characterization of cellulases using pure cellulosic substrates

Biomass Refining CAFI Auburn

University

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


Ce

llob

iose

Glu

cose

Ce

llob

iose

Glu

cose


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


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


(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



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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


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


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


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



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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



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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

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Wood chip

Bagasse

Rice straw

Sawdust

characterization of cellulases using pure cellulosic substrates

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