Cellobiose

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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 - PowerPoint PPT Presentation

Transcript of Cellobiose

Cellobiose

GlucoseLow DP

Cello-oligosaccharides(LD-COS)

High DP Cello-oligosaccharides

(HD-COS)Not picked in chromatograph

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CottonAvicelFilter Paperα-Cellulose

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CottonAvicelFilter Paperα-Cellulose

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CottonAvicel

Filter Paperα-Cellulose

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CottonAvicelFilter Paperα-Cellulose

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.

Cotton NCC

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 (m2/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 andNon-Crystalline Cellulose (NCC)

XRD plots for different Cellulosic Substrate

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Inte

nsity

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

gmol of reducing ends

Average mol.wt

Average DP

5min 20minCotton 0.050 0.065 1.57E-08 3191318.2 19699NCC 6.420 13.140 2.03E-06 24607.9 152Avicel 4.390 11.225 1.39E-06 35988.2 222

alpha cell 1.250 1.795 3.95E-07 126426.8 780filter paper 0.085 0.335 2.67E-08 1869470.4 11540

Absorbance

Enzymatic Hydrolysis of NCC

Enzyme Loading:

High Loading: 0.1 ml / g GlucanLow Loading: 0.005 ml / g Glucan

Enzymatic Hydrolysis of Crystalline Substrate

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Low Loading

Excess amount of β-G added after 72hrs

Glucose

Cellobiose

LD-COS

HD-COS

Enzyme Loading: 0.1 ml / g Glucan

Glucose

Cellobiose HD-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 actionon NCC

HPLC Chromatograph of Cello-Oligosaccharide(COS was prepared in our lab by acid Hydrolysis of Cotton)

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Low DP Cello-oligosaccharides

(LD-COS)

CellobioseGlucose

DP Retention time

Difference in retention time between two

successive DP COSArea Height Concentration

(g/l)

7 7.97 0.33 1322.25 61.73 1.706 8.30 0.50 997.58 21.66 1.285 8.80 0.53 0.004 9.33 0.83 407.74 13.37 0.523 10.17 1.42 403.11 12.55 0.522 11.58 2.52 320.24 9.21 0.411 14.10 250.05 7.58 0.32

4.74SUM

Quantification of COS

Actual COS concentration in solution: 9 g/LCOS 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 sugarsProduced by Enzymatic hydrolysis of NCC

(HPLC Column used: Biorad Aminex P-Column)

(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)