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SUPPLEMENTARY MATERIALS
Enzyme-Enabled Responsive Surfaces for AntiContamination Materials
Songtao Wu1,2,#, Andreas Buthe1,#, Hongfei Jia2,*, Mindy Zhang2, Masahiko Ishii3, Ping Wang1,*
1
Department of Bioproducts and Biosystems Engineering and Biotechnology Institute, University
of Minnesota, St. Paul, MN, 55108; 2Toyota Research Institute of North America, Toyota Motor
Engineering and Manufacturing North America, Inc., Ann Arbor, MI, 48105; 3Vehicle Material
Engineering Division, Toyota Motor Corporation, Toyota City, Aichi, 471-8572, Japan
#
These authors contributed equally to this work; * Corresponding authors
S-1.
Additional details of activity assays
S-2.
Microscopic imaging analysis of bioactive coatings
S-3.
The anti-contamination activity of bioactive coatings studied by using high
performance liquid chromatography (HPLC)
-S1-
S-1
Additional details of activity assays
The activity of α-amylase-coatings was measured by a standard colorimetric assay
(Fischer and Stein 1961), of which the amount of reducing groups released from starch as
amylolytic product is determined via a reaction with 3,5-dinitrosalicylic acid. The substrate
solution comprised of 1% w/v potato starch in 20 mM sodium phosphate buffer with 6.7 mM
sodium chloride (pH 6.9). To measure coating activity, one piece of film was incubated in 2 ml
of substrate solution. In case of native enzyme, 10 μl of the enzyme solution (0.2 mg/ml) were
admixed to the substrate solution. After exact 3 min at RT, 3,5-dinitrosalicylic acid was added
and the solution was moved into boiling water bath for 15 min, and then transferred to ice bath.
The equivalent of reducing sugar was determined on a UV-Vis spectrometer at 540 nm. One
activity unit is defined as 1.0 mg reducing sugar (calibrated using maltose as the standard)
released from starch in 3 min at RT.
Proteolytic activity of Thermoase C160 was determined by measuring tyrosine equivalent
(Folin and Ciocalteu 1927). The assay substrate solution was prepared by mixing 1 ml of 2%
(w/v) of casein (0.05 M sodium phosphate, pH 7.5) with 0.2 ml of sodium acetate buffer (10
mM, pH 7.5) containing 5 mM of calcium acetate. The substrate solution was pre-incubated in
the water bath for 3 min to reach 37°C, followed by the addition of one piece of bioactive
coating to trigger the reaction. The reaction mixture was shaken at 200 rpm until reaction was
stopped by adding 1 mL of 110 mM tricholoroacetic acid, while the reaction mixture was further
incubated for 30 min at 37°C prior to centrifugation. The supernatant (0.4 ml) was mixed with
0.2 ml of 25% (v/v) Folin-Ciocalteau reagent and 1 mL of 0.5 M sodium carbonate, and then
analyzed on a UV-Vis spectrophotometer at 660 nm for tyrosine equivalent. One activity unit is
defined as the amount of enzyme hydrolyzing casein to produce absorbance equivalent to 1.0
µmol of tyrosine per min at 37°C.
-S2-
S-2
Microscopic imaging analysis of bioactive coatings
The cross-section of bioactive coatings was investigated by a JSM 5800 model (JEOL
Co., Tokyo, Japan) scanning electron microscope (SEM) to visualize the dispersed enzyme
aggregates within the coatings. Cross-sectionized samples were prepared by coating on the
aluminium foil, following the same protocol as given for the aluminium panels. The fully cured
coatings were torn and the resulting cross-section of the fractured polymer and surface were
sputtered with Au-Pd, mounted on a thin specimen split mount for SEM characterization.
Enzymes in the coating were further analyzed by confocal laser scanning microscopy
(CLSM) after being specifically labeled with a fluorescent dye (Pinto and Macías 1995). First, a
dye solution of 50 µmole was obtained by dissolving Oregon Green 488 Maleimide in 2 mL of
phosphate buffer (pH 7.0, 10 mM). Then dye solution was used to immerse a small piece of the
enzyme coating. The reaction was conducted at RT for 2 hours and avoided from light by
wrapping with aluminum foil. After the reaction, free dye molecules were washed away with
phosphate buffer (pH 7.0, 10 mM) at RT for 2 hours under shaking at 200 rpm. After dried at RT
for 1 hour, the film was mounted with cover glass by Prolong Gold antifade media, and observed
by CLSM (Zeiss LSM 510-META, Thornwood, NY) equipped with an inverted microscope. An
objective lens (63×/water immersion) was utilized. The excitation wavelength was set at 488 nm
with an emission maximum at approximately 524 nm. Laser intensity and detection settings were
maintained constant during imaging of all samples.
S-3
The anti-contamination activity of bioactive coatings studied by using high
performance liquid chromatography (HPLC)
Quantitative analysis of the biocatalytic action against starch stains on α-amylasecontaining coating surface was conducted via HPLC analysis, following a reported procedure
(Sutthirak et al. 2005). Wheat starch solution with concentration of 10% w/v was prepared in
-S3-
deionized water and 100 µl of stain solution was applied onto one α-amylase-containing coating
for a specific period, and BSA-containing coating was used as control. Subsequently the spots
were washed off from the coating panels using 9.9 mL of deionized water. The washing solution
was immediately recovered, diluted 100 times followed by centrifugation and filtration over
NanoSEP filter (Pall, East Hills, NY) with a cut-off 30 KDa at 16,000 rpm for 1 min, and finally
analyzed by HPLC chromatograph (Varian, Walnut Creek, CA). Separation was achieved using
a Varian Metacarb 87P (300×7.8 mm) column (Varian, Walnut Creek, CA) with HPLC grade
water as eluent (flow rate 0.4 mL/min; 80°C; running time 45 min), analytes were detected via a
ELSD (Polymer Laboratories 2100 ELSD, Amherst, MA). Calibration was done by using pure
10 min
0.4
60 min
0 min
0.2
90 min
0
Relative peak height (-)
a
0.6 0.8 1.0
carbohydrates.
5
10
15
Retention time (min)
20
25
b
Enzyme
different periods (0-90
min) loadings (%)
5.0
2.5
1.25
0.625 0.125
0
0 20 40 60 80 100
Relative path length (%)
Relative surface activity (%)
0 20 40 60 80 100
Figure S1. HPLC analysis of starch stains degradation time course on an α-amylase coating for
The activity of α-amylase coatings when contacted with wet starch stains for different
duration (0-90 min) is demonstrated in Figure S1. At 0 min, there is only one peak with a
retention time at 10.3 min on the chromatograph. Because all samples were filtrated prior to
0
1
2
3
4
5
HPLC analysis, this peak should correspond to the water soluble (MW≤30 KDa) content of the
Enzyme loading (%)
Relative activity (%)
40
80
120
Relative activity (%)
40
80
120
starch. Once starch stain was in contact with the α-amylase coating, the area of the peak at 10.3
c
min first increased and then decreased over time, along with emergence of peaks with longer
retention time corresponding to fractions of lower MW. This observation basically confirmed
0
0
-S4-
0
30
60
90
120
0
10
20
30
40
that, due to enzymatic catalysis, starch stain was first digested into high MW compounds, which
were then further broken down into smaller molecules.
References
Fischer EH, Stein EA. 1961. α-Amylase from human saliva. Biochem. Prep. 8:27-33.
Folin O, Ciocalteu V. 1927. On tyrosine and tryptophane determinations in proteins. J. Biol.
Chem. 73:627-650.
Pinto MC, Macías P. 1995. Determination of intraparticle immobilized enzyme distribution in
porous support by confocal scanning microscopy. Biotechnol. Tech. 9: 481-486.
Sutthirak P, Dharmsthiti S, Lertsiri S. 2005. Effect of glycation on stability and kinetic
parameters of thermostable glucoamylase from Aspergillus niger. Process Biochem. 40:
2821-2826.
-S5-
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