Cellulase Assays 纤维素酶活性测定

1

Chapter 14

Cellulase Assays

Y.H. Percival Zhang, Jiong Hong, and Xinhao Ye

Summary

Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase,

exoglucanase (cellobiohydrolase), and b-glucosidase to work together. We summarize the most

commonly used assays for individual enzymes and cellulase mixture.

Key words:

b-Glucosidase, Cellobiase, Cellobiohydrolase, Cellulose, Cellulase assay, Endoglucanase,

Sugar assay

2

3

4

5

6

7

8

9

1. Introduction

Cellulose, which is the most abundant renewable biological

resource, is produced mainly by plant photosynthesis. Cellulose

biodegradation mediated by cellulases or cellulolytic microor-

ganisms releases organic carbon in plant, animal, and microbial

sediments back to the atmosphere as carbon dioxide and methane.

Complete enzymatic crystalline cellulose hydrolysis requires three

types of enzymes (endoglucanase, exoglucanase or cellobiohy-

drolase (CBH), and b-glucosidase) to work together (1–4).

Physical heterogeneity of the cellulosic materials and the com-

plexity of cellulase enzyme systems (synergy and/or competition)

on solid enzyme-accessibility-limited substrate surfaces present

some challenges for cellulase activity assays (5–8). A number of

cellulase activity assays have been summarized (5, 6). In this

chapter, we describe the common cellulase activity assays including

the total cellulase assays, b-glucosidase assays, endoglucanase

assays, and exoglucanase (CBH) assays. This chapter will provide

some useful guidance, especially in Subheading 4.

Jonathan R. Mielenz (ed.), Biofuels: Methods and Protocols, Methods in Molecular Biology, vol. 581

DOI 10.1007/978-1-60761-214-8_14, © Humana Press, a part of Springer Science + Business Media, LLC 2009

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214 Zhang, Hong, and Ye

2. Materials

2.1. Total Cellulase

Assays

2.1.1. Filter Paper Activity

Assay

2.1.2. Anaerobic Cellulase

Assay Using Avicel

2.2. b-Glucosidase

Assays

2.2.1. b-Glucosidase Assay

Using p-Nitrophenyl-

b-D-Glucoside (pNPG)

DNS (3,5-dinitrosalicylic acid) reagent. Dissolve 10.6 g of DNS

and 19.8 g of NaOH in 1,416 ml of distilled water. After com-

plete dissolution, add 360 g of Rochelle salts (sodium potassium

tartrate), 7.6 ml of melted phenol (at 50°C) (

8.3 g of sodium metabisulfite, and then mix well. Titrate 3 ml

see Note 1), and

of the DNS reagent using 0.1 M HCl using the phenolphthalein

endpoint pH check. It should take 5–6 ml of HCl for a transi-

tion from red to colorless. Add NaOH if required (2 g of NaOH

added = 1 ml of 0.1 M HCl used for 3 ml of the DNS reagent)

(see Note

monohydrate in 750 ml of distilled water, and add 50–60 g solid

Citrate

2).

buffer (1 M, pH 4.5). Dissolve 210 g of citric acid

NaOH until pH is 4.3. Dilute the solution to nearly 1,000 ml and

check the pH. If necessary, add NaOH to adjust the pH to 4.5.

(pH 4.5) by adding 19 times distilled water.

Citrate buffer (50 mM, pH 4.8). Dilute 1 M citrate buffer

Whatman No. 1 paper strips with a paper cutter (

Filter paper strip (50 mg, 1.0 × 6.0 cm). Cut 1.0 × 6.0 cm

Glucose standard stock solution – 10 g/l (see

see

Note

Note

4).

3).

1. Tris–HCl

optional 1.5% NaN

buffer (0.5 M Tris, pH 7.0, 0.1 M CaCl

2

, and

(pH 7.0), dissolve 11.1 g of CaCl

3

). Prepare 0.5 l of 1 M Tris–HCl buffer

distilled water to make up to 1 l.

2

and 15 g NaN

3

, and add

2. Dithiothreitol (DTT, 0.5 M). The DTT solution can be stored

at 4°C for at least a half year. Less costly cysteine can replace

DTT (9).

3. Avicel

completely dry Avicel (FMC 105 or Sigmacell 20) in 820 ml

suspension solution (24.4 g/l). Suspend 20 g of

of distilled water with a magnetic stirrer.

4. Glucose standard solution – 1 g/l.

5. Phenol aqueous solution (5% w/v). Store at 4°C in dark-

ness.

6. Sulfuric acid ~98% w/w.

1. Sodium acetate buffer, 0.1 M, pH 4.8.

2. p

in 100 ml acetate buffer.

NPG (5 mM) in acetate buffer. Dissolve 0.1576 g of p NPG

3. Clycine buffer (0.4 M) pH 10.8. Dissolve 60 g of glycerin in

1,500 ml of distilled water, add 50% w/v NaOH until the pH

is 10.8, and then dilute to 2 l.

4. p-Nitrophenol (pNP; 20 g/l) in acetate buffer (see Note 5).

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2.2.2. b-Glucosidase Assay

Using Cellobiose

2.3. Endoglucanase

Assays

2.3.1. Endoglucanase

Assay Using Carboxymeth-

ylcellulose (CMC)/DNS

2.3.2. Endoglucanase

Assay Using CMC/

Bicinchoninic Acid (BCA)

2.3.3. Endoglucanase

Assay Using CMC/Viscosity

2.3.4. Semiquantitative

Endoglucanase Assay

Based on Dye Release

Microbe-Secreted

Endoglucanase Assay

on Agar Medium

Endoglucanase Assay

on Agarose Gel

Endoglucanase Assay

on Polyacrylamide Gel

2.4. Exoglucanase

Assays

2.4.1. Exoglucanase Assay

Using Avicel

Cellulase Assays 215

1. Cellobiose (15 mM) in citrate buffer (freshly made substrate

69

solution).

70

2. Citrate buffer (50 mM, pH 4.8).

71

1. Citrate buffer (50 mM, pH 4.8).

72

2. CMC (2% w/v) in citrate buffer (above).

73

3. DNS reagent (above).

74

4. Glucose standard (2 g/l).

75

1. Citrate buffer (50 mM, pH 4.8).

76

2. CMC solution (0.05% w/v) in the citrate buffer.

77

3. BCA

78

(97.1 mg) in a solution of 2.714 g of Na

Solution A. Dissolve disodium 2,2¢-bicinchoninate

79

NaHCO

2

CO

3

and 1.21 g of

80

stable for 4 weeks at 4°C in darkness.

3

with a final volume of 50 ml. Solution A will remain

81

4. BCA Solution B. Dissolve CuSO

(63.1 mg) in 50 ml of water. Solution B will remain stable for

4

.5H

2

O (62.4 mg) and

l

-serine

82

83

4 weeks at 4°C in darkness.

84

5. Working BCA reagent. Mix equal volumes of solution A and B.

85

The reagent is to be made immediately before use.

86

6. Glucose standard solution (0.9 g/l, 5 mM).

87

1. Sodium acetate buffer (50 mM, pH 5.0).

88

2. CMC solution (0.5% w/v, medium viscosity, degree of sub-

89

stitution of 0.5–0.7) in acetate buffer.

90

1. Congo red solution (1 g/l) prepared by dissolving 100 mg

91

Congo red in 99 ml water and 1% ethanol.

92

2. NaCl (1 M) solution.

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3. Sodium phosphate buffer (0.1 M, pH 6.5).

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1. CMC (1% w/v, low viscosity) in 1.5% agar medium. Dissolve

95

CMC before adding agar and autoclave.

96

1. CMC (1% w/v, low viscosity) in 0.8% agarose. Dissolve CMC

97

completely before adding agarose.

98

1. CMC (1% w/v) in sodium phosphate buffer whose pH is cho-

99

sen depending on the specific cellulase.

100

1. Avicel (FMC PH 101 or PH 105 or Sigmacell 20).

101

2. Sodium acetate buffer (0.1 M, pH 4.8).

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3. Phenol (5%) solution.

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4. Sulfuric acid, ~98%.

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216 Zhang, Hong, and Ye

2.4.2. Exoglucanase Assay

Using Regenerated

Amorphous Cellulose

(RAC)

3. Methods

3.1. Total Cellulase

Assays

3.1.1. Filter Paper Assay

(FPA)

Procedure

1. Sodium acetate buffer (1 M, pH 4.5).

2. Phenol (5%) solution.

3. Sulfuric acid (~98%).

4. RAC (1% w/v). RAC preparation is given below.

A

endoglucanases,

total cellulase system consists of three

cellulase activities are always measured using insoluble substrates,

exoglucanases, and b-

enzymatic activities:

d

-glucosidases. Total

including pure cellulosic substrates such as Whatman No. 1 filter

paper, cotton linter, microcrystalline cellulose, bacterial cellulose,

algal cellulose, as well as cellulose-containing substrates such as

dyed

The two most common assays (filter paper assay and anaerobic

cellulose, a-cellulose, and pretreated lignocellulose (2).

cellulase assay) are described here.

FPA

mended

is the most common total cellulase activity assay

Chemistry

by

activity (FPA) assay that differs from most enzyme assays based

(IUPAC)

the International

(6). IUPAC

Union

recommends

of Pure

a

and

filter

Applied

recom-

paper

on soluble substrate for initial reaction rates. This assay is based

on a fixed degree of conversion of substrate, i.e. a fixed amount

(2 mg) of glucose (based on reducing sugars measured by the DNS

assay) released from 50 mg of filter paper (i.e., both amorphous

and crystalline fractions of the substrate are hydrolyzed) within a

fixed time (i.e., 60 min). In part due to the solid heterogeneous

substrate, reducing sugar yield during hydrolysis is not a linear

function of the quantity of cellulase enzyme in the assay mixture.

That is, twice the amount of enzyme does not yield two times the

reducing sugar within equal time. Total cellulase activity is described

in

(undiluted)

terms of “filter-paper

that

enzyme solution.

units”

The

(FPU)

strengths

per milliliter

of this

of

assay

original

are

is reasonably susceptible to cellulase activity. However, the FPA

(1) the substrate is widely available and (2) the substrate

has long been recognized for its complexity and susceptibility to

operator errors (10).

1. Place a rolled filter paper strip into each 13 × 100 test tube.

2. Add 1.0 ml of 50 mM citrate buffer (pH 4.8) to the tubes; the

paper strip should be submerged in the buffer.

3. Prepare the enzyme dilution series, of which at least two dilu-

tions must be made of each enzyme sample, with one dilution

releasing slightly more than 2.0 mg of glucose (

one slightly less than 2.0 mg of glucose (1.9 mg) (

~2.1 mg) and

see Note 6).

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Calculation

Cellulase Assays 217

4. Prepare the dilute glucose standards (GSs) as below:

146

GS1: 1.0 ml of glucose standard + 4.0 ml buffer = 2 mg/ml

147

148

GS2: 1.0 ml of glucose standard + 2.0 ml buffer = 3.3 mg/ml

(1.0 mg/0.5 ml).

149

150

GS3: 1.0 ml of glucose standard + 1.0 ml buffer = 5 mg/ml

(1.65 mg/0.5 ml).

151

152

GS4: 1.0 ml of glucose standard + 0.5 ml buffer = 6.7 mg/

(2.5 mg/0.5 ml).

153

154

Add 0.5 ml of GS1–4 solutions to 13 × 100 mm test tubes,

ml (3.35 mg/0.5 ml).

155

and add 1.0 ml of 0.050 M citrate buffer.

156

5.

157

Reagent blank (RB): 1.5 ml of 50 mM citrate buffer.

Prepare the blank and controls.

158

159

0.5 ml enzyme dilution series whose enzyme concentrations are

Enzyme controls (EC1–5): 1.0 ml of 50 mM citrate buffer +

160

the same as those from E1 to E5 (see Note

161

162

filter paper strip.

Substrate control (SC): 1.5 ml of 50

7

mM

).

citrate buffer +

163

6. Prewarm

equilibrium.

the enzyme solutions, blank, and controls until

164

165

7. Add 0.5 ml of the enzyme dilution series to the tubes with

filter

166

dilution

paper substrate (E1–5); add 0.5 ml of

167

(EC1–5).

series to the tubes without filter paper

the

substrate

enzyme

168

169

8. Incubate the tubes of E1–5, GSs, RB, EC1–5, and SC in a

50°C water bath for exactly 60 min.

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171

9. Add 3.0 ml of the DNS reagent to stop the reaction, and mix

well.

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1 0. Boil all tubes for exactly 5.0 min (see Note 8).

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1 1. Transfer the tubes to an ice-cold water bath.

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1 2. Withdraw

centrifuge tubes and centrifuge at

~0.5 ml of the colored solutions into 1.5-ml micro-

~10,000 g for 3 min.

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1 3. Add 0.200 ml of the supernatant into 3-ml spectrophotometer

178

cuvette tubes, add 2.5 ml of water, and mix well by using a

pipette or by inversion several times.

179

180

1 4. Measure absorbance at 540 nm, where the absorbance of RB

181

is used as the blank.

182

1. Draw a standard sugar curve (sugar along the x-axis vs.

183

absorbance at 540 along the y-axis), as shown in Fig. 1.

184

2. Calculate

(

the delta absorbance of dilute enzyme solutions

185

ance of EC1–5 and SC.

DE1–4) for E1–5 by subtraction of the sum of the absorb-

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3. Calculate the real glucose concentrations released by E1–5

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according to a standard sugar curve.

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218 Zhang, Hong, and Ye

3.1.2. Anaerobic Cellulase

Assay Using Avicel

1x10

−2

0.8

G standard

E5

0.009

enzymes

E4

0.008

S4

0.007

0.006

m

0.6

E3

0.005

n

0

S3

n

o

5

i

4

E2

0.004

t

u

l

i

e

d

c

n

a

0.4

0.003

e

m

b

E1

S2

y

r

o

z

s

n

b

E

A

0.2

0.002

S1

0.0

RB

−3

01234

1x10

Glucose standard (mg/0.5 mL)

Fig. 1 The relationship of absorbance at 540 nm for the DNS assay and EDRs in terms

of glucose concentration.

4. Draw the relationship between the real glucose concentrations

and their respective enzyme dilution rates (EDRs) (Fig. 1).

5. Link the points less than 2 mg and greater than 2 mg by a line,

and

based on the line

identify the EDR

(Fig.

by

1).

using the point for 2-mg glucose

6. Calculate

solution in terms of FPU/ml:

the FPA of the original concentrated enzyme

FPA

=

0.37

EDR

where 2 mg glucose = 2 mg/(0.18 mg/

0.37 mmol/min/ml (see Notes 9, 10).

mmol) × 0.5 ml × 60 min =

Some cellulases or cellulosomes isolated from anaerobic environ-

ments need the presence of a reducing agent and some metal ions,

such as calcium, to exert maximal hydrolysis ability, for example,

the

Clostridium

cellulosome

a turbidometric method based on the change of 0.6 g/l Avicel

thermocellum

from the

(11

thermophilic

). Johnson et

anaerobic

al. (11) established

bacterium

(FMC

and sodium carboxymethylcellulose, but the results often suffer

RC-591), which is a blend of microcrystalline cellulose

from

modified

large

initial hydrolysis period

on

variations.

the basis of

The anaerobic cellulosome assay was

(12, 13

the soluble

).

sugar release during the

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Procedure

Phenol–Sulfuric Acid Assay

(A Linear Range from

Sugars in the Samples

from 0.005 to 0.1 g/l)

3.2. b-Glucosidase

Assays

Cellulase Assays 219

1. Add 4.10 ml of the well-suspended Avicel solution into

212

16 × 125 mm Hungate tubes, and add 0.50 ml of Tris–HCl

213

buffer (each sample needs triplicate tubes).

214

2. Add the rubber stopper and seal the tubes.

215

3. Vacuum and flush the headspace gas by

nitrogen at least three times.

~5 psi (ultra) pure

216

217

4. Add 0.10 ml of 0.5 M DTT solution using a syringe with a

218

23G needle before enzyme activity assay.

219

5. Prewarm the tubes in a water bath at 60°C.

220

6. Prepare the enzyme solution.

221

7. Add 0.30 ml of the dilute enzyme solution series into the

222

tube using a syringe with a 23G needle.

223

8. After the first 10 min of adsorption and reaction, withdraw

224

~0.5 ml of well-suspended sample using a syringe with a

225

21G needle as the starting point for enzymatic hydrolysis.

226

The larger gauge needle is needed for homogeneous sampling

227

of cellulose slurry.

228

9. Shake the tubes continuously or manually mix them every

229

10–15 min.

230

1 0. Withdraw another 0.50 ml

231

sion every 1 h using a syringe with a 21G needle into the

of well-mixed Avicel suspen-

232

precooled 1.5-ml microcentrifuge tubes or stop the reaction

233

after 1 h by transferring to an ice-cooled water bath.

234

1 1. Centrifuge the samples in 1.5-ml microtubes at 13,000 g for

235

3 min.

236

12. Measure total soluble sugars in the supernatants by the phenol–

237

sulfuric acid assay.

238

1 3. Calculate the net soluble sugar release during the hydrolysis

239

process by subtraction of the sugar at the starting point.

240

1 4. Determine enzyme activity at a linear range between sugars

241

released and enzyme concentrations.

242

1. Add 0.7 ml of sugar-containing solution to 13 × 100 mm

243

disposable

solution.

glass tubes, and mix with 0.7 ml of 5% phenol

244

245

2. Add

mixing (

3.5

see

ml

Note

of concentrated

11).

sulfuric acid with vigorous

246

247

3. Read absorbance at 490 nm after cooling to room tempera-

248

ture (e.g., 20–30 min).

249

b-Glucosidase can cleave b-1,4-glucosidic bonds of soluble

250

substrates, including cellobiose, longer cellodextrins with a DP

251

from 3 to 6, and chromogenic substrates, such as

252

enyl-b-

-glucoside, p-nitrophenyl b-

p-nitroph-

dd

-1,4-glucopyranoside,

253

220 Zhang, Hong, and Ye

3.2.1. b-Glucosidase

Assay Using pNPG

Procedure

3.2.2. b-Glucosidase

Assay Using Cellobiose

Procedure

b-naphthyl-b-

d

-glucopyranoside, 6-bromo-2-naphthyl-b-

d

-

glucopyranoside, and 4-methylumbelliferyl-

(

b-

d

-glucopyranoside

enzyme’s broad substrate specificity.

2). The term “cellobiase” is often misleading because of this key

This pNPG method is an initial reaction rate assay (6).

1. Add 1.0 ml of

into test tubes.

pNPG solution and 1.8 ml of acetate buffer

2. Equilibrate at 50°C.

3. Prepare the enzyme dilution series.

4. Add 0.2 ml of diluted enzymes into the tubes containing the

substrate, and mix well.

5. Enzyme blanks: Add 0.2 ml of diluted enzymes into the tubes

containing 2.8 ml of acetate buffer, and mix well; Substrate

blank: Add 1.0 ml of pNPG solution and 2.0 ml of acetate

buffer into test tubes.

6. Incubate all tubes at 50°C for 15 or 30 min.

7. Add 4.00 ml of glycine buffer to stop the reaction.

8. Measure the absorbance of liberated products of

at 430 nm based on the substrate blank.

p-nitrophenol

9. Read

subtracting readings of the enzyme blanks.

the net absorbance of the enzyme solutions by

1 0. Determine

concentration of

p-nitrophenol release on the basis of the known

p-nitrophenol diluted by glycine at 430 nm.

1 1. Calculate the enzyme activity on the basis of the linear range

between absorbance and enzyme concentrations.

The

IUPAC is based on a fixed amount (1 mg) of glucose formation

b-glucosidase based on cellobiose assay recommended by

from cellobiose

reaction

enzyme dilutions. One dilution should release slightly more and

mixture

(6)

are

. The glucose concentrations in the cellobiose

determined using at least two different

one slightly less than 1.0 mg (absolute amount) of glucose in the

reaction conditions.

1. Dilute the enzyme solution by citrate buffer in a series. At least

two dilutions must be made of each enzyme sample inves-

tigated. One dilution should release slightly more and one

slightly less than 1.0 mg (absolute amount) of glucose in the

reaction conditions (i.e., 0.5 mg glucose released/ml).

2. Add 1 ml of diluted enzyme solution (DES) to the tubes.

3. Equilibrate the enzyme solutions and substrate solutions at

50°C.

4. Add 1.0 ml of substrate solution into the tubes containing

the enzyme solution.

254

255

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293

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295

Calculation

3.2.3. b-Glucosidase

Assay Using Cellobiose

3.3. Endoglucanase

Assays

3.3.1. Endoglucanase

Assay Using CMC/DNS

Cellulase Assays 221

5. Incubate at 50°C for exactly 30 min.

296

6. Immerse the tubes in boiling water for exactly 5.0 min to

297

stop the reaction.

298

7. Transfer the tubes to a cold water bath.

299

8. Substrate Blank: A mixture of 1.0 ml of cellobiose solution

300

and 1.0 ml of citrate buffer. Enzyme Blanks: A mixture of

301

1.0 ml of cellobiose solution and 1.0 ml of DESs. Treat

302

substrate and enzyme blanks identically as the experimental

303

tubes (i.e., equilibrate at 50°C, heat, boil, and cool).

304

9. Determine

oxidase kit (GOD) or a glucose hexose kinase and glucose-6

glucose release using a commercial glucose

305

306

phosphate dehydrogenase kit (HK/G6PDH) or HPLC.

307

1 0. Measure the absorbance of all solutions based on the substrate

308

blank.

309

1. Calculate the delta absorbance of dilute enzyme solutions by

310

subtracting absorbance of the respective enzyme blanks.

311

2. Calculate the real glucose concentrations released according

312

to a standard glucose curve by the enzyme kit.

313

3. Link the points less than 1 mg and greater than 1 mg by a

314

line,

supposed to produce 1 mg glucose.

and determine the EDR by using the point that is

315

316

4. Calculate cellobiase solution activity (IU/ml) (see Note 12):

317

Cellobiase

=

0.0926

.

318

EDR

b-Glucosidase activity can be measured on the basis of initial

319

reaction

320

Subheading

rates

3.2.1

of

and

cellobiose

3.2.2. The hydrolysis product – glucose –

by combining the methods of

321

can be measured by the glucose HK/G6PDH kit (14).

322

Endo-

323

intermolecular

b-1,4-D-glucanase (EC 3.2.1.4) randomly cleaves accessible

324

Because insoluble cellulose has very low accessible fractionation of

b-1,4-glucosidic bonds on the surface of cellulose.

325

b-glucosidase bonds to cellulase (3, 8, 15), water-soluble cellulose

326

derivatives

327

commonly used for endoglucanase activity assays. The hydrolysis

such as CMC and hydroxyethylcellulose (HEC) are

328

can be determined by measuring the changes in reducing sugars or

329

viscosity or color. Since CMC is an anionic substrate, its properties

330

change with pH. Nonionic substrates such as HEC are recom-

331

mended sometimes.

332

The IUPAC-recommended endoglucanase (CMCase) assay is a

333

fixed conversion method, which requires 0.5 mg of

334

glucose released under the reaction condition

335

end concentration is measured by the DNS method.

(6). The reducing

absolute

336

222 Zhang, Hong, and Ye

Procedure

3.3.2. Endoglucanase

Assay Using CMC/BCA

1. Prepare

dilutions

the

dilution releasing slightly more than 0.5 mg of glucose and

must

enzyme

be made

dilution

of each

series,

enzyme

of which

sample,

at least

with

two

one

one slightly less than 0.5 mg of glucose.

2. Add 0.5 ml of the DESs into test tubes with a volume of at

least 25 ml.

3. Equilibrate

50°C.

the enzyme solution and substrate solution at

4. Add 0.5 ml of the CMC solution to the test tubes and mix

well.

5. Incubate at 50°C for 30 min.

6. Add 3.0 ml of DNS solution and mix well.

7. Boil for exactly 5.0 min in vigorously boiling water.

8. Place the tubes in an ice-cooled water bath to quench the

reaction.

9. Add 20 ml of distilled water and seal with parafilm or by a

similar method. Mix by inverting the tubes several times.

1 0. Read

blank.

the absorbance at 540 nm based on the substrate

1 1. Prepare the substrate blank (0.5 ml of CMC solution + 0.5

ml of citrate buffer) and the enzyme blanks (0.5 ml of CMC

solution + 0.5 ml of dilute enzyme solutions). Treat substrate

and enzyme blanks identically as the experimental tubes.

1 2. Prepare the glucose standards:

GS1 – 0.125 ml of 2 mg/ml glucose + 0.875 ml of buffer.

GS2 – 0.250 ml of 2 mg/ml glucose + 0.750 ml of buffer.

GS3 – 0.330 ml of 2 mg/ml glucose + 0.670 ml of buffer.

GS4 – 0.500 ml of 2 mg/ml glucose + 0.500 ml of buffer.

1 3. Calculate the glucose released by the enzyme solutions with

deduction

glucose standard curve.

of the enzyme blank absorbance based on the

1 4. Draw the relationship between the real glucose concentra-

tions and their respective EDRs.

1 5. Link the points less than 0.5 mg and greater than 0.5 mg by

a line, and identify the EDR by using the point for 0.5 mg

glucose.

1 6. Calculate the CMCase activity of the original concentrated

enzyme solution in terms of IU/ml:

CMCase

=

0.185

EDR

This initial reaction rate enzymatic assay can be conducted at a

very low enzyme concentration. The reducing end concentration

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

Procedure

3.3.3. Endoglucanase

Assay Using CMC/Viscosity

Procedure

Cellulase Assays 223

is measured by the BCA method, in which the color development

378

reaction is conducted at 75°C in order to avoid b-glucosidic bond

379

cleavage during the color-development process (16).

380

1. Dilute

using the 50 mM citrate buffer and prepare the dilute enzyme

the enzyme solution extensively (e.g., 1,000-fold)

381

382

solution series.

383

2. Add 1.8 ml of CMC solution into 13 × 100 mm test tubes.

384

3. Equilibrate at 50°C water bath.

385

4. Add 0.2 ml of DES and mix well.

386

5. Incubate at 50°C for 10 min.

387

6. Add 2 ml of working BCA reagents and mix well.

388

7. Incubate at 75°C for 30 min.

389

8. Read absorbance at 560 nm after subtracting the readings for

390

the enzyme blanks and the substrate blank.

391

9. Calculate the enzyme activity based on a linear range between

392

reducing end concentrations and enzyme concentrations.

393

394

buffer; enzyme blanks: 1.8 ml of CMC solution + 0.2 ml of dilute

Substrate blank: 1.8 ml of CMC solution + 0.2 ml of citrate

395

enzyme

396

samples.

solutions. Treat blanks identically as the experimental

397

398

citrate buffer by 100-fold to 50

Glucose standard: 1 ml of 5 mM glucose diluted by 50 mM

399

prepare the sugar standards as below:

mM glucose standard solution;

400

GS1 – 0.4 ml of 50

401

GS2 – 0.8 ml of 50

m

402

GS3 – 1.2 ml of 50

m

M glucose + 1.6 ml of buffer.

M glucose + 1.2 ml of buffer.

403

GS4 – 1.6 ml of 50

m

404

GS5 – 2.0 ml of 50

m

M glucose + 0.8 ml of buffer.

m

M glucose + 0.4 ml of buffer.

M glucose.

405

This initial reaction rate assay is based on the reduction in specific

406

viscosity of soluble cellulose derivatives such as CMC and HEC

407

Both endoglucanase and exoglucanase can release new reducing

(2).

408

sugar

409

hydrolysis, endoglucanase can decrease specific viscosity greatly,

ends from soluble substrates. Within a limited degree of

410

and exoglucanase can decrease specific viscosity slowly (7).

411

1. Add 6.0 ml of prewarmed CMC solution in a water bath at

412

30°C into an Ostwald viscometer (water flow time of 15 s at

413

30°C) (see Note 13).

414

2. Add 1.0 ml of the prewarmed DES (see Note 14).

415

3. Determine the flow rates every 5 or 10 min.

416

4. Calculate specific viscosity (h

sp

):

417

h

sp

=

t

−

t

0

t

418

0

224 Zhang, Hong, and Ye

3.3.4. Semiquantitative

Endoglucanase Assay

Based on Dye Release

Microbe-Secreted

Endoglucanase Assay

on Agar Medium

Procedure

Endoglucanase Assay

on Agarose Gel

Procedure

where

time of the buffer (s).

t is the effluent time of the buffer (s) and t

0

is the efflux

5. Plot

viscosity against the enzyme concentration; a linear relation

the increasing rate of the reciprocal of the specific

should be obtained.

6. Calculate unit of activity

ship

reciprocal of the viscosity of the CMC solution (

between enzyme concentration/rate

arbitrarily from the linear relation-

of

see

increase

Note 15).

of

Endoglucanase

solid

because these dyes are adsorbed only by long chains of polysac-

supports by

activity

staining

can be

polysaccharides

detected semiquantitatively

with various dyes

on

charides.

numbers of samples but differences in enzyme activity levels of

These methods are suitable for monitoring large

less than twofold are difficult to detect by eye. A linear relationship

between the halo diameter and the logarithm of endoglucanase

activity can be established as

the diameter,

D = K × log(A) + N, where the D is

determined by the standard curve of the known enzyme activity

A is the enzyme activity, and K and N are parameters

solutions. The activity of unknown samples can be calculated on

the basis of the standard curve. Three procedures are described

involving in vivo as well as in vitro endoglucanase detection.

1. Inoculate

the

the

solid

the endoglucanase-secreted microorganisms on

(see Note

growth rate of the microorganism and enzyme activity

CMC medium. The growth time depends on

16).

2. Stain a 9-cm Petri dish by adding 20 ml of Congo red solution

at room temperature for 30 min.

3. Rinse the residual dye on the dish using distilled water.

4. Destain Congo red with

the halos are not clear, destain the dish by another

~20 ml of 1 M NaCl for 30 min. If

NaCl solution.

~20 ml of

5. Detect the clear, weak yellow halos for endoglucanase activity

with the red background.

6. Option: In order to increase halo contrast, add

acetate acid or 1 M HCl to the plate at room temperature for

~20 ml of 5%

10 min, and pour off. The background of the plate will turn

blue.

1. Pour

into a 9-cm Petri dish.

~20 ml of the melted CMC agarose solution (~50°C)

2. Drill wells on the solidified agarose gel with a cork borer, and

remove the agarose particles in the wells by suction or a pair

of tweezers (see Note 17).

3. Add 10–20 ml of the enzyme solution into the holes.

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

Endoglucanase Assay

on Polyacrylamide Gel

Procedure

3.4. Exoglucanase

Assays

Cellulase Assays 225

4. Put the plate in the incubator (37°C or desired temperature)

462

for several hours or even overnight.

463

5. Wash the plate with distilled water.

464

6. Add 10 ml of the Congo red solution and incubate at room

465

temperature for 30 min.

466

7. Wash the residual dye on the plate by distilled water.

467

8. Destain the dye by using 20 ml of 1 M NaCl solution at room

468

temperature

Note 18).

for 30 min, and decant the destained solution

469

(see

470

9. Detect the clear yellow halo with the red background.

471

This method can separate mixed protein components by electro-

472

phoresis and then detect endoglucanase activity on polyacrylamide

473

gels

474

cellulase activity must be detected after SDS removal and protein

(SDS PAGE or native PAGE). If SDS-PAGE is used,

475

re-naturation.

476

1. Separate the protein mixtures by native or SDS PAGE.

477

2. Rinse the gel in distilled water for 5 min.

478

3. Soak the gel in the sodium phosphate buffer with gentle

479

shaking for 20 min, and repeat the washing procedure three

480

times to remove the SDS.

481

4. Transfer the gel into the CMC/phosphate buffer for 30 min.

482

5. Rinse the gel with distilled water.

483

6. Incubate the gel in 0.1 M sodium phosphate buffer at 40°C

484

for 1 h.

485

7. Stain the gel with the Congo red solution at room temperature

486

for 30 min.

487

8. Wash the gel with distilled water, and destain the gel in 1 M

488

NaCl solution at room temperature for 30 min (see Note 19).

489

Exoglucanase

490

and/or

(CBH,

491

reesei

cellobiose from

EC

ends

3.2.1.91)

of cellulose

can release

chains.

either

Trichoderma

glucose

492

end and the non-reducing end of cellulose chains, respectively.

CBH1 and CBH2 cleave cellobiose units from the reducing

493

In contrast to endoglucanase and b-

d

494

are difficult to measure due to the lack of specific substrates and

-glucosidase, exoglucanases

495

interference from other cellulase components. Accordingly, exoglu-

496

canases have to be assayed in the purified form. The activity of

497

purified exoglucanases is often estimated using Avicel. Avicel is

498

a

499

highest ratio of end/accessibility

good substrate for exoglucanase activity assay

500

is regarded as synonymous with exoglucanase or CBH. In addition,

(3, 7). To some extent, Avicelase

because of its

501

amorphous

502

nase activity.

cellulose can be used for determining of exogluca-

503

226 Zhang, Hong, and Ye

3.4.1. Exoglucanase Assay

Using Avicel

Procedure

3.4.2. Exoglucanase Assay

Using RAC

RAC Preparation

Procedure (17)

1. Suspend 1.25% (w/v) Avicel in acetate buffer (see Note 20).

2. Add 1.6 ml of Avicel suspension solution into the tubes.

3. Dilute a series of enzyme solutions by acetate buffer.

4. Equilibrate

bath at 50°C.

the substrate and enzyme solutions in a water

5. Add

substrate and mix well.

0.4 ml of the dilute enzyme solutions to the Avicel

6. Incubate at 50°C for 2 h.

7. Stop

water bath.

the reaction by submerging the tubes in ice-cooled

8. Withdrew

and centrifuge the sample at 13,000 g for 3 min.

~1 ml of hydrolysate into microcentrifuge tubes

9. Prepare enzyme blanks (0.4 ml of diluted enzymes and

1.6

(0.4 ml of 0.1 M acetate buffer and 1.6 ml of 1.25% (w/v)

ml of 0.1 M acetate buffer) and substrate blank

Avicel suspension buffer).

1 0. Determine

the Phenol–Sulfuric Acid assay where the absorbance of the

the total soluble sugars in the supernatant by

substrate blank is used as the blank (

Acid assay in Subheading 3.1.2).

see the Phenol-Sulfuric

1 1. Calculate the enzyme activity on the basis of a linear rela-

tionship between the total soluble sugar release and enzyme

dilution.

the

One unit of exoglucanase activity is

glucose equivalent per minute from Avicel.

amount of enzyme that releases one micromole

defined as

of

1. Microcrystalline

centrifuge tube, and 0.6 ml distilled water is added to form

cellulose (0.2 g) is added to a 50-ml

a suspended cellulose slurry.

2. Ten milliliters of ice-cold 86.2% H

added to the slurry with vigorous stirring. Before adding the

3

PO

4

is slowly and carefully

last

solution must be thoroughly mixed. The cellulose mixture

2 ml of phosphoric acid, the cellulose suspension

turns transparent within several minutes, and should be held

for ca. 1 h on ice with occasional stirring.

3. Approximately 40 ml of ice-cold water is added at the rate

of approximately 10 ml per addition with vigorous stirring

between additions, resulting in a white cloudy precipitate.

4. The precipitated cellulose is centrifuged at

for 20 min.

~5,000 g and 4°C

5. The

followed

pellet is

containing

by centrifugation

suspended in about

to

45 ml ice-cold water

times.

phosphoric acid; this

remove

step is

the

repeated

supernatant

four

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

Assay Procedure

3.5. Summary

Cellulase Assays 227

6. Approximately 0.5 ml of 2 M Na

2

CO

3

is added to neutralize

547

the

distilled water is used to suspend the cellulose pellet.

residual phosphoric acid, and then 45 ml of ice-cold

548

549

7. After centrifugation, the pellet is suspended in distilled water

550

and centrifuged twice or until the pH reaches 5–7.

551

8. The carbohydrate concentration of RAC is calibrated by the

552

Phenol-Sulfuric Acid method and diluted to 1%.

553

9. Addition of 0.2%, w/v sodium azide is optional for extended

554

RAC storage at 4°C (see Note 21).

555

1. 0.5 ml of 1% (w/v) RAC and 0.05 ml of 1 M citrate buffer

556

plus 0.25 ml water in the tubes.

557

2. Dilute

buffer.

the enzyme solution series with 50 mM acetate

558

559

3. Equilibrate the tubes containing the enzyme and substrate

560

solutions at 50°C.

561

4. Add 0.2 ml of the DESs and mix well.

562

5. Incubate at 50°C for 10–30 min.

563

6. Place the tubes in an ice-cold water bath.

564

7. Centrifuge the hydrolysate sample at 10,000 g for 3 min.

565

8. Prepare enzyme blanks (0.2 ml of the DES, 0.05 ml of 1 M

566

citrate buffer, and 0.75 ml of distilled water) and substrate

567

blanks (0.5 ml of 1% w/v RAC, 0.45 ml of distilled water,

568

and 0.5 ml of 1 M citrate buffer).

569

9. Measure the total soluble sugar concentration in the super-

570

natants

571

the absorbance at 490 nm using the absorbance of the sub-

by the Phenol–Sulfuric Acid method and measure

572

strate blank as the blank; (

573

in Subheading 3.1.2).

see the Phenol–Sulfuric Acid assay

574

1 0. A linear relationship between the total soluble sugar release

575

and

576

activity. One unit of exoglucanase activity is defined as the

enzyme dilution is used for calculating the enzyme

577

amount of enzyme that releases one micromole of glucose

578

equivalent per minute from Avicel.

579

A number of cellulase activity assays have been developed over

580

several

581

cellulase activity assays here. Heterogeneity of insoluble cellulose,

decades, but we have presented only the most popular

582

complicated

583

exoglucanase,

synergy/competition

584

formidable

and changes in ratio

among

of enzyme/substrate

endoglucanase

pose

and

585

(

586

(such as limited accessibility to enzyme, degree of polymerization

2, 7, 8). Keeping

challenges

special

in developing

properties

cellulase

of insoluble

activity

substrates

assays

587

(DP), etc.) in mind

588

the hydrolysis rates obtained on soluble and insoluble substrates,

(8), there is no clear relationship between

589

228

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

Zhang, Hong, and Ye

mainly because of large variations in limited solid substrate

accessibility to cellulase (7). A functionally based model has been

developed to suggest the complexity among endoglucanase,

exoglucanase, their ratio, cellulose accessibility, DP, enzyme

concentration, and reaction time (7). The model suggests the

challenges in applying the results of total cellulase activity assay

measured on one solid substrate to a different solid substrate.

Researchers must state clearly all parameters of their assay

conditions and resist the temptation to compare their results

to those of other researchers using different substrates, experi-

mental conditions, etc. An understanding of enzymatic cellulose

hydrolysis mechanisms among substrate characteristics is urgently

needed, as well as development of enzyme activities to evaluate

cellulase performance on insoluble cellulosic materials, especially

for pretreated lignocellulosic materials.

605

4. Notes

1. Be careful to handle the phenol safely.

2. The DNS reagent can be stored in darkness at 4°C for at

least 1 month. It could lose its reducing ability after long

storage (18). The freshness of the DNS reagent is often

ignored (18).

3. It is important to check each paper strip weight to ensure that

the weight variation is less than 1 mg per strip because FPA

is subject to the filter paper weight. Handle the paper with

forceps or gloved hands.

4. Aliquots of the standard glucose solution can be tightly sealed

and stored frozen. The solution should be mixed well after

thawing.

5. 4-Methylumberliferyl-b-glucoside can replace pNPG, which

results in an assay with higher sensitivity.

6. Take commercial concentrated cellulase solution as an

example. Dilute the enzyme solution 20-fold using 50 mM

citrate buffer DES, and then prepare a series of dilutions from

E1 to E5 with different dilution rates as below:

E1: 0.10 ml of DES + 1.90 ml of citrate buffer (dilute rate

= 0.0250).

E2: 0.15 ml of DES + 1.85 ml of citrate buffer (dilute rate

= 0.0375).

E3: 0.20 ml of DES + 1.80 ml of citrate buffer (dilute rate

= 0.0500).

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

Cellulase Assays 229

631

632

633

634

E4: 0.30 ml of DES + 1.70 ml of citrate buffer (dilute rate

= 0.0750).

E5: 0.35 ml of DES + 1.65 ml of citrate buffer (dilute rate

= 0.0850).

7. Commercial enzyme solutions can contain a significant

635

636

amount of reducing sugars (19).

8. The boiling condition should be severe, and the volume of

the boiling water bath should be maintained above the level

of the total liquid volume of the test tubes to promote full

color development.

637

638

639

640

9. International Unit (IU) is defined as 1 mmol/min, based on

641

the initial hydrolysis rate, and is different from FPU assay,

642

643

which is a fixed conversion assay.

10. The b-

d

-glucosidase level present in the cellulase mixture

greatly influences the FPA assay (2, 5, 6, 20) because the

DNS readings are strongly influenced by the reducing end

ratio of glucose, cellobiose, and longer cellodextrins (16).

644

645

646

647

648

650

651

652

653

654

11. The Phenol–Sulfuric Acid assay is an extremely exothermic

649

reaction; so be cautious not to spill the liquid.

12. 0.5 mg of glucose produced/ml × 2 ml volume = 1 mg of

glucose produced = 5.56 mmol of glucose produced = 2.78

mmol of cellobiose consumed. Since reaction time is 30 min,

0.0926 IU of b-glucosidase can produce 1 mg of glucose

from cellobiose within 30 min.

13. A convenient viscometer such as capillary tubing could be

655

656

used to replace the Ostwald viscometer.

14. Constant temperature during the viscosity measurement is

657

important because viscosity is greatly influenced by tempera-

658

ture change.

659

15. Exact endoglucanase activity (mmol bond cleavage/min)

based on changes in specific viscosity can be calculated

through a relationship between the viscosity and the DP of

CMC. Viscosity of the substrate is strongly associated with

substrate DP, and medium-viscosity CMC is recommended.

660

661

662

663

664

665

16. If CMC inhibits microorganism growth, a second layer of

CMC solid medium can be applied to the primary medium

666

containing other carbon sources or nutrients.

667

17. Strong enzyme activity or the short distance between the

668

wells results in the fused halos, which may be difficult to

669

differentiate or measure.

670

18. Often, the halo can be observed in 5 min; if the halo is not clear,

671

destain again by adding 20 ml of the NaCl solution. If the band is

672

not clear, destain the gel by using the NaCl solution again.

673

674

675

230

676

677

678

679

680

681

682

683

684

685

686

687

688

Zhang, Hong, and Ye

19. For native PAGE, a one-time soak is enough. Do not use a

potassium phosphate buffer because potassium precipitates

in the gel.

20. Since Avicel powder could contain approximately 4–8%

moisture, weight adjustment is needed.

21. RAC, different from phosphoric acid-swollen cellulose (PASC),

is a homogeneous amorphous cellulose that is precipitated

from dissolved cellulose. RAC has a constant quality because

it is regenerated from homogeneous dissolved cellulose, has

easy handling and transferring properties, is a homogeneous

substrate, and has high substrate reactivity (17). Take care

if azide is used because of both toxicity and the explosive

nature of powders.

689

Acknowledgments

This work was made possible in part by support from the

Biological Systems Engineering Department of Virginia

Polytechnic Institute and State University and USDA-CSREES

(2006–38909–03484) and DOE BioEnergy Science Center

(BESC).

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