Sol-gel materials as efficient enzyme protectors: Preserving the activity of phosphatases under extreme pH conditions

Article abstract of DOI:10.1021/ja0507719

By entrapment in (surfactant modified) silica sol-gel matrixes, alkaline phosphatase (AIP) - naturally with optimum activity at pH 9.5 - was kept functioning at extreme acidic environments as low as pH 0.9, and acid phosphatase (AcP) - naturally with opti

Full text of DOI:10.1021/ja0507719

Published on Web 05/14/2005
Sol-Gel Materials as Efficient Enzyme Protectors: Preserving
the Activity of Phosphatases under Extreme pH Conditions
Hagit Frenkel-Mullerad and David Avnir*
Contribution from the Institute of Chemistry, The Hebrew UniVersity of Jerusalem,
Jerusalem 91904, Israel
Received February 5, 2005; E-mail: david@chem.ch.huji.ac.il
Abstract: By entrapment in (surfactant modified) silica sol-gel matrixes, alkaline phosphatase (AlP) s
naturally with optimum activity at pH 9.5 s was kept functioning at extreme acidic environments as low as
pH 0.9, and acid phosphatase (AcP) s naturally with optimum activity at pH 4.5 s was kept functioning at
extreme alkaline environments, up to pH 13.0. Propositions are offered as to the origin of the ability of the
matrixes to provide such highly efficient protection and as to the origin of the synergetic enhancing effect
when both the silica and the surfactants are used as a combined entrapping environment. It was found
that the protectability of the enzymes against harsh pH values is dependent on the nature of the surfactant.
molecules,17 ease of their heterogenization,18 compatibility with
opposing reagents,19 the convenience of tailoring the chemical
Background
As the name of the enzyme alkaline phosphatase (AlP)
implies, its catalytic activity (hydrolysis of phosphoesters to
phosphate and to the corresponding alcohol or phenolate) is
optimal at basic pH values (9-101,2). Here, we show that by
utilizing the protective features of sol-gel materials, one can
keep this alkaline enzyme active under extreme acidic condi-
tions, going down the pH scale to as low as pH 0.9!, and that
when, for comparison purposes, the acidic enzyme acid phos-
phatase (AcP, optimal performance at pH 4.5-6.02) is entrapped
in these materials, it is kept active under extreme alkaline
conditions, as high as pH 13. Silica-based sol-gel materials,
with and without surfactant modification, were the key to these
unusually large effects, which amount to practical alteration of
the classical phosphatases’ properties.
and physical properties as needed for specific bioapplications,17
improved endurance of the entrapped proteins to denaturing
thermal conditions,17 to long-term storage conditions,20,21 and
to organic solvents22,23 are but some of the reasons for this fast
growth. Here, we show an extreme pH-protectability of enzymes
provided both by silica sol-gel matrixes and through synergism
between matrix and surfactant interactions, thus utilizing yet
another observation, namely, that the properties of dopants can
be tailored and modified by the coentrapment of surfactants
within sol-gel materials.24,25 Finally, we note that the interaction
of enzymes with surfactants in solution was studied in various
contexts, such as providing enzymes with hydrophobic working
environments26,27 and shifting the optimal pH for activity.28,29
We recall that sol-gel materials have proven in the last two
decades to be versatile carriers of active dopants.3-5 Diverse
reactive functionalities have been introduced into these materials
by either direct physical doping6-8 or covalent attachment.9-11
Of the various families of functional sol-gel materials that have
been developed, one, which has progressed particularly fast,
has been the family of sol-gel materials with biochemical and
biological activities.12-16 Enhanced stability of entrapped bio-
Results and Discussion
The activity of AlP entrapped in three types of sol-gel
matrixes is shown in Figure 1A, and in Figure 1B, it is compared
to the activity in solution.
(12) Wei, Y.; Dong, H.; Xu, J.; Feng, Q. ChemPhysChem 2002, 9, 802-807.
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Synth. Catal. 2003, 345, 717-728.
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S. M. J. Mater. Chem. 2004, 14, 2311-2316.
(1) Fernley, H. N. Mammalian Alkaline Phosphatases. In The Enzymes, 3rd
ed.; Boyer, P. D., Ed.; Academic Press: New York, 1971; Vol. IV, pp
417-447.
(2) McComb, R. B.; Bowers, G. N.; Posen, S. Alkaline Phosphatase; Plenum
Press: New York, 1979.
(3) Ciriminna, R.; Pagliaro, M. Chem.sEur. J. 2003, 9, 5067-5073.
(4) Dave, B. C.; Ottosson, J. E. A. J. Sol-Gel Sci. Technol. 2004, 31, 303-
307.
(16) Nassif, N.; Bouvet, O.; Rager, M. N.; Roux, C.; Coradin, T.; Livage, J.
Nat. Mater. 2002, 1, 42-44.
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2003, 15, 737-745.
(18) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater.
Lett. 1990, 10, 1-5.
(19) Gelman, F.; Blum, J.; Avnir, D. New J. Chem. 2003, 27, 205-207.
(20) Shtelzer, S.; Braun, S. Biotechnol. Appl. Biochem. 1994, 19, 293-305.
(21) Besanger, T. R.; Chen, Y.; Deisingh, A. K.; Hodgson, R.; Jin, W.; Mayer,
S.; Brook, M. A.; Brennan, J. D. Anal. Chem. 2003, 75, 2382-2391.
(22) Li, J.; Tan, S. N.; Oh, J. T. J. Electroanal. Chem. 1998, 448, 69-77.
(23) vanUnen, D.; Engberson, J. F. J.; Reinhoudt, D. N. Biotechnol. Bioeng.
2001, 75, 154-158.
(5) Barbe, C.; Bartlett, J.; Kong, L.; Finnie, K.; Lin, H. Q.; Larkin, M.; Calleja,
S.; Bush, A.; Calleja, G. AdV. Mater. 2004, 16, 1959-1966.
(6) Gelman, F.; Blum, J.; Schumann, H.; Avnir, D. J. Sol-Gel Sci. Technol.
2003, 26, 43-46.
(7) Zhang, J.; Au, K. H.; Zhu, Z. Q.; O’shea, S. Opt. Mater. 2004, 26, 47-55.
(8) Gutierrez, J. A. R.; Dominguez, M. D. P.; Macias, J. M. P. Anal. Chem.
Acta 2004, 524, 339-346.
(24) Rottman, C.; Grader, G.; Hazan, Y. D.; Melchior, S.; Avnir, D. J. Am.
Chem. Soc. 1999, 121, 8533-8543.
(25) Badjic, J. D.; Kostic, N. M. J. Phys. Chem. B 2001, 105, 7482-7489.
(26) Gladilin, A. K.; Levashov, A. V. Biochemistry (Moscow) 1998, 63, 345-
356.
(9) Brusatin, G.; Innocenzi, P.; Guglielmi, M.; Babonneau, F. J. Sol-Gel Sci.
Technol. 2003, 26, 303-306.
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(11) Fireman-Shoresh, S.; Avnir, D. Langmuir 2001, 17, 5958-5963.
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A R T I C L E S
Frenkel-Mullerad and Avnir
and high pH scale ends), the cationic CTAB spoils it (Figure
1A). In solution (Figure 1B), CTAB lowers the activity of AlP
(Table 1) but does not quench it altogether. A nonionic
surfactant, Brij 56, was tested as well, and while it did show
some added protectability, the effect is much smaller (not
shown), thus supporting, as we shall see, the ionic mechanism
of the surfactant operation suggested below.
To see if this enhanced stability phenomenon is applicable
beyond the specific case of AlP, we took acid phosphatase (AcP)
not only because we wanted to see if one can “symmetrically”
protect it against extreme alkalinity but also because the
mechanism of operation of this enzyme, as well as the details
of its active site, are completely different from those of AlP.30
It was found that whereas this enzyme loses all of its activity
in solution under high alkaline conditions (Figure 2B), it remains
active within the sol-gel matrixes at pH values of 10.0, 11.0,
12.0, and as high as 13.0 (Figure 2A).
Unlike AlP, matrix isolation did not protect AcP activity
below pH 3. As for the surfactants effect, a “mirror” behavior
was observed. This time, CTAB was the surfactant that was
found to enhance significantly the activity, compared to entrap-
ment in pure silica (Figure 2A), while AOT poisoned the
enzyme completely, both in the entrapped form and in solution
(data not shown).
Another relevant observation, to be discussed below, is that
the type or presence of the surfactant affected the amount of
the entrapped enzyme (Table 2).
In brief, the main features of Table 2 are good entrapment
with CTAB (somewhat better for AcP), somewhat lower
entrapment efficiency without any surfactant, and about 50%
lower AlP entrapment in the presence of AOT.
Figure 1. The activity of alkaline phosphatase (expressed in terms of
turnover number (T.O.N.)) under extreme acidic and alkaline conditions.
(A) Immobilized in three types of silica sol-gel matrixes (with the
surfactants AOT or CTAB, or with no surfactant); and (B) comparison to
solutions (with and with no surfactants).
We shall now offer some interpretations for these striking
phenomena, starting with the protectability provided by the pure
silica sol-gel cage. The accumulated observations on the stabili-
zation of enzymes within silica sol-gel matrixes have pointed
to physical cage confinement as a major source of this stabili-
zation. Thus, it has been proposed31-35 that the rigidity of the
ceramic cage does not allow the protein to undergo denaturing
unfolding-refolding motions, and once the enzyme is held intact
from that point of view, its activity is preserved. Here, we pro-
pose to consider a new possible explanation for the entrapment-
protectability against steep pH gradients, which adds to the cage
yet another role in the protection mechanism (Scheme 1).
To begin with, we note that the relatively free rotation of
sol-gel entrapped enzymes31,32 implies that the entrapping cage
still has some free space. That free space, the space between
the outer surface of the protein and the silica surface of the
cage, is composed of very few, perhaps as low as one or two,
water molecule layers. Let us focus then on this layer and
evaluate what do large pH changes actually mean in such very
small local environments? Suppose, for the sake of demonstrat-
ing the point, that the water layer is a small reservoir of 100
water molecules surrounding the protein as a blanket against
the silica cage wall, and that the external pH is very acidic, say
pH 0, and that the hydronium ions penetrate that reservoir until
It is seen that while in solution the enzyme loses all of its
reactivity at pH 3.0, when entrapped in the sol-gel matrixes
(silica and silica/AOT), this alkaline enzyme remains active at
extreme pH values of 3.0, 2.2, and as low as 0.9, namely, a 30
billion times higher hydronium concentration compared to the
optimal pH in solution (pH 9.5). At pH 3.5, where some activity
is still detectable in solution, the entrapped enzyme activity is
4 times higher at that pH. Comparison of the activity at pH 2.4
to the activity at the optimal pH of 9.5 is provided in Table 1.
Entrapment leads to increases in Km and decreases in Vmax, indi-
cating trends which are consistent with diffusional limitations.
It is immediately evident from Table 1 that while pH 9.5 is
still optimal for AlP@SG, moving 7 pH units down the scale
does take its toll, but that toll is not heavy, particularly when
noticing that in solution, the enzyme is totally inactive at that
pH. Interestingly, the matrix also offers efficient protectability
at the very high alkaline pH range (Figure 1), with significant
AlP activity at pH 12.0 and even at pH 13.0. The activities of
AlP@SG at pH 12.0 and 13.0 are 2-3 and 40 times higher
than those in solution, respectively; in fact, in solution, the
enzyme is practically inactive at pH 13. It is also seen (Figure
1A) that while the anionic surfactant, AOT, improves the
extreme pH durability of the entrapped enzyme (both at the low
(30) Hollander, V. P. Acid Phosphatase. In The Enzymes, 3rd ed.; Boyer, P. D.,
Ed.; Academic Press INC: New York and London, 1971; Vol. IV, pp 449-
495.
(31) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 1-36.
(32) Eggers, D. K.; Valentine, J. S. J. Mol. Biol. 2001, 314, 911-922.
(33) Badjic, J. D.; Kostic, N. M. Chem. Mater. 1999, 11, 3671-3679.
(34) Flora, K. K.; Brennan, J. D. Chem. Mater. 2001, 13, 4170-4179.
(35) Nguyen, D. T.; Smit, M.; Dunn, B.; Zink, J. I. Chem. Mater. 2002, 14,
4300-4306.
(27) Reynolds, J. A. The Role of Micelles in Protein-Detergent Interactions.
Methods in Enzymology; Academic Press: New York, 1979; Vol. 61, pp
58-61.
(28) Levashov, A. V.; Khmelnitsky, Y. L.; Klyachko, N. L.; Martinek, K.
Reversed Micellar Enzymology. In Surfactants in Solution; Mittal, K. L.,
Lindman, B., Eds.; Plenum Press: New York, 1984; Vol. 2.
(29) Nametkin, S. N.; Kabanov, A. V.; Levashov, A. V. Biochem. Mol. Biol.
Int. 1993, 29, 103-111.
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Sol−Gel Materials as Efficient Enzyme Protectors
A R T I C L E S
Table 1. Effects of Surfactants on the Activity of Entrapped Alkaline Phosphatase at pH 9.5 and 2.4
Entrapped Enzyme
Enzyme in Solution
pH 9.5
pH 2.4
pH 9.5
pH 2.4
a
Vmax
M/s)
Km
T.O.N.b
Vmax
Km
T.O.N.
Vmax
M/s)
Km
T.O.N.
Vmax
M/s)
Km
T.O.N.
1
1
1
1
surfactant
(
µ
(mM)
(s-
)
(
µ
M/s)
(mM)
(s-
)
(
µ
(mM)
(s-
)
(
µ
(mM)
(s-
)
none
AOT
CTAB
0.12
0.15
0.04
0.167
0.331
0.216
23.5
58.8
7.5
0.04
0.04
no activity
0.119
0.267
7.8
15.7
0.45
0.37
0.13
0.059
0.048
0.0004
81.8
67.3
23.6
no activity under any condition
a Michaelis-Menten constant. b Calculated by dividing the rate of reaction at substrate saturation by the concentration of the entrapped enzyme. We refer
to enzyme concentration within the gels as the amount used for entrapment minus the various washings.
ment with hydronium ions, promoting efficient denaturing
reactions, the confinement within a small cage containing a small
reservoir of water molecules shunts this process and renders a
local nominal value of high acidity benign. A similar proposed
argument holds for the protectability at the alkaline side of the
pH scale. Finally, a word of caution is in order regarding the
terminology used for this molecular level description; it is, of
course, not possible to use terms which belong to the realm of
very large (thermodynamic relevant) assemblies, such as pH.
Therefore, the picture of what happens inside a small pore
should indeed be described in terms of the small numbers
involved, that is, 2 protons in an environment of 100 molecules,
in our example; yet, as we have shown here, the shift from
thermodynamic concepts to very small numbers is an eye
opener.
For AlP, this effect, as mentioned above, is moderately
enhanced by the use of coentrapped AOT but quenched when
CTAB is used (Figure 1). We also mention again the “mirror”
behavior found for AcP, namely, that CTAB enhanced signifi-
cantly the activity (by a factor of 130 at pH 10) and that AOT
poisons the enzyme completely both in solution and in the
entrapped form. It is thus evident that the nature of the surfactant
plays a crucial rule in affecting the reactivity of the entrapped
enzymes. We recall that in solution, surfactant binding onto a
protein surface is a mixed event. It usually starts with
electrostatic interactions between the polar head of the surfactant
and polar moieties on the protein surface, and when these
Figure 2. The activity of acid phosphatase under extreme alkaline
become saturated, the interaction reverses and the hydrophobic
conditions. (A) Immobilized in two types of silica sol-gel matrixes (with
chains of the surfactant bind by van der Waals interactions with
or with no CTAB); and (B) comparison to solutions (with and with no
hydrophobic moieties on the protein.36 However, when this
CTAB).
system is confined within a solid matrix, the interactions are
further complicated by competing with the binding sites of the
Table 2. Amount of Enzyme Entrapped in the Matrixes
solid substrate.37,38 In the case of the sol-gel matrixes, the AOT
and CTAB differ in their modes of interaction both with the
surfaces of the two proteins (although their pKi values are quite
similar, 5.0 for AcP39 and 5.7 for AlP1) and with the two
entrapping silica surfaces. This is so because the entrapment
procedures, which take place at two different pH values, charge
the silica surface and the proteins surfaces differently. Thus,
AlP, which is entrapped at pH 9.5, becomes rich in negatively
charged moieties, and these sites adsorb strongly the cationic
CTAB to the level that this tight adsorption blocks the ability
of pNPP to penetrate the active site. CTAB also adsorbs strongly
to the negatively charged silica cage surface (SiO- at this pH),
adding to the closed packing around the enzyme in a (disor-
surfactant
enzyme
entrapment (%)
none
AlP
AcP
AlP
AcP
AlP
AcP
93.5
94.6
46.7
a
96.4
98.7
AOT
CTAB
a Since AcP is not active when exposed to AOT in solution, it is not
possible to determine leaching by measuring the enzymatic activity of the
washings (as done for all other entries in the table).
equilibrium is reached, and a nominal “pH 0” is obtained there,
too. What does this mean from the point of view of the protein?
It means that the protein gets protonated by only two protons!
(Recall that “pH 0” means ∼2 moles H3O+ for each 100 moles
water.) So, on one hand, two protons are enough to compensate
for the extreme pH gradient, but on the other hand, for the
protein itself, these two protons pose no stress at all. Perhaps
two basic amino acid residues are protonated, and that is all. In
other words, whereas in solution pH 0 means constant bombard-
(36) Goddard, E. D. Interactions of Surfactants with Polymers and Proteins;
CRC Press: Boca Raton, FL, 1993.
(37) Atkin, R.; Craig, V. S. J.; Wanless, E. J.; Biggs, S. AdV. Colloid Interface
2003, 103, 219-304.
(38) Green, R. J.; Su, T. J.; Lu, J. R.; Webster, J. R. P. J. Phys. Chem. B 2001,
105, 9331-9338.
(39) Uehara, K.; Fujimoto, S.; Taniguchi, T. J. Biochem. (Tokyo) 1974, 75, 627-
638.
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A R T I C L E S
Frenkel-Mullerad and Avnir
Scheme 1. Schematic View of the Entrapped Enzyme with a Few Water Molecules Inside a Pore, Two of which are Protonated: The
Nominal ″pH″ is Very Low
dered) bilayer arrangement. The anionic AOT, on the other hand,
“suffers” from an unfriendly environment of negative charges
on the surfaces of both the enzyme and the silica cage. AlP is
thus held quite loose by this surfactant blanket, as indeed evident
both by the fact that about 50% is washed away during
entrapment (compared to 5% in the case of CTAB; see Table
2) and by the very fact that it is free enough to remain reactive.
The added stability against a steep hydronium gradient is
probably provided by the negative head of AOT, acting as a
hydronium sponge,18,40 and by the difficulty of the hydronium
ions to penetrate the hydrophobic portions of the protecting
blanket. The added protectability against hydroxyls can be
attributed to the negative charge repulsion of the whole
system: protein, surfactant, and silica.
Similar arguments, although in “symmetrically” reverse
charge picture, hold for AcP. Since the entrapment was carried
out at pH 4, CTAB molecules adsorb on the silica through the
positive nitrogen (CTAB interacts strongly with either SiO- or
SiOH41) but to a much lesser degree on the mostly positively
charged protein surface, thus coating it loosely with the
hydrophobic chains of the surfactant molecules that allow the
penetration of pNPP. As already shown by Rottman et al.,40
entrapped micellar structures exhibit enhanced hydrophobicity,
and this may account for the significant synergetic effect of
both the silica cage and surfactant in protecting the AcP against
extreme alkaline pH values (Figure 2). In fact, this effect is
much stronger here than for AlP@AOT/silica, and we attribute
this difference to the different pH values at which the proteins
were entrapped (9.5 for AlP and 4.5 for AcP), resulting in the
known more spacious network and cages for the latter.42 This
interpretation is corroborated by the lower entrapment of AlP
compared to that of AcP (Table 2), by its higher T.O.N. values
(Table 1), and also by the inability of the matrix to protect AcP
activity below pH 3. Also in support for the ionic mechanism
involved with the surfactants in both cases is the observation
mentioned above, that is, the nonionic surfactant Brij 56 provides
only minimal added value.
In conclusion, we have revealed an unusually high level of
protection of phosphatases against harsh pH conditions by
entrapment in silica sol-gel matrixes and have shown the added
value of coentrapment of surfactants. The main objective of this
research has been to answer the need for having enzymes
capable of operating under unorthodox environments as may
be required for sensors and for biocatalysts operating under non-
native conditions. We believe that this objective has been
achieved by showing its feasibility in the case of an alkaline
enzyme operating at pH 0.9 and of an acidic enzyme operating
at pH 13.0.
(41) Iler, R. K. The Chemistry of Silica; Wiley: New York, 1979.
(42) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: New
York, 1990.
(40) Rottman, C.; Avnir, D. J. Am. Chem. Soc. 2001, 123, 5730-5734.
9
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Sol−Gel Materials as Efficient Enzyme Protectors
A R T I C L E S
Acknowledgment. Various parts of this research project were
supported by the Ministry of Science, the Eshkol Scholarship
(to H.F.-M.), the Infrastructure Project (Tashtiot), and the
French-Israeli AFIRST Program.
Supporting Information Available: Experimental details.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA0507719
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