A novel support for enzyme adsorption: Properties and applications of aerogels in low water media

Article abstract of DOI:10.1016/S0040-4039(00)01522-7

Aerogels, because of their porosity, have a great ability to adsorb water, and this characteristic was exploited to extend the applicability of aerogels for the adsorption and entrapment of hydrolases to be used in organic media. The applicability of aero

Full text of DOI:10.1016/S0040-4039(00)01522-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8627–8630
A novel support for enzyme adsorption: properties and
applications of aerogels in low water media
a
a
a
a,
a
Alessandra Basso, Luigi De Martin, Cynthia Ebert, Lucia Gardossi, * Alida Tomat,
b
b
Maurizio Casarci and Ornella Li Rosi
a
Dipartimento di Scienze Farmaceutiche, Universit a` degli Studi, Piazzale Europa 1, 34127 Trieste, Italy
b
ENEA, C.R. Casaccia, Via Anguillarese 301, Rome, Italy
Received 5 May 2000; accepted 7 September 2000
Abstract
Aerogels, because of their porosity, have a great ability to adsorb water, and this characteristic was
exploited to extend the applicability of aerogels for the adsorption and entrapment of hydrolases to be
used in organic media. The applicability of aerogels as solid supports for immobilisation of the enzymes
PGA, thermolysin and a-chymotrypsin was assayed. By controlling the distribution of water between the
catalyst, the support and the reaction medium, aerogel preserves the catalytic activity of the enzymes and
prevents hydrolytic reactions. © 2000 Elsevier Science Ltd. All rights reserved.
Aerogels are advanced materials that consist normally of more than 96% air, while the
remaining 4% is a matrix of silicon dioxide. Aerogel, consequently, is one of the lightest solids
ever conceived and it has unique properties such as high porosity, large surface area, low density
and low thermal conductivity. Moreover, silica aerogels present an extremely large number of
accessible hydroxyl groups, and consequently show strong hydrogen-bonding effects. All these
properties make silica aerogels effective materials, in for instance the production of microfiltra-
1
tion membranes or adsorbents.
Studies on the adsorption of water on porous materials indicate that motion of water
2–5
molecules in silica based matrixes strongly depends on the pore dimensions. There are two
4
types of water in the pores. One type of water behaves like bulk water, probably present in the
central region of the pores, and the other type is water perturbed strongly by the silica surface.
Consequently, pores of different sizes have different relative amounts of bound and free
3
–5
water.
Recently we demonstrated that penicillin G amidase (PGA) and proteases such as thermolysin
and a-chymotrypsin are active in low-water media, at controlled water activity, when adsorbed
*
Corresponding author. Fax: +39-040-52572; e-mail: linda@univ.trieste.it
0
040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01522-7

8628
6
on hydrated Celite R-640, a porous silica matrix which can adsorb large quantities of water
7
(
>100% of its weight).
Aerogels, because of their porosity, have a great ability to adsorb water. The aim of this study
is to verify the applicability of aerogels, for the adsorption and entrapment of hydrolases to be
used in organic media at controlled water activity.
The applicability of aerogel as a solid support for immobilisation of the enzymes PGA,
thermolysin and a-chymotrypsin was assayed.
Preparation and characterisation of the aerogel
†
The aerogel was prepared in three main steps:
1
. Alcogel synthesis: a base catalysed (NH OH, NH F) polymerisation of the tetraethyl
4
4
orthosilicate (TEOS) with water in ethanol.
(
Si(OCH CH ) +2H O“SiO
+4HOCH CH3(liq.))
2
3 4(liq.)
2
2(solid)
2
2
. Aging: the gel is covered with an alcohol/water (3/1) mixture for at least 72 hours to allow
a strengthening of the silica network.
3
. Drying, aerogel formation and solvent removal under vacuum.
The aerogel prepared following this procedure was characterised. The surface area of aerogel
2
2
(
(
672 m /g) and the micropore surface (11 m /g) were calculated using the B.E.T. technique
Brunauer–Emmet–Teller). The data obtained indicate a micropore percentage of 1.64% (diam-
eter<30 A). The shape and dimension of the fragments of aerogel are not homogeneous (300
,
mm–3 mm).
Aerogel (200 mg) in 1 mL of dry toluene leads to a water activity of 0.4 due to the percentage
of water that cannot be removed during the drying step. Upon addition of water to the solvent,
the silica matrix presents a great ability to adsorb water, maintaining the a between 0.75 and
w
0
.87, namely from 20 to 200 mL in 1 mL of toluene.
PGA adsorbed onto silica aerogels
‡
The aerogel was previously hydrated with 50 mL of buffer or ultrapure water in toluene.
After 24 h, the enzyme, solubilised in either 100 mL of ultrapure water or aqueous phosphate
buffer (pH 8), at two different concentrations (0.1 and 1 M), was adsorbed onto the siliceous
matrix by adding the enzymatic solution to the toluene containing the aerogel, according to a
6
method previously described. After 24 h of equilibration the observed values of water activity
were in agreement with the necessary hydration to maintain PGA active in organic solvent: i.e.
8
aw=0.83–0.87.
The synthetic activity of the enzymatic preparation was studied in the acylation of
L
-TyrOEt
(
1) with methyl phenylacetate (2) in toluene. All the preparations obtained have a good activity
in toluene and all reactions reached complete conversion in 5–10 hours and no hydrolytic
reaction was observed. The enzymatic preparation adsorbed using a 0.1 M phosphate buffer, to
−
1
−1
solubilise the enzyme, gave an initial rate of 2.09 mmol h U . It is noteworthy that PGA
6
−1
adsorbed on Celite R-640 following the same procedure leads to an initial rate of 0.98 mmol h
†
Experimental conditions: TEOS 30 mL, ultrapure water 21 mL, ethanol 60 mL, NH OH 0.37 mL, NH F 0.36 mL.
4
4
‡
Experimental conditions: reaction volume=1 mL of dry toluene, T=30°C; [1]=80 mmoles, [2]=100 mmoles, 6.2
mg/mL of lyophilised PGA (activity=9.6 U/mg of protein).

8629
−
1
U , in the same range of water activity. This difference is most probably due to the larger
2
2
surface area of aerogel (672 m /g), if compared to Celite R-640 (65 m /g). Adsorption on
aerogels translates into an improvement of the dispersion of the enzyme and of the enzyme-sub-
strate interaction.
Proteases adsorbed onto silica aerogels
The activity of thermolysin and a-chymotrypsin, adsorbed onto the same matrix, in toluene,
was studied. The activity of both enzymes in organic media is deeply affected by the degree of
6
hydration. A major limit to the achievement of high yields is due to the production of water
9
,10
during these reactions.
In this connection, the aerogel has been used as an alternative method
for removing the excess water produced during the reactions because of its high water-binding
capacity. The hydrolases were adsorbed onto the aerogel using the same procedure of adsorp-
tion as described for PGA. The reaction between the acyl donor Zꢀ
L
-PheꢀCO H and some
2
nucleophiles was studied (Table 1).
a
Table 1
b
c
Enzyme
[ZꢀL
-PheꢀCO H], mmoles
Nucleophile
aw
Conversion (144 h)
2
d
Thermolysin
Thermolysin
Thermolysin
a-Chymotrypsin
a-Chymotrypsin
100
100
100
80
L
L
L
-PheꢀOEtd
0.80
0.83
0.83
0.82
0.83
84
45
77
54
30
-TyrꢀOEt
d
-LeuꢀNH2
e
MeOH
-TyrꢀOEt
d
100
L
a
Experimental conditions: reaction volume=1 mL of dry toluene, T=30°C.
b
c
5
mg/mL of enzyme (activity of thermolysin=42 U/mg, activity of a-chymotrypsin=68.6 U/mg).
Conversions were evaluated by titration of the Zꢀ -PheꢀCO H with 0.01 M NaOH.
L
2
d
e
[
Nucleophile]=80 mmoles.
[
MeOH]=100 mmoles.
In contrast with the reactions using enzymes adsorbed onto Celite R-640, in all the reactions
6
described no complete conversion was achieved after 144 hours of reaction time. This can be
probably ascribed to diffusion limits in the reaction system between the enzyme and the
substrates. It is remarkable that the a of the systems remained at a constant value (a =0.80–
w
w
0
.83) during the course of the reactions, making the silica aerogels suitable as supports in
synthetic reactions liberating water.
PGA entrapped in aerogel
In order to exploit the innovative potential of aerogel as a support for biocatalysts, the
§
entrapment of PGA on aerogel was also studied.
To entrap PGA in the aerogel, 29.4 mg of the enzyme was solubilised in ultrapure water (21
mL) and added during alcogel synthesis step. The obtained enzymatic preparation, assayed in
water, showed a ten time decrease of enzymatic activity, when compared to the native enzyme.
The same enzymatic preparation gave no reaction at all in toluene, even working at a =0.83–
w
§
Experimental conditions: reaction volume=1 mL of dry toluene, T=30°C, [1]=80 mmoles, [2]=100 mmoles, 200 mg
of PGA/aerogel.

8630
0
.87. The a of the system was adjusted by adding 100 mL of water and phosphate buffer at
w
different pH (pH 7, pH 8 pH 9). The loss of activity of the PGA entrapped within aerogel is
probably due to diffusion barriers, since the entrapped enzyme is not easily accessible by the
organic solvent and substrates.
Conclusions
Silica aerogel is a solid matrix suitable for the adsorption of enzymes applicable in organic
media, whereas the entrapment of the enzyme within the aerogel causes loss of catalytic activity,
both in toluene and water. Silica aerogels are hygroscopic supports and their adsorption
capacity can be exploited in synthetic reactions, liberating water to prevent the competition of
hydrolytic reactions (esterification of amino acids and peptide synthesis). The degree of
fragmentation of aerogel must be optimised, in order to obtain aerogel samples with repro-
ducible properties (porosity, shape, adsorption capacity). These promising preliminary results
induce to pursue the study of the properties of aerogels. In particular, the porosity of these
supports and their great capacity of adsorbing water make this type of porous support
potentially applicable in biotransformations in non-aqueous media at controlled water activity.
Acknowledgements
Thanks are due to M.U.R.S.T. (Roma) for financial support to C.E.
References
1. Gesser, H. D.; Goswami, P. C. Chem. Rev. 1989, 89, 765–788.
2. Clifford, J.; Zettlemoyer, A. C. Water: A Comprehensive Treatise; Franks, F., Ed.; Plenum Press: New
York–London, 1975; pp. 75–133.
3
4
5
6
7
8
. Allen, S. G.; Stephenson, P. C. L.; Strange, J. H. J. Chem. Phys. 1998, 108, 8195–8198.
. Overloop, K.; Van Gerven, L. J. Magn. Reson. A 1993, 101, 147–156.
. Takamuku, T.; Yamagami, M.; Wakita, H.; Masuda, Y.; Yamaguchi, T. J. Phys. Chem. B 1997, 101, 5730–5739.
. Basso, A.; De Martin, L.; Ebert, C.; Gardossi, L.; Linda, P. Chem. Commun. 2000, 467–468.
. De Martin, L.; Ebert, C.; Garau, G.; Gardossi, L.; Linda, P. J. Mol. Catal. B: Enzymatic 1999, 6, 437–445.
. Basso, A.; De Martin, L.; Ebert, C.; Gardossi, L.; Linda, P.; Zlatev, V. J. Mol. Catal. B: Enzymatic 2000, 11
(1–6), 000.
9. Aldercreutz, P. Eur. J. Biochem. 1991, 199, 609–614.
10. Reslow, M.; Aldercreutz, P.; Mattiasson, B. Eur. J. Biochem. 1988, 177, 313–318.
.
.