C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5

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Influence of the support surface properties on the proteinloading and activity of lipase/mesoporous carbon biocatalysts

M. Quiros, A.B. Garcıa, M.A. Montes-Moran *

Instituto Nacional del Carbon, CSIC, Apartado 73 E-33080 Oviedo, Spain

A R T I C L E I N F O A B S T R A C T

Article history:

Received 1 July 2010

Accepted 20 September 2010

Available online 24 September 2010

0008-6223/$ - see front matter � 2010 Elsevidoi:10.1016/j.carbon.2010.09.037

* Corresponding author: Fax: +34 985 297662.E-mail address: miguel@incar.csic.es (M.A

Three mesoporous carbons were selected as supports for two lipases from Candida antarctica

(CALA and CALB). A commercially available mesoporous carbon (MB) was used as starting

material. Two additional supports were prepared from MB by heat treatment at 1773 K and

oxygen plasma, bringing about materials with marked differences in both textural and

surface chemical properties. Heterogenisation of lipases was performed at different immobi-

lisation pH (4–8 range). The lipase/mesoporous carbons showed an enhancement of the cat-

alytic activity in the kinetic resolution of (±)-1-phenylethanol when compared to that of the

unsupported enzymes. For CALA, enzyme loadings were controlled by enzyme–support elec-

trostatic interactions, whereas adsorption of CALB was ruled by specific interactions related

to the enzyme and support surface chemistry. No direct correlation was found between the

protein loads and catalytic activity. The catalytic activity of CALA/carbon systems was found

to depend on both immobilisation pH and selected support more strongly than CALB biocata-

lysts. Changes observed in the support textural properties affect less to protein adsorption

and catalytic activity. Operational stability tests were carried out with selected biocatalysts,

with no significant activity loss being observed after 10 consecutive cycles of use and

recovery.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Biocatalysis has become a technology of incomparable value,

especially when the synthesis of optically active compounds

is required. Owing to the important technological advances

in the field, enzymes are available as efficient and selective

catalysts for virtually any chemical reaction, either in water

or organic solvents, thus avoiding the environmental impact

and toxicity caused by most chemical catalysts [1–3]. This ex-

plains the increasing demand for optimal biocatalysts by sev-

eral industrial sectors such as pharmaceutical, agrochemical,

food and fine chemicals. However, there are still problems to

successfully meet this demand, mainly related to the lack of

a long time operational stability of the biocatalysts, the

er Ltd. All rights reserved. Montes-Moran).

difficulty for recovering and reusing the native enzyme due

to its solubility in water and/or denaturalization, as well as

their cost. Therefore, the development of an economically

attractive process that allows the heterogenisation of the en-

zyme, preserving its catalytic properties and improving oper-

ational stability and reuse is a challenge of great interest [4–6].

The immobilization of the enzymes in suitable solid supports

comes up as a way to achieve their heterogenisation [4,7].

A great variety of materials have been used as enzyme

supports, both inorganic (clay, silica, alumina, metal oxides)

and organic (natural or synthetic polymers) [5,8]. Mesoporous

materials show unique textural properties that make them

very attractive as enzyme supports, namely pore diameters

which are close to the diameter of the enzyme molecules,

.

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5 407

high pore volumes (up to 1 cm3g�1) and surface areas

(>800 m2 g�1). Furthermore, the materials pore structure can

be tailored to suit the enzyme immobilisation needs [9–12].

Previous studies in the literature about enzyme immobili-

sation on mesoporous silica are extensive [13–15]. However,

only few reports in relation to the use of mesoporous carbon

materials as enzyme supports have been published so far

[16–18]. As compared to silica materials, carbon materials

have superior textural properties, higher water stability and

relatively lower cost [17,19]. In this context, Vinu et al. sug-

gested recently that mesoporous carbons were much better

adsorbents for biocompounds than mesoporous silicates [18].

In this work, different mesoporous carbon materials have

been tested as supports for the immobilisation of lipases by

physical adsorption in aqueous media [4,20,21]. Activated car-

bon beads of controlled size have been preferred over carbon

powders on the basis of biotechnology industry requirements,

among which the shape and size of the support are very

important parameters in relation with handling, process

monitoring, recovery and reuse [4,22,23]. The intended pur-

pose was to obtain, in a simple way, efficient enzyme/carbon

material biocatalysts with a long-life regarding to their opera-

tional stability and reuse. Lipases were selected for immobili-

sation due to their high degree of selectivity in a broad range

of synthetic applications of industrial importance, including

kinetic resolutions, aminolysis, esterification, etc. [2,24,25].

Furthermore, the immobilisation of lipases on mesoporous

materials is scarcely reported [11].

The influence of the textural and surface chemical proper-

ties of the carbon material on the immobilisation of lipases is

discussed with special emphasis on the catalytic activity of

the resulting enzyme/carbon material system. This approach

is stressed all through the paper since an inadequate immobi-

lisation could lead to the enzyme de-activation, even though

high enzyme loadings are eventually achieved. Special inter-

est is also placed on the influence of the immobilisation pH,

which is one of the relevant parameters having an effect on

enzyme heterogenisation by physical adsorption [4,6,7]. The

immobilisation pH determines the net charge of both the en-

zyme and the material surface, thus affecting the enzyme-

support interactions and, consequently, the conformation

adopted by the enzyme on the support. Moreover, it is known

that the enzyme ‘‘remembers’’ the pH of the last aqueous

solution in which it was dissolved [26], so its ionization state

will remain essentially unchanged (unless acids or bases are

present in the reactant solution) once the water has been re-

moved and either the enzyme or the enzyme/support system

is transferred to an organic medium (i.e., for the measure-

ment of their catalytic activity).

Table 1 – Selected properties of CALA and CALB [25,28].

CALA CALB

Dimensions (nm) n.a. 3 · 4 · 5Molecular weight (kD) 45 33Isoelectric point (IEP) 7.5 5–8pH optimum 7 7Interfacial activation Yes No

2. Experimental

2.1. Carbon supports

Commercially available mesoporous carbon beads (MB) of

particle size between 0.5 and 1 mm were used as starting sup-

ports. They are phenolic resin derived activated carbons with

a bi-modal porous texture [27]. The carbon material MB was

heated for 1 h at 1773 K in a graphite electrical furnace, at a

heating rate of 10 K min�1 in flowing argon (MB-1500). Oxida-

tion of MB (MB-LTA) was carried out in an Emitech K1050X

plasma reactor where oxygen was excited using radiofre-

quency (RF) energy (13.56 MHz). The plasma was maintained

at 90 Pa by flowing oxygen into the reaction chamber. During

the treatment, the temperature of the sample was below

323 K. Batches of approx. 1 g of MB were treated using a RF

power of 75 W and 10 min exposure. Three successive treat-

ments were carried out per batch in order to attain an homog-

enous carbon surface oxidation, beads being stirred manually

between treatments.

2.2. Enzymes and reagents

Crude lipases CALA (lyophilised; specific activity of 32 U mg�1

solid) and CALB (lyophilised; specific activity of 40.1 U mg�1

solid) from Candida antarctica were provided by Biocatalytics,

Inc. (Pasadena, CA, USA), stored at 277 K, and used as-re-

ceived. Some characteristics of both enzymes are collected

in Table 1 [25,28].

The (±)-1-phenyletanol and vinyl acetate substrates for the

enzyme catalysed reaction as well as the dried solvent tert-

buthylmethylether (HPLC grade, stored under freshly acti-

vated 0.4 nm molecular sieve) were purchased from Aldrich.

The inorganic salts to prepare the buffers: potassium hydro-

gen phosphate, potassium dihydrogen phosphate, sodium

acetate and TRIS (tris(hydroxymethyl)aminomethane) were

of analytical grade from VWR-Prolabo.

2.3. Carbon supports characterisation

The surface area, pore volume and pore size distribution of

MB, MB-1500 and MB-LTA materials were derived from the

N2 adsorption–desorption isotherms performed at 77 K in a

Micromeritics ASAP 2420 volumetric adsorption system. Prior

to measurement, samples were outgassed overnight by heat-

ing at 523 K under vacuum. Specific surface areas (SBET) were

calculated using the Brunauer–Emmett–Teller (BET) method,

taking 16.2 nm2 for the cross-sectional area of the nitrogen-

adsorbed molecule. Total micropore volumes (VDRN2) were

assessed by applying the Dubinin–Radushkevich (DR) equa-

tion to the suitable adsorption data. Total pore volumes (Vt)

were determined by the amount of N2 adsorbed at

p/p0 = 0.99. Pore size distributions were obtained by applying

the DFT (Density Functional Theory) method to the N2

adsorption isotherms. Water vapour adsorption isotherms

were carried out in a Hydrosorb volumetric system (Quanta-

chrome), at 303 K. Prior to measurements, samples were out-

gassed for 2 h at 353 K. XPS surface chemical analysis of the

carbon supports was carried out in a SPECS Phoibos 100

408 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5

analyser using Mg Ka X-rays (1486.6 eV) at a power of 120 W

and in a residual vacuum of 10�7 Pa. Measurements were

made with the analyser in fixed transmission mode and nor-

mal to the plane of the sample. Analyser pass energy of 80 eV

has been used to collect broad scan spectra (0–1100 eV). The

atomic percentages (atom%) of the different elements present

in the approx. 10 nm upper layer probed by XPS were calcu-

lated from the survey spectra by considering the integrated

areas of the main XPS peaks. Representative samples of the

mesoporous carbon supports (around 150 mg) were tested.

Typical standard deviation of the measurements is within

±0.5 atom% of the reported values. SEM micrographs were ob-

tained in a FE-SEM Ultra Plus (Zeiss). The carbon beads were

placed on a graphite conductive tape adhered to an alumin-

ium stub. No further coating was required. Images were col-

lected using both conventional (out-lens) and in-lens

secondary electron detectors. Zeta potential measurements

of the supports were performed on a Laser Zee Meter (Model

501, Pen Kern Company) following the principle of microelec-

trophoresis. The electrophoretic mobility was converted into

zeta potential according to Smoluchowski’s equation. For

each determination, 0.1 g of sample was dispersed in 200 ml

of each buffered solution (pH 4–8). The suspension was then

magnetically stirred to equilibrium for at least 18–24 h before

measurement.

2.4. Enzyme immobilisation and protein loadingmeasurements

Immobilisation experiments were assayed in buffers at differ-

ent pH values ranged from 4 to 8, namely, 25 mM potassium

phosphate at pH 6 and 7, 25 mM sodium acetate at pH 4

and 5, and 25 mM TRIS pH 8. Parameters such as amount of

support (40 mg), temperature (303 K), buffer ionic strength

(25 mM) and agitation (220 rpm) were set for all experiments.

Preliminary tests were carried out using the material MB as

support in order to establish the amount of enzyme to immo-

bilise, the volume of buffer used and the incubation time. In

view of the activity results obtained, it was decided to per-

form all the adsorption experiments using 14 mg of CALA

and 16 mg of CALB in 2 ml buffer solutions. Incubation time

of the enzyme with the support in the corresponding buffer

was set to 68 h for CALB and 95 h for CALA.

In a typical immobilisation experiment, lipase (14 mg of

CALA and 16 mg of CALB) dissolved in the selected buffer

(2 ml; 25 mM; pH range from 4 to 8) was mixed with the adsor-

bent material (40 mg of carbon support) in a screw top vial.

Prior to its utilisation, the support was subsequently washed

with 1-propanol and water, and finally equilibrated for 1 h

OH

O

OLipase/Carbon material

rac -1

Fig. 1 – Test reaction to evaluate the activit

with the corresponding immobilisation buffer. The mixture

was shaken at 220 rpm on a rotary shaker at 303 K. Samples

from the supernatant solution were withdrawn periodically

for immediate analysis and then returned to the mixture.

The enzyme content of the supernatant was measured using

UV absorption at 280 nm (Shimadzu UV-2401 PC scanning

spectrophotometer). After adsorption equilibrium, the solid

enzyme/carbon material system was removed from the sus-

pension, and the supernatant was analysed for protein con-

centration. The enzyme/carbon material system was then

subjected to different washes: buffer (50 mM), distilled water,

cold acetone and tert-buthylmethylether (the solvent used for

the enzymatic reactions). The enzyme/carbon material sys-

tem was then dried at 303 K in a vacuum oven (80 mbar) for

4–5 h. Once dried, it was either used directly or stored at

277 K for later use. The amount of protein immobilised on

the support was calculated by subtracting the protein concen-

tration in the supernatant remaining after removal of the en-

zyme/carbon biocatalysts, from the concentration of initial

protein in the reaction mixture. The protein concentration

in the initial and final solutions was determined spectropho-

tometrically according to the Bio-Rad protein assay (Bio-Rad

Laboratories., Catalog No. 500-0002), based on the Bradford

dye-binding procedure [29], measuring the UV–Vis absor-

bance at 595 nm. Bovine serum albumin was used as the stan-

dard protein for preparing the calibration curves.

2.5. Activity of the carbon supported biocatalysts

The kinetic resolution of racemic 1-phenylethanol (0.3 mmol;

36.2 ll) using vinyl acetate (0.9 mmol; 83 ll) as acyl donor was

used as model reaction (Fig. 1). The enzyme/carbon material

system was added to a suspension of the substrates in 3 ml

of dry tert-butylmethylether in a screw top vial. The reaction

vial was shaken at 220 rpm on a rotary shaker at 303 K. Ali-

quots (30 ll) were periodically withdrawn and dissolved with

hexane/propan-2-ol (90:10 v/v) to 1 ml final volume, filtered

and then analysed by HPLC as described below in order to

monitor the progress of the reactions. All the experiments

were repeated at least three times to ensure the reproducibil-

ity of the results.

Chiral HPLC analyses were carried out on a Hewlett Packard

Separations module equipped with a Chiralcel-ODH column

(Daicel, 250 · 4.6 mm). Conditions for analyses were as follows,

flow-rate: 1 ml min�1, solvent composition hexane: 2-PrOH

(90:10), temperature: 298 K, UV detection, kmax: 210 nm, reten-

tion times: (S)-1-phenylethanol, tR = 6.45 min, (R)-1-phenyleth-

anol tR = 5.94 min, (S)-1-phenylethyl acetate, tR = 4.45 min,

(R)-1-phenylethyl acetate, tR = 4.22 min.

OH O

O

(S)-1 (R)-2

y of carbon supported lipase catalysts.

0.0 0.2 0.4 0.6 0.8 1.05

10

15

20

25

30

35

Qua

ntity

ads

orbe

d (m

mol

g-1)

Relative pressure (p / p0)

MB MB-1500 MB-LTA

Fig. 2 – N2 adsorption isotherms (77 K) on the carbon supports.

Table 2 – Textural parameters of the carbon supports.

Sample Textural propertiesSBET

(m2 g�1)Vt

(cm3 g�1)VDRN2

(cm3 g�1)VMESO

(cm3 g�1)aMicro(%)b

MB 1294 1.141 0.506 0.635 44MB-1500 894 0.990 0.345 0.645 35MB-LTA 1251 1.111 0.490 0.621 44a Mesopore volume = Vt � VDRN2.b % of Micropore volume (VDRN2) over the total N2 pore volume Vt

(at p/p0 = 0.99).

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5 409

2.6. Recovery and reuse of the biocatalysts

After the first reaction cycle, the lipase/carbon material bio-

catalysts were removed from the reaction media, washed sev-

eral times with tert-butylmethylether in order to extract the

residual products and then dried at 303 K in a vacuum oven

(80 mbar) for 4 h. Once dried, they were either used directly

in the next reaction cycle or stored at 277 K for later use.

3. Results and discussion

3.1. Support surface properties

The nitrogen adsorption isotherms of the three supports are

shown in Fig. 2. As expected, the original MB combines a high

adsorption capacity in the micropore region (p/p0 < 0.1) with a

desorption hysteresis loop, typical of mesoporous materials.

Textural parameters of MB (Table 2) show relatively high mi-

cro and mesopore volumes. LTA oxidation of MB causes little

changes in the textural parameters, differences being within

experimental error (Table 2). Heat treatment reduces signifi-

cantly the BET surface area at the expense of the micropore

volume, i.e., mesopore volume of MB remains virtually un-

changed after the treatment. A reasonable percentage of

microporosity (35%) is nevertheless retained in MB-1500

(Table 2).

DFT calculations on the N2 adsorption isotherms rendered

bi-modal pore size distributions for the three the carbon sup-

ports, with a mesopore distribution centred at approx 34 nm

(Fig. S1). This pore size should, in principle, ensure an ade-

quate loading of the biomolecules, according to their molecu-

lar dimensions (Table 1.). It has been suggested that the pore

diameter should be at least 1.5-fold greater than the main

axis of the protein molecule [4,7,12]. On the other hand,

SEM images (Fig. S2) show a considerable increase in the

support surface roughness after the LTA treatment. An

enhancement of macropores in that particular sample is also

clearly observed at higher magnifications, whereas a smooth-

er, less macroporous surface is characteristic of MB-1500,

when compared to the original support.

Unlike universal dispersive interactions between the

protein molecules and the solid support, specific interactions

depend on the surface chemistry of both enzyme and sup-

porting material [4,6,21]. For a given enzyme, at a given

immobilisation conditions, these latter interactions could be

enhanced by changing the hydrophobic/hydrophilic character

of the support [6,20,30–32]. Lipases have a great affinity to-

wards hydrophobic surfaces, generally allowing the protein

to maintain their active conformation [33–35]. In the case of

carbon materials, hydrophilicity is strongly dependent on

the type and amount of oxygen functional groups present at

their surface [36,37]. XPS surface analysis (Table 3) reveals

compositional differences of the outermost layers of MB after

both treatments. Plasma oxidation increases the amount of

surface oxygen dramatically, whereas the heat treatment

reduces more than a half the original 5% of atomic oxygen

present in MB. This latter treatment also removes other

Table 3 – XPS compositional analysis of the carbon supports.

Sample Atom%

C O N F Na Si Fe

MB 91.5 5.0 0.3 0.9 1.4 1.0 –a

MB-1500 98.3 1.7 –a –a –a –a –a

MB-LTA 70.4 25.8 0.8 –a 1.4 1.3 0.3a Not detected.

Table 4 – Zeta potential of the carbon materials at differentpHs.

pH Zeta potential (mV)

MB MB-1500 MB-LTA

4 8 �4 �105 �8 �5 �146 �18 �17 �247 �26 �23 �308 �27 �31 �37

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

4 77 5 90 6 81 7 778 73

(c) CALA/MB-LTApH %Prot.

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

4 775 726 63 7 59 8 55

(b) CALA/MB-1500pH %Prot.

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

4 56 5 766 667 628 56

(a) CALA/MBpH %Prot.

Fig. 3 – Catalytic activity of CALA immobilised, at different

pH, on MB (a), MB-1500 (b) and MB-LTA (c) for the kinetic

resolution of (±)-1-phenylethanol. Also shown are the

results obtained using as received lyophilised CALA powder

(h). Enzyme loading (%Prot.) at each pH is also indicated.

410 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5

heteroatoms bringing about a highly pure carbon surface (%

C > 98%). It is thus expected that the hydrophilicity of the sup-

ports should increase as follows: MB-1500 < MB < MB-LTA.

This behaviour has been partially confirmed by water adsorp-

tion measurements carried out at 303 K (Fig. S3) [37].

Electrostatic interactions between the protein and the sup-

port are also prone to occur. An adequate balance between

such interactions and hydrophobic ones is decisive in deter-

mining the protein loading as well as the protein orientation

and conformation, which is critical for the catalytic activity

of the resulting enzyme/carbon system [31,38–41]. Zeta po-

tential measurements of the carbon supports provide valu-

able information to estimate their surface charge density at

different pH. The isoelectric point of the original MB support

is within 4–5 pH units (Table 4). The modified supports exhibit

negative surface charge densities in the whole range of pH. Fi-

nally, MB-LTA is clearly more (negatively) charged than any of

the other two supports for a given pH value. Differences be-

tween MB and MB-1500 at pH P 5 are not significant.

3.2. Carbon supported CALA biocatalysts

The protein loading and catalytic activity of CALA lipase sup-

ported on the three mesoporous carbons under study are

shown in Fig. 3. For every carbon support, five heterogeneous

biocatalysts were prepared using buffers of different pH

(within the 4–8 unit range). Results obtained for the catalytic

activity of the as received lyophilised CALA powder are also

shown in Fig. 3a. Overall, the catalytic activity of the free li-

pase under the current experimental conditions was remark-

ably enhanced after immobilisation in any of the carbon

support considered. It should be reminded that 50% is the

maximum conversion attainable in a racemic resolution

(Fig. 1). Moreover, enzyme enantioselectivity was not altered

at all after heterogeneisation. Another essential feature that

should be pointed out after first inspection of Fig. 3 is that

there was no correlation between the amount of protein

adsorbed and the catalytic activity of the CALA supported

on the carbon materials under study. In principle, for a giving

system, it should be expected that a higher protein loading

brings about an increase in the catalytic activity displayed

[4,6]. To help in discussing the results obtained, the effect of

both the carbon surface properties and immobilisation pH

on the amount of protein adsorbed is initially dealt with.

Then, the influence of such properties in the catalytic activity

is addressed. The strong effect of the immobilisation pH on

protein loading suggests that protein-surface electrostatic

interactions are playing a crucial role in the adsorption pro-

cess of CALA on the carbon supports. This would contrast

with the observation of Vinu et al. in their adsorption studies

of lysozyme on ordered mesoporous carbons [18]. They con-

clude that protein adsorption was mainly controlled by pro-

tein–protein interactions rather than protein-surface ones.

In our case, textural properties such as BET surface area or

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5 411

total pore volume (Table 2) seem to affect less the amount of

enzyme supported. In addition to the significant differences

in protein charge for a given support when the loading is

carried out at different pH, the highest protein loads were ob-

tained for the MB-LTA support, with a VMESO and mean mes-

opore size very similar to those of MB and MB-1500 (Table 2).

In the case of the CALA/MB system (Fig. 3a), the sequence

of protein loading was as follows: pH 5 > pH 6 > pH 7 > pH

8 � pH 4. The IEP of the crude CALA is 7.5 (Table 1.), which

means that it will have a positive charge in the pH range from

4 to 7 and a negative charge at pH 8. On the other hand,

the surface of MB will be positively charged at pH 4 and

increasingly negatively charged in the pH range 5–8 (Table

4). Therefore, while at pH 5–7 enzyme-support electrostatic

interactions will be favoured, thus increasing the protein

loading, electrostatic repulsions should reduce enzyme

adsorption at pHs 4 and 8.

The loading sequence detailed in Fig. 3b for CALA/MB-1500

is different from that already discussed for CALA/MB. In this

case, the maximum protein uptake was achieved at the low-

est immobilisation pH (pH 4) and it decreases as the pH in-

creases up to pH 8. MB-1500 does not have an IEP in the pH

range under consideration and the negative value of the zeta

potential increases monotonically with pH (Table 4). The en-

zyme-support electrostatic interactions should be also fa-

voured with the exception of those immobilizations at

pHs > 7.5 at which both CALA and carbon support have a

net negative charge (Tables 1 and 4). The protein loading of

CALA/MB and CALA/MB-1500 systems was very similar for

all immobilisation pH but pH 4. These results would confirm

the relevance of the electrostatic interactions in the adsorp-

tion process of CALA on those two particular carbon materials

since differences of zeta potential values between them are

negligible at pH P 5 (Table 4).

As mentioned above, MB-LTA was the support that seizes

the highest enzyme loads at any immobilisation pH. Actually,

the lowest protein loading on MB-LTA (73% at pH 8) is almost

as high as the highest loads attained in MB (76% at pH 5) and

MB-1500 (77% at pH 4). These values are also well above most

of the lipase loads attained when using mesoporous silicas

[42,43]. This behaviour is expected assuming the relevance

of the electrostatic interactions discussed so far. Thus, zeta

potential values of MB-LTA (Table 4) are strongly negative in

the whole pH range thus maximising the enzyme-support

interactions at pH < 7.5. Attending just to charges interaction,

a sequence of enzyme loading similar to that of MB-1500, i.e.,

an increase of loading as the buffer pH decrease would be pre-

dicted. However, the protein loadings seem to indicate that

additional contributions to the adsorption of CALA on this

particular support are also significant. Based only in electro-

static interactions, the relatively high loading attained at pH

8 is unexpected as the surface of MB-LTA withstands the high-

est negative charge at that particular pH value (Table 4), i.e.,

the electrostatic repulsions with CALA should be maximum.

Similarly, if the reasoning just presented for the CALA/MB-

1500 prevails, the enzymatic loading in MB-LTA should have

been optimal at pH 4, which was not the case (Fig. 3c).

MB-LTA is the carbon support with the highest surface

oxygen concentration (Table 3). Plasma oxidation is known

to increase both acidic and basic oxygen functional groups

on the surface of carbons [44]. These groups can establish

interactions with other functional groups of the protein

[39,40,45], thus promoting adsorption. This could explain

the high enzyme loads achieved on this support at pH 8 when

compared with MB and MB-1500 materials. In fact, such spe-

cific interactions between appropriate functional groups

might also contribute to the protein adsorption in the whole

range of immobilisation pH, thus leading to the high enzy-

matic loads attained on the MB-LTA support. The drop ob-

served in the amount of CALA immobilised on MB-LTA at

pH 4 could be attributed to the presence of acidic oxygen

groups (carboxylic) on the carbon surface not being fully dis-

sociated at such pH value. The possibility of chemical interac-

tions between those carboxylic groups and the protonated

amine groups of the lipases would be substantially reduced

[46,47].

Dealing with differences in activity between the biocata-

lytic systems is more complex than with enzyme adsorption.

The enzyme conformation, which is crucial for its enzymatic

activity, may vary with pH of the immobilisation media lead-

ing to changes in the microenvironment of some amino acid

residues which are involved in the catalytic process [4,6,38].

Therefore, regardless of the amount of enzyme loaded in

the carbon material, there is an optimum immobilisation

pH at which the enzyme has a proper conformation thus

showing the maximum catalytic activity. Similarly, certain

pHs may have a negative effect on those important amino

acids residues. Moreover, random immobilisation might hin-

der the accessibility to the active site of the enzyme

[48].Among the different pHs, the CALA/carbon material sys-

tems prepared at pH 6 showed the highest catalytic activity

(approx. 45% of conversion after 6 h, for the three supports)

(Fig. 3). Accordingly, 6 seems to be the optimum pH for the

immobilisation process of the CALA enzyme even though

the conversions achieved with the CALA/MB-1500 at pHs 6

and 7 are comparable (Fig. 3b). The lowest conversions were

obtained when lipases were immobilised at the extreme pH

values, i.e., pH 8 and, specially, pH 4. The effect of the immo-

bilisation pH on the catalytic activity was particularly notice-

able for MB and MB-LTA supports (Fig. 3a and c), whereas the

conversion values of the biocatalytic systems based on MB-

1500 were less affected (Fig. 3b). This may be related to the

higher hidrophobicity of the MB-1500 support (Table 3). It is

well know that most lipases display a large increase in activ-

ity when adsorbed on hydrophobic supports [33–35]. This

characteristic has been shown to be associated with confor-

mational changes in the enzyme upon adsorption, creating

an open substrate-accessible active site. Thus, lipases recog-

nise hydrophobic surfaces similar to those of their natural

substrates and they undergo interfacial activation during

immobilization. CALA is a lipase that exhibits a well-defined

lid domain and a modest degree of interfacial activation

(Table 1) [49].

Fig. 4 shows better a comparison of the activity of CALA

biocatalysts prepared from the three carbon supports, at

three different immobilisation pH. Different trends are clearly

depicted. At the optimum immobilisation pH (= 6) (Fig. 4b), al-

most identical conversions were attained for the three sys-

tems in the whole time span, regardless differences in

protein loading, or textural and chemical characteristics of

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

CAL/MBCALA/MB-LTACALA/MB-1500

(c) pH 8

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

CALA/MBCALA/MB-LTACALA/MB-1500

(b) pH 6

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

CALA/MBCALA/MB-LTACALA/MB-1500

(a) pH 4

Fig. 4 – Catalytic activity of CALA immobilised on MB, MB-

1500 and MB-LTA, at pH 4 (a), pH 6 (b), and pH 8 (c) for the

kinetic resolution of (±)-1-phenylethanol.

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

4 87 5 90 6 89 7 90 8 88

(c) CALB/MB-LTApH %Prot.

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

4 73 5 73 6 767 75 8 74

(b) CALB/MB-1500%Prot.pH

Time (h)0 1 2 3 4 5 6

% C

onve

rsio

n

0102030405060

4 785 856 837 83 8 81

(a) CALB/MBpH %Prot.

Fig. 5 – Catalytic activity of CALB immobilised, at different

pH, on MB (a), MB-1500 (b) and MB-LTA (c) for the kinetic

resolution of (±)-1-phenylethanol. Also shown are the

results obtained using as received lyophilised CALB powder

(h). Enzyme loading (%Prot.) at each pH is also indicated.

412 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5

the supports. This behaviour should be ascribed to the partic-

ularly active conformation of CALA at this pH. Far from this

optimum pH, CALA is expected to lower its activity. The ex-

tent of such a decrease would be tempered by an appropriate

environment when the lipase is supported on the mesopor-

ous carbons. Thus, at pH 4 or 8 (Fig. 4a and c), the maximum

activity would be determined by the combination of electro-

static interactions and the hydrophobicity of the supports,

making MB-LTA and MB-1500 based biocatalysts to yield high-

er catalytic activity than CALA/MB.

3.3. Carbon supported CALB biocatalysts

Results of the immobilisation of the lipase B from C. antarctica

(CALB) on the three carbon supports are reported in Fig. 5. As

for CALA, the activity of CALB increases after heterogenisa-

tion. Substantial differences are however noticeable when

comparing Figs. 3 and 5. Moreover, it should be kept in mind

that, in contrast to many other lipases, CALB has not lid

covering the access to the active site and shows no interfacial

activation [50,51]. The role of the support hydrophobicity is

thus expected to be minimised for this particular enzyme.

Starting with the enzyme adsorption process, the % of CALB

immobilised on a given support was always greater than that

of CALA. CALB loads on the different supports seem, in gen-

eral, to be independent of the immobilisation pH, especially

in the case of the MB-modified supports (Fig. 5b and c), for

which an average of 74% and 89% of protein loading was

found in the whole pH range, respectively. CALB/MB systems

showed an average loading of 83% at 5 6 pH 6 8, but a signif-

icant lower value at pH 4 (78%) (Fig. 5a).

There are a number of possible reasons for these observed

outcomes. Although it has been traditionally assumed that

the IEP of the CALB appeared at pH 6, recent investigations

have reported that this particular lipase has no titratable

groups in the pKa range 5–8, thus leading to an unusually

broad range in which the CALB remains uncharged [28]

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5 413

(Table 1). If so, the electrostatic interactions that were so rel-

evant in the immobilisation of CALA are no longer imperative

when supporting CALB. Other specific interactions related to

the surface chemistry of both enzyme and support should

be now responsible of differences observed. Therefore, the

higher loadings were obtained for the MB-LTA support, fol-

lowed by MB and MB-1500, which correlates with their

amount of surface oxygen (Table 3). In spite of all this, it is

worth to notice that the significant drop in the % of protein

adsorbed at pH 4 on the MB material (Fig. 5a) coincides with

the change of zeta potential sign for the carbon support

(Table 4). The role of the textural properties on the adsorption

capacity is, for CALB immobilisation, still minimised attend-

ing to the enzyme loads of MB and MB-LTA and results shown

above (Fig. 2, Table 2). Differences in the catalytic activities of

the CALA (Fig. 3) and CALB (Fig. 5) supported systems are also

easy to point out at first sight. Thus, a much lower influence

of the support is observed in the CALB systems, the differ-

ences of the activities reached with the three supports not

being very significant, for a given pH. In addition, the immo-

bilisation pH affects to a lesser extent the activity of the CALB

biocatalysts, with the exception of those prepared with the

modified carbon supports studied at pH 6 (Fig. 5b and c). Actu-

ally, CALB immobilisation on any of the mesoporous carbons

at pH 6 leads to materials yielding the lowest catalytic activi-

ties. There may be several reasons as to why the biocatalytic

systems from pH 6 were not as efficient as expected. It is well

established that any immobilisation process might distort the

native structure of the enzyme in three possible ways [4]:

reinforcement of the structure of the enzyme, loss of native

enzyme structure or no appreciable change. Studies have re-

vealed that the orientation of a physically adsorbed enzyme

can also affect its activity [38,52].

Apart from the distinctive low activity of the CALB/meso-

porous carbons prepared at pH 6, CALB/MB-LTA was the only

support where small differences in the catalytic activity of

materials immobilised at different pH values were detected

(Fig. 5c). The lower catalytic activity of the CALB/MB-LTA sys-

tems prepared at pH 4 and 8 suggests that, as it was discussed

for CALA, the ionization state of the functional groups of both

the MB-LTA and the enzyme could be altered by the immobi-

lization pH, thus having a negative effect on the catalytic

activity of the resulting biocatalytic system. For example, at

pH 8 some of the amino end terminal groups of the protein

will be mostly unprotonated and consequently interactions

with the carboxylate groups of the MB-LTA support would

be disrupt [47].

3.4. Operational stability tests

In view of the results discussed above, it can be stated that the

MB, MB-1500 and MB-LTA mesoporous carbons are promising

supports for the immobilisation of the lipases CALA and CALB.

In order to accomplish a widespread applicability and make its

implementation in large-scale processes attractive, an

adequate operational stability of the biocatalytic systems is

required. Therefore, the operational stability of the CALA/

MB-LTA and CALB/MB-LTA biocatalytic systems immobilised

at pHs 6 and 7, respectively, were tested by performing

subsequent reaction cycles of the kinetic resolution of (±)-1-

phenylethanol (Fig. 1). Between each reaction cycle, the usual

workup including catalyst recovery, washing and drying opera-

tion, was carried out (see Section 2.5). Then, the catalytic

activity was monitored to investigate the possible catalyst

de-activation over all reaction cycles. Interestingly, there was

no significant loss of activity during the 10 cycles carried out.

This is a valuable outcome from the viewpoint of a potential

industrial application.

4. Conclusions

Changes on the textural and surface chemistry properties of a

commercial mesoporous carbon material were brought about

by different methods including heat treatment and cold

oxygen plasma oxidation. The mesoporous carbon materials

obtained has been successfully used as carriers for the immo-

bilization of lipase A and B from C. antarctica by physical

adsorption in aqueous solutions. Results showed that both

enzyme loading and catalytic activity of the enzyme/carbon

material systems can be tuned and enhanced by combining

the surface chemistry of the carbon material and the pH of

the immobilisation media. It was demonstrated that the cata-

lytic activity of a given enzyme/carbon system does not always

correlates with the amount of protein adsorbed on the sup-

port. Moreover, determination of zeta potential of the carbon

materials enables the prediction of protein affinity to the

different carriers and the selection of an optimum immobilisa-

tion pH. On the other hand, the oxidised mesoporous carbon

MB-LTA showed the best enzyme adsorption capabilities and

high catalytic activities of the resulting enzyme/carbon mate-

rial systems, proving that the density of binding functional-

ities on the support surface affects the enzyme loading and

the conformational flexibility of the protein, critical for the

catalytic activity. Both oxidative and high temperature treat-

ments did not change significantly the mesoporous volume

nor the mesopore size distribution of the carbon supports.

Hence, differences in the textural properties of the materials

have been found not be very determinant in both enzyme

adsorption and biocatalytic activity. Finally, the preparation

of biocatalytic systems with 80–90% enzyme loading that can

be easily recovered and reused is emphasised from the view-

point of their application in scale-up processes. Specifically,

the biocatalytic systems CALA/MB-LTA and CALB/MB-LTAwere

used in 10 consecutive cycles without a significant loss of

catalytic activity, proving their excellent operational stability.

Another important aspect is that the commercial lipases, used

as received, did not show hardly any activity under the tested

conditions.

Acknowledgements

This work was partially funded by PCTI-Principado de Asturias

(IB09-002C2 and COF09-25 projects) and MICINN (CTM2008-

06869-C02-01/PPQ).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.carbon.2010.09.037.

414 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 6 – 4 1 5

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