1. Cellobiose Glucose Low DP Cello-oligosaccharides (LD-COS) High DP Cello-oligosaccharides (HD-COS) Not picked in chromatograph Introduction Various forms of pure cellulosic substrates were utilized to study the reaction mechanism in cellulase reaction. The substrates employed were micro-crystalline cellulose (Avicel), α- cellulose, filter paper, cotton, and non-crystalline cellulose (NCC). These substrates were first characterized with respect to Degree of polymerization (DP), crystallinity, surface area and other physical properties. NCC is a product of our laboratory. It is highly amorphous cellulose with crystallinity index less than 10%. When hydrolyzed with cellulase (Spezyme CP supplied by Genencore Int.), it produces significant amount of cello-oligosaccharides as reaction intermediates, along with glucose and cellobiose. Cello-oligosaccharides (COS) were categorized into two separate fractions: Low DP cello-oligosaccharides (LD-COS) and high DP cello-oligosaccharides (HD-COS). LD-COS, from DP 1-7, are detected by HPLC whereas HD-COS are detected only after secondary hydrolysis. On the basis of the profiles of these sugars during enzymatic hydrolysis, individual actions of Exo-glucanase (Exo-G), Endo- glucanase (Endo-G) and β-glucosidase (β-G) the overall reaction patterns are proposed. The major findings on the function of individual cellulase components are as follows: (1) Exo-G and Endo-G do not hydrolyze COS. (2) β-G hydrolyzes cellobiose and LD-COS. (3)Exo-G is responsible for LD-COS production and Endo-G for HD-COS. Crystallinity of substrates primarily affects the initial rate of cellulose hydrolysis due to preference of Endo-G towards the accessible amorphous region in cellulose. CottonNCC Acid Hydrolysis Amorphous domain (Substrate for Endo-glucanase) Reducing Ends (Susbtrate for Exo-glucanase) Amorphous domain (Substrate for Endo-glucanase) Reducing Ends (Susbtrate for Exo-glucanase) BET Surface Area (m 2 /g): 1.232 2.311 DP : > 2000 100-150 Initial hydrolysis rate : 3.4 37 (% glucan conversion/hr) Enzyme loading: 3FPU/g glucan Schematic Presentation SEM Pictures Comparison between Cystalline Cotton and Non-Crystalline Cellulose (NCC) Cotton(77% Crystalline) α-Cellulose(65% Crystalline) NCC(8% Crystalline) Filter Paper(88% Crystalline) Crystallinity of Pure Cellulosic Substrate Degree of Polymerization (DP) of Pure Cellulosic Susbtrate Calculated by measuring the absorbance at @540nm after reaction of 50mg of substrate with DNS reagent Enzymatic Hydrolysis of NCC Enzyme Loading: High Loading: 0.1 ml / g Glucan Low Loading: 0.005 ml / g Glucan Enzymatic Hydrolysis of Crystalline Substrate High Loading Low Loading High Loading Low Loading Excess amount of β-G added after 72hrs Glucose Cellobiose LD-COS HD-COS Enzyme Loading: 0.1 ml / g Glucan Glucose CellobioseHD-COS Excess amount of β-G added after 72hrs SOLID LIQUID Low DP NCC HD-COS LD-COS CELLOBIOSE GLUCOSE β-G NCC Exo-G Endo-G Proposed Mechanism of Cellulase action on NCC HPLC Chromatograph of Cello-Oligosaccharide (COS was prepared in our lab by acid Hydrolysis of Cotton) 14.1 Low DP Cello-oligosaccharides (LD-COS) Cellobiose Glucose Quantification of COS Actual COS concentration in solution: 9 g/L COS not shown in HPLC : approx. 50% ------ HD-COS Summary: Enzymatic hydrolysis of NCC produces glucose(G1), cellobiose (G2) and cello-oligosaccharides (COS). This is in contrast to the crystalline cellulose, which produces only G1 and G2. In NCC reaction with very low enzyme loading, G2 and LD-COS accumulate. Upon addition of external β-G, both are hydrolyzed to glucose. This proves that β-G not only works on cellobiose but also on LD-COS. In NCC reaction, formation of cellobiose is proportional to LD-COS, a proof that LD-COS is a product of Exo-G. When Exo-G hydrolyzes NCC, it produces cellobiose by sequential action. This process, however, ceases when DP goes below certain level, leaving unhydrolyzed LD-COS as one of the end-products. The LD-COS is not observed during hydrolysis of crystalline subs- trates because the unreacted LD-COS is tightly bound to cellulose by hydrogen bonding. HD-COS is mainly produced in the earlier phase of the reaction by Endo-G and not consumed by any of the cellulase components. Endo-G attacks on insoluble substrates at amorphous site, cleaving the glycosidic bond. Of the cleaved chains, if one part is below certain DP, it becomes solublized and not further hydrolyzed by Endo-G. Production of HD-COS from NCC is several times that from crystalline substrate. It is clear evidence that Endo-G activity is very high against NCC since HD-COS generation is solely by Endo-G. Its high reactivity is due to high amorphous nature of NCC. Within crystalline substrates, formation of HD-COS is higher with α-cellulose than with filter paper, Avicel, or cotton. This has to do with the fact that α-cellulose has the lowest crystallinity among these substrates. LD-COS Not Detected Glucose + Cellobiose HPLC Chromatograph of Soluble sugars Produced by Enzymatic hydrolysis of NCC (HPLC Column used: Biorad Aminex P-Column) Acknowledgement US Department of Energy for funding the project (US/DOE No. DE-PS36-00GO10482) Members of CAFI-II team Genencor International Inc. for supplying enzyme ( Picture of Enzyme cartoon has been taken from the Video on “cellulase mechanism” distributed by NREL)