616

generally observed. TNBS should produce a similar effect with alldetergents as new hydrophobic groups are introduced. However, insome instances this modification reduces, while in other instancedincreases, the effects of the detergents on enzyme activity. Glu-taraldehyde should increase the enzyme rigidity (if some inter orintramolecular crosslinkings are formed) [47,54] and that can makethe enzyme less responsive to the action of the detergents. How-ever, in this study, this has been observed in some cases whilein other cases glutaraldehyde modification increased mainly thenegative effects for the detergents.

3.5. Thermal stability

Massive chemical modification of the protein surface may pro-duce some unexpected changes in enzyme stability [30]. Thosechanges on enzyme stability may depend on the inactivation con-ditions (e.g., pH value), mainly considering the changes in enzymeionization or hydrophobicity/hydrophobilicity balance [29]. Table 2shows the half-lives of the different preparations under differentconditions. Temperature was selected accordingly to the stabilityof the enzyme under the different pH values.

At pH 7 and 55 ◦C, the covalent preparation is slightly more sta-ble than the octyl-Lecitase (half lives of 63 and 48 min), while atpH 5 and 50 ◦C, octyl-agarose is much more stable (half lives of60 and 267 min) and at 53 ◦C and pH 9, both preparations stabil-ities are almost identical (around 40 min). It has been describedfor many immobilized lipases that interfacially immobilized lipasesare more stable than even lipases immobilized via multipoint cova-lent attachment [61], and the CNBr preparation has been preparedin a fashion that should not favor a very intense multipoint cova-lent attachment [25]. However, the effect of the immobilization ofLecitase on octyl-agarose does not seem as positive as for otherlipases, only at pH 5 the octyl preparation is clearly more stablethan the covalent preparation.

Amination increased the half-lives of the octyl-Lecitase (by a 3-to 4-fold factor at pHs 7 and 9, and by 1.5 at pH 5), while the ami-nation of the CNBr-Lecitase had no effect on the enzyme stability(e.g., pHs 7 and 5) or this effect was slightly negative (e.g., pH 9).

Modification with TNBS presented a quite negative effect onenzyme stability using the octyl-Lecitase, mainly at pH 5 (almost a10-fold factor). However, the covalent preparation after the TNBSmodification retained the stability at pH 5, this treatment decreasedit by a 2-fold factor at pH 7, while at pH 9 it was increased by a12-fold factor.

The treatment with glutaraldehyde also produced mixed resultson enzyme stability. Octyl-Lecitase increase the stability by a 15-fold factor at pH 9 and by a 3-fold factor at pH 7, but reducedthe stability by a 2-fold at pH 5. This effect of glutaraldehydeon enzyme stability could be attributed to a poor intramolecularcrosslinking of the enzyme. To check if at least there are someintermolecular crosslinking, the preparation was submitted to aSDS-PAGE (Fig. 7), that revealed many aggregates (protein bandsof an apparent higher molecular weight), confirming that at leastmany molecules were intermolecularly covalently crosslinked. Per-haps the negative effect of the hydrophobization of the Lys chain(already visualized after TNBS modification) could not be reversedby the positive effects of the crosslinking, and it may be that manyintermolecular crosslinkings had not been formed (modificationwas quite rapid, jus 30 min before reduction). Using the CNBr prepa-ration, stability was not altered by glutaraldehyde treatment at pH9, it decreased by a 2-fold factor at pH 7 and increased by a 2-foldfactor at pH 5.

Modification of aminated enzymes with TNBS also gave com-pletely diverse effects. Using aminated octyl-Lecitase, enzymestability is reduced to values similar to those of the TNBS-octyl-Lecitase at all pH values. However, using the CNBr preparation,

C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616 613

Table 2Thermal stability of the different modified enzyme preparations given as half-lives in minutes. Temperatures were 55 ◦C at pH 7, 53 at pH 9 and pH 5 and 49 ◦C at pH 9. Otherspecifications are described in Section 2.

Biocatalyst/pH Chemical modification

None EDA TNBS GLUTA EDA-TNBS EDA-GLUTA TNBS-EDA GLUTA-EDA

Octyl/pH5 267 ± 15 406 ± 20 28 ± 1 120 ± 5 36 ± 1 315 ± 10 65 ± 4 366 ± 20Octyl/pH7 48 ± 3 180 ± 10 22 ± 1 154 ± 10 26 ± 1 191 ± 10 34 ± 2 279 ± 20Octyl/pH 9 37 ± 3 112 ± 9 22 ± 12 511 ± 30 27 ± 4 278 ± 15 37 ± 2 239 ± 15

± 1

± 2

± 3

sttumiuo

nhe(ednauHeCmp

tnaap

FbmLg

CNBr/pH 5 60 ± 3 60 ± 4 60 ± 5 11CNBr/pH 7 63 ± 3 56 ± 3 30 ± 2 30CNBr/pH 9 40 ± 3 29 ± 2 542 ± 30 42

tability of the aminated enzyme increased after TNBS modifica-ion (4- to 5-fold factor at pH 7 and 5, 8 times at pH 9), leavinghis double modified enzyme as more stable preparation than thenmodified one by very similar factors. This occurred although theodification was only positive using the native enzyme at pH 9. The

ncrement of TNBS molecules incorporated to the enzyme whensing the aminated enzyme can completely alter the interactionsn the enzyme surface.

If the modification is performed using glutaraldehyde, ami-ated octyl-Lecitase increased the stability by 2.5-folds at pH 9 andad no significant effect at pH neither 5 nor 7. And this occurredven though existed a quite intense intermolecular crosslinkingsee Fig. 7). Curiously, this figure shows that the just aminatednzyme has a higher electrophoretical mobility than the unmo-ified enzyme. Considering that other aminated enzymes haveot showed this behavior [30,37], this higher mobility could bessociated to some intermolecular crosslinking that avoid the fullnfolding of the enzyme molecule (cause by the carbodiimide).owever, it is hard to ensure this even considering the stabilizingffects found in some cases of the amination treatment. AminatedNBr-Lecitase increased the stability after glutaraldehyde treat-ent by a 2-fold factor at pH 7, by 6 times at pH 5 and only a 50% at

H 9.Amination of TNBS modified octyl-Lecitase permitted to par-

ially recover the values of stability at all pH values, but it wasot enough to fully eliminate the negative effect of the TNBS. The

mination of the glutaraldehyde treated octyl-Lecitase is positivet pH 5 (by a 2.5-folds), and 7 (by 1.8-folds), and is negative atH 9 (by 2 times). Using CNBr-Lecitase, the amination of TNBS

ig. 7. SDS-PAGE analysis of different octyl-Lecitase preparations. Experiments haveeen performed as described in Section 2. Lane 1: molecular markers, Lane 2: com-ercial Lecitase suspension, Lane 3: octyl-Lecitase, Lane 4: aminated-Octyl-Lecitase,

ane 5: glutaraldehyde-octyl-Lecitase, Lane 6: aminated octyl-Lecitase treated withlutaraldehyde, Lane 7: glutaraldehyde-Lecitase treated with ethylenediamine.

311 ± 20 364 ± 25 34 ± 3 110 ± 9230 ± 20 120 ± 10 40 ± 3 44 ± 4259 ± 20 41 ± 4 43 ± 2 41 ± 3

modified enzyme produced a slight stabilization at pH 7, but atpH 5 the stability decreased by almost a 2-fold factor and alsoeliminated all the positive effects if TNBS modification at pH 9, infact, the final preparation is similar in stability to the unmodifiedpreparation.

Thus, as in activity, the effects of the chemical modificationon the enzyme stability depend on the immobilization strategyemployed and on the previous modifications performed, and it isnot possible to give any general rule. Even glutaraldehyde crosslink-ing has not always a positive effect on enzyme stability.

Glutaraldehyde, even though intermolecular crosslinking maybe detected and some intramolecular crosslinking cannot be dis-carded, has only a moderate positive effect on enzyme stability inmany inactivation conditions (no giving stabilization factors higherthat 10-folds), and even in certain cases the effect of the modifica-tion was found to be negative.

3.6. Specificity of the different preparations

Chemical modification of enzymes has proved to be a tool toalter the enzyme specificity [29,30]. Thus, we have evaluated theactivity of the different enzyme preparations versus some othersubstrates under different conditions and Table 3 shows the activityof the different preparations versus R methyl mandelate and methylphenylacetate.

The first point to be observed is that the two immobilizedpreparations already presented a very different pattern. Whileoctyl-Lecitase is much more active versus pNPB (see Table 1),CNBr-Lecitase has 5 (versus methyl phenylacetate) and 8 (versus R-methyl mandelate) times more activity that the octyl preparation.At pH 7 and 8.5 the CNBr preparation gave almost the same activity,while at pH 5 the activity decreased about 6-folds for methyl man-delate and only about 10% for methyl mandelate. Octyl-Lecitasehas clearly more activity at pH 7 than at pH 8.5 (activity decreasesaround a 20–30%) and than at pH 5 (activity decrease by a 60%),for both substrates. This pH/activity curve is completely differentto that found using pNPB (see above).

The strong changes in lipase specificity and on the influence ofthe experimental variables of the lipases after immobilization havebeen already described in many papers [24] and may be expectedwhen comparing two preparations of Lecitase as different as thetwo used in this study, but differences are much larger than thoseusually found.

Using this “dissimilar” starting material, the effects of the chem-ical modifications have been analyzed.

Amination of the CNBr-Lecitase preparation produced a strongdecrease in enzyme activity at pH 7 and 8.5 versus R methyl man-delate (to 15%), at pH 5 the effect is also negative but more limited(50%). This is quite different to the result using pNPB, where the

more negative effect was at pH 5 and at alkaline pH, the effectwas even positive. Using methyl phenylacetate, the activity wasnot detectable under the experimental conditions at any pH value(less than 1% of hydrolysis after 24 h of reaction). The amination of

614 C. Garcia-Galan et al. / Process Biochemistry 49 (2014) 604–616

Table 3Activity of different Lecitase preparations versus different substrates. Experimental details may be found in Section 2. RM, R methyl mandelate; MPA, methyl phenylacetate.XXX, activity too low to be determined. Activity is given as U/mg.

Biocatalyst/pH/substrate(R/S activity ratio)

Chemical modification

None EDA TNBS GLUTA EDA-TNBS EDA-GLUTA TNBS-EDA GLUTA-EDA

Octyl/pH5/R-MM 7.5 ± 0.5 1.0 ± 0.09 1.3 ± 01 18.4 ± 1 2 ± 0.1 4.8 ± 0.03 2.2 ± 0.15 2 ± 0.01(VR/VS) (0.82 ± 0.1) (0.72 ± 0.1) (0.77 ± 0.1) (4.6 ± 0.4) (0.38 ± 0.07) (0.74 ± 0.1) (0.77 ± 0.1) (0.61 ± 0.1)Octyl/pH7/R-MM 19.7 ± 2 12.7 ± 1 2.5 ± 0.1 3.2 ± 0.2 3.7 ± 0.1 23 ± 1 2.7 ± 0.2 24 ± 2(VR/VS) (0.73 ± 0.1) (0.4 ± 0.07) (0.72 ± 0.1) (1.1 ± 0.1) (0.38 ± 0.05) (0.48 ± 0.07) (0.83 ± 0.1) (0.71 ± 0.1)Octyl/pH 8.5/R-MM 13.7 ± 2 12.1 ± 0.9 4.8 ± 0.3 8.7 ± 0.3 8.6 ± 0.4 11.6 ± 1 8.2 ± 1 7 ± 1(VR/VS) (0.68 ± 0.1) (0.37 ± 0.05) (1.2 ± 0.2) (1.3 ± 0.1) (0.52 ± 0.7) (0.5 ± 0.5) (0.76 ± 0.15) (0.49 ± 0.06)Octyl/pH5/MPA 0.15 ± 0.02 0.24 ± 0.02 0.78 ± 0.05 0.21 ± 0.02 0.7 ± 0.1 0.96 ± 0.07 0.69 ± 0.02 0.36 ± 0.03Octyl/pH7/MPA 0.4 ± 0.05 XXX 0.63 ± 0.05 0.18 ± 0.01 0.99 ± 0.1 0.65 ± 0.04 0.57 ± 0.02 0.36 ± 0.03Octyl/pH 8.5/MPA 0.22 ± 0.03 0.19 ± 0.02 0.52 ± 0.03 0.14 ± 0.01 0.64 ± 0.05 0.63 ± 0.02 0.36 ± 0.02 0.34 ± 0.02CNBr/pH 5/R-MM 24 ± 3 22.3 ± 1 10.6 ± 0.5 36.2 ± 2 2.1 ± 0.2 4.8 ± 0.3 2.2 ± 0.1 21.2 ± 0.9(VR/VS) (0.64 ± 0.1) (0.99 ± 0.1) (0.98 ± 0.1) (0.7 ± 0.1) (0.38 ± 0.05) (0.41 ± 0.05) (0.52 ± 0.1) (0.55 ± 0.1)CNBr/pH 7/R-MM 160 ± 10 22.3 ± 1 142.4 ± 12 125 ± 10 23.6 ± 2 17.8 ± 1.5 25.6 ± 2 10.9 ± 1(VR/VS) (0.93 ± 0.1) (1 ± 0.1) (0.96 ± 0.1) (0.83 ± 0.15) (0.38 ± 0.04) (0.71 ± 0.5) (1.3 ± 0.15) (1.4 ± 0.1)CNBr/pH 8.5/R-MM 143 ± 10 19 ± 2 542 ± 30 126 ± 13 26.2 ± 2 15.1 ± 1.5 21.5 ± 2 101.8 ± 3(VR/VS) (1,22 ± 0.15) (1.2 ± 0.1) (0.96 ± 0.1) (0.76 ± 0.1) (0.52 ± 0.1) (0.9 ± 0.15) (1.15 ± 0.15) (1.2 ± 0.1)

1.83 ±0.62 ±0.71 ±

tboaiav

eaitahv7tapw

vaLm8

dseUTsti5ah

pTwi

CNBr/pH 5/MPA 1.7 ± 0.2 XXX 0.77 ± 0.05

CNBr/pH 7/MPA 2.2 ± 0.1 XXX 0.75 ± 0.03

CNBr/pH 8.5/MPA 2.0 ± 0.15 XXX 0.77 ± 0.04

he octyl-Lecitase increased the activity versus methyl mandelatey a 30% at pH 5 and decreased it at pH 7 (by a 30%) and pH 8.5 (bynly a 10%). Using as substrate methyl phenylacetate, the activityt pH 7 was so low that it was not possible to detect it, but activityncreased compared to the unmodified enzyme at pH 5 by a 50%nd decreased at pH 8.5 by a 10%. Again, using pNPB, results wereery different, with increments of activities under all conditions.

Modification of CNBr-Lecitase with TNBS had almost no effect onnzyme activity versus methyl mandelate at pH 7, while at pH 5 thectivity decreased more than a 2-fold factor and at pH 8.5 the activ-ty is reduced by around 30%. Using methyl phenylacetate, at pH 7here was neither a significant effect, while at pH 8.5 and pH 5 thectivity decreased to around 35/45%. The modification with TNBSas an even more negative effect on octyl-Lecitase on the activityersus methyl mandelate, decreasing the activity at pH between

and 5 by a 6- to 8-fold factor while at pH 8.5 this decrement iso 40%. However, using methyl phenylacetate, a clear increment ofctivity is observed at the 3 pH values, with the new optimal pH atH 5 (here the increment of activity is by a 5-fold). Again, resultsere very different using pNPB.

Modification with glutaraldehyde of CNBr produced neitherery relevant increment nor decreases in activity (no more than

50%), depending on the pH and the substrate. Using the octyl-ecitase preparation, at pH 5 activity decrease to 45% using methylandelate while increase a 40% using methyl phenylacetate. At pH

.5 the activity for both substrates is reduced to 60–70%.Amination of TNBS-CNBr-Lecitase produced a very significant

ecrease of the biocatalyst activity versus methyl mandelate, butlightly smaller than the amination of the unmodified immobilizednzyme at pH 7 and 8.5 while at pH 5 the activity decrease to 10%.sing methyl phenylacetate as substrate, at pH 5 the activity of theNBS modified enzyme increased after the amination to becomeimilar to that of the unmodified enzyme, while at pH 7 and 8.5,he activity decreased. Using the octyl TNBS preparation, aminationncreased the activity versus methyl mandelate by 1.7 times at pH

and 8.5, and only by a 10% at pH 7, using methyl phenylacetatectivity slightly decrease in all the range of studied pHs, with aigher decrement at pH 8.5 (by 30%).

The amination of the glutaraldehyde modified CNBr-Lecitase

roduced a slight decrease in activity versus both substrates.he situation was different using glutaraldehyde octyl-Lecitase,here using methyl mandelate a 30% increment of enzyme activ-

ty was detected at pH 7 while activity decreased at pHs 5 and 8.5.

0.1 1.8 ± 0.2 0.27 ± 0.02 1.51 ± 0.1 1 ± 0.05 0.05 0.62 ± 0.05 0.51 ± 0.03 1.43 ± 0.1 1.8 ± 0.08 0.1 0.71 ± 0.02 0.37 ± 0.02 0.62 ± 0.04 1.6 ± 0.03

However, using methyl phenylacetate, the activity increased by a2- to 2.3-fold factor after the amination of the modified enzyme.

TNBS modification of aminated CNBr-Lecitase slightly improvedthe activity versus methyl mandelate at pH 7 and 8.5, while hav-ing a very negative effect at pH 5 (by a 6-fold factor). Using methylphenylacetate, it is now possible to detect again the enzyme activ-ity, and at pH 5 is even higher than that of the unmodified enzyme.Aminated octyl-Lecitase after modification with TNBS decreasedthe activity versus methyl mandelate, mainly at pH 5 and 7, how-ever the activity versus methyl phenylacetate increased, becomingthe most active octyl preparation versus this compound at pH 7and 8.5 (at pH 5 is alike with just the TNBS modified biocatalystin second position). TNBS modification of the immobilize enzymealready provided good hyperactivation versus this compound.

Glutaraldehyde treatment of aminated CNBr-Lecitase has a neg-ative effect on the activity of the enzyme versus methyl mandelate,to 35% in the most drastic case (pH 5). Using methyl mandelate,activity is detected again, but with values lower than 25% of theunmodified preparation. Using aminated octyl-Lecitase, the situa-tion is quite different. Activity increased using methyl mandelateat pH 7 (by a 80%) and decreased at pH 5 (by a 50%), while at pH8.5 there was no significant change. The most relevant changes arein the activity versus the ester of the phenylacetic acid, now theactivity is detectable under all the range of pHs and even is themost active octyl preparation at pH 5.

3.7. Enantiospecificity of the different preparations

Table 3 also shows the VR/VS ratios. As the reactions is not oforder 1, it is not directly associated to enantiospecificity, but is stillvalid to detect changes in the activity of the enzyme versus differentenantioisomers.

CNBr-Lecitase has very similar activity using both isomers atpH 7, at pH 5 the S isomer was preferred (0.65) and at pH 8.5 the Risomer was preferred (1.2). The octyl preparation hydrolyzed fasterthe S isomer at the 5 pH values, but with poor VR/VS ratio (bestresults was 0.68 at pH 8.5). Thus, both Lecitase preparations werealmost not enantioselective.

Even in these unfavorable conditions, the chemical modification

shows that some improvements could be achieved if using a batteryof modifications is used. CNBr-Lecitase improved the specificity forthe S isomer after amination and further TNBS modification (to0.38) at pH 7, while amination of the TNBS and glutaraldehyde

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odified enzyme produce preparations with some significant pref-rence for the R isomer (1,3/1,4). At pH 5, the most relevant result is

ratio of 4.7 for the glutaraldehyde modified enzyme and of 0.38 forhe aminated and TNBS modified enzyme. This last was again thene with a more significant ratio at pH 8.5, 0.5 (while the unmodi-ed enzyme preferred the R isomer). Using the octyl-Lecitase, onlyhe aminated enzyme at pH 7 (ration 0.4) and at pH 8.5 (ratio 0.38)mproved the preference for the S isomer. The only octyl prepa-ation with a significant preference for the R isomer is the TNBSodified enzyme (1.2).

. Conclusions

The results presented in this paper shows that the propertiesf Lecitase may be easily altered via immobilization or chemicalodification. The activity, specificity or stability, even the effect

f the detergents on enzyme activity may be modulated by theseechniques. However, it has been shown that trying to give generalules to rationalize the effects is very complicated with the availablenowledge, the effect of a chemical modification is very differentepending on the immobilized preparation used. Moreover, theffect of the experimental conditions on the enzyme features is alsoltered via immobilization and chemical modification: a change inhe experimental conditions that have a positive effect on enzymeeature in one biocatalyst may have a negative effect using othermmobilized preparation with the same modification.

Thus, immobilization and chemical modification seem to be aowerful to improve the Lecitase properties, although currentlyhis will be based in a trial and error strategy, the final results veryikely may be positive if the battery of biocatalysts is large enough62].

cknowledgments

We gratefully recognize the support from the Spanish Govern-ent, grant CTQ2009-07568, grant VIE cod 5706 (UIS-Colombia)

nd CNPq (Brazil). The predoctoral fellowships for Ms. García-GalánSpanish Government) and Mr dos Santos (CNPq, Brazil) are alsoecognized. Dr Pereira thanks Fundación Carolina and UNIFAL-MG.he authors wish to thank Mr. Ramiro Martínez (Novozymes, Spain)or kindly supplying the enzymes used in this research. The helpnd comments from Dr. Ángel Berenguer (Instituto de Materiales,niversidad de Alicante) are kindly acknowledged.

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