Characterization of Cellulases Using Pure Cellulosic Substrates
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Characterization of Cellulases Using
Pure Cellulosic Substrates
Suma Peri, Rajesh Gupta, and Y. Y. LeeDepartment of Chemical Engineering
Auburn University, AL 36849
AIChE Annual Meeting, Cincinnati, OH, November 1, 2005
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Presentation Outline
• Overview of current cellulase activity test method.
• Discussion on synergetic actions of cellulase.
• Use of cello-oligsacchrides and non-crystalline cellulose for study of cellulases.
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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.
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• Filter Paper (Whatman No.1) & Avicel (PH-101).• HPLC is used for measurement of released sugar.• G1 & G2 are converted to glucan for conversion
calculation. • Uses slope-method (multiple points) for initial rate.
Modified method used in this work
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Initial Sugar Release (Enzyme loading: 0.112 ml Sp CP-A/ g glucan)
Glucose
0%
1%
2%
3%
4%
5%
6%
7%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
% G
luca
n eq
uiva
lent
Avicel
Filter paper
Cellobiose
0%
1%
2%
3%
4%
5%
6%
7%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
% G
luca
n eq
uiva
lent
Avicel
Filter paper
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Estimation of Initial Slopes (G1 + G2)
Avicel Filter paper
Sp CP-A (0.06ml/g glucan)
y = 0.0579x + 0.0356
R2 = 0.9892
0%
5%
10%
15%
20%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
%G
luca
n Eq
uiva
lent
Sp CP-A (0.06ml/g glucan)
y = 0.0734x + 0.0044
R2 = 0.9549
0%
5%
10%
15%
20%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)%
Glu
can
Equi
vale
nt
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Relative activities of cellulasesAvicel
Filter paper
ml/g glucan Sp CP-A Sp CP-B GC2200.06 1 1.495 2.3310.15 1 1.531 2.4210.3 1 1.872 2.645
Avg 1 1.633 2.466
ml/g glucan Sp CP-A Sp CP-B GC2200.06 1 1.474 2.2570.15 1 1.210 1.9940.3 1 1.358 2.085
Avg. 1 1.347 2.112
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“Cellulase” is composed of:
• Endo-Glucanase
• Exo-Glucanase
• β-Glucosidase
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GlucoseReducing ends
Cellobiose
Crystalline region Amorphous region
Avicel
Filter Paper
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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.
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Additional Substrates
• Cello-oligosaccharides
• Non Crystalline Cellulose
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Acid Hydrolysis of Cello-oligosaccharides
Cello-oligosaccharides
Ce
llob
iose
Glu
cose
Glu
cose
4% H2SO4, 121C, 1 hr
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Enzymatic Hydrolysis of Cello-oligosaccharides
Cello-oligosaccharides
Ce
llob
iose
Glu
cose
Ce
llob
iose
Glu
cose
Cello-oligosaccharides
Enzymatic Hydrolysis Loading: 15FPU/ g glucan
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Enzymatic Hydrolysis of Cello-oligosaccharides
Sp CP-A (3 FPU/g glucan)
0%2%4%6%8%
10%12%14%
0 20 40 60 80 100 120
Time (Hours)
% G
luc
an
Eq
uiv
ale
nt
Glucose
Cellobiose
Sp CP-A (15 FPU/g glucan)
0%2%4%6%8%
10%12%14%
0 20 40 60 80 100 120
Time (Hours)
% G
luc
an
Eq
uiv
ale
nt
Glucose
Cellobiose
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Hydrolysis of cello-oligosaccharide by β-glucosidase (Novo188)
Hydrolysis of Cello-oligosaccharides
0%
5%
10%
15%
20%
25%
30%
35%
0 20 40 60 80 100 120
Time (Hours)
% G
luc
an E
qu
iva
len
t)
Glucose (15 FPU+30 CBU)
Cellobiose (15 FPU+30 CBU)
Glucose (30 CBU)
Cellobiose (30 CBU)
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Endo-Glucanase
Exo-Glucanase
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Non-Crystalline Cellulose (NCC)
• Amorphous cellulose made in our laboratory
from crystalline cellulose.
• Hydrogen-bonds in cellulose are disrupted.
• Crystallinity is essentially removed.
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a-Cellulose NCC (Freeze-Dried)
SEM
(1000X)
(3000X)
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• X-Ray Diffraction Patterns of MicroCrystalline Cellulose),a-Cellulose & Non-Crystalline Cellulose
20 25 30 35 40 45 50
2
Inte
nsi
ty
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DSC curves for a–Cellulose [----- ] & NCC [ - - - ] Melting Pt. : NCC= 260 oC, a-Cellulose = 340 oC
-6
-4
-2
0
2
4
Hea
t Flo
w (
W/g
)
0 50 100 150 200 250 300 350 400
Temperature (°C)
––––––– HH_cell1a.001– – – – HH_cell2.002
Exo Up Universal V3.1E TA Instruments
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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)
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Enzymatic Hydrolysis ofNCC
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NCC HydrolysateLoading: 0.01 ml Sp CP-A/ g-Glucan, 1 hr
Ce
llo-
olig
os
ac
ch
arid
es
Ce
llob
ios
e
Glu
co
se
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Initial sugar release from different substrates
Enzyme loading: 0.112 ml Sp CP-A/g-glucan,15 min.
Substrate Cellobiose Glucose OligomersCellobiose+
GlucoseTotal
% % % % %Avicel 3.77 1.08 0 4.85 4.85Filter Paper 1.85 0.63 0 2.48 2.48NCC 12.48 9.36 7.52 21.84 29.36
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NCC hydrolysis with different cellulase(Enzyme loading: 0.01 ml/g glucan)
Cellobiose released
0%2%4%6%8%
10%12%14%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
% G
luca
n E
quiv
alen
t Sp CP-A
Sp CP-B
GC220
Glucose released
0%2%4%6%8%
10%12%14%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
% G
luca
n E
quiv
alen
t Sp CP-A
Sp CP-B
GC220
Oligosaccharide released
0%
1%2%
3%4%
5%6%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
% G
luc
an
E
qu
iva
len
t
Sp CP-A
Sp CP-B
GC220
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Hydrolysis of NCC with β-glucosidase
(Enzyme loading: 7 CBU/ g glucan)
Release of Glucose in NCC and Cellobiose
0%
4%
8%
12%
16%
20%
0 0.2 0.4 0.6 0.8 1
Time (Hours)
%G
luc
an
Eq
uiv
ale
nt
NCC
Cellobiose
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Cellobiose
Soluble Cellodextrins
Insoluble Cellodextrins
Reducing ends
NCC Structure
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Cellulase Reaction Pathways
Cellobiose
Cello-oligosaccharides
Non-crystalline Cellulose
(NCC)
GlucoseExo-G
Endo-G
β-G
β-G
Crystalline Cellulose
Disrupted Cellulose Cellobiose GlucoseEndo-G Exo-G β-G
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Glucose and Cellobiose released by NCC with different Enzymes
(Glucose+Cellobiose) released with Sp CP-A
y = 0.1143x + 0.0188
y = 0.0742x + 0.0132
0%2%4%6%8%
10%12%
0 0.1 0.2 0.3 0.4 0.5 0.6Time (hours)
%G
luca
n Eq
uiva
lent
0.01 ml/ g glucan0.005 ml/ g glucan
(Glucose+Cellobiose) released with Sp CP-B
y = 0.1218x + 0.0151
y = 0.0719x + 0.013
0%2%4%6%8%
10%12%
0 0.1 0.2 0.3 0.4 0.5 0.6Time (hours)
%G
luca
n Eq
uiva
lent
0.01 ml/ g glucan0.005 ml/ g glucan
(Glucose+Cellobiose) released with GC220
y = 0.1815x + 0.0228
y = 0.1191x + 0.0182
0%2%4%6%8%
10%12%
0 0.1 0.2 0.3 0.4 0.5 0.6
Time (hours)
%G
luca
n
Eq
uiv
alen
t
0.01 ml/ g glucan0.005 ml/ g glucan
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Relative activity of Exo-glucanase in different cellulases
Enzyme Loading
( ml/ g glucan)
0.01 1.00 1.07 1.59
0.005 1.00 0.97 1.61
Average 1.00 1.02 1.60
Sp CP-A Sp CP-B GC220
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Relative Endo-glucanase Activity
Oligosaccharide released
0%
1%2%
3%4%
5%6%
0 0.2 0.4 0.6 0.8 1 1.2
Time (Hours)
% G
luca
n
Eq
uiv
alen
t
Sp CP-A
Sp CP-B
GC220
Enzyme loading: 0.01ml /g glucan
Sp CP-A Sp CP-B GC220
1.00 1.18 1.22
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Hydrolysis of Cellobiose with different cellulase
Glucose released with SpCP-A
y = 0.0107x + 0.0055
y = 0.005x + 0.0043
0.0%0.2%0.4%0.6%0.8%1.0%1.2%1.4%1.6%1.8%
0 0.2 0.4 0.6 0.8 1 1.2
Time (hours)
%G
luca
n E
quiv
alen
t
0.01 ml/ g glucan0.005 ml/ g glucan
Glucose released with SpCP-B
y = 0.0084x + 0.0057
y = 0.003x + 0.0047
0.0%0.2%0.4%0.6%0.8%1.0%1.2%1.4%1.6%1.8%
0 0.2 0.4 0.6 0.8 1 1.2
Time (hours)
%G
luca
n E
quiv
alen
t
0.01 ml/ g glucan0.005 ml/ g glucan
Glucose released with GC220
y = 0.0239x + 0.008
y = 0.0119x + 0.0053
0.0%0.5%1.0%1.5%2.0%2.5%3.0%3.5%
0 0.2 0.4 0.6 0.8 1 1.2
Time (hours)
%G
luc
an
Eq
uiv
ale
nt
0.01 ml/ g glucan0.005 ml/ g glucan
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Relative activity of β-glucosidase in different cellulases
Enzyme Loading
( ml/ g glucan)
0.01 1.00 0.79 2.23
0.005 1.00 0.60 2.38
Average 1.00 0.69 2.31
Sp CP-A Sp CP-B GC220
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Summary of Relative Activities
SubstrateComponent of
Enzyme Sp CP-A Sp CP-B GC220
Avicel Overall 1 1.63 2.46Filter Paper Overall 1 1.35 2.11
NCC Endo-glucanase 1 1.18 1.22NCC Exo-glucanase 1 1.02 1.6
Cellobiose β-glucosidase 1 0.69 2.31
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oo
Summary • 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 reliable measurement of initial rates.
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• 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).
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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. Formation of COS from NCC may be taken as an approximate measure of endo-glucanase activity.
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Future Work
• Verification of end-glucanase activity.
• Find ways to determine the number of reducing ends in the solid to accurately quantify endo-G reaction.
• Determine the kinetic parameters from the time-course data using kinetic model and parameter estimation.
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Acknowledgements US Department of Energy Office of the Biomass
Program, Contract DE-FG36-04GO14017 Genencor International CAFI Team:
Dartmouth College; Michigan State, Purdue, and Texas A&M; the University of British Columbia; and the National Renewable Energy Laboratory
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Questions?
Corn stover
Corn stover
Wood chip
Bagasse
Rice straw
Sawdust