Not Really About Hemicellulose
The Cole-Fort Research Group
1
Want to Live to Be 140?
Start with calorie restriction (CR):
 Calorie restriction means a low calorie diet
containing all necessary nutrients
 CR extends the life of C. elegans,
Drosophila, mice, and some primates
 Good health is maintained
Resveratrol
 Found in red wine (pg/mL)
 Claimed to be responsible
for the “French paradox”
 Mimics the effect of calorie
restriction
2
OK, you’re a fat mouse, and we
feed you resveratrol:
 Insulin sensitivity increases
 Insulin-like growth factor (IGF-1)
decreases
 Peroxisome proliferator increases
 Motor function improves
 Alzheimer’s peptide decreases
Result:
 No type II diabetes
 Reduced risk of cancer
 Lower blood lipids
 You make the mouse Olympics
More: http://chemistry.umeche.maine.edu/CHY431/Resveratrol.html
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So we went out in the woods, cut down a bunch of
trees, stripped the bark, and made conifer bark tea
 Grind up the bark
 Place in the cup of a
Soxhlet extractor
 Boil alcohol and water
in the bottom
 Go home for 24 hours
Why the bark?
Conifers store antioxidants in the bark to guard
against fungi and bark beetles
Fungi use oxidative enzymes (laccase, peroxidase) to
degrade bark; beetles attack partially degraded bark
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Quantitation by HPLC-MS, GC-MS+SIM
Source
Resveratrol, mg/g
Norway spruce
567
Black Spruce
439
White spruce
382
Eastern White Pine
7.1
Japanese knotweed
523
Grape skins
32.5
Low bush blueberries
12.8
Heirloom tomatoes
1.8
Unsweetened chocolate
1.24
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Our work with formaldehyde, starts here: Katrina
and the infamous FEMA trailers
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Raw (untreated) wood emits formaldehyde,
at room temperature and above!
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Collecting Formaldehyde Emissions
10
Temperature vs. Formaldehyde Emissions
2000
μg H2CO/g OD wood
1800
1600
1400
1200
1000
800
600
400
200
0
0
50
100
150
200
250
Temperature (oC)
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Trapped as 2,4,6-trichlorophenylhydrazide, gc-ms+sim
analysis
Trap
22°C
200°C
1
3.45
1847
2
1.88
7.74
3
2.08
2.25
Total
7.40
1857
ppm formaldehyde, by mass, oven dried wood
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Higher plants do not make free formaldehyde!
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Most formaldehyde comes from lignin.
How?
Trap
Crystalline
Cellulose
Alcell
Lignin
1
681
1943
2
21.7
28.9
3
17.3
15.2
Total
719
1987
ppm by mass, air-dried material
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OH
O
O
OH
HO
OH
HO
OH
HO
CH2OH
OH
HO
H = 75.7 kcal/mol
Glucose
O
HO
+
+
CH2OH
O
OCH3
OCH3
OH
OH
Lignin model
H = 57.6 kcal/mol
(energies from ab initio calculations)
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OH
HO
OH
O
O
OCH3
+
OCH3
HO
CH2O
HO
OCH3
OCH3
H = 60.1 kcal/mol
Lignin model
CH2O
+
CH2O
H = - 61.7 kcal/mol
CH3OH
+
H2C O
Formaldehyde
(energies from ab initio calculations)
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Enzymatic Hydrolysis of Xylans and
Cellulose
 The goal is to convert these wood
components to sugars under mild conditions
 Immobilization of enzymes on polymer
beads would make the enzymatic processes
more economical
 Immobilization also would allow a
continuous flow process, rather than batch
processing
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Multiple Enzymes Will Be Required
Xylanase
OAc
O
O
HO
HO
O
O
O
AcO
O
O
OH
O
OH
α-Glucuronidase
HO
H3CO
O
COOH
18
Hydrolysis of Birch Xylan with the Xylanase from
Trichoderma Viride
Homology model of T. viride xylanase, based on xylanase from T. reesei
19
Computer “Docking” of a Model Substrate
Glu178
Glutamic Acid
Glu86
H O
H 2N
C
C OH
CH2
CH2
CO2H
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Hydrolysis of Birch Xylan
4% Sulfuric Acid, 119 oC
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Enzymatic Hydrolysis, pH 4.5, 35 oC
Hardwood hemis hydrolyzed in “green liquor”
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Another Source of Sugars for Energy
OH
OH
OH
HO
O
O
O
O
HO
OH
HO
O
OH
O
O
O
O
HO
OH
OH
OH
Cellulose
OH
OH
HO
HO
O
O
HO
O
OH
OH
OH
Cellobiose
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Enzymes
Homology model of
glucohydrolase from
Aspergillus niger
Glucohydrolase from
Trichoderma reesei
24
Lignin-related Molecules Tested As Inhibitors
Vanillin
p-Coumaric Acid
Acetovanillone
Vanillic Acid
25
Inhibitors were evaluated by classical enzyme
kinetics, using the Lineweaver-Burke technique
Average Lineweaver-Burk plots of three replicated trials with and
without vanillic acid inhibitor
1 / Velocity (Lh/mol)
30000
y = 16.291x + 11833
R2 = 0.9578
25000
20000
y = 13.983x + 8642.7
R2 = 0.9836
15000
10000
5000
0
-1000
-500
0
500
1000
1500
1 / Cellobiose con.(L/mol)
Km, the binding constant, and kcat, the turnover
number can be determined from these plots
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Results of Kinetic Studies
For the enzyme from A. niger, the inhibitors tested had
no effect within experimental error
The enzyme from T. reesei did suffer inhibition of the
order of 30-35% with some materials; for example,
vanillic acid
We now are conducting computational docking studies
to explore the mode of interaction of the inhibitors
with the enzymes
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Example Docking
Cellobiose
(substrate) docked
to T. versicolor
enzyme
p-Coumaric
acid docked
to A. niger
enzyme
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Formaldehyde
Resveratrol
Ashley Hellenbrand (MS)
Hannah Cole (OHS)
Anna Weigang UM
Andria Peña-Melendez (REU)
Joel Strothers (REU)
Regan LeBlanc (MS)
Melody Rhine (REU)
Dustin Neidt (BS)
Panduka Piyaratne
Enzymatic Hydrolysis
LeRae Graham (PhD)
Gamini Rupasinghe (MS)
Francis and Robin Avery, Brian Perkins
$$ USDA, NSF-EPSCoR, WBC, FBRI
Prof. Barbara Cole
Prof. Joe Genco
Prof. Ray Fort
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Extra:
How does
Resveratrol
Work?
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Extra: How Does Resveratrol Work?
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