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Not Really About Hemicellulose The Cole-Fort Research Group 1.

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Presentation on theme: "Not Really About Hemicellulose The Cole-Fort Research Group 1."— Presentation transcript:

1 Not Really About Hemicellulose The Cole-Fort Research Group 1

2 2 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

3 3 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:

4 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

5 5 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

6 6

7 7 Source Resveratrol,  g/g Norway spruce567 Black Spruce439 White spruce382 Eastern White Pine7.1 Japanese knotweed523 Grape skins32.5 Low bush blueberries12.8 Heirloom tomatoes1.8 Unsweetened chocolate1.24 Quantitation by HPLC-MS, GC-MS+SIM

8 8 Our work with formaldehyde, starts here: Katrina and the infamous FEMA trailers

9 9 Raw (untreated) wood emits formaldehyde, at room temperature and above!

10 10 Collecting Formaldehyde Emissions

11 11

12 12 Trap22°C200°C 13.451847 21.887.74 32.082.25 Total7.401857 ppm formaldehyde, by mass, oven dried wood Trapped as 2,4,6-trichlorophenylhydrazide, gc-ms+sim analysis

13 13 Higher plants do not make free formaldehyde!

14 14 TrapCrystalline Cellulose Alcell Lignin 16811943 221.728.9 317.315.2 Total7191987 Most formaldehyde comes from lignin. How? ppm by mass, air-dried material

15 15

16 16

17 17 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

18 18 Xylanase α-Glucuronidase Multiple Enzymes Will Be Required

19 19 Hydrolysis of Birch Xylan with the Xylanase from Trichoderma Viride Homology model of T. viride xylanase, based on xylanase from T. reesei

20 20 Glu178 Glu86 Glutamic Acid Computer “Docking” of a Model Substrate

21 21 Hydrolysis of Birch Xylan 4% Sulfuric Acid, 119 o C

22 22 Enzymatic Hydrolysis, pH 4.5, 35 o C Hardwood hemis hydrolyzed in “green liquor”

23 23 Another Source of Sugars for Energy

24 24 Enzymes Homology model of glucohydrolase from Aspergillus niger Glucohydrolase from Trichoderma reesei

25 25 Lignin-related Molecules Tested As Inhibitors Vanillin Acetovanillone p-Coumaric Acid Vanillic Acid

26 26 Inhibitors were evaluated by classical enzyme kinetics, using the Lineweaver-Burke technique K m, the binding constant, and k cat, the turnover number can be determined from these plots

27 27 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

28 28 p-Coumaric acid docked to A. niger enzyme Example Docking Cellobiose (substrate) docked to T. versicolor enzyme

29 29 FormaldehydeResveratrol Ashley Hellenbrand (MS) Regan LeBlanc (MS) Hannah Cole (OHS)Melody Rhine (REU) Anna Weigang UMDustin Neidt (BS) Andria Peña-Melendez (REU)Panduka Piyaratne Joel 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 GencoProf. Ray Fort

30 30 Extra: How does Resveratrol Work?

31 31 Extra: How Does Resveratrol Work?

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