Characterization of Cellulases Using Pure Cellulosic Substrates

1. Characterization of Cellulases Using Pure Cellulosic Substrates Suma Peri, Rajesh Gupta, and Y. Y. LeeDepartment of Chemical EngineeringAuburn University, AL 36849 AIChE Annual Meeting, Cincinnati, OH, November 1, 2005

2. Presentation Outline • Overview of current cellulase activity test method. • Discussion on synergetic actions of cellulase. • Use of cello-oligsacchrides and non-crystalline cellulose for study of cellulases.

3. 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. Cellulase Activity Determination

4. Modified method used in this work • Filter Paper (Whatman No.1) & Avicel (PH-101). • HPLC is used for measurement of released sugar. • G1 & G2 are converted to glucan for conversion calculation. • Uses slope-method (multiple points) for initial rate.

5. Initial Sugar Release (Enzyme loading: 0.112 ml Sp CP-A/ g glucan)

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

7. Relative activities of cellulases Avicel Filter paper

8. “Cellulase” is composed of: • Endo-Glucanase • Exo-Glucanase • β-Glucosidase

9. Avicel Cellobiose Glucose Reducing ends Amorphous region Crystalline region Filter Paper

10. 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.

11. Additional Substrates • Cello-oligosaccharides • Non Crystalline Cellulose

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

13. Cello-oligosaccharides Cello-oligosaccharides Enzymatic Hydrolysis of Cello-oligosaccharides Glucose Cellobiose Cellobiose Glucose Enzymatic Hydrolysis Loading: 15FPU/ g glucan

14. Enzymatic Hydrolysis of Cello-oligosaccharides

15. Hydrolysis of cello-oligosaccharide by β-glucosidase (Novo188)

16. Endo-Glucanase Exo-Glucanase

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

18. a-Cellulose NCC (Freeze-Dried) SEM (1000X) (3000X)

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

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

21. FTIR graph for Treated & Untreated a-Cellulose-- A (Untreated a-cellulose), 1.019 (Without baseline correction)----- B (Treated a-cellulose), 2.165 (Baseline correction from 1800 cm-1 to 847.27 cm-1)

22. Enzymatic Hydrolysis ofNCC

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

24. Initial sugar releasefrom different substratesEnzyme loading: 0.112 ml Sp CP-A/g-glucan,15 min.

25. NCC hydrolysis with different cellulase(Enzyme loading: 0.01 ml/g glucan)

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

27. NCC Structure Cellobiose Soluble Cellodextrins Insoluble Cellodextrins Reducing ends

28. Cellulase Reaction Pathways Crystalline Cellulose Disrupted Cellulose Cellobiose Endo-G Exo-G β-G Glucose Non-crystalline Cellulose (NCC) Cellobiose β-G Exo-G Glucose Endo-G Cello-oligosaccharides β-G

29. Glucose and Cellobiose released by NCC with different Enzymes

30. Relative activity of Exo-glucanase in different cellulases

31. Relative Endo-glucanase Activity Enzyme loading: 0.01ml /g glucan

32. Hydrolysis of Cellobiose with different cellulase

33. Relative activity of β-glucosidase in different cellulases

34. Summary of Relative Activities

35. Summary • 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 reliable measurement of initial rates.

36. 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).

37. 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. • Formation of COS from NCC may be taken as an approximate measure of endo-glucanase activity.

38. Future Work • Verification of end-glucanase activity. • Find ways to determine the number of reducing ends in the solid to accurately quantify endo-G reaction. • Determine the kinetic parameters from the time-course data using kinetic model and parameter estimation.

39. Acknowledgements • US Department of Energy Office of the Biomass Program, Contract DE-FG36-04GO14017 • Genencor International • CAFI Team: Dartmouth College; Michigan State, Purdue, and Texas A&M; the University of British Columbia; and the National Renewable Energy Laboratory

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