Biotechnology
Speciality Chemicals Magazine June 2009 53
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enzyme was able to decontaminate paraoxon once the coating
was rehydrated. If the instability of the OPH enzyme contributed
to the loss of activity after extended ageing, then it would have
been expected to remain inactive after being rehydrated and
should have failed to hydrolyse the OP substrate. This was not the
case, which validates the OPH stability in the polymer.
Lipase hydrolysis activity
To observe the hydrolysis of the ester bonds of a water-insoluble
substrate, the lipid-hydrolysing enzyme lipase was incorporated
into a commercial latex coating. To determine if the rate of con-
version was a function of total surface area, 3 wt% porcine pan-
creas lipase was blended with latex paint and drawn onto
polypropylene sheets at 200 �m wet thickness.
The films were dried for 30 hours at 25�C, then removed and
sectioned into 1, 2 and 3 cm2 areas. The films were challenged
with the model substrate p-nitrophenyl acetate 1.45 mM in 100
mM Tris at pH 7.1.
The rate of catalysis was measured by monitoring the hydrol-
ysis product p-nitrophenol ( 400 = 13,500 M-1 cm-1, pH 7.1). The
observed catalytic rate of hydrolysis increases with increasing
sample surface area (Figure 4a). The highest catalytic conversion
rate was observed in the 3 cm2 sample, with the 1 cm2 sample
having the lowest observed activity.
The observed increased hydrolysis of ester bonds as a function
of surface area was investigated further to determine if bulk vol-
ume was able to contribute to the total catalytic rate of ester
hydrolysis.
To determine the bulk phase catalytic rate of ester hydrolysis,
3 wt% porcine pancreas lipase was blended with commercial
latex and applied to aluminium panels at 50, 100, 150 or 200 �m
wet thickness. Control panels were prepared similarly without
lipase enzyme. The films were dried at 25�C for 30 hours prior
to cutting into 3 cm2 samples.
To observe any effect that bulk properties contribute to the
hydrolysis of ester substrates, the films were not removed from
the aluminum panels. Therefore, any substrate introduced to the
sample would diffuse through one surface. Observed catalytic
rates of 15.82 � 0.33 g/hr/m2 in the lipase-latex films indicate that
the activity is limited by surface area and independent of bulk vol-
ume (Figure 4b).
It is possible that the low solubility of the substrate in latex films
restricts reagent diffusion into the bulk phase to interact, react or
hydrolyse with embedded biocatalysts. Although the catalytic rate
of ester hydrolysis is surface area-dependent in latex films, the
bulk volume-dependant catalytic rates observed in PMMA films
indicates that porcine pancreas lipase can be used for ester
hydrolysis. By understanding biocatalyst interactions with sub-
strates in relation to the polymer matrix, stimuli-responsive solid
phase technologies can be achieved by engineered functional
coatings.
Summary
The ester linkage, a common linkage in several chemical warfare
agents and pesticides, was hydrolysed with self-decontaminating
bioenhanced coatings. Enzyme modification and engineering
continually produces more efficient catalysts with effective sub-
strate specificity.
It is expected that specific enzymes can be harvested or engi-
neered for transforming a wide variety of selected substrates.
Some variants are engineered to have increased stability, while
others have been designed for increased catalytic rates against
selected substrates. As both rational and random methods of
enzyme improvement are developed, enzymes are increasingly
being engineered to play an expanding role in the coatings
industry.
As progress continues in the development of bioactive mole-
cules, efforts are being made to optimise their incorporation into
a variety of coatings. The effectiveness of the biomolecules is
influenced not only by the coating polymer chemistry but also by
the large number of components generally present in coatings.
The interactions of biomolecules with resins, cross-linkers, sol-
vents, and additives are being evaluated independently and in
combination to determine which possess optimal interactions that
maximise enzyme efficiency and activity retention in thin films.
Understanding the effect of combined parameters broadens the
utility for self-decontamination and reactive coatings.
with coating technology
References:
1. S.C. McDaniel, J.
McDaniel, M.E. Wales & J.R.
Wild, Prog. Org. Coat. 2006,
55, 182-188
0.24
0.20
0.16
0.12
0.08
0.04
0.00
mPp-NP
0 50 100 150 200 250
Time (minutes)
1x1 cm latex-3Lip
1x2 cm latex-3Lip
1x3 cm latex-3Lip
1x3 cm control
Blank
0.05
0.04
0.03
0.02
0.01
250200150100500
p-NP(mM/cm2
)
Time (minutes)
10
20
30
40
50
60
70
80
p-NP(g/m2
)
AMV100-3Lip 2mil
AMV100-3Lip 4mil
AMV100-3Lip 6mil
AMV100-3Lip 8mil
AMV100-0Lip 2mil
AMV100-0Lip 4mil
AMV100-0Lip 6mil
AMV100-0Lip 8mil
Control
For more information, please contact:
Steve McDaniel
Reactive Surfaces Ltd.
300 West Avenue, Suite 1316
Austin
TX-78701
USA
Tel : +1 512 472 8282
E-mail: smcdaniel@reactivesurfaces.com
Website: www.reactivesurfaces.com
Figure 4 - Lipase mediated
hydrolysis of p-nitrophenyl
acetate via surface area
dependant assay using 1, 2
and 3 cm2 free films (a) &
bulk volume dependant
assay using 3 cm2 latex
coated aluminum panels (b)
a
b

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