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Measurement of Endo and Exo-Glucanase Activities in Cellulase Using Non-Crystalline Cellulose

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 yylee@eng.auburn.edu AIChE Annual Meeting, San Francisco, CA November 16, 2006. Outline.

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Measurement of Endo and Exo-Glucanase Activities in Cellulase Using Non-Crystalline Cellulose

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  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 yylee@eng.auburn.edu AIChE Annual Meeting, San Francisco, CA November 16, 2006

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

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

  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. EthanolBoom • 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 2005-2012. • Direct spending and indirect impact will add $200 billion to GDP over 2005-2012. • 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 Difficultiesin Cellulosic Ethanol • Heterogeneous Feedstock • Solid Handling • Slow Reaction • Non-reusable Catalyst (Enzyme)

  9. Cellulose & Cellulase

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

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

  12. 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

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

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

  15. 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.

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

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

  19. NCC Cotton

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

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

  22. 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)

  23. 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.

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

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

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

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

  28. Measurement of Endo-Glucanase and Exo-Glucanase Activities from NCC 0.5ml 2% sonicated NCC Solution with Buffer 0.5ml 2% sonicated NCC Solution with Buffer 0.5ml of diluted Enzyme Solution 0.5ml of diluted Enzyme Solution Incubate at 50oC for 30min. Incubate at 50oC for 30min. Stop the reaction by boiling it for 5min. & Dilute it 3 times by cold DI water 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 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) Take liquid sample for HPLC sugar analysis 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 ) 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. Find the Glucose and Cellobiose concentration Determine Glucose and Cellobiose concentration

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

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

  31. Summary of Activity Test Relative Activities Based on Volume A: Conventional B: Proposed in this work

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

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

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

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

  36. 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.

  37. 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.

  38. 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.

  39. 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).

  40. Acknowledgements • US Department of Energy Office of the Biomass Program (Contract DE-FG36-04GO14017) • US EPA-TSE Program (No. RD-83164501) • 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

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

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

  43. Simplified Process Schematics Enzyme Corn Stover Feed Handling 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 Syrup Ethanol Biogas & Sludge S/L Sep Cake Steam Utilities Burner/Boiler Turbogenerator Storage Electricity

  44. Economic Summary Sheet

  45. Enzymatic Hydrolysis of Cello-oligosaccharides

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

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

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