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 Overview on Cellulosic Ethanol Significance of Cellulase Enzymes Cellulase Action and Activities

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

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.

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).

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.

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.

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

Cellulose & Cellulase

Cellulose Hydrolysis as a Rate Process Non-Reaction Resistances Reaction

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

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.

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

Activity Determination by initial slope method Substrate: Filter Paper

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.

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

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

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

NCC Cotton

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

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

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)

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.

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

Initial sugar release from different substrates Enzyme loading: 0 Initial sugar release from different substrates Enzyme loading: 0.112 ml Sp CP-A/g-glucan, 15 min.

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

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

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 )

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

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

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

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

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

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

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

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.

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.

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.

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).

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

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

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

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

Economic Summary Sheet

Enzymatic Hydrolysis of Cello-oligosaccharides

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

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