Protection of mesopore-adsorbed organic matter from enzymatic degradation

Article abstract of DOI:10.1021/es035340+

Synthetic mesoporous alumina and silica minerals with uniform pore geometries, and their nonporous analogues, were used to test the role of mineral mesopores (2-50 nm diameter) in protecting organic matter from enzymatic degradation in soils and sediments

Full text of DOI:10.1021/es035340+

Environ. Sci. Technol. 2004, 38, 4542-4548
OM (8) and soil OM (9), suggest that formation of OM-
Protection of Mesopore-Adsorbed
Organic Matter from Enzymatic
Degradation
mineral complexes can stabilize labile forms of OM against
microbial attack (10, 11). The biodegradation and bioreme-
diation of organic contaminants in soils are also retarded by
mineral sorption (12, 13). Organic contaminants have been
found to become less bioavailable with “aging” time following
initial adsorption (14, 15). The processes responsible for this
behavior within soils, sediments, and aquifers are not well
understood.
A N D R E W R . Z I M M E R M A N , * , † , §
J O N C H O R O V E R , ‡
K E I T H W . G O YN E , ‡ A N D
S U S A N L . B R A N T L E Y†
Cycling of organic carbon in sediments and soils has been
characterized by complex kinetics in which fast and slow
processes of carbon degradation are observed (2). Fast cycling
has been related to biodegradation of readily available OM,
whereas slow cycling has been attributed to the occurrence
of a less bioavailable portion of the OM. This dual availability
character has been attributed both to recalcitrant versus labile
fractions and to sorbed versus dissolved fractions (8, 16).
However, decreased biological degradation of organic com-
pounds has also been attributed to the presence of pores in
soils and test materials (14, 17, 18). Because mineral surface
areas can be dominated by the internal surfaces of pores
ranging 2-50 nm in diameter (19, 20), some workers have
suggested that mineral mesopores play a major role in the
sequestration and preservation of sedimentary OM (19, 21).
This may occur by physical occlusion of OM within mineral
pores, thus protecting OM from degradative attack by bacteria
and their extracellular enzymes (19, 21, 22). Others have even
suggested that OM sorption to soil minerals may be a
prerequisite to mineralization because of the preferential
colonization of surfaces by microorganisms (23). Here, we
directly test the viability of the “mesopore protection”
hypothesis with in vitro combinations of either mesoporous
or nonporous mineral analogues and a model organic
substrate-enzyme pair.
In our initial tests of the mesopore protection hypothesis,
amino acid monomers and polymers were sorbed onto and
desorbed from fabricated mesoporous and nonporous
alumina and silica in batch aqueous experiments (24). Each
mineral pair was of similar surface chemistry (site density
and charge properties) and differed only in the presence or
absence of intraparticle mesoporosity (25). All amino acid
monomers and polymers smaller than about one-half of the
pore diameter exhibited significantly greater surface area-
normalized adsorption to mesoporous alumina and silica as
compared to nonporous analogues. Proteins of sizes similar
to or larger than the mesopores exhibited diminished
adsorption to porous relative to nonporous solids, indicating
sorptive exclusion from the internal pore surfaces. Further,
evidence for enhanced retention of OM within mesopores
was found in the increased desorption hysteresis for me-
soporous versus nonporous mineral-sorbed amino acid
compounds. Although the mechanism of pore affinity
remains unclear, it is plausible that a unique chemical
environment exists therein (e.g., electric double layer overlap
or water exclusion), to favor stronger sorption. Nonetheless,
the observationsthat small organic molecules may be
strongly retained in mesopores whereas enzyme-sized mol-
ecules may be excludedssuggests the protective capacity of
mesopores.
Department of Geosciences, The Pennsylvania State
University, University Park, Pennsylvania 16802, and
Department of Soil, Water and Environmental Science,
University of Arizona, Tucson, Arizona 85721
Synthetic mesoporous alumina and silica minerals with
uniform pore geometries, and their nonporous analogues,
were used to test the role of mineral mesopores (2-50
nm diameter) in protecting organic matter from enzymatic
degradation in soils and sediments. Dihydroxyphenylalanine
(L-DOPA), a model humic compound, was irreversibly sorbed
to both mineral types. The surface area-normalized
adsorption capacity was greater for the mesoporous
minerals relative to their nonporous analogues. The
degradation kinetics of free and mineral-sorbed L-DOPA
by the enzyme laccase was monitored in a closed cell via
oxygen electrode. Relative to freely dissolved L-DOPA,
nonporous alumina-sorbed substrate was degraded, on
average, 90% more slowly and to a lesser extent (93%), likely
due to laccase adsorption to alumina. In contrast,
relative to free L-DOPA, degradation of nonporous silica-
sorbed L-DOPA was enhanced by 20% on average. In the
case of mesoporous alumina and silica-sorbed L-DOPA,
the enzyme activity was 3-40 times lower than that observed
for externally sorbed substrate (i.e., L-DOPA sorbed to
nonporous minerals). These results provide strong evidence
to support the viability of the mesopore protection
mechanism for sequestration and preservation of sedimentary
organic matter and organic contaminants. Nanopore
adsorption/desorption phenomena may aid in explaining
the slow degradation of organic contaminants in certain soils
and sediments and may have implications for environmental
remediation and biotechnological applications.
Introduction
Despite its lability, it has been observed that some fraction
of biomolecular organic matter (OM) and degradable con-
taminants remain preserved in soils and sediments, appar-
ently unavailable for microbial utilization (1, 2). Direct
correlations between organic carbon and specific surface
area in many soils and sediments (3, 4-7), and the slower
mineralization of sorbed versus desorbed marine sedimentary
The goal of this study was to directly test the mesopore
protection hypothesis by comparing the enzyme-mediated
degradation rate of an organic compound sorbed to non-
porous versus mesoporous minerals. A diphenol, 3,4-L-
dihydroxyphenylalanine (L-DOPA), was chosen as a model
small organic compound as it possesses moieties commonly
found in complex natural OM and phenolic xenobiotics
* Corresponding author phone: (352)392-0070; fax: (352)392-9294;
e-mail: azimmer@geology.ufl.edu.
† The Pennsylvania State University.
‡ University of Arizona.
§
Present address: University of Florida, Department of Geological
Sciences, 241 Williamson Hall, P.O. Box 112120, Gainesville, FL 32611-
2120.
9
4 5 4 2 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 17, 2004
10.1021/es035340+ CCC: $27.50
2004 Am erican Chem ical Society
Published on Web 07/23/2004

(carboxylic, amine, hydroxyl). L-DOPA was incubated with
laccase, a member of the phenoloxidase class of enzymes
that are capable of catalyzing the oxidation of aromatic
compounds using molecular oxygen (26). Although the
particular laccase used was derived from Trametes villosa
fungi, many soil microorganisms including bacteria and other
fungi are known to produce phenoloxidases (27). The results
indicate that small organic molecules are not accessible to
enzymatic degradation when sorbed within mineral meso-
pores, and, therefore, we provide direct evidence for the
viability of the proposed mineral mesopore protection
mechanism for sequestration and preservation of natural
and contaminant organic compounds.
Enzym e Activity Assays. Fungal-derived extracellular
laccase (T. villosa, EC 1.10.3.2) was provided by Novo Nordisk
(Danbury, CT). The activity of laccase, with free and adsorbed
L-DOPA as substrate, was determined using a biological
oxygen monitor (BOM model 5300; Yellow Spring Instruments
Co., Yellow Springs, OH) equipped with a Clark oxygen
electrode (5357 Oxygen Probe; Yellow Spring Instruments
Co.). Mineral-sorbed L-DOPA was prepared and quantified
as above. After L-DOPA adsorption, minerals were rinsed in
5 mL of background electrolyte solution for 5 min to remove
the easily desorbable fraction, and the mass of sorbate
retained was calculated. Five milliliter aliquots of dissolved
L-DOPA solution, or varying masses of mineral with sorbed
L-DOPA and 5 mL of background solution, were then sealed
without air bubbles into the BOM sample chamber and
incubated at 25 °C while stirring with a Teflon-coated
magnetic bar. After the vessel was sealed and the oxygen
probe was equilibrated, 0.1 mL of laccase solution (15 µg
mL-1) was introduced through a side port next to the electrode
via syringe. Oxygen concentration in the vessel was recorded
every 30 s until oxygen consumption ceased, after no more
than 5 min in all cases. Following incubations, sorbents from
BOM vessels were transferred quantitatively into preweighed
pans, dried (80 °C), and weighed to record the mass of sorbent
material so that the mass of sorbed L-DOPA present in the
reactor could be calculated.
Materials and Methods
Mineral Sorbents. Amorphous mesoporous alumina (Al2O3)
and silica (SiO2) minerals were synthesized by the neutral
template route as described by Komarneni et al. (28) and
Pauly and Pinnavaia (29), respectively. Briefly, dodecylamine
was stirred in water and ethanol while aluminum isopro-
poxide or tetraethyl orthosilicate was slowly added (0.27:
29.6:9.09:1.0 and 0.25:127.5:10.3:1.0 molar ratios, for alumina
and silica, respectively). After 20 h of aging (while stirred at
65 °C in the case of silica) followed by air-drying, the material
was calcined (540 °C, 6 h) to remove the organic template
leaving only the inorganic support. Mesoporous alumina and
silica were of unimodal pore size distribution with mean
pore diameters of 8.2 and 4.0 nm, respectively, and specific
surface areas of 242 and 962 m2 g-1, respectively, as
determined by N2 sorptometry. On the basis of N2 sorp-
tometry, we also estimate that 96.5% and 99.7% of the
fabricated alumina and silica surface area, respectively, is
located within pores between 2 and 20 nm in size (25).
Nonporous alumina (γ-Al2O3) and silica, with specific
surface areas of 37 and 7.5 m2 g-1, respectively, were
purchased from Alfa Aesar (Ward Hill, MA; stock Nos. 40007
and 89709, respectively) and were chosen for their similarity
in surface charge properties to their mesoporous analogues.
Following washing of the nonporous alumina (0.02 M CaCl2,
24 h, 5 times), all minerals, porous as well as nonporous,
were found to be free of organic carbon, that is, below
detection limits. All were used as powders with particle sizes
of roughly 0.1 µm. Additional information on the morphology
and chemistry of the sorbents is published elsewhere (24,
25).
Sorption and Desorption Methods. L-DOPA (Sigma-
Aldrich; St. Louis, MO) was used as received and reacted
with mesoporous and nonporous minerals in an aqueous
batch suspension, as described previously (24). Briefly, surface
area-normalized batch reactions were conducted by adding
80 m2 of mineral to 50 mL polypropylene centrifuge tubes
containing 25 mL of aqueous background solution (0.02 M
CaCl2, and 200 mg L-1 HgCl2 as a bactericide). A pH of 6.5
was maintained by addition of HCl for alumina, and Ca-
(OH)2 for silica minerals. After 3 d of end-over-end rotation
at 8 rpm, tubes were centrifuged (4500 rpm, 1 h), the
supernatant solution was removed and replaced with 25 mL
of OM-free background solution, and the particles were
redispersed on an agitator for 1 min prior to a 3 d desorption
period with the same rotational mixing. The surface excess
(sorbed µmol) of L-DOPA and its change during desorption
were calculated from the difference between L-DOPA con-
centration in a sorbent-free control and that of the suspension
supernatant solution. L-DOPA concentrations were measured
on a fluorometer after the addition of a fluorescent derivative
(30). Laccase adsorption isotherms were determined similarly
except a 5 min adsorption period was used (to match the
time period of enzyme experiments, below) and solution
concentrations were measured using a Bio-Rad protein assay
(UV detection at 595 nm), calibrated with albumin standards.
Using energy minimization modeling techniques (24) and
other published sources, we estimate the spherical molecular
diameters of laccase (66 kDa) (31) and L-DOPA to be about
7 and 0.5 nm, respectively. Thus, on the basis of our previous
findings (24), we expect that L-DOPA will be able to enter
both alumina and silica mesopores (8.2 and 4.0 nm mean
diameters, respectively) and laccase will largely be excluded
from the pores.
Data Analysis. Adsorption data were fit to the Langmuir-
Freundlich (LF) isotherm that takes the form
NbC m
1 + bC m
q )
(1)
and describes the equilibrium relationship between the
surface excess of sorbate, q, and the equilibrium concentra-
tion of adsorptive in solution, C (32). N, b, and m are fitting
parameters that represent adsorption maximum, affinity, and
site heterogeneity, respectively. The LF equation is useful
when modeling adsorption of organic compounds to ener-
getically heterogeneous materials (32, 33). The LF isotherm
equation was fit to the experimental data following the
method of Umpleby et al. (33) in which the solver function
of Microsoft Excel 2002 is used to maximize the coefficient
of determination (R 2) through iterative variation of the fitting
parameters. R 2 is calculated from the sum of residuals, that
is, the difference between experimental and model-predicted
values of q. Methods used to calculate a hysteresis index are
detailed elsewhere (24).
The kinetics of the conversion of free (i.e., dissolved)
L-DOPA to the product dopaquinone (Figure 3, inset 1) was
modeled by the Michaelis-Menten equation
V
maxSo
vo )
(2)
So + b
where So, vo, and Vmax are the initial substrate concentration,
the initial reaction rate, and the maximum initial reaction
rate, respectively. Systems obeying Michaelis-Menten ki-
netics are linear when So and vo are plotted in double-
reciprocal form. This Lineweaver-Burk plot has an intercept
of 1/ Vmax and a slope of Km/ Vmax where Km is the so-called
half-saturation constant or substrate affinity.
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VOL. 38, NO. 17, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4 5 4 3

FIGURE 1. L-DOPA adsorption/desorption to (a) nonporous alumina
(filled symbols), and (b) mesoporous alumina (open symbols).
Adsorption data are shown as circles, and desorption data are
shown as triangles. Error bars represent 95% confidence intervals
for each data point. Lines are best-fit Langmuir-Freundlich isotherms
(equations shown) for adsorption data only.
FIGURE 2. L-DOPA adsorption/desorption to (a) nonporous silica
(filled symbols), and (b) mesoporous silica (open symbols). Other
designations are the same as in the Figure 1 caption.
Results
Substrate Adsorption-Desorption. The adsorption-de-
sorption of L-DOPA on nonporous and mesoporous alumina
(NP-Al and MP-Al, respectively) and silica (NP-Si and MP-
Si, respectively) sorbents were similar to that of amino acid
monomers and dimers investigated previously (24). First,
adsorption isotherm data were not well-fit by the common
Langmuir and Freundlich equations but were better predicted
by the hybrid LF eq 1 (Figures 1 and 2). Second, adsorption
maxima, N, expressed on a surface area-normalized basis,
were similar or greater for mesoporous versus nonporous
mineral analogues (0.53 and 0.46 µmol m-2 for MP-Al and
NP-Al, respectively, and 1.29 and 0.07 µmol m-2, for MP-Si
and NP-Si, respectively). In addition, the mesoporous
sorbents exhibited greater surface area-normalized adsorp-
tion at all equilibrium solution concentrations of adsorptive,
C. The binding affinity, b, of L-DOPA was 2 times greater for
MP-Al versus NP-Al but 3 times smaller for MP-Si versus
NP-Si. Last, the magnitude of desorption hysteresis was
greater for mesoporous versus nonporous mineral-sorbed
L-DOPA (Figures 1 and 2); hysteresis indices (24) were 2 times
(MP-Al) and 4 times (MP-Si) higher than those for the
corresponding nonporous sorbents.
These sorption data highlight the unique nature of internal
mesopore adsorption of small organic compounds as com-
pared to those sorbed to external surfaces. Relatively strong
adsorptive interaction and/ or inhibited desorption of com-
pounds from mesopores have been variously attributed to
increased intramolecular interaction of sorbate molecules
due to surface curvature (34), superposition of interaction
potentials on opposing pore walls (35), electric double layer
overlap (36), or pore-filling (24). Although the mechanism
for enhanced adsorption to mesoporous minerals remains
FIGURE 3. Kinetics of enzyme (laccase)-mediated conversion of
free L-DOPA. Inset 1 shows the reaction. Inset 2 is the Lineweaver-
Burk plot.
unclear, it is evident that L-DOPA and other small organic
molecules can be strongly sorbed, sometimes irreversibly, to
internal mesopore surfaces.
Substrate Incubations with Laccase. Conversion of free
L-DOPA to the diketone product was well-modeled by eq 2,
yielding Vmax and Km values of 1.06 µmol of O2 consumed
min-1 (stoichiometrically equivalent to 2.12 µmol of L-DOPA
min-1) and 2.20 µmol of L-DOPA, respectively (Figure 3).
These kinetic constants are in the range of those reported for
the conversion of L-DOPA or similar phenols by laccase or
other phenoloxidases (37, 38). The mass of L-DOPA present
was linearly related to total O2 consumption for free L-DOPA
less than 1.5 µmol. We calculate that 0.77 ((0.07) mol of O2
was consumed for each mole of dissolved L-DOPA present
rather than the stoichiometrically predicted factor of 0.5 mol
of O2 mol-1 of L-DOPA. This may be because (i) the incubation
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TABLE 1. Enzyme Degradation of Alumina-Sorbed L-DOPA
Experimental Data
initial enzyme
total consumed
mineral-sorbed
L-DOPA (mmol)
mineral
mass (g)
activity (µmol
oxygen (µmol
of O min-1
)
of O )
2
2
Nonporous Alumina
0.837
0.000
0.000
0.876
0.876
1.061
1.491
1.559
1.649
2.515
2.544
2.660
2.995
5.591
5.813
5.927
6.154
6.814
9.398
13.676
15.925
17.437
0.001
0.010
0.072
0.064
0.077
0.000
0.124
0.072
0.017
0.023
0.090
0.123
0.147
0.112
0.102
0.073
0.062
0.129
0.095
0.128
0.077
0.001
0.017
0.340
0.219
0.342
0.001
0.437
0.282
0.075
0.081
0.469
0.439
0.498
0.454
0.522
0.442
0.427
0.402
0.342
0.483
0.283
0.389
0.116
0.119
0.144
1.190
0.211
1.293
0.780
0.822
0.360
0.275
0.512
0.771
0.825
0.813
0.900
0.861
1.254
1.461
1.599
Mesoporous Alumina
0.000
0.000
1.584
2.470
2.580
2.652
6.172
6.305
6.459
8.192
8.670
9.191
14.217
23.349
29.580
0.128
0.009
0.001
0.002
0.000
0.005
0.009
0.028
0.019
0.032
0.019
0.018
0.014
0.020
0.012
0.011
0.017
0.004
0.007
0.003
0.021
0.029
0.115
0.079
0.198
0.188
0.092
0.033
0.067
0.027
0.031
0.255
0.160
0.250
0.143
0.137
0.126
0.131
0.108
0.135
0.143
0.108
0.170
0.279
0.348
FIGURE 4. (a) Initial and (b) total activity of laccase-mediated
degradation of L-DOPA sorbed to nonporous (closed symbols) and
mesoporous (open symbols) alumina. Incubations displaying the
greatest apparent degree of activity suppression (discussed in text)
are circled. The dashed line is the regression line for the conversion
of free dissolved substrate.
solution was not fully O2 saturated, (ii) less than the total 5
mL of solution was in contact with the added laccase, or (iii)
additional free substrate would have been converted had
further time been allowed.
Relative to the rate and extent of solution-phase substrate
conversion observed for free L-DOPA, incubations of laccase
with alumina-sorbed L-DOPA (porous or nonporous) resulted
in lower enzyme activity and less total substrate converted
(Figure 4; Table 1). For example, whereas the half-saturation
substrate amount of 2.2 µmol of L-DOPA resulted in an initial
enzyme activity of 0.53 µmol of O2 min-1, the same mass of
NP-Al-sorbed L-DOPA resulted in a maximum observed rate
of 0.12 µmol of O2 min-1. Fractional loss of initial enzyme
activity due to adsorption to NP-Al-sorbed L-DOPA varied
from 4% to 34% (mean of 10%). The dashed line shown in
Figure 4b is the regression line in the low concentration region
of free dissolved substrate conversion. All data points underlie
this line and therefore indicate reduced conversion. On
average, the extent of conversion of NP-Al-sorbed L-DOPA
was 7% that of free L-DOPA.
In contrast, the rates and extents of laccase conversion
of NP-Si-sorbed L-DOPA (Figure 5; Table 2) were similar to
or greater than values obtained for equivalent masses of free
L-DOPA. On average, excluding the two incubations with the
most sorbed L-DOPA, initial enzyme activity and total L-DOPA
converted using NP-Si-sorbed substrate were both 1.2 times
that of dissolved substrate. Enhanced enzyme activity of NP-
Si-sorbed substrate is indicated by points above the dashed
line in Figure 5b.
4a) was 5-27 times greater for NP-Al relative to MP-Al, and
total laccase activity (Figure 4b) was 4-36 times greater. Rates
(Figure 5a) and extents (Figure 5b) of conversion of NP-
Si-sorbed L-DOPA were enhanced over their MP-Si-sorbed
analogues to a similar degree (7-16 times for both). For both
sets of sorbents, initial and total enzyme activity increased
steeply with substrate loading, eventually reaching a maxi-
mum and then decreasing with even higher substrate loading.
Plausible reasons for this convex upward curve and differ-
ences in laccase-mediated degradation as a function of
mineral morphology are discussed in the following section.
Discussion
Enzym atic Degradation of Nonporous Mineral-Sorbed
Substrate. The comparative enzymatic degradation of free
versus adsorbed substrate is complicated by the possibility
that the enzyme itself may become sorbed to the particle
surfaces, which can affect its activity. Many studies have
examined the kinetic properties of mineral-adsorbed en-
zymes. Early work in this area focused on layer silicate clays
(39-41), while, more recently, mesoporous materials have
attracted attention for possible biotechnology applications
because of their ability to stabilize enzymes over longer time
periods (42, 43). In all of these studies, however, enzymes
were adsorbed or reagent-immobilized on surfaces prior to
substrate addition. Our work is, to our knowledge, the first
to show examples of both reduced and enhanced enzyme
activity for sorbed versus free substrate.
Sorption to mesoporous alumina and silica resulted in
much lower rates and extents of enzyme-mediated substrate
conversion relative to those observed for nonporous sorbents.
At similar L-DOPA loadings, initial laccase activity (Figure
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VOL. 38, NO. 17, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4 5 4 5

due to enzyme interaction with mineral surfaces (44). For
example, the activity of acid phosphatase sorbed to mont-
morillonite and Al hydroxide was found to be reduced relative
to its free activity by 80% and 65%, respectively (44). Likewise,
the residual activity of different laccases adsorbed to several
types of bentonite, kaolinite, and quartz was found to be
reduced by 0-89%, 50-70%, and 43-65%, respectively (39).
These reductions in enzyme activity may be attributable to
steric hindrance, conformational changes of the enzyme
induced by adsorption to the mineral surface (40), or pH
changes close to the mineral surface that alter enzyme activity
(39).
Cases of unchanged or increased enzyme activity upon
mineral interaction, such as those that we observed for silica,
are less common in the literature, although some have been
noted (41). For example, Claus and Filip (39) observed little
change in peroxidase and tyrosinase (both phenoloxidases)
activity upon adsorption to quartz sand (residual activities
of 106% and 101%, respectively). Many studies have examined
reagent-immobilized enzymes on mesoporous silicas, and,
in most cases, enzyme activity is lost (45, 46). However, Lei
et al. (47) documented that the activity of entrapped
organophosphorus hydrolase was twice that of the free
enzyme. It is possible that silica binds L-DOPA in such a way
as to make it more accessible to enzymatic degradation.
However, due to its associated H+ ions, the pH is likely lower,
closer to the negatively charged silica surface than in the
bulk solution (48). We therefore hypothesize that enhanced
laccase activity for NP-Si-sorbed versus free substrate may
be because the pH is closer to the optimum pH for fungal
laccase activity (i.e., pH 3-5) (49) close to silica’s surface.
The kinetics observed in the mineral-sorbed L-DOPA
systems cannot be modeled using the Michaelis-Menten
equation. Instead, initial activity versus substrate curves for
both nonporous minerals exhibit a hyberbolic shape and
may be indicative of substrate inhibition. This is a special
case of enzyme uncompetitive inhibition in which substrate
molecules compete for active enzyme sites at high substrate
concentrations (50). Our data fit the theoretical model
describing substrate inhibition according to Cleland (50) and
Whitwam and Tien (51) (see Supporting Information).
However, this mechanism is unlikely because incubations at
high substrate concentrations with no mineral present
showed no evidence of declining enzyme activity. We note,
however, that incubations displaying the greatest apparent
degree of activity suppression (those circled in Figures 4a
and 5a) were often those in which the greatest mineral surface
area was added. For example, the eight NP-Al incubation
data points circled in Figure 4a are among the 11 cases in
which more than 0.81 g of mineral (>30 m2) was used. Two
other incubations using >30 m2 of mineral resulted in almost
no enzyme activity. Similarly, five of the six NP-Si incubations
showing the greatest signs of activity suppression (circled in
Figure 5a) are among the eight incubations with greater than
20 m2 of mineral surface area added. We therefore hypoth-
esize that suppressed laccase activity was due to laccase
adsorption to mineral surfaces and this effect was greater in
incubations with higher particle surface area.
FIGURE 5. (a) Initial and (b) total activity of laccase-mediated
degradation of L-DOPA sorbed to nonporous (closed symbols) and
mesoporous (open symbols) silica. The circled data and dashed
line are explained in Figure 4.
TABLE 2. Enzyme Degradation of Silica-Sorbed L-DOPA
Experimental Data
initial enzyme
total consumed
mineral-sorbed
L-DOPA (mmol)
mineral
mass (g)
activity (µmol
oxygen (µmol
of O min-1
)
of O )
2
2
Nonporous Silica
0.072
0.104
0.216
0.216
0.220
0.270
0.290
0.303
0.309
0.334
0.439
0.449
0.704
0.832
0.855
1.067
1.084
2.587
0.026
0.021
0.325
0.221
0.166
0.145
0.228
0.145
0.235
0.171
0.201
0.400
0.286
0.287
0.288
0.100
0.115
0.059
0.045
0.733
0.523
0.338
0.307
0.482
0.306
0.509
0.415
0.550
0.840
0.647
0.666
0.649
0.235
0.246
3.733
2.664
2.664
1.493
1.834
2.413
2.054
2.575
4.120
3.662
3.045
2.438
2.880
2.960
6.135
3.440
To explore this hypothesis further, we conducted laccase
adsorption experiments for each mineral phase using the
same conditions as imposed during enzyme activity mea-
surements. That is, a constant mass of laccase (1.5 µg) was
added to 5 mL solutions with varying amounts of mineral for
a period of 5 min. For each sorbent, plots of adsorbed laccase
versus mineral surface area (Figure 6) were modeled using
a simple exponential function, q ) k(S.A.)n, where k and n
are fitting parameters. The results indicate that at the
conditions of the enzyme activity experiments, assuming no
influence of mineral-sorbed L-DOPA, the majority (80-100%)
of the laccase added was likely to have adsorbed to NP-Al
Mesoporous Silica
0.000
0.645
1.661
0.093
0.004
0.011
0.011
0.012
0.026
0.029
0.029
0.014
1.335
0.025
0.031
0.028
1.339
1.334
1.338
1.359
0.103
0.116
0.125
0.125
0.163
0.116
0.111
5.140
10.348
10.877
21.179
24.467
Most workers have shown decreased enzyme activity
(lower Vmax and higher Km, i.e., reduced substrate affinity)
9
4 5 4 6 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 17, 2004

size and/ or steric constraints. This is consistent with our
previous finding that proteins of molecular dimensions
similar to, or larger than, mesopore openings are inhibited
from entering pores, resulting in lower surface area-normal-
ized adsorption capacities (24). L-DOPA is of a molecular
size (about 0.5 nm) likely to be sequestered within both the
alumina and the silica mesopores (8.2 and 4.0 nm, respec-
tively), while laccase (about 7 nm) was inhibited from entering
most mesopores of both minerals.
Adsorption of organic compounds to mineral surfaces (9,
13, 56, 57) and entrapment within intraparticle mineral pores
(12, 14, 17, 57) are believed to be responsible for the reduction
of OM biodegradation rates in experiments conducted with
live bacteria. Using enzymes and comparing nonporous and
mesoporous minerals of similar surface chemistry, this study
identifies a mechanism likely responsible for these observa-
tions. Although adsorption to mineral surfaces can enhance
or retard enzymatic degradation of organic compounds,
occlusion within nanometer-sized pores may lead to long-
term preservation of even extremely labile OM and organic
contaminants due to enzyme exclusion. We conclude that
both the extent of enzyme adsorption to mineral surfaces
and the degree of mineral nanoporosity are important factors
in determining the likelihood of OM preservation in sediment
and soil. Although this study utilizes a single enzyme-
substrate system and synthetic minerals, it serves as a model
from which studies utilizing more complex OM assemblages
and mineral surface types may be extended. Furthermore,
mesopore-adsorbed substrate systems may prove useful as
delivery vehicles for organic compounds to agricultural,
medical, or environmental systems.
FIGURE 6. Laccase adsorption to alumina and silica minerals as
a function of surface area. Lines are fits to an exponential model
described in text.
where at least 10 m2 of mineral surface area was present.
Only 5-20% was likely to have sorbed to NP-Si. This may
explain the greater suppression of laccase activity during
experiments with alumina versus silica as well as the loss of
enzyme activity in incubations with greater mineral surface
area available for adsorption of laccase.
The above conclusion is also supported by prior reports.
Suppression of montmorillonite-sorbed-tyrosinase activity,
also a phenoloxidase, has been shown to vary directly with
the degree of hydroxyaluminum coatings (52). Although
ligand exchange and electrostatic and hydrophobic interac-
tions are all considered important for protein-mineral
adsorption (53-55), the former two are likely of greater
importance for enzyme interaction with hydroxylated alu-
mina surfaces and are, apparently, more apt to result in the
observed enzyme activity suppression due to conformational
changes.
Enzym atic Degradation of Mesoporous Mineral-Sorbed
Substrate. While enzymatic degradation ofL-DOPA was either
somewhat reduced or enhanced by nonporous mineral
adsorption, degradation was greatly reduced by adsorption
to minerals with intraparticle mesoporosity. Importantly, the
effect was present both for alumina, an activity-suppressing
surface, and for silica, an activity-enhancing surface. This
stronger suppression cannot be attributed to greater adsorp-
tion of laccase to the mesoporous minerals because, for both
alumina and silica, the mesoporous materials were found to
adsorb less laccase on a surface area-normalized basis (Figure
6). As an aside, it should be noted that under the adsorption
conditions used, laccase covered only a small fraction
(<0.01%) of the surfaces of the materials tested; thus, the
effect of excluded mesoporous surface on adsorption reduc-
tion was not greater.
Another possibility is that L-DOPA was more readily
desorbed from nonporous mineral surfaces during the
incubations, leading to measurement of greater laccase
activity. However, the amount of L-DOPA we would expect
to desorb (10% and 30% maximum from alumina and silica,
respectively, based on 3 d adsorption/ desorption isotherms)
could not support the observed differences in enzyme activity.
The difference between substrate desorption from meso-
porous versus nonporous analogues would be even smaller
than the total desorbable fraction. Further, by rinsing the
minerals prior to the start of the degradation experiment,
the rapidly desorbing fraction was removed. These observa-
tions also argue against the oxidation of desorbed substrate
having a strong effect on further desorption.
Acknowledgments
We thank Drs. Sridhar Komarneni and Jean-Marc Bollag for
use of their laboratories during mesoporous mineral synthesis
and enzyme activity incubations, respectively. The laboratory
assistance of Dr. Mi-Youn Ahn is especially appreciated.
Acknowledgment is also made to the Penn State Bio-
geochemical Research Initiative for Education (BRIE) spon-
sored by NSF (IGERT) Grant DGE-9972759 and to the donors
of the American Chemical Society Petroleum Research Fund
for their support of this research.
Supporting Information Available
One figure showing Lineweaver-Burk plots and substrate
inhibition models of enzyme activity. This material is available
free of charge via the Internet at http:/ / pubs.acs.org.
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Received for review December 2, 2003. Revised manuscript
received May 11, 2004. Accepted May 18, 2004.
ES035340+
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