Not Really About Hemicellulose The Cole-Fort Research Group 1.
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Transcript of Not Really About Hemicellulose The Cole-Fort Research Group 1.
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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
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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
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
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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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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
Quantitation by HPLC-MS, GC-MS+SIM
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0 50 100 150 200 2500
200
400
600
800
1000
1200
1400
1600
1800
2000
Temperature vs. Formaldehyde Emissions
Temperature (oC)
μg H
2CO
/g O
D w
ood
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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
Trapped as 2,4,6-trichlorophenylhydrazide, gc-ms+sim analysis
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Trap CrystallineCellulose
AlcellLignin
1 681 1943
2 21.7 28.9
3 17.3 15.2
Total 719 1987
Most formaldehyde comes from lignin. How?
ppm by mass, air-dried material
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OOH
OHHOHO
OH
OOH
OHHOHO CH2OH+
Glucose H = 75.7 kcal/mol
OHO
OCH3
OH
O
OCH3
OH
CH2OH+
Lignin modelH = 57.6 kcal/mol
(energies from ab initio calculations)
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OHO
OH
OCH3
HO
OCH3
O
OH
OCH3
HO
OCH3
CH2O+
Lignin model H = 60.1 kcal/mol
CH2O CH2O+ CH3OH H2C O+
FormaldehydeH = - 61.7 kcal/mol
(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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Xylanase
α-Glucuronidase
OOHO O
HOOAc
O
OOAcO O
OHO
O
OH
HOH3CO COOH
Multiple Enzymes Will Be Required
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Hydrolysis of Birch Xylan with the Xylanase from Trichoderma Viride
Homology model of T. viride xylanase, based on xylanase from T. reesei
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OO O
OO
OO
OO
OH
OH
OH
OH
HOOH
HOOH
HOOH
HOOH
OHOHO
OHO
OH
OOH
OHHO
OH
Cellulose
Cellobiose
Another Source of Sugars for Energy
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Enzymes
Homology model of glucohydrolase fromAspergillus niger
Glucohydrolase from Trichoderma reesei
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Lignin-related Molecules Tested As Inhibitors
Vanillin Acetovanillone
p-Coumaric Acid Vanillic Acid
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Average Lineweaver-Burk plots of three replicated trials with and without vanillic acid inhibitor
y = 13.983x + 8642.7
R2 = 0.9836
y = 16.291x + 11833
R2 = 0.9578
0
5000
10000
15000
20000
25000
30000
-1000 -500 0 500 1000 1500
1 / Cellobiose con.(L/mol)
1 /
Vel
oci
ty (
Lh
/mo
l)
Inhibitors were evaluated by classical enzyme kinetics, using the Lineweaver-Burke technique
Km, the binding constant, and kcat, the turnover number can be determined from these plots
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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
Results of Kinetic Studies
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p-Coumaric acid docked to A. niger enzyme
Example Docking
Cellobiose (substrate) docked to T. versicolor enzyme
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Formaldehyde Resveratrol
Ashley Hellenbrand (MS) Regan LeBlanc (MS)Hannah Cole (OHS) Melody Rhine (REU)Anna Weigang UM Dustin Neidt (BS)Andria Peña-Melendez (REU) Panduka PiyaratneJoel Strothers (REU)
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