1. Measurement of Endo and Exo-Glucanase Activities in Cellulase Using Non-Crystalline Cellulose
Rajesh Gupta and Y. Y. Lee
Department of Chemical Engineering
Auburn University, AL 36849
AIChE Annual Meeting, San Francisco, CA
November 16, 2006

2. Outline Overview on Cellulosic Ethanol
Significance of Cellulase Enzymes
Cellulase Action and Activities

3. BIOFUEL Ethanol (E-10, E-85) Biodiesel Biocrude Methane Hydrogen
“Sustainable transportation fuel made from renewable feedstock”
Ethanol (E-10, E-85)
Biodiesel
Biocrude
Methane
Hydrogen

4. Why Biofuels?
Biofuels present opportunities for new industry and improved farm economy.
The fuel ethanol industry alone adds more than $20 billion/year to the US economy.

5. Ethanol Boom
US produced 3.9 billion gallons of Bioethanol in 2005 (about 3% of US gasoline consumption) exclusively from corn.
94 ethanol refineries nationwide (over 4 billion gallons/year capacity).
30 new plants and 9 expansions are currently under way(=additional 1.8 billion gallons/year capacity).

6. Economic Impact of Corn Based Ethanol
Goals of Renewable Fuel Standard (2005):
8 billion gallons by 2012.
Ethanol industry will spend $70 billion over
Direct spending and indirect impact will add $200 billion to GDP over
Reduce trade deficit by $64 billion.
Long term goal: 20 billon gallons by 2020.

7. Constraints of Corn-Based Ethanol
Upper limit?
8 billion gallons/year represents 1/3 of total corn production.
Marketability of byproducts (DDG).
Feedstock other than corn must be utilized.

8. Inherent Engineering Difficulties in Cellulosic Ethanol
Heterogeneous Feedstock
Solid Handling
Slow Reaction
Non-reusable Catalyst (Enzyme)

9. Cellulose & Cellulase

11. Cellulose Hydrolysis as a Rate Process
Non-Reaction
Resistances
Reaction

12. Cellulase Endo-Glucanase Exo-Glucanase (CBH) β-Glucosidase
Cellulose → Scarred Cellulose
Exo-Glucanase (CBH)
Scarred Cellulose → Cellobiose
β-Glucosidase
Cellobiose → Glucose
(Courtesy of NREL)

13. Cellulase Activity Determination
FPU Method (Goshe, 1987)
Uses filter paper (Whatman No.1) as the standard
substrate.
Initial rate is measured by one data-point.
Released sugars are measured in terms of reducing ends by DNS reagent (does not distinguish G1 and G2).
Repeatability is poor because of several factors in
the procedure that are error-prone.

14. Proposed Modification
HPLC for measurement of released sugar
G1 & G2 are converted to glucan for conversion calculation.
Slope-method (multiple points) for initial rate

15. Activity Determination by initial slope method
Substrate: Filter Paper

16. Beyond the FPU?
Observation of G1 & G2 is not sufficient to characterize the cellulase.
Different combination of the three cellulase
components may give same FPU.
Use of substrates with different properties may provide additional information.

17. Additional Substrates
Non-Crystalline Cellulose (NCC)
Cello-oligosaccharides

18. Non-Crystalline Cellulose (NCC)
Amorphous cellulose made in our laboratory
from crystalline cellulose.
Hydrogen-bonds in cellulose are disrupted.
Crystallinity is essentially removed.

19. α-Cellulose NCC (Freeze-Dried)
SEM
(1000X)
(3000X)
α-Cellulose NCC (Freeze-Dried)

20. NCC
Cotton

21. X-Ray Diffraction Patterns of MicroCrystalline Cellulose),
a-Cellulose & Non-Crystalline Cellulose

22. DSC curves for a–Cellulose [----- ] & NCC [ - - - ] Melting Pt
DSC curves for a–Cellulose [----- ] & NCC [ ] Melting Pt. : NCC= 260 oC, a-Cellulose = 340 oC

23. FTIR graph for Treated & Untreated a-Cellulose -- A (Untreated a-cellulose), (Without baseline correction) B (Treated a-cellulose), (Baseline correction from 1800 cm-1 to cm-1)

24. NCC as a Substrate for Activity Measurement
Highly amorphous ( suitable for Endo-G activity measurement)
Low DP ( suitable for Exo-G activity measurement)
Higher surface area than crystalline cellulose
Extremely high initial hydrolysis rate (short reaction time)
Endo and Exo-G activities can be measured simultaneously in one experiment.
Both activities can be measured using single substrate.

25. NCC Hydrolysate Loading: 0.01 ml Sp CP-A/ g-Glucan, 1 hr Cellobiose
Glucose
Cello-oligosaccharides
Glucose

26. Initial sugar release from different substrates Enzyme loading: 0
Initial sugar release from different substrates Enzyme loading: ml Sp CP-A/g-glucan, 15 min.

27. Schematic Representation of Structural Differences between Crystalline Cellulose & NCC
Amorphous domain
(Substrate for Endo-glucanase)
Reducing Ends
(Susbtrate for Exo-glucanase)
Amorphous domain
(Substrate for Endo-glucanase)
Reducing Ends
(Susbtrate for Exo-glucanase) E P
k’ = k[S] dP =
K’[E] dt
E + S P k dP =
k[E][S] dt

28. Hydrolysis of NCC SOLID LIQUID Endo-G Exo-G NCC Β-G Low DP NCC COS
CELLOBIOSE
Β-G
GLUCOSE

29. Endo-Glucanase and Exo-Glucanase Activities from NCC
Measurement of
Endo-Glucanase and Exo-Glucanase Activities from NCC
0.5ml 2% sonicated NCC Solution with Buffer
0.5ml of diluted Enzyme Solution
Stop the reaction by boiling it for 5min.
&
Dilute it 3 times by cold DI water
Stir the content and take two 0.5ml sample
containing
uniform mixture liquid and solid
Take liquid sample
for HPLC
sugar analysis
Determine Glucose and Cellobiose
concentration
Incubate at 50oC
for 30min.
Add 3ml DNS reagent in one sample and
centrifuge other sample & then add 3ml of DNS in liquid
part only. Boil both the sample for 5min and cool
Immediately afterward. Centrifuge the sample
and find the absorbance of the
Solid+Liquid part and the Liquid part.
0.5ml 2% sonicated NCC Solution with Buffer
0.5ml of diluted Enzyme Solution
Stop the reaction by boiling it for 5min.
&
Dilute it 3 times by cold DI water
Stir the content and take two 0.5ml sample
containing
uniform mixture liquid and solid
Take liquid sample
for finding the sugar concentration
(From HPLC)
Find the Glucose and Cellobiose
concentration
Incubate at 50oC
for 30min.
Add 3ml DNS reagent in one sample as it is and
centrifuge other sample & then add 3ml of DNS in liquid
part only. Boil both the sample for exact 5min and cool
the content Immediately. Centrifuge both the sample again
and find the absorbance of both
( Solid+Liquid part and only Liquid part )

30. Activity Determination
Sample calculation
* HPLC results represent mg of sugars generated from the reaction.

31. Exo & Endo-G Activity Determination
from NCC
Run 2: Low enzyme Loading (<0.1 FPU/g-glucan)

32. Summary of Activity Test Relative Activities Based on Volume
Overall
Endo-G
Exo-G
β-G
Substrate
Filter Paper
CMC
NCC
Avicel
Cellobiose
Method A B
Spezyme CP-A
1.0
Spezyme CP-B
0.98
0.89
1.07
0.86
1.12
1.08
0.60
GC-220
1.65
1.31
1.51
1.46
1.42
1.77
2.31
A: Conventional
B: Proposed in this work

33. Acid Hydrolysis of Cello-oligosaccharides
Glucose
Cellobiose
Glucose
4% H2SO4, 121C, 1 hr

34. Enzymatic Hydrolysis of Cello-oligosaccharides
Glucose
Cello-oligosaccharides
Cellobiose
Cellobiose
Glucose
Enzymatic Hydrolysis
15FPU/g-glucan

35. Hydrolysis of cello-oligosaccharide by β-glucosidase (Novo-188)

36. Hydrolysis of NCC and Cellobiose with β-glucosidase (Enzyme loading: 7 CBU/ g glucan)

37. Summary
The economic, environmental, and strategic benefits of cellulosic ethanol are great.
Time is ripe for commercial production of cellulosic ethanol.
Cost of the enzyme is a significant cost factor in the cellulosic ethanol process.

38. There is a room for improvement in the conventional
FPU method.
The points to be addressed:
HPLC in place of reducing sugar.
Calculate the extent of reaction in terms of the
glucan equivalent of combined G1 and G2.
Filter paper is still preferred over α-cellulose or
Avicel because of consistency in property.
Multiple-point (slope) method is preferred over
one-point method for higher accuracy.

39. Non-crystalline cellulose (NCC) can be used as a
substrate to determine the relative activities of
individual components of cellulase.
Hydrolysis of NCC by cellulase produces
G1,G2,and cello-oligosacchrides (COS).
Formation of G1 and G2 from NCC may be taken
as relative activity of exo-glucanase.
Increase of reducing sugar in NCC may be taken
as a measure of endo-glucanase activity.

40. Cello-oligosaccharides (COS) can be used as a substrate for identification of cellulase reactions.
COS is hydrolyzed only by β-glucosidase.
(Endo and Exo-G cannot hydrolyze COS.)
Hydrolysis of COS by cellulase is much slower
than NCC.
β-Glucosidase works only on soluble substrates
(G2 & oligomeres).

41. Acknowledgements
US Department of Energy Office of the Biomass Program (Contract DE-FG36-04GO14017)
US EPA-TSE Program (No. RD )
AU Team: Rajesh Gupta, Suma Peri, Yongming Zhu, Hatem Harraz, T. H. Kim, Clayton Smith
CAFI Team:
Dartmouth College; Michigan State, Purdue, Texas A&M; University of British Columbia; and the National Renewable Energy Laboratory

42. Questions? Corn stover Rice straw Wood chip Corn stover Bagasse
Sawdust

43. Cellulase Endo-Glucanase Cellulose → Scarred Cellulose
Exo-Glucanase (CBH)
Scarred Cellulose --- Cellobiose
Beta-Glucosidase
Cellobiose --- Glucose

44. Simplified Process Schematics
Enzyme
Feed Handling
Corn Stover
Pretreatment
Simultaneous
Saccharification
& Co-Fermentation
Hydrolyzate
Broth
Recycle Water
Waste Water
Recycle &
Condensate
Solids
Liquor
Conditioning
Distillation &
StillageTreatment
Steam
Waste Water
Treatment
Waste Water
Waste Water
Biogas & Sludge
Syrup
Ethanol
S/L Sep
Cake
Steam
Utilities
Burner/Boiler
Turbogenerator
Storage
Electricity

45. Economic Summary Sheet

46. Enzymatic Hydrolysis of Cello-oligosaccharides

47. Enzymatic Hydrolysis of Crystalline and Non-Crystalline Cellulose
Treated a-Cell.
a-Cellulose

48. Estimation of Initial Slopes (G1 + G2)
Avicel
Filter paper