Interfacially activated lipases against hydrophobic supports: Effect ofthe support nature on the biocatalytic properties

Gloria Fernandez-Lorente b, Zaida Cabrera a, Cesar Godoy a,Roberto Fernandez-Lafuente a, Jose M. Palomo a,*, Jose M. Guisan a,*aDepartamento de Biocatalisis, Instituto de Catalisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, SpainbDepartamento de Microbiologıa, Instituto de Fermentaciones Industriales, c/Juan de la Cierva 3, 2006 CSIC, Madrid, Spain

1. Introduction

Lipases are the most used enzymes in biocatalysis and organic

chemistry [1–5]. They recognize a wide variety of substrates while

exhibiting high regioselectivity and enantiospecificity in many

instances [6,7]. Lipases present a specific catalytic mechanism of

action, existing in two structural forms, the closed one, where a

polypeptide chain (lid or flat) isolates the active center from the

medium, and the open form, where this lid moves and the active

center isexposed[8–10].Thisequilibriumisshiftedtowardstheopen

form in the presence of hydrophobic surfaces (e.g., droplets of oils),

where the lipasebecomes adsorbedby the largehydrophobic pocket

around their active center and the internal face of the lid [11,12].

Moreover, lipases may become adsorbed to other hydrophobic

surfaces followinga similarmechanism:hydrophobic proteins – like

hydrophobins–[13],other lipasemolecules [14–17]oronthesurface

of hydrophobic supports [18–20]. This particular mechanism of

catalysis has permitted to develop specific protocols for the

immobilization of lipases. Thus, the immobilization of lipases via

interfacial activation on hydrophobic supports at low ionic strength

has been reported to be a very simple and efficient method to

immobilize and purify lipases [18]. This protocol fixes the open form

of lipases via interactions between the hydrophobic surroundings of

their active centre – the internal face of the lid and the area of the

lipase around the active center that interacts with it – and the

hydrophobic surface of the support. These immobilized biocatalysts

are very active against hydrophobic substrates having small-

medium size, even showing higher activity than that of the soluble

enzymewhenactingonfullysolublesubstrates[21].Moreover, these

immobilized enzyme preparations are very stable under different

experimental conditions [18]. The adsorption of the lipase on the

support is quite strong, but it is still reversible, permitting to recover

and reuse the support after enzyme inactivation [18].

Process Biochemistry 43 (2008) 1061–1067

A R T I C L E I N F O

Article history:

Received 25 March 2008

Received in revised form 16 May 2008

Accepted 19 May 2008

Keywords:

Interfacial activation

Modulation of lipase properties

Lipase purification

Hydrophobic supports

Lecitase

A B S T R A C T

Different lipases (lipase B from Candida Antarctica, CAL-B, lipase from Thermomyces lanuginose, TLL and

lipase from Bacillus thermocatenulatus, BTL) and a phospholipase (Lecitase1 Ultra) were immobilized by

interfacial activation on four different hydrophobic supports (hexyl- and butyl-toyopearl and butyl- and

octyl-agarose) and their properties were compared. The results suggested that selection of different

supports yielded very different results in terms of recovered activity (ranging from a sevenfold

hyperactivation to almost fully inactive biocatalysts), stability, specificity and adsorption strength. Even

more interestingly, the enantioselectivity of the enzymes in the hydrolysis of (�)-2-O-butyryl-2-

phenylacetic acid was strongly dependent on the support utilized. For example, BTL immobilized on octyl-

agarose was fully enantiospecific towards the hydrolysis of (S)-2-O-butyryl-2-phenylacetic acid (E > 100),

whereas when immobilized on hexyl-toyopearl, the enantiomeric value of the immobilized lipase was only

E = 8. However, there is not an optimal support; it depends on the lipase and on the studied parameter. In the

asymmetric hydrolysis of phenylglutaric acid diethyl diester, BTL immobilized on hexyl-toyopearl was the

most enantioselective catalyst with ee > 99% (A factor >100) in the production of S-monoester product,

whereas the enzyme immobilized on butyl-toyopearl only exhibited an A factor of 3.

Finally, butyl-agarose was chosen as the most specific support on the lipase adsorption – compared to

other proteins – at low ionic strength yielding the best purification of BTL from crude preparations.

ß 2008 Elsevier Ltd. All rights reserved.

* Corresponding authors. Tel.: +34 91 585 4809; fax: +34 91 585 4760.

E-mail addresses: josempalomo@icp.csic.es (J.M. Palomo), jmguisan@icp.csic.es

(J.M. Guisan).

Contents lists available at ScienceDirect

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1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2008.05.009

Following this immobilization mechanism, the active center of

the lipase is in close contact with the support surface, which will

generate a hydrophobic environment around the active center.

Thus, changes in the support nature (hydrophobicity, internal

morphology, etc.) could alter the strength of the lipase adsorption

on the support and even the structure of the active center

surroundings and, therefore, the final lipase catalytic features. It

has been reported that due to the large changes in the lipase

conformation during catalysis, it is quite simple to modulate their

catalytic properties during immobilization (varying the orienta-

tion, rigidity, microenvironment, etc.), greatly altering the

enantiospecificity [22–27] or regioselectivity [28,29] of the

immobilized enzymes.

Here, the results of a study of the support effects on the

properties of lipases (selectivity, specificity, activity, etc.) are

presented. In this case, the immobilization of lipases follows the

same mechanism: interfacial activation against hydrophobic

supports [18]. The lipase active center will be in very close contact

with the support surface. However, considering the hydrophilic

nature of the lipases out of the active center surroundings, scarce

additional influences of the lipase with the support should be

expected. The close contact between the support and the active

center may produce large differences in the final structure of the

enzyme active center and, therefore, on the final enzyme catalytic

properties. Thus, the nature of the support that produces that

interfacial activation could determine the final properties of the

immobilized enzyme.

Moreover, the selectivity in the protein adsorption on the

different supports and the adsorption strength of the lipase to the

different supports has been analyzed. Two supports with very

different internal morphology were used.

Agarose beads are formed bywide trunks [30]. Compared to the

lipase size, an almost planar surface will be available to interact

with the enzyme. Agarose is very hydrophilic, but if coated with

octyl or butyl chains, it offers a hydrophobic surface suitable for the

lipase interfacial activation [18]. Toyopearl is an acrylic support

formed by amild crosslink (supplier information), giving quite thin

fibers, even smaller than the size of a lipase molecule. The support

matrix has a certain hydrophobic character that may be reinforced

by coating the fibers surface with butyl or hexyl groups. Previous

results suggested that the adsorption of lipases to a hydrophobic

support may be driven by reasons more complex that the mere

hydrophobicity of the support. Usually, the more hydrophobic the

support, the higher the amount of lipases that becomes adsorbed

on it [18]. However, in some cases the use of moderately

hydrophobic supports permits the adsorption of lipases that did

not adsorb on supports with high hydrophobicity, for example

pancreas porcine lipase did not adsorb on octadecyl Sepabeads or

octyl-agarose but it was strongly adsorbed on phenyl agarose (a

support with lower hydrophobicity) [31]. Thus, a more systematic

study may help to understand on the reasons for the adsorption of

lipases on hydrophobic supports.

In this study, we have used three different lipases: those from

Bacillus thermocatenulatus (BTL) [14], Candida antarctica B (CAL-B)

[32,33] and from Thermomyces lanuginose (TLL) [34]. Moreover, we

have included in this study a commercial phospholipase, Lecitase1

Ultra, which is used in degumming processes [35] and that has

been described to behave as a lipase in many aspects. This enzyme

presents a certain interest in fine chemistry [36,37].

2. Materials and methods

2.1. Materials

Lecitase1 Ultra, CAL-B and TLL were obtained from Novozymes (Denmark).

Butyl- and octyl-agarose 4BCL was purchased from GE healthcare (Uppsala,

Sweden). BTL cloned in E. coliwas produced as previously described [14]. Butyl- and

hexyl-toyopearl were from Tosoh Corporation (Tokyo, Japan). R/S mandelic acid,

Triton X-100, hexadecyltrimethylammonium bromide (CTAB), and p-nitrophenyl

butyrate (pNPB), were from Sigma. 2-O-Butyryl-2-phenylacetic acid (1) was

synthesized as previously described [38].

2.2. Lipase activity determination

The standard assay was performed by measuring the increase in absorbance at

348 nm (isoblastic point of p-nitrophenol) produced by the releasing of p-

nitrophenol in the hydrolysis of 0.4 mM p-nitrophenyl butyrate (pNPB) in 25 mM

sodium phosphate at pH 7 and 25 8C, using a thermostatized spectrophotomer with

magnetic stirring. To start the reaction, 0.1 mL of lipase solution or suspension was

added to 2.5 mL of substrate solution. An international unit of pNPB activity is

defined as the amount of enzyme necessary to hydrolyze 1 mmol of pNPB/min (IU)

under the conditions described above.

2.3. Lipase immobilization

Ten grams of support were added to 100 mL of lipase solution (0.01 mg prot/mL

or 1 mg/mL) in 5 mM sodium phosphate pH 7 at 25 8C under very mild stirring.

Periodically, samples of supernatant and suspension were withdrawn and the

activities were determined as described above. After immobilization, the

immobilized enzymes were washed with an excess of distilled water and stored

at 4 8C. The preparations with low enzyme loading, where diffusion problems were

avoided, were used to determine the activity with pNPB and inactivation activity.

The preparations with high enzyme loading were used in the hydrolysis of 2-O-

butyryl-2-phenylacetic acid and phenylglutaric acid diethyl diester.

2.4. Lipase desorption

One gramof immobilized lipase preparationwas re-suspended in 10 mL of 5 mM

sodium phosphate pH7 and 25 8C and detergent (Triton X-100, except for TLLwhere

CTABwas used due to the inhibition caused by Triton X-100 [39]) was progressively

added [18]. The immobilized enzymes were incubated under gentle stirring for

30 min before measuring the enzyme activity in the supernatant. Afterwards,

whenever necessary, new additions of detergent were performed. References with

soluble enzymes submitted to identical treatment were used to determine the

effect of the detergent on the enzyme activity.

2.5. Lipase inactivation experiments

Immobilized enzyme suspensions were incubated at the indicated temperature

and at pH 7. Samples were periodically withdrawn and the activity was determined

as previously described.

2.6. Synthesis of 3-phenylglutaric acid diethyl diester (2)

A solution of ethanol (70 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodii-

mide (3.6 mmol) with dimethylamino-pyridine (0.72 mmol) in diethyl ether (10 mL)

was added drop-wise over a stirred solution of 3-phenylglutaric acid (1.44 mmol) in

diethyl ether (25 mL) The mixture was stirred at 25 8C for 5 h and the reaction was

followed by HPLC. After that, the mixture was extracted with 100 mMNaCl solution

and after re-extraction of the water phase with diethyl ether. The combined organic

solvents were dried with NaSO4 and the solvent was evaporated. The crude extract

waswashed several timeswith cold ether (5� 2 mL) and dried in vacuum. Yield: 98%.1HNMR(400MHzCDCl3):d = 7.33–7.20 (m, 5H, Ph); 4.52 (m,1H,CH), 4.13 (m,4H,2�

CH2), 2.54 (m, 4H, 2� CH2), 1.29 (t, 6H, 2� CH3).

2.7. Enzymatic hydrolysis of substrates 1 and 2

One gram of immobilized lipase (prepared using 10 mg of protein per g of

support) was added to 10 mL of 5 mM (1) in 25 mM sodium acetate at pH 5 and

25 8C or 1 mM (2) in 25 mM sodium phosphate at pH 7 and 25 8C.

During the reaction, the pH value was maintained constant by automatic

titration using a pH-stat Mettler Toledo DL50 graphic. The degree of hydrolysis was

confirmed by reverse-phase HPLC (Spectra Physic SP 100 coupled with an UV

detector Spectra Physic SP 8450) on a Kromasil C18 (25 cm � 0.4 cm) column

supplied by Analisis Vinicos (Spain). At least triplicates of each assay were made.

The elution was isocratic with a mobile phase of acetonitrile (35%) and 10 mM

ammonium phosphate buffer (65%) at pH 2.95 and a flow rate of 1.5 mL/min. The

elution was monitored by recording the absorbance at 225 nm (substrate 1) and

205 nm (substrate 2). The enzymatic activity was measured in mmol of substrate

hydrolyzed per hour per mg of immobilized protein.

2.8. Determination of enantiomeric excess

At different conversion degrees, the enantiomeric excess (ee) of the acid (in the

hydrolysis of 1) or the formed monoester (in the hydrolysis of 2) was analyzed by

G. Fernandez-Lorente et al. / Process Biochemistry 43 (2008) 1061–10671062

chiral reverse phase HPLC. The column was a Chiracel OD-R. In the case of 1, the

mobile phase was an isocratic mixture of 5% acetonitrile and 95% NaClO4/HClO4

0.5 M at pH 2.3 and the analyses were performed at a flow of 0.5 mL/min by

recording the absorbance at 225 nm. For 2, the mobile phase was acetonitrile (25%)

and 10 mM ammonium phosphate buffer (75%) at pH 2.95 and the analyses were

performed at a flow of 0.7 mL/min by recording the absorbance at 205 nm.

2.9. Calculation of E value and A factor

The enantiomeric ratio (E) was defined as the ratio between the percentage of

hydrolyzed R and S isomers (from racemic mixture) at hydrolysis degrees between

15 and 20%.

The asymmetric factor (A) of the producedmonoester isomerswas defined as the

ratio between the percentage of produced R and S isomers of monoester. The

absolute configuration was assigned in agreement with the results of Ostaszewski

and co-workers [40].

3. Results and discussion

3.1. Immobilization of BTL on different hydrophobic supports

After 1 h, BTL had been fully immobilized on all hydrophobic

supports. In all cases, the effect of the immobilization of the lipase

on its activity against pNPB was positive (Table 1). BTL increased

the activity by a twofold factor when it was immobilized on butyl-

or octyl-agarose supports, or hexyl-toyopearl, and by a 1.5-fold

factor when the support was butyl-toyopearl.

The different adsorbed BTL-preparations were incubated in

growing concentrations of detergents and the amount necessary to

desorb 100% of the lipase was taken as a measure of the adsorption

strength (Table 2). Curiously, BTL adsorbed on both butyl- and

octyl-agarose supports – support having larger surfaces and where

protein-support multi-interactions could be more easily obtained

– could be desorbed from the supports at the lowest concentration

of detergent. However, toyopereal adsorbed more strongly the

enzyme, although the fibers are very thin and it is difficult to

establish many enzyme–support interactions (Table 2).

It should be considered that themechanism of adsorption of the

lipases in hydrophobic supports is not via a conventional

hydrophobic adsorption (where the surface offered by the support

to give multiple interactions may be a key to define the strength of

the adsorption) [22]. Now, the only part of the lipase that needs to

interact with the support is the large hydrophobic pocket around

the active center of the lipase [18]. Thus, agarose offers a plane for

the interaction with the lipase [30], but has a reduced geometrical

congruence with themore or less internal pocket formed by the lid

and the hydrophobic areas around the active center and the layer

of hydrophobic groups in the support. The use of toyopearl

supports made it necessary to double the concentration of

detergent to release the lipase from the support, suggesting a

much stronger interaction between the thin fibers that compose

this support and the lipase. It may be assumed that the

hydrophobic pocket of the lipasemay present a higher geometrical

congruence with these thin fibers than with large planar surfaces,

involving more groups of the enzyme in the adsorption that may

adapt to these thin fibers. In none of the supports utilized, was

possible it to detect a relevant effect of using more hydrophobic

moieties coating the support. In fact, in both cases, the BTL

adsorbed on the supports activated with the less hydrophobic

group required the higher amount of detergent to be desorbed.

Results suggest that not only the hydrophobicity of the support

surface but also the exact morphology of the support surface may

define the adsorption of a lipase on a support [31].

The thermal stability of the different BTL preparations was

again quite dissimilar. Octyl-agarose-BTL was the most stable

preparation, being the secondmost stable butyl-agarose-BTLwhile

both toyopearl preparations presented very similar stability

(Fig. 1).

Next, the hydrolysis of (�)-2-O-butyryl-2-phenylacetic acid (1)

(Scheme 1) by the different BTL preparations was studied (Table 3).

All the BTL preparations presented very similar activity value.

Curiously, the octyl-agarose-BTL preparationwas the only one having

around 60% of the activity (in the case of pNPB this preparation was

the most active). Although all the enzymes preparations preferred to

hydrolyze (S)-1, the agarose-BTL preparations were the most

enantiospecific in this reaction, with a E value over 100 using

octyl-agarose-BTL and over 60 using butyl-agarose-BTL. The E values

for the other preparations were much lower. The different BTL

preparations were also assayed against di-ethyl phenylglutarate (2)

(Scheme 1), a prochiral diester (Table 4). The activities against this

substratewere two orders of magnitude lower than using substrate 1,

and the specific activities were quite similar for all preparations,

ranging from 0.0010 U/mg protein (using butyl-agarose-BTL) to

Table 1

Effect on the enzyme activity of the immobilization of BTL on different hydrophobic

supports

Support Relative activity (%)a

Octyl-agarose 200

Butyl-agarose 200

Hexyl-toyopearl 200

Butyl-toyopearl 150

a Relative activity considers 100 the activity of the soluble enzyme.

Table 2

Amount of detergent necessary to fully release BTL adsorbed on different

hydrophobic supports

Support Triton X-100 (%)

Octyl-agarose 0.2

Butyl-agarose 0.3

Hexyl-toyopearl 0.4

Butyl-toyopearl 0.6

Experimental conditions are described in Section 2.

Fig. 1. Inactivation of different preparations of Bacillus thermocatenolatus lipase.

Inactivations were performed at pH 7 and 50 8C. Squares, octyl-sepharose-BTL;

circles, butyl-sepharose-BTL; triangles, butyl- or hexyl-toyopearl-BTL.

Scheme 1. Model compounds used to evaluate the selectivity of different lipase

preparations. Racemic 2-O-butyryl-2-phenylacetic acid (Rac 1); 3-phenylglutaric

acid diethyl diester (2).

G. Fernandez-Lorente et al. / Process Biochemistry 43 (2008) 1061–1067 1063

0.0015 U/mg protein (using octyl-agarose-BTL), with the activities

displayed for both toyopearl preparations in between. The differences

in enantioselectivity values were much higher. For example, the

butyl-toyopearl-BTL gave only an asymmetric factor value of 3, while

hexyl-toyopearl-BTL permitted a value over 100, producing only the

(S)-monoester product.

Therefore, the results showed clear differences in the behaviour

of BTL depending on the hydrophobic support employed. In all

cases the enzyme was immobilized via interfacial activation on a

hydrophobic support, but the change in the internal morphology

(plane versus thin fiber) or the groups coating the support may

greatly alter the enzyme properties. However, the effects are not

easy to predict, the optimal catalyst depends on the specific

parameter studied, in some aspects one preparation was the most

adequate (e.g., octyl-agarose-BTL for the resolution of 1), while in

other cases the best preparation was other (e.g., hexyl-toyopearl-

BTL for the asymmetric hydrolysis of 2). Thus, due to the close

contact between the enzyme active center and the support, it

seems that the active center of the lipase may be easily altered by

just changing the support morphology.

3.2. Immobilization of other lipases on different hydrophobic supports

In order to check if these results achieved using BTL were just a

peculiarity of BTL or a more general phenomenon, similar studies

using CAL-B, TLL and Lecitase were performed.

In all cases, immobilization yield of the enzymes was over 80%

in only 1 h. Next, we show the results where the largest differences

were found between the enzymes immobilized on the different

supports.

3.2.1. Effect on enzymatic activity

CAL-B slightly increased its activity when it was immobilized

on octyl-agarose while the immobilization on both toyopearl

supports produced a reduction on the enzyme activity by around

50% (Table 5). TLL increased the activity by a 7.5-fold factor when it

was immobilized on octyl-agarosewhile reducing it to 60%when it

was immobilized on butyl-toyopearl (Table 5). Lecitase increased

the activity by a twofold factorwhen immobilized on octyl-agarose

but the activity was reduced by 50% when the support was butyl-

toyopearl. Thus, octyl-agarose afforded the highest activity for the

three enzymes studied. Immobilization on toyopearl gave the

lowest activities, this could be related to a partial blocking of the

active center of the lipases by an ‘‘excessive’’ congruence between

the thin fibers that form the support and the hydrophobic pocket

formed by the surroundings of the active center of the lipase.

3.2.2. Strength of lipase adsorption on different hydrophobic supports

The support that requires the highest amount of detergent to

desorb TLL was hexyl-toyopearl whereas in the case of Lecitase,

was butyl-toyopearl (Table 6). Toyopearl was the support that gave

the strongest adsorption. This suggested again that there is a better

congruence between the pocket of the lipase and these thin fibers

than with large and plane surfaces, in opposition to any other

immobilization based in multipoint adsorption [22].

The fact that the adsorption strength is different depending on

the lipase used, suggests that the adsorption strength is

determined not only by the support (e.g., hydrophobicity of the

support surface and size of the fiber of the support) but also by the

lipase features (size of the lid and hydrophobic residues number).

All the supports – after the lipase desorption – may be utilized

to immobilize fresh enzyme, permitting their reuse by several

adsorption–desorption cycles without detecting any effect on the

immobilization parameters: immobilization ratio and yield was

maintained, the amount of detergents to desorb the enzyme was

identical, and the catalytic features of the lipase were maintained

within the error limits.

3.2.3. Thermal stability of lipases immobilized on different

hydrophobic supports

All the lipases studied resulted much more stable after

immobilization than in their soluble form [18].

The different lipases exhibited very different results in terms of

thermal stability when the obtained immobilized preparations

using the different supports were compared. For example, TLL

immobilized on hexyl-toyopearl was the most stable, being the

enzyme immobilized on butyl-toyopearl the less stable (Fig. 2).

However, using CAL-B or Lecitase, all the immobilized enzyme

preparations presented almost identical stability (see as an

example in Fig. 3 the results with CAL-B).

Table 3

Effect on the enzyme enantioselectivity of the immobilization of BTL on different

hydrophobic supports in the hydrolysis of substrate 1

Support Activitya E Isomer

Octyl-agarose 0.25 >100 S

Butyl-agarose 0.4 66 S

Hexyl-toyopearl 0.4 8 S

Butyl-toyopearl 0.38 18 S

Other details are described in Section 2.a Activity in mmol� gÿ1

cat �minÿ1 at 15% conversion.

Table 4

Asymmetric hydrolysis of 2 catalyzed by immobilized BTL on different hydrophobic

supports

Support Activitya A factor (S)

Octyl-agarose 0.0015 44

Butyl-agarose 0.0010 29

Hexyl-toyopearl 0.0012 116

Butyl-toyopearl 0.0014 3

Experiments were performed as described in Section 2.a Activity in mmol� gÿ1

cat �minÿ1.

Table 5

Effect on the enzyme activity of the immobilization of lipases on different

hydrophobic supports

Enzyme Support Relative activitya (%)

CAL-B Octyl-agarose 130

Hexyl-toyopearl 40

TLL Octyl-agarose 750

Butyl-toyopearl 60

Lecitase Octyl-agarose 220

Butyl-toyopearl 50

Activity was determined against pNPB as described in Section 2.a Relative activity considers 100 the activity of the soluble enzyme.

Table 6

Amount of detergent necessary to fully release the lipases adsorbed on different

hydrophobic supports

Enzyme Support Detergent Concentration (%)

CAL-B Octyl-agarose Triton X-100 0.2

Butyl-toyopearl 0.2

TLL Octyl-agarose CTAB 0.3

Hexyl-toyopearl 0.8

Butyl-toyopearl 0.4

Lecitase Octyl-agarose Triton X-100 0.2

Butyl-toyopearl 0.6

Experimental conditions are described in Section 2.

G. Fernandez-Lorente et al. / Process Biochemistry 43 (2008) 1061–10671064

Again, depending on the enzyme, the effect of the hydrophobic

support (in this case on the enzyme stability) was completely

different.

3.2.4. Enantioselectivity of lipases immobilized on different

hydrophobic supports

The different lipase immobilized preparations were used in the

hydrolytic resolution of 1. Again, the lipase features depended on

the support used to immobilize it (Table 7).

The octyl-agarose-lipase preparations were the most active

catalysts for all enzymes against pNPB. However, in the hydrolysis

of 1, the adsorption on hexyl-toyopearl permitted to obtain the

most active catalyst for TLL and Lecitase. On the other hand, TLL

and Lecitase immobilized on butyl-toyopearl were almost inactive

against this substrate, although the activity against pNPB was

significantly high.

Even more interesting was the fact that the enantiomeric ratio

(E) of the different preparations also depended on the support

used, CAL-B preferred the isomer R, while the other lipases

displayed a preference towards the hydrolysis of S-isomer.

CAL-B-octyl-agarose preparation showed the highest E value of

60 compared to the enzyme immobilized on butyl-toyopearl

(E = 3). However, TLL and Lecitase presented similar enantioselec-

tivity using the different hydrophobic supports (E = 5–6 by TLL and

E = 8–10 by Lecitase).

3.3. Selectivity of the lipase adsorption

CAL-B, TLL and Lecitasewere quite pure lipase preparations that

became fully purified after immobilization on any of the supports

used in this work, e.g., octyl-agarose [18]. However, BTL extract

was a quite crude preparation and other proteins – apart from the

lipase – were adsorbed on octyl-agarose at low ionic strength,

reducing the selectivity of the adsorption [41].

Here, we have compared the proteins adsorbed on the different

supports. In all cases the purification of the lipase was very high

just during the adsorption step, but Fig. 4 shows that the amount of

contaminant proteins that became adsorbed on the support

increased when changing butyl-agarose, by octyl-agarose or

hexyl-toyopearl, but decreased when butyl-toyopearl was used.

These results suggested that hexyl-toyopearl was slightly less

selective for lipase adsorption that octyl-agarose while butyl-

agarose was significantly the most selective.

4. Conclusions

Lipases may become adsorbed in many different hydrophobic

supports, most likely by interfacial activation. The immobilization

is very efficient, permitting the full and rapid immobilization of the

lipase. The adsorption is strong enough to use the enzyme

preparation as an immobilized one, although the enzyme may be

desorbed by using detergents, therefore, this immobilization

strategy retains the reversibility that is one of the advantages of

physical adsorptions versus covalent immobilization.

However, depending on the nature of the support that produces

the lipase activation, all the properties of the immobilized lipase

changed, and the optimal preparation was different depending on

the parameter or the lipase studied. Considering that all the

Fig. 2. Inactivation courses of different preparations of TLL. Inactivations were

performed at pH 7 and 65 8C. Circles, hexyl-toyopearl-BTL; triangles, octyl-

sepharose-BTL; squares, butyl-toyopearl-BTL.

Fig. 3. Inactivation courses of different preparations of CAL-B. Inactivations were

performed at pH 7 and 65 8C. Circles, octyl-sepharose CAL-B; squares, hexyl-

toyopearl-CAL-B; triangles, butyl-toyopearl-CAL-B.

Table 7

Activity and enantioselectivity on the hydrolysis of 1 of lipases immobilized on

different hydrophobic supports

Enzyme Support Activitya Hydrolyzed isomer Eb

CAL-B Octyl-agarose 17 R 60

Butyl-toyopearl 27 R 3

TLL Octyl-agarose 0.22 S 5

Hexyl-toyopearl 0.24 S 6

Butyl-toyopearl 0.001 S –

Lecitase Octyl-agarose 0.85 S 10

Hexyl-toyopearl 1.25 S 8

Butyl-toyopearl 0.001 S –

Other details are described in Section 2.a Activity was defined as: UI/mgprotein � 10ÿ3. It was calculated at 10–15%

conversion.b E was calculated when 20% of the substrate had been consumed.

Fig. 4. SDS-PAGE of the proteins adsorbed on different hydrophobic supports after

incubating the supports with crude extract of BTL. Lane 1: molecular weight

markers; lane 2: BTL cloned in E. coli crude extract; lane 3: proteins adsorbed on

octyl-agarose; lane 4: proteins adsorbed on hexyl-toyopearl; lane 5: proteins

adsorbed on butyl-toyopearl; lane 6: proteins adsorbed on butyl-agarose.

G. Fernandez-Lorente et al. / Process Biochemistry 43 (2008) 1061–1067 1065

immobilized enzymes become adsorbed on the support via

interfacial activation, the results suggest that the close contact

between the support and the active center of the enzymemay fully

alter the enzyme properties.

Thus, octyl-agarose seemed to be the support that permitted

the higher activity using simple and small substrates [21]. This

could be due to the internal morphology of the support; agarose is

formed by moderately thick trunks of agarose polymers [30] that

reduce the geometrical congruence between the enzyme active

center and the support, and therefore reducing the blockage of the

active center of the enzyme after enzyme adsorption by the

surroundings of the active center. Toyopearl is formed by fine

fibers that permit a higher geometrical congruence between the

pocket formed by active center surroundings and the support,

promoting a more significant blockage of the enzyme active center

by the support [21]. This could explain also that lipases may

become more strongly adsorbed on these supports than in octyl-

agarose, but simultaneously butyl-toyopearl seems to be the most

selective for lipase adsorption.

Finally, the use of different supports to interfacially activate the

lipases permits to change the specificity and enantiospecificity of

the lipases, in some cases in a very significant way. For example,

BTL may be fully enantioselective in the hydrolysis of (�)-2-O-

butyryl-2-phenylacetic acid hydrolyzing the S-isomer when it was

adsorbed on octyl-agarose, while the E value greatly decreased when

it was adsorbed on the other supports utilized in this study and

previously described. In the asymmetric hydrolysis of phenylglutaric

acid diethyl diester, BTL immobilized on hexyl-toyopearl was the

most enantioselective catalyst with ee > 99% (A factor >100) in the

production of S-monoester product, whereas the enzyme immobi-

lized on butyl-toyopearl only exhibited an A factor of 3.

Therefore, even though the hydrophobic supports are able to

immobilize the open form of the lipases, depending on the support

nature, the final features of the immobilized enzyme may be fully

different. That way, the selection of the support may be a critical

step in the design of a lipase biocatalyst.

Acknowledgments

The authors gratefully recognize the support from the Spanish

CICYT (project. BIO-2005-8576). We gratefully recognize CSIC by

I3P contracts for Dr. Palomo (foundedwith FEDER founds), AECI for

a fellowship for Ms. Cabrera and MEC for a Ph.D. fellowship for Mr.

Godoy and for a RyC contract for Dr. Fernandez-Lorente. The author

would also like to thank Mrs. Alicia Palomo D.P.S.I.C (official

translator and proof-reader) for the English proof-reading of the

manuscript. The help and suggestions from Dr. Angel Berenguer

(University of Cambridge) are gratefully acknowledged.

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