1

Not Really About Hemicellulose

The Cole-Fort Research Group

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

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

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

7

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

8

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

9

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

10

Collecting Formaldehyde Emissions

11

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

12

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

13

Higher plants do not make free formaldehyde!

14

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

15

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)

16

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)

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

Xylanase

α-Glucuronidase

OOHO O

HOOAc

O

OOAcO O

OHO

O

OH

HOH3CO COOH

Multiple Enzymes Will Be Required

19

Hydrolysis of Birch Xylan with the Xylanase from Trichoderma Viride

Homology model of T. viride xylanase, based on xylanase from T. reesei

20

Glu178

Glu86

H2N C C OH

OH

CH2

CH2

CO2H

Glutamic Acid

Computer “Docking” of a Model Substrate

21

Hydrolysis of Birch Xylan4% Sulfuric Acid, 119 oC

22

Enzymatic Hydrolysis, pH 4.5, 35 oC

Hardwood hemis hydrolyzed in “green liquor”

23

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

24

Enzymes

Homology model of glucohydrolase fromAspergillus niger

Glucohydrolase from Trichoderma reesei

25

Lignin-related Molecules Tested As Inhibitors

Vanillin Acetovanillone

p-Coumaric Acid Vanillic Acid

26

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

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

p-Coumaric acid docked to A. niger enzyme

Example Docking

Cellobiose (substrate) docked to T. versicolor enzyme

29

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

30

Extra:How doesResveratrolWork?

31

Extra: How Does Resveratrol Work?