Cellobiose
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Cellobiose Glucose Low DP Cello-oligosaccharides (LD-COS) High DP Cello-oligosaccharides (HD-COS) Not picked in chromatograph 0% 5% 10% 15% 20% 0 20 40 60 80 100 120 Tim e(hr) % G lucan Equivalent Cotton Avicel FilterPaper α-Cellulose 0% 2% 4% 6% 8% 10% 0 20 40 60 80 100 120 Tim e(hr) % G lucan Equivalent Cotton Avicel FilterPaper α-Cellulose 0% 20% 40% 60% 80% 0 20 40 60 80 100 120 Tim e(hr) % G lucan Equivalent Cotton Avicel FilterPaper α-Cellulose 0% 20% 40% 60% 80% 0 20 40 60 80 100 120 Tim e(hr) % G lucan Equivalent Cotton Avicel FilterPaper α-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) a (m 2 /g): 1.232 2.311 : > 2000 100-150 ate : 3.4 37 /hr) /g glucan Schematic Presentation SEM Pictures Comparison between Cystalline Cotton and Non-Crystalline Cellulose (NCC) XR D plots fordifferentC ellulosic Substrate 0 500 1000 1500 2000 2500 3000 3500 4000 4500 10 15 20 25 30 35 40 45 50 2θ Intensity 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 gm olof reducing ends A verage m ol.wt A verage DP 5min 20min Cotton 0.050 0.065 1.57E-08 3191318.2 19699 NCC 6.420 13.140 2.03E-06 24607.9 152 Avicel 4.390 11.225 1.39E-06 35988.2 222 alpha cell 1.250 1.795 3.95E-07 126426.8 780 filter paper 0.085 0.335 2.67E-08 1869470.4 11540 A bsorbance 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 0% 20% 40% 60% 80% 100% 0 30 60 90 120 Tim e(hrs) % G lucan E quivalent 0% 5% 10% 15% 20% 25% 0 30 60 90 120 Tim e(hrs) % G lucan E quivalent 0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 0 30 60 90 120 Tim e(hrs) % G lucan E quivalent 0% 4% 8% 12% 16% 20% 0 30 60 90 120 Tim e(hrs) % G lucan E quivalent High Loading Low Loading High Loading High Loading High Loading Low Loading Low 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 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 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 DP R etention tim e D ifference in retention tim e betw een tw o successive D P C O S A rea H eight C oncentration (g/l) 7 7.97 0.33 1322.25 61.73 1.70 6 8.30 0.50 997.58 21.66 1.28 5 8.80 0.53 0.00 4 9.33 0.83 407.74 13.37 0.52 3 10.17 1.42 403.11 12.55 0.52 2 11.58 2.52 320.24 9.21 0.41 1 14.10 250.05 7.58 0.32 4.74 SUM 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 t 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 hydrolyze 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 sequenti action. This process, however, ceases when DP goes below cert level, leaving unhydrolyzed LD-COS as one of the end-products • The LD-COS is not observed during hydrolysis of crystalline s trates because the unreacted LD-COS is tightly bound to cellu by hydrogen bonding. • HD-COS is mainly produced in the earlier phase of the reactio 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 p 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 activ 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. T to do with the fact that α-cellulose has the lowest crystalli 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) (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
description
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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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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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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Glucose
Cellobiose
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Enzyme Loading: 0.1 ml / g Glucan
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Cellobiose HD-COS
Excess amount of β-G added after 72hrs
SOLID
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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)
14 .1
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)
