EFFECTS OF NIPAM POLYMER ADDITIVES ON THE ENZYMATIC HYDROLYSIS OF
AVICEL AND PRETREATED MISCANTHUS
Katherine J. Mackenzie and Matthew B. Francis*
Department of Chemistry, University of California, Berkeley, and Lawrence Berkeley National
Laboratory,
Materials Sciences Division, Berkeley, CA 94720-1460 (U.S.A.)
mbfrancis@berkeley.edu
Supporting Information
General Procedures and Materials
Unless otherwise noted, all chemicals and solvents used were of analytical grade and were used
as received from commercial sources. Analytical thin layer chromatography (TLC) was
performed on EM Reagent 0.25 mm silica gel 60-F254 plates with visualization by ultraviolet
(UV) irradiation at 254 nm, ninhydrin, or potassium permanganate stain. Purifications by flash
chromatography were performed using EM silica gel 60 (230-400 mesh). The eluting system for
purification was determined by TLC. Room temperature and 4 ºC centrifugations were
conducted either with a Sorvall RC 5C (Sorvall, USA) Plus for samples greater than 50 mL, a
Sorvall LEGEND Mach 1.6R for samples between 1 and 50 mL, or an Eppendorf Mini Spin Plus
for samples less than 1 mL (Eppendorf, USA). Centrifugations above room temperature were
performed on a Hettich Rotofix 46 H (GMI, Ramsey, MN). Samples were lyophilized using a
LAB CONCO FreeZone 1L (Lab Conco, USA). Regular scale UV-Vis spectroscopic
measurements were conducted in a Varian Cary 50 spectrophotometer (Agilent, USA).
Absorbance and fluorescence measurements of samples in 96 well plates were obtained on a
SpectraMax M2 (Molecular Devices, Sunnyvale, CA).
Instrumentation and Sample Analysis Preparation
Gel Permeation Chromatography (GPC). GPC was performed on a Waters system, including
a Waters 515 pump, Waters 717 autosampler, and Waters 2414 differential refractive index (RI)
detector. SEC was performed at 1.0 mL/min in a PLgel Mixed B (10 μm) and a PLgel Mixed C
(5 μm) column (Polymer Laboratories, both 300 × 7.5 mm), in that order, using a mobile phase
of DMF with 0.2% LiBr and linear PMMA (690-194,400 MW) as the calibration standards. The
columns were kept at 70 ºC.
Experimental
poly(NIPAm-co-MEPO) (S1). This was synthesized multiple times following a previously
reported procedure on a 2 g scale.1Analysis of the 1H-NMR
spectrum showed the molar ratio of MEPO:NIPAm to be from
O
NH
O
NH
1:10.2 to 1:11.3 depending on the batch, or 8.1-8.9% incorporation
of MEPO. GPC analysis using PMMA standards indicated the
m
n
NH
batches were of similar mass distribution, approximately
O
Mn=68,300, Mw=163,000, and PDI=2.4.
ONH
2
Small-Molecule Modification of S1. Solutions of 10 mg/mL S1 and 60 mM acetone,
formaldehyde, or 4-hydroxy butanone, or 600 mM mannose were prepared in 50 mM NaOAc
(pH 4.5) and reacted for 24 h at rt. Unreacted small molecules were removed by exchanging the
modified polymer into pure water through 8 rounds of ultrafiltration (10 kDa MWCO). The
polymers were lyophilized and analyzed by NMR spectroscopy to confirm modification of all
aminooxy groups.1
Protein Expression and Activity Assays
Expression and Purification of EGPh. The endoglucanase from Pyrococcus horikoshii (EGPh)
was expressed and purified following a previously reported procedure.1
Preparation of Miscanthus. The Miscanthus used for activity assays was obtained from the lab
of Prof. Douglas Clark at UC Berkeley. The substrate (Miscanthus giganteus) had been cut into
approximately 1-inch pieces, then subjected to acid pretreatment at 25% biomass loading with
1.5% (w/w) sulfuric acid at 190 °C for approximately 1 min. After a subsequent steam explosion
step the solids were pressed to remove liquids, then washed extensively with deionized water
until the filtrate had no detectable glucose and neutral pH. The solids were dried for 24 h at
104 °C, ground into a fine powder with a mortar and pestle, then stored at rt until use.
Other Additives. The homopolymer of NIPAm was prepared following the procedure for S1, at
1/5 the scale and without MEPO. GPC analysis using PMMA standards indicated Mn=49,575,
Mw=155,535, PDI=3.1. Bovine Serum Albumin (BSA) was desalted before use. Poly(ethylene
glycol) (PEG) (Mn=20k; Sigma-Aldrich) and poly(acrylamide) (Mw= 1,000k; Polysciences, Inc.)
were washed extensively with deionized water before use. Tween 20 was used as received.
Other Enzymes. Celluclast was obtained from Sigma-Aldrich and desalted before use. An
endoglucanase from Aspergillus niger and cellobiohydrolase I (CBHI) from Trichoderma
longibrachiatum were obtained from Megazyme International (Bray, Ireland) and used as
received.
General EGPh Activity Assays. All assays were performed in triplicate. Unless otherwise
indicated, assays were performed in 1.3 mL Eppendorf tubes on a 0.5 mL scale, in 50 mM
NaOAc (pH 4.5), with 1% (w/v) Avicel pH 101 microcrystalline cellulose (Sigma-Aldrich) or
acid-pretreated and steam-exploded Miscanthus, 0.2 μM EGPh, for 12 h in a 40 °C water bath.
Mannose-modified S1 was included at 2 mg/mL unless otherwise stated, and was allowed to mix
thoroughly with the substrate before addition of enzyme. Controls contained no additive.
Samples were stirred by a magnetic stir plate at a rate sufficient to fully suspend the substrate but
not so vigorously as to cause a vortex. After 12 h, the tubes were centrifuged at 13.2k rpm for
two 5 min intervals, with a 180° rotation in between. An aliquot of the clarified supernatant was
transferred to a 0.6 mL Eppendorf tube and frozen on dry ice, then stored at -20 °C until soluble
reducing sugar was quantified. For time-point experiments, the total volume of each sample was
1.3 mL, with identical concentrations as indicated above. At each indicated time point, the
reaction tubes were removed from the water bath and shaken to ensure even distribution of the
contents. A 100 μL aliquot was transferred to a clean, empty Eppendorf tube, and the reaction
tube returned to the water bath. The removed aliquot was centrifuged for 10 min at 13.3k rpm
and the clarified supernatant was transferred to a new tube and frozen until later analysis.
Soluble Reducing Sugar Analysis. Substrate hydrolysis was determined by measuring soluble
reducing sugar following a previously reported method, using the paired glucose oxidasehorseradish peroxidase assay with OxiRed as the fluorogenic substrate.1 All samples were
measured in triplicate using clear-bottom plastic 96-well plates, with an internal standard curve
of 250, 200, 150, 100, 50, and 0 μM glucose. Samples of 100 and 50 μM cellobiose were also
included to confirm full hydrolysis to glucose. Frozen aliquots from activity assays were thawed
on ice and diluted with pH 4.5 buffer, then 8 uL aliquots were mixed with 8 uL of β-glucosidase
(5 mg/mL in 10 mM NaOAc pH 4.6) in a 96-well plate and incubated at 37 °C for 60 min to
hydrolyze any soluble oligosaccharides. Glucose was then quantified by adding 65 μL of glucose
oxidase (1.25 U/mL), horseradish peroxidase (1.25 U/mL), and OxiRed (60 μM) in 125 mM
phosphate buffer (pH 7.45) and incubating at rt for 15 min in the dark. The amount of Resorufin
formed was measured on an optical plate reader with excitation at 535 nm and emission
detection at 590 nm. The internal standards were used to make linear standard curves for each
plate (r2 > 0.98) which were used to calculate the glucose equivalents present in each well. The
triplicate measurements of each supernatant sample were averaged, then the values of the
triplicate assay samples were averaged to calculate each data point.
HPLC. Analysis of glucose, cellobiose, and cellotriose was performed for a range of enzymes
assayed on Avicel. Samples of cleared supernatant were filtered through 0.45 μM PTFE filters,
then injected onto a 3 x 250 mm PA200 column (Dionex, now Thermo Fisher Scientific) with a
guard column (3 x 50 mm) of the same material and analyzed using a ICS-3000 chromatography
system (Dionex). Compounds were eluted at 30 °C with a flow rate of 0.4 mL/min and mobile
phase buffer A (0.1 M NaOH) and buffer B (0.1 M NaOH, 1 M NaOAc) with the following
gradient: in 25 min from 0% B to 13.4% B, then in 0.1 min to 30% B, 2.9 min isocratic, then 0%
B for 7 min. Compounds were detected using pulsedamperometric detection with the
carbohydrate standard 4-potential waveform. Glucose, cellobiose (Sigma), and cellotriose
(Seikagaku Biobusiness, Tokyo, Japan) standards were used for calibration. The results of this
analysis are shown in Figure S1.
References
1. Mackenzie K J, Francis M B. (2013) Recyclable thermoresponsive polymer-cellulase
bioconjugates for biomass depolymerization. J. Am. Chem. Soc. 135: 293-300.
glucose equivalents from each species
copol.
glucose
avg. std. dev.
cellobiose
avg. std. dev.
cellotriose
avg. std. dev. total
glucose
as a % of total glucose equivalents equivalents
glucose
cellobiose cellotriose from OxiRed
EGPh
−
+
49.9
356.9
1.2
5.5
562.2
2716.3
42.0
47.4
104.7
134.5
7.7
21.2
717
3208
7.0%
11.1%
78.4%
84.7%
14.6%
4.2%
689
2678
A. niger
−
+
16.6
211.0
3.8
1.9
114.1
626.8
8.8
48.3
135.7
820.9
25.4
31.8
266
1659
6.2%
12.7%
42.8%
37.8%
50.9%
49.5%
236
1238
T. long.
−
+
108.7
296.4
5.8
9.5
2493.5
3450.1
69.6
136.9
142.5
181.1
6.9
13.9
2745
3928
4.0%
7.5%
90.8%
87.8%
5.2%
4.6%
2418
3439
Celluclast
−
+
823.6
11.8
6718.4
90.5
75.3
14.8
7617
10.8%
88.2%
1.0%
6852
1606.3 111.6
7801.5
746.8
120.4
9.6
9528
16.9%
81.9%
1.3%
8957
Figure S1. HPLC analysis of hydrolysis products from enzymes assayed on Avicel. Cellobiose
and cellotriose values have been converted into glucose equivalents assuming full hydrolysis.
Copolymer (mannose-capped S1) was included at 2 mg/mL if indicated. Glucose equivalent
values from OxiRed assay analysis shown here are the values from Figure 2a.
relative glucose equivalents
3.5
(a)
3.0
2.5
2.0
1.5
O
O
1.0
O
n
0.0
1
2
(b)
3
4
5
6
sample designation
7
poly(N-isopropylacrylamide)
R groups derived from:
8
OH OH O
O
(c)
NH
O
n
3.5
X= –(CH 2)3NHCO–
O
HO
O
NH
X
acetone
N
R2
H
OH OH
m
O
R3
H
3.0
mannose
O
H
formaldehyde
OH
4-hydroxy-2-butanone
2.5
2.0
1
2
3
4
1.5
1.0
control (no additive)
PEG
poly(acrylamide)
poly(NIPAm)
NIPAm
copolymer
relative glucose equivalents
4.0
n
n
poly(acrylamide) poly(ethylene glycol) [PEG]
0.5
H
N
O
NH2
5
6
7
8
acetone modified
mannose modified
formaldehyde modified
4-hydroxy-2-butanone modified
0.5
0.0
1
3
4
6
1
5
sample designation
7
8
Figure S2. Comparison of the effect various NIPAm-related polymers have on (a) 12 h Avicel
hydrolysis, (b) 12 h Miscanthus hydrolysis, and (c) 16 h Miscanthus hydrolysis by EGPh. The Nisopropyl group is required for activity enhancement. The small molecule chosen to modify the
aminooxy group has little effect on the level of activity enhancement.