Cellobiose

1. Amorphous domain (Substrate for Endo-glucanase) Cellobiose High DP Cello-oligosaccharides (HD-COS) Not picked in chromatograph Amorphous domain (Substrate for Endo-glucanase) Filter Paper(88% Crystalline) Glucose α-Cellulose(65% Crystalline) Low DP Cello-oligosaccharides (LD-COS) NCC(8% Crystalline) Cotton(77% Crystalline) Reducing Ends (Susbtrate for Exo-glucanase) Reducing Ends (Susbtrate for Exo-glucanase) Cotton NCC Low Loading SEM Pictures High Loading Acid Hydrolysis Glucose LD-COS High Loading Low Loading Low Loading High Loading Schematic Presentation Excess amount of β-G added after 72hrs Low Loading High Loading Cellobiose HD-COS BET Surface Area (m2/g): 1.232 2.311 DP : > 2000 100-150 Initial hydrolysis rate : 3.4 37 (% glucan conversion/hr) Enzyme loading: 3FPU/g glucan HPLC Chromatograph of Soluble sugars Produced by Enzymatic hydrolysis of NCC (HPLC Column used: Biorad Aminex P-Column) LD-COS Not Detected Investigation of cellulase reaction mechanism using pure cellulosic susbstrate Rajesh Gupta & Y Y Lee Department of Chemical Engineering Auburn University 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. HPLC Chromatograph of Cello-Oligosaccharide (COS was prepared in our lab by acid Hydrolysis of Cotton) Proposed Mechanism of Cellulase action on NCC Comparison between Cystalline Cotton and Non-Crystalline Cellulose (NCC) (HPLC Column used: Biorad Aminex P-Column) Low DP Cello-oligosaccharides (LD-COS) Low DP NCC Endo-G SOLID LIQUID HD-COS Cellobiose Glucose 14.1 LD-COS NCC Exo-G CELLOBIOSE Quantification of COS β-G GLUCOSE ( Picture of Enzyme cartoon has been taken from the Video on “cellulase mechanism” distributed by NREL) • 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. Crystallinity of Pure Cellulosic Substrate Actual COS concentration in solution: 9 g/L COS not shown in HPLC : approx. 50% ------ HD-COS Enzymatic Hydrolysis of NCC Enzyme Loading: High Loading:0.1 ml / g Glucan Low Loading: 0.005 ml / g Glucan 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 Crystalline Substrate Enzyme Loading: 0.1 ml / g Glucan Glucose Glucose + Cellobiose Excess amount of β-G added after 72hrs Cellobiose HD-COS • 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