characterization of thermobifida fusca cutinase-cbm...

1

1

2

Characterization of Thermobifida fusca Cutinase-CBM 3

Fusion Proteins and Their Potential Application in 4

Bioscouring 5

Yao Zhang1,

2, Sheng Chen

1, 2, Meng Xu

1, 2, Artur Cavoco-Paulo

3, Jing Wu

1, 2*

, 6

and Jian Chen1, 2*

7

1State Key Laboratory of Food Science and Technology,

2School of Biotechnology and 8

Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 9

Wuxi, 214122, China, 3

Department of Textile Engineering, University of Minho, 4800 10

Guimaraes, Portugal. 11

12

13

14

15

16

* Corresponding author. Mailing address: State Key Laboratory of Food Science and 17

Technology, Jiangnan University, Wuxi 214122, China. Jing Wu, Phone: 18

86-510-85327802. Fax: 86-510-85327802. E-mail: [email protected] or Jian 19

Chen, Phone: 86-510- 85329031. Fax: 86-510-85918309. Email: [email protected]

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00896-10 AEM Accepts, published online ahead of print on 20 August 2010

on July 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from


.asm.org/

Dow

nloaded from


.asm.org/

Dow

nloaded from

http://aem.asm.org/

http://aem.asm.org/

http://aem.asm.org/

2

ABSTRACT 21

Cutinase from Thermobifida fusca is thermally stable and has potential application 22

in the bioscouring of cotton in the textile industry. In the present study, the 23

carbohydrate-binding module (CBM) from T. fusca cellulase Cel6A (CBMCel6A) and C. 24

fimi cellulase CenA (CBMCenA) were fused, separately, to the carboxyl-terminus of T. 25

fusca cutinase. Both fusion enzymes, cutinase-CBMCel6A and cutinase-CBMCenA, were 26

expressed in E. coli and purified to homogeneity. Enzyme characterization showed that 27

both of them displayed similar catalytic properties and pH stability to T. fusca cuinase. 28

In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal 29

temperature of 50 °C. Compared with T. fusca cutinase, in the absence of pectinase, the 30

binding activity on cotton fiber was enhanced by 2% for cutinase-CBMCel6A and 28% 31

for cutinase-CBMCenA; while in the presence of pectinase, the binding activity was 32

enhanced by 40% for the former and 45% for the latter. Notably, dramatic increase of up 33

to 3-fold was observed in the amount of released fatty acids from cotton fiber by both 34

cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first 35

report of improving the scouring efficiency of cutinase by fusing it with CBM. The 36

improvement in activity and the strong synergistic effect between the fusion proteins 37

and pectinase suggests that they may have better applications in textile bioscouring than 38

the native cutinase.39


.asm.org/

Dow

nloaded from

http://aem.asm.org/

3

INTRODUCTION 40

Cotton fiber has a multilayered structure with its outermost surface being the 41

cuticle which is crosslinked to the primary cell wall of cotton fiber by esterified pectin 42

substances. The major component of the cuticle is cutin, an insoluble polyester 43

composed mainly of saturated C:16 and C:18 hydroxy and epoxy fatty acids (15, 17, 37, 44

48). During the process of scouring in the textile industry, the cuticle layer has to be 45

removed in order to improve the wettability of cotton fiber, which then facilitates 46

uniform dyeing and finishing. Traditionally, this process is performed by hot hydrolysis 47

in alkaline medium, which not only consumes large quantities of water and energy but 48

also causes severe pollution and fiber damage (21, 22, 43). Therefore, 49

environment-friendly scouring methods based on biocatalysts have been actively sought 50

(2, 41, 46). 51

Cutinase is a multi-functional esterase capable of degrading the cutin component of 52

the cuticle. Earlier reports showed that the fungal cutinase from Fusarium solani pisi 53

has potential use for cotton cuticle degradation and exhibits a good synergistic effect 54

with pectinase, an enzyme utilized to degrade pectin, in the scouring of cotton fiber (1, 8, 55

9, 15). Moreover, site-directed mutagenesis has been performed to substitute the 56

specific amino acid residues near the active site of cutinase (3) to improve its hydrolytic 57

activity towards polyesters. More recently, a cutinase from the thermophilic bacterium 58

Thermobifida fusca has been identified and overexpressed in E. coli in our laboratory 59

(11). The good thermal stability and alkali resistance of this recombinant T. fusca 60

cutinase make it potentially more amenable to textile bioscouring (11). 61


.asm.org/

Dow

nloaded from

http://aem.asm.org/

4

To further improve the applicability and/or catalytic efficiency of T. fusca cutinase, 62

the present study attempts to engineer a novel cutin-degrading enzyme, based on 63

analysis of the surface structure of cotton fiber. It has been observed that, in addition to 64

cutin, pectin, proteins and other components, there is also a large amount of cellulose on 65

the surface layer of cotton fiber (31). Thus it is tempting to hypothesize that if the 66

enzyme can be engineered to specifically bind to cellulose through a “gain of function” 67

modification, its concentration on the surface of cotton fiber could increase significantly. 68

Subsequently, its catalytic efficiency for cutin breakdown could be improved due to 69

proximity effect. In order to design such an enzyme, a fusion protein strategy in which a 70

cellulose binding protein/module will be attached to cutinase is considered. 71

It is well known that cellulase is capable of binding specifically to cellulose (35, 72

42). This enzyme has two separate modules, a catalytic module and a 73

carbohydrate-binding module (CBM) (12). The two modules are discrete structural and 74

functional units usually connected by a flexible linker (5, 18, 39). CBM has high 75

specific capacities for cellulose binding. Previously, it has been reported that CBM is 76

able to be fused to a chosen target protein by genetic manipulation (46), resulting in 77

enhanced binding of this fusion protein to cellulose (7, 40). For example, fusion 78

proteins were constructed by fusing CBM to β-glucosenucleotide enzyme (GUS) (14) or 79

β-glycosidase (BglA) (20), which facilitates biochemical analysis of scouring efficiency 80

for cotton fabrics. 81

In the present study, the CBM from T. fusca cellulase Cel6A (abbreviated as 82

CBMCel6A) and the CBM from C. fimi cellulase CenA (abbreviated as CBMCenA) were 83


.asm.org/

Dow

nloaded from

http://aem.asm.org/

5

fused, separately, to the carboxyl terminus of T. fusca cutinase. The resulting fusion 84

enzymes were compared with the native cutinase in terms of their biochemical 85

properties as well as the catalytic efficiency in cutin breakdown on cotton fiber. This is 86

the first report of improving the scouring efficiency of cutinase by fusing it with CBM. 87

88

MATERIALS AND METHODS 89

Bacterial strains, plasmids, and culture conditions 90

The strain T. fusca, plasmid pET20b(+)-Tfu_0883, and pBSK-CBMCenA were lab 91

stocks (4,11). Plasmid pET20b(+)-Tfu_0883 was used as the gene source of T. fusca 92

cutinase. The strain T. fusca and plasmid pBSK-CBMCenA were used as the gene source 93

of CBMCel6A and CBMCenA, respectively. E. coli strains BL21 (DE3) was used as the 94

expression host, and pET20b(+) was used as the cloning and expression vector. Cells 95

were grown in Luria-Bertani medium at 37 °C and, if necessary, ampicillin in a final 96

concentration of 100 µg/ml was added to the medium. 97

Construction of cutinase-CBM fusion protein expression vectors 98

The gene encoding cutinase (NCBI accession number YP_288944) and CBMCenA 99

(NCBI accession number AAA23084.1) was amplified using plasmid 100

pET20b-Tfu_0883 and pBSK-CBMCenA as the template, respectively. The gene 101

encoding CBMCel6A and its linker (NCBI accession number YP_289135) was amplified 102

from T. fusca genomic DNA. Overlapping PCR was used to fuse the T. fusca CBMCel6A 103

or C. fimi CBMCenA to the C-terminus of T. fusca cutinase. Sequences of the primers are 104

given in Table 1. The procedure of fusion is shown in Figure 1. An NcoI restriction site 105


.asm.org/

Dow

nloaded from

http://aem.asm.org/

6

(boldface) was introduced at the 5' end of P1 while an EcoR1 restriction site (boldface) 106

was introduced at the 3' end of P4/P4'. The overlapping area was underlined. The 107

primers were synthesized by Shanghai Sangon Biological Engineering Technology & 108

Services Co. Ltd. 109

The PCR was performed using 35 successive cycles as following: denaturation at 110

94 °C for 0.5 min, annealing at 56 °C for 0.5 min, and primer extension at 72 °C for 1.5 111

min. PrimeSTAR HS DNA Polymerase was utilized during the process. The 112

amplification product was isolated and ligated into the vector pMD18T-simple. The 113

ligation mixture was used to transform chemically competent E. coli JM109. The 114

plasmid isolated from these transformants was verified by restriction analysis and the 115

gene sequence was confirmed by DNA sequencing. The plasmids with the correct 116

sequences of cutinase-CBMCel6A and cutinase-CBMCenA were named as 117

pMD18T/cutinase-CBMCel6A and pMD18T/cutinase-CBMCenA, respectively. The 118

resulting plasmids were digested with NcoI and EcoRI and ligated into the similarly 119

digested expression vector pET20b(+). The ligation mixture was used to transform 120

chemically competent E.coli JM109 cells. The plasmid isolated from these 121

transformants was verified by restriction analysis and the gene sequence was confirmed 122

again by DNA sequencing. The plasmids with the correct sequences of 123

cutinase-CBMCel6A and cutinase-CBMCenA were named as pET20b/cutinase-CBMCel6A 124

and pET20b/cutinase-CBMCenA, respectively. 125

Enzymes used for DNA manipulations were purchased from TakaRa 126

Biotechnology Co. Ltd. DNA sequencing was performed by Shanghai Generay 127


.asm.org/

Dow

nloaded from

http://aem.asm.org/

7

Biotechnology Co. Ltd. Genomic DNA extraction was performed following the method 128

of Sambrook et al. (1989). Plasmid DNA was extracted by using the Sangon EZ-10 Spin 129

Column Plasmid Mini-Preps kit. Plasmid and PCR products were recovered from 130

agarose gel using Sangon purification kit. 131

Expression and purification of the cutinase-CBM fusion proteins 132

E.coli BL21(DE3) cells harboring pET20b/cutinase-CBMCel6A or 133

pET20b/cutinase-CBMCenA were grown in TB medium containing ampicillin (100 134

µg/ml) at 37 °C. When the culture reached an A600 of 1.5 to 2.0, 135

isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 136

0.5 mM. The culture after 18 h of induction was centrifuged (10,000 × g, 30 min, 4 137

°C) and the supernatant was collected. 138

The above supernatant was treated with 70% (w/v) saturated ammonium sulphate 139

solution and the solution was kept at 4 °C overnight. Precipitates were collected by 140

centrifugation and dissolved in 50 ml buffer A (20 mM sodium phosphate, 0.5 M NaCl, 141

and 20 mM imidazole, pH 7.4). The solutions were subsequently dialyzed against 2 142

liters of buffer A overnight and applied to a nickel affinity column pre-equilibrated with 143

buffer A. The samples were allowed to bind with Ni-NTA agarose at a flow rate of 1 144

ml/min, followed by washing with buffer A until the UV baseline was reached. Elution 145

was performed using a linear gradient from 0 to 500 mM imidazole in buffer A over 100 146

min. The fractions containing p-nitrophenyl butyrate (pNPB) hydrolase activity were 147

pooled and dialyzed against 2 liters of buffer B (20 mM Tris-HCl, pH 8.0) at 4 °C 148

overnight. The purified enzyme was concentrated by ultrafiltration and stored at -80°C. 149


.asm.org/

Dow

nloaded from

http://aem.asm.org/

8

Enzyme characterization of cutinase-CBMs 150

For enzyme characterization, all the values presented in graphs and tables are the 151

means of three replications. Esterase activity, cutinase activity, and lipase activity were 152

assayed as described previously (11), with pNPB, cutin and triolein as the substrates, 153

respectively. 154

Temperature optima of the fusion and native enzymes were measured at 155

temperatures ranging between 20-70 °C. The reaction was performed in a buffer 156

containing 20 mM Tris-HCl, 10 mM NaCl, and 50 mM sodium taurodeoxycholate at pH 157

8.0 using pNPB as substrates. Since the pH of Tris buffer is temperature dependent, the 158

buffers were adjusted to pH 8.0 at the desired temperature. Enzyme activity was 159

measured by pre-incubating the buffer at a desired temperature for 2 min to allow it to 160

reach the final pH of 8.0. The thermostability of the enzymes was determined by 161

incubating the enzymes in 20 mM Tris-HCl (pH 8.0) at 50 °C. At different intervals, 162

samples were taken and assayed for residual activity using pNPB as the substrate. 163

pH optima of the fusion and native enzymes was investigated between pH 6.0 to 164

9.0 using either potassium phospate buffer (pH 6.0-7.0) or Tris-HCl buffer (pH 7.0-9.0). 165

To determine the pH stability, 20 mM concentrations of the following buffers were used: 166

sodium acetate (pH 4.0-6.0), potassium phospate (pH 6.0-7.0), Tris-HCl (pH 7.0-9.0), 167

and glycine-NaOH (pH 9.0-11.0). The enzymes were pre-incubated in the various 168

buffers at 37 °C for 24 h, followed by the determination of residual activity using pNPB 169

as the substrate. 170

The Michaelis-Menten parameters, Vmax and Km, were determined from 171


.asm.org/

Dow

nloaded from

http://aem.asm.org/

9

Michaelis-Menten plots of specific activities at various substrate concentrations. Rates 172

were measured in triplicate using pNPB (100-2000 µM) as the substrate by continuous 173

spectrophotometric analysis. Initial reaction velocities were calculated from the linear 174

region (~60s) of the reaction progress curve and measured in triplicate by varying the 175

concentration of the substrate. Apparent kinetic constant Km was calculated using the 176

Graph Pad Prism program. 177

Protein quantification and SDS-PAGE analysis 178

Protein concentrations were determined using the Bio-Rad protein assay kit 179

(Bio-Rad), with purified bovine serum albumin (Promega) as the standard. Sodium 180

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 10% 181

acrylamide gels and the proteins were visualized by staining with Coomassie brilliant 182

blue R-250. 183

Binding and catalytic activity of cutinase-CBMs towards cotton fiber 184

The adsorption property of the enzymes was tested with cotton fiber as described 185

previously with minor modifications (4). Prior to treatment, raw cotton fiber was boiled 186

for 1 min, and then dried at 40 °C overnight. 1 g of pretreated cotton fiber was mixed 187

with 0.1% penetrant and 0.5% bovine serum albumin (BSA) in Tris-HCl buffer (20 mM, 188

pH 8.0) at 25 °C. The mixture was incubated for 30 min to avoid non-specific binding. 189

Equal units of native cutinase or fusion enzyme (10 U/ml toward pNPB) was then added 190

to the above solution and incubated for another 1 h with shaking at 60 rpm. The reaction 191

mixtures were centrifuged at 3,000 ×g for 2 min and the amount of unbound enzyme 192

was estimated from the residual activity in the supernatant. The amount of cotton 193


.asm.org/

Dow

nloaded from

http://aem.asm.org/

10

fiber-bound enzyme was calculated from the difference between the initial enzyme 194

activity and unbound enzyme activity. Control assays were performed under the same 195

conditions, except in the absence of raw cotton fiber. Pectinase was added when needed. 196

The desorption property of the enzymes was performed according to a method 197

described previously (4). After 2 h of incubation, the above reaction solution was 198

diluted (1:20) with Tris-HCl buffer and the mixture was incubated for another 60 min 199

with shaking at 200 rpm at 25 °C. The amount of desorbed enzyme was estimated from 200

the enzymatic activity in the supernatant. All assays were performed in triplicate. 201

For the determination of released fatty acids catalyzed by cutinase or 202

cutinase-CBM, a 10 ml reaction mixture containing 1 g of raw cotton fiber and 50 µM 203

of native or fusion cutinase was incubated in 20 mM Tris-HCl buffer with or without 204

pectinase. The reaction mixture was shaken at 200 rpm for 18 h at 50 °C. At various 205

times, samples were removed and subjected to titration using 20 mM NaOH. 206

207

RESULTS 208

Construction and purification of cutinase-CBM fusion proteins 209

Cutinase-CBM fusion proteins were generated by fusing either the CBM of 210

cellulase Cel6A from T. fusca (CBMCel6A) or the CBM of cellulase CenA from C. fimi 211

(CBMCenA) to the C-terminus of T. fusca cutinase through overlapping PCR 212

amplification. The fused genes were subsequently inserted into the expression vector 213

pET-20b(+) which contains a C-terminal His6 tag and an N-terminal signal peptide PelB 214

to allow the expressed proteins to be secreted. The resulting constructs 215


.asm.org/

Dow

nloaded from

http://aem.asm.org/

11

pET20b/cutinase-CBMCel6A and pET20b/cutinase-CBMCenA were used for protein 216

expression in E.coli BL21(DE3). The pNPB hydrolyzing activity in the culture 217

supernatant was 94 U/ml for cutinase-CBMCel6A and 76 U/ml for cutinase-CBMCenA, 218

which was 230-fold and 190-fold to that of the control culture (in which E.coli cells 219

carried the vector pET-20b(+)), respectively. Both fusion enzymes were purified by 220

ammonium sulfate fraction and Ni-Sepharose affinity chromatography (Supplemental 221

Table S1 and S2). SDS-PAGE results demonstrated that they were purified to 222

homogeneity with the same molecular mass of about 45 kDa (Figure 2). In addition, the 223

purified enzymes were active with a specific activity of 20000 U/µmol protein 224

(Supplemental Table S1) for cutinase-CBMCel6A and 15000 U/µmol protein 225

(Supplemental Table S2) for cutinase-CBMCenA. 226

Enzymatic properties of cutinase-CBM fusion proteins 227

The optimal temperature and pH of the fusion enzymes were determined at a 228

temperature range of 20-70 °C (Supplemental Figure S3A) and a pH range of 6-9 229

(Supplemental Figure S3C). For comparative purpose, T. fusca cutinase was also 230

subjected to similar analysis. The results showed that both cutinase-CBMCel6A and 231

cutinase-CBMCenA exhibited an optimal temperature at 50 °C, whereas the native 232

cutinase displayed an optimal temperature at 60 °C. Not surprisingly, all native and 233

fusion enzymes exhibited the same optimal pH of 8.0. Subsequently, the thermostability 234

was determined at the temperature of 50 °C (Supplemental Figure S3B), while the pH 235

stability was determined at pH values between 4 to 11 (Supplemental Figure S3D). The 236

half-life of the native cutinase was 70 h at 50 °C, while those of cutinase-CBM fusion 237


.asm.org/

Dow

nloaded from

http://aem.asm.org/

12

enzymes were both 53 h at 50 °C. Similar pH stability at a pH range of 6-9 was 238

observed for all three enzymes. 239

Previously, it has been shown that T. fusca cutinase has broad substrate specificity 240

against cutin, soluble esters and insoluble triglycerides (11). The activity of 241

cutinase-CBMs towards soluble ester had been confirmed in the above purification and 242

characterization analysis using pNPB as a substrate. For the insoluble triglycerides, both 243

cutinase-CBMCel6A and cutinase-CBMCenA were found to be capable of hydrolyzing 244

triolein with specific activities corresponding to 128% and 111% of the native cutinase, 245

respectively. Furthermore, their cutin hydrolyzing activities were evaluated under their 246

individual optimal temperature and pH. As shown in Table 2, the C16 and C18 family 247

fatty acid monomers released after enzymatic reaction were 61% for cutinase-CBMCel6A, 248

64% for cutinase-CBMCenA, and 59% for the native cutinase. The hydroxy fatty acids, 249

which are specific in cutin, were 3.6% for cutinase-CBMCel6A, 3.7% for 250

cutinase-CBMCenA, and 3.1% for the native cutinase. These results demonstrated that 251

both cutinase-CBMCel6A and cutinase-CBMCenA can hydrolyze cutin as efficiently as the 252

native cutinase. 253

The kinetics of the fusion enzymes was analyzed using pNPB as the substrate 254

(Table 3). Their Km values were similar to that of the native cutinase, while their 255

catalytic efficiency (Kcat/Km) was about 94% (cutinase-CBMCel6A) and 85% 256

(cutinase-CBMCenA) of the native cutinase. 257

Binding and hydrolytic activity of cutinase-CBMs towards cotton fiber 258

Adsorption of the enzyme on the surface of cotton fiber is the first step for cutinase 259


.asm.org/

Dow

nloaded from

http://aem.asm.org/

13

to perform its hydrolysis towards cutin (47). The binding experiments (Figure 3A) were 260

performed under the conditions with or without the presence of pectinase, an enzyme 261

utilized to remove pectin in bioscouring. The results showed that, compared with T. 262

fusca cutinase, the binding of cutinase-CBMCel6A was enhanced by 2% in the absence of 263

pectinase and 40% in the presence of pectinase, while the binding of cutinase-CBMCenA 264

was enhanced by 28% in the absence of pectinase and 45% in the presence of pectinase. 265

After dilution and re-equilibration, almost all of the bound enzymes were desorbed. 266

Thus, the binding of both cutinase and cutinase–CBMs with cotton fiber appeared to be 267

reversible. 268

In addition to binding, their hydrolytic efficiency towards cotton fiber was also 269

compared (Figure 3B). In the absence of pectinase, the amount of released fatty acids 270

was similar to that of the native cutinase for cutinase-CBMCel6A, but was 1.8-fold higher 271

for cutinase-CBMCenA. In the presence of pectinase, however, both fusion enzymes 272

released almost the same amount of fatty acids and exhibited a catalytic efficiency 273

3-fold higher than that of the native cutinase. This result is consistent with that of the 274

above binding experiment. The increased binding capability resulted in enhanced cutin 275

hydrolytic efficiency. 276

277

DISCUSSION 278

Previously, site-directed mutagenesis of the amino acid residues surrounding the 279

active site has been performed in order to obtain higher catalytic efficiency for cutinase 280

(3). In the present study, a fusion protein approach in which cutinase was fused with 281


.asm.org/

Dow

nloaded from

http://aem.asm.org/

14

CBM for improved affinity to cotton fiber was developed. By taking advantage of the 282

CBM’s specific binding to cellulose on the surface of cotton fiber where cutin also 283

exists, the concentration of fusion cutinase around cutin would be increased, which may 284

then result in enhanced enzyme catalytic efficiency due to proximity effect. Considering 285

the conditions of textile scouring process, the cutinase-CBM fusion protein has to meet 286

the following requirements: (1) no significantly decreased cutin hydrolyzing activity 287

when comparing with the native cutinase; (2) good thermal stability and alkali 288

resistance; (3) good binding affinity to cotton fiber; and (4) improved scouring effect for 289

cotton fiber. 290

In order to meet the above requirements, the choice of a suitable CBM is critical. 291

To date, characteristics of CBMs have been explored extensively (23, 45). They are 292

found mainly in carbohydrate degrading enzymes from fungi (e.g. Trichoderma reesei) 293

and bacteria (e.g. Cellulomonas fimi), including cellulase, xylanase, mannanase and a 294

number of non-hydrolytic proteins (33). Considering the source of the cutinase used in 295

the present study, naturally a CBM was first selected from the genome of T. fusca. 296

As identified by the CAZy ModO database (http://www.afmb.cnrs-mrs.fr/CAZY/), 297

the genome of T. fusca encodes a total of 17 hydrolytic enzymes that possess a CBM 298

(supplemental Table S3), with 6 of them experimentally characterized (underlined in 299

supplemental Table S3) (36). In addition, CBMs are divided into 59 different families 300

(http://www.afmb.cnrs-mrs.fr/CAZY/) and those from the second family were found to 301

be able to ease crystalline cellulose without significant fiber damage (10). Among the 302

six characterized CBMs from T. fusca, except for the CBM3 of Cel9A and the CBM4 of 303


.asm.org/

Dow

nloaded from

http://aem.asm.org/

15

Cel9B, all the others were identified to belong to the second family and thus were 304

candidates for fusing with cutinase. 305

As for the directionality of the fusion, the homology structural model of cutinase 306

showed that both the N- and C-termini of the enzyme were exposed to the solvent side 307

(11). Considering the presence of the N-terminal PelB signal sequence for 308

transmembrane localization, it appears that the CBM is better to be fused to the 309

C-terminus of cutinase. Another consideration is a possible linker sequence between the 310

cutinase and the CBM. For example, it has been reported that the linker between the 311

CBM and catalytic domain of a bacterial cellulase is composed entirely of the Pro-Thr 312

repetitive sequence (19). Such a linker in cellulases would possess certain flexibility and 313

avoid possible structural hindrance, which ensures the uniform movement of the two 314

domains on the fiber surface (19, 39, 44). Therefore, an appropriate linker between the 315

cutinase and the CBM is desired. 316

Putting together the above considerations, the CBM from T. fusca cellulase Cel6A, 317

which belongs to the second family of CBMs and has a 28-residue linker region, was 318

chosen to be fused to the C-terminus of cutinase. 319

Cutinase-CBMCel6A was well expressed and purified, and shown to be able to 320

hydrolyze not only cutin but also insoluble triglycerides (triolein) and soluble esters 321

(pNPB). In addition, it shares similar pH stability as cutinase and displayed optimal 322

temperature at 50°C and half-life of 53 h at 50 °C. When their binding and catalytic 323

efficiency towards cotton fiber were compared, cutinase-CBMCel6A did not appear to 324

have a significantly better performance than the native cutinase. However, in the 325


.asm.org/

Dow

nloaded from

http://aem.asm.org/

16

presence of pectinase, cutinase-CBMCel6A exhibited significant improvement in binding 326

and a dramatic 3-fold increase in catalytic efficiency. This sharp contrast is likely 327

because most of the cellulose on the surface of cotton fiber is not well exposed to the 328

solvent and is embedded in the epidermis full of pectins, proteins and other components, 329

thus limiting the binding of CBM to cotton fiber. When pectinase was added in the 330

reaction mixture, removal of pectin by this enzyme may lead to the exposure of 331

cellulose, resulting in increased adsorption of cutinase-CBMCel6A, which eventually led 332

to higher scouring efficiency of cotton fiber. 333

In addition to CBMCel6A, we also examined the possibility of using other CBMs 334

that have been experimentally characterized. The CBM of endoglucanase A (CenA) 335

from C. fimi, which also belongs to the second family of CBMs, was shown to have 336

high affinity to cellulose (13, 16, 49) and appears to be a suitable candidate. 337

Subsequently, CBMCenA was fused to the C-terminus of cutinase using the same linker 338

from T. fusca Cel6A. As expected, cutinase-CBMCenA displayed similar substrate 339

specificity and catalytic properties as the native cutinase. Interestingly, although the 340

CBMCenA is from a mesophilic bacterium, this fusion enzyme still retained decent 341

thermostability, which may be due to the presence of a disulfide bond in CBMCenA (16). 342

Notably, significant improvement in the binding and catalytic efficiency towards cotton 343

fiber was observed for cutinase-CBMCenA when compared to the native cutinase and 344

cutinase-CBMCel6A. In addition, similar to cutinase-CBMCel6A, strong synergistic effect 345

with pectinase was also observed with cutinase-CBMCenA. Thus, it appears that the 346

scouring effect of cutinase-CBMCenA is better than cutinase-CBMCel6A. 347


.asm.org/

Dow

nloaded from

http://aem.asm.org/

17

In conclusion, cutinase-CBM fusion proteins were successfully created by fusing a 348

CBM to the C-terminus of T. fusca cutinase. Compared with the native cutinase, both 349

fusion proteins, cutinase-CBMCel6A and cutinase-CBMCenA, share similar stability and 350

catalytic properties, but showed greatly enhanced binding and hydrolytic activity 351

towards cotton fiber. These improvements as well as the synergistic effect between the 352

fusion proteins and the pectinase suggests that they may have better application 353

potential in textile bioscouring. 354

355

ACKNOWLEDGMENT 356

This work was supported financially by the National High-tech Research and 357

Development Program of China (2009AA02Z204), the National Natural Science 358

Foundation of China (30970057), the National Outstanding Youth Foundation of China 359

(20625619), Research Program of State Key Laboratory of Food Science and 360

Technology (SKLF-MB-200802), the Key Program of National Natural Science 361

Foundation of China (20836003), Program of Innovation Team of Jiangnan University 362

(2008CXTD01) and the Self-determined Research Program of Jiangnan University (Yao 363

Zhang)364


.asm.org/

Dow

nloaded from

http://aem.asm.org/

18

REFERENCE 365

1. Agrawal*, P. B., V. A. Nierstrasz, G. H. Bouwhuis, and M. M. C. G. 366

Warmoeskerken. 2008. Cutinase and pectinase in cotton bioscouring: an 367

innovative and fast bioscouring process. Biocatalysis and Biotransformation 368

26:412-421. 369

2. Araujo, R., M. Casal, and A. Cavaco-Paulo. 2008. Application of enzymes 370

for textile fibres processing. Biocatal Biotransfor 26:332-349. 371

3. Araujo, R., C. Silva, A. O'Neill, N. Micaelo, G. Guebitz, C. M. Soares, M. 372

Casal, and A. Cavaco-Paulo. 2007. Tailoring cutinase activity towards 373

polyethylene terephthalate and polyamide 6,6 fibers. J Biotechnol 128:849-57. 374

4. Azevedo, H., D. Bishop, and A. Cavaco-Paulo. 2000. Effects of agitation 375

level on the adsorption, desorption, and activities on cotton fabrics of full 376

length and core domains of EGV (Humicola insolens) and CenA 377

(Cellulomonas fimi). Enzyme Microb Technol 27:325-329. 378

5. Black, G. W., J. E. Rixon, J. H. Clarke, G. P. Hazlewood, L. M. Ferreira, D. 379

N. Bolam, and H. J. Gilbert. 1997. Cellulose binding domains and linker 380

sequences potentiate the activity of hemicellulases against complex substrates. 381

J Biotechnol 57:59-69. 382

6. Blanco, J., J. J. Coque, J. Velasco, and J. F. Martin. 1997. Cloning, 383

expression in Streptomyces lividans and biochemical characterization of a 384

thermostable endo-beta-1,4-xylanase of Thermomonospora alba ULJB1 with 385

cellulose-binding ability. Appl Microbiol Biotechnol 48:208-17. 386


.asm.org/

Dow

nloaded from

http://aem.asm.org/

19

7. Bolam, D. N., A. Ciruela, S. McQueen-Mason, P. Simpson, M. P. 387

Williamson, J. E. Rixon, A. Boraston, G. P. Hazlewood, and H. J. Gilbert. 388

1998. Pseudomonas cellulose-binding domains mediate their effects by 389

increasing enzyme substrate proximity. Biochem J 331 ( Pt 3):775-81. 390

8. Carvalho, C. M., M. R. Aires-Barros, and J. M. Cabral. 1999. Cutinase: 391

from molecular level to bioprocess development. Biotechnol Bioeng 66:17-34. 392

9. Cavaco-Paulo, A. 1998. Processing textile fibers with enzymes: an overview. 393

ACS Symposium Series:180-189. 394

10. Cavaco-Paulo, A., J. Morgado, J. Andreaus, and D. Kilburn. 1999. 395

Interactions of cotton with CBD peptides. Enzyme and Microbial Technology 396

25:639-643. 397

11. Chen, S., X. Tong, R. W. Woodard, G. Du, J. Wu, and J. Chen. 2008. 398

Identification and characterization of bacterial cutinase. J Biol Chem 399

283:25854-62. 400

12. Ciolacu, D., J. Kovac, and V. Kokol. 2010. The effect of the 401

cellulose-binding domain from Clostridium cellulovorans on the 402

supramolecular structure of cellulose fibers. Carbohydr Res 345:621-630. 403

13. Damude, H. G., S. G. Withers, D. G. Kilburn, R. C. Miller, Jr., and R. A. 404

Warren. 1995. Site-directed mutation of the putative catalytic residues of 405

endoglucanase CenA from Cellulomonas fimi. Biochemistry 34:2220-4. 406

14. Degani, O., S. Gepstein, and C. G. Dosoretz. 2004. A new method for 407

measuring scouring efficiency of natural fibers based on the cellulose-binding 408


.asm.org/

Dow

nloaded from

http://aem.asm.org/

20

domain-beta-glucuronidase fused protein. J Biotechnol 107:265-73. 409

15. Degani, O., S. Gepstein, and C. G. Dosoretz. 2002. Potential use of cutinase 410

in enzymatic scouring of cotton fiber cuticle. Appl Biochem Biotechnol 411

102-103:277-89. 412

16. Din, N., I. J. Forsythe, L. D. Burtnick, N. R. Gilkes, R. C. Miller, Jr., R. A. 413

Warren, and D. G. Kilburn. 1994. The cellulose-binding domain of 414

endoglucanase A (CenA) from Cellulomonas fimi: evidence for the 415

involvement of tryptophan residues in binding. Mol Microbiol 11:747-55. 416

17. Fett, W. F., H. C. Gerard, R. A. Moreau, S. F. Osman, and L. E. Jones. 417

1992. Screening of Nonfilamentous Bacteria for Production of 418

Cutin-Degrading Enzymes. Appl Environ Microbiol 58:2123-2130. 419

18. Gilkes, N. R., B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. 420

Warren. 1991. Domains in microbial beta-1, 4-glycanases: sequence 421

conservation, function, and enzyme families. Microbiol Rev 55:303-15. 422

19. Gilkes, N. R., D. G. Kilburn, R. C. Miller, Jr., and R. A. Warren. 1989. 423

Structural and functional analysis of a bacterial cellulase by proteolysis. J Biol 424

Chem 264:17802-8. 425

20. Ha, J. S., Y. M. Lee, S. L. Choi, J. J. Song, C. S. Shin, J. H. Kim, and S. G. 426

Lee. 2008. Thermostable beta-glycosidase-CBD fusion protein for 427

biochemical analysis of cotton scouring efficiency. J Microbiol Biotechnol 428

18:443-8. 429

21. Hardin, I. R., Li, Y., Akin, D. 1998. Cotton wall structure and enzymatic 430


.asm.org/

Dow

nloaded from

http://aem.asm.org/

21

treatments. ACS Sysmposium Series 687:190-203. 431

22. Hartzell, M. M., Hsieh, Y.L. 1998. Enzymatic scouring to improve cotton 432

fabric wettability. J.Textile Res. 68:233-241. 433

23. Hashimoto, H. 2006. Recent structural studies of carbohydrate-binding 434

modules. Cell Mol Life Sci 63:2954-67. 435

24. Hilge, M., S. M. Gloor, W. Rypniewski, O. Sauer, T. D. Heightman, W. 436

Zimmermann, K. Winterhalter, and K. Piontek. 1998. High-resolution 437

native and complex structures of thermostable beta-mannanase from 438

Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5. 439

Structure 6:1433-44. 440

25. Irwin, D., E. D. Jung, and D. B. Wilson. 1994. Characterization and 441

sequence of a Thermomonospora fusca xylanase. Appl Environ Microbiol 442

60:763-70. 443

26. Irwin, D., D. H. Shin, S. Zhang, B. K. Barr, J. Sakon, P. A. Karplus, and D. 444

B. Wilson. 1998. Roles of the catalytic domain and two cellulose binding 445

domains of Thermomonospora fusca E4 in cellulose hydrolysis. J Bacteriol 446

180:1709-14. 447

27. Irwin, D. C., M. Cheng, B. Xiang, J. K. Rose, and D. B. Wilson. 2003. 448

Cloning, expression and characterization of a family-74 xyloglucanase from 449

Thermobifida fusca. Eur J Biochem 270:3083-91. 450

28. Irwin, D. C., S. Zhang, and D. B. Wilson. 2000. Cloning, expression and 451

characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca. 452


.asm.org/

Dow

nloaded from

http://aem.asm.org/

22

Eur J Biochem 267:4988-97. 453

29. Jung, E. D., G. Lao, D. Irwin, B. K. Barr, A. Benjamin, and D. B. Wilson. 454

1993. DNA sequences and expression in Streptomyces lividans of an 455

exoglucanase gene and an endoglucanase gene from Thermomonospora fusca. 456

Appl Environ Microbiol 59:3032-43. 457

30. Jung, H., D. B. Wilson, and L. P. Walker. 2003. Binding and reversibility of 458

Thermobifida fusca Cel5A, Cel6B, and Cel48A and their respective catalytic 459

domains to bacterial microcrystalline cellulose. Biotechnol Bioeng 84:151-9. 460

31. Krakhmalev, V. A., Paiziev,A.A. 2006. Spiral structures of cotton fiber. 461

Cellulose 13:45-52. 462

32. Lao, G., G. S. Ghangas, E. D. Jung, and D. B. Wilson. 1991. DNA 463

sequences of three beta-1,4-endoglucanase genes from Thermomonospora 464

fusca. J Bacteriol 173:3397-407. 465

33. Levy, I., and O. Shoseyov. 2002. Cellulose-binding domains: 466

biotechnological applications. Biotechnol Adv 20:191-213. 467

34. Li, Y., D. C. Irwin, and D. B. Wilson. 2007. Processivity, substrate binding, 468

and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl 469

Environ Microbiol 73:3165-72. 470

35. Linder, M., J. Winiecka-Krusnell, and E. Linder. 2002. Use of recombinant 471

cellulose-binding domains of Trichoderma reesei cellulase as a selective 472

immunocytochemical marker for cellulose in protozoa. Appl Environ 473

Microbiol 68:2503-8. 474


.asm.org/

Dow

nloaded from

http://aem.asm.org/

23

36. Lykidis, A., K. Mavromatis, N. Ivanova, I. Anderson, M. Land, G. 475

DiBartolo, M. Martinez, A. Lapidus, S. Lucas, A. Copeland, P. Richardson, 476

D. B. Wilson, and N. Kyrpides. 2007. Genome sequence and analysis of the 477

soil cellulolytic actinomycete Thermobifida fusca YX. J Bacteriol 478

189:2477-86. 479

37. Nini, L., L. Sarda, L. C. Comeau, E. Boitard, J. P. Dubes, and H. 480

Chahinian. 2001. Lipase-catalysed hydrolysis of short-chain substrates in 481

solution and in emulsion: a kinetic study. Biochim Biophys Acta 1534:34-44. 482

38. Posta, K., E. Beki, D. B. Wilson, J. Kukolya, and L. Hornok. 2004. Cloning, 483

characterization and phylogenetic relationships of cel5B, a new endoglucanase 484

encoding gene from Thermobifida fusca. J Basic Microbiol 44:383-99. 485

39. Quentin, M., M. Ebbelaar, J. Derksen, C. Mariani, and H. van Der Valk. 486

2002. Description of a cellulose-binding domain and a linker sequence from 487

Aspergillus fungi. Appl Microbiol Biotechnol 58:658-62. 488

40. Richins, R. D., A. Mulchandani, and W. Chen. 2000. Expression, 489

immobilization, and enzymatic characterization of cellulose-binding 490

domain-organophosphorus hydrolase fusion enzymes. Biotechnol Bioeng 491

69:591-6. 492

41. Sae-be, P., U. Sangwatanaroj, and H. Punnapayak. 2007. Analysis of the 493

products from enzymatic scouring of cotton. Biotechnol J 2:316-25. 494

42. Sakka, K., G. Takada, S. Karita, and K. Ohmiya. 1996. Identification and 495

characterization of cellulose-binding domains in xylanase A of Clostridium 496


.asm.org/

Dow

nloaded from

http://aem.asm.org/

24

stercorarium. Ann N Y Acad Sci 782:241-51. 497

43. Sawada, K., and M. Ueda. 2001. Enzyme processing of textiles in reverse 498

micellar solution. J Biotechnol 89:263-9. 499

44. Shen, H., M. Schmuck, I. Pilz, N. R. Gilkes, D. G. Kilburn, R. C. Miller, 500

Jr., and R. A. Warren. 1991. Deletion of the linker connecting the catalytic 501

and cellulose-binding domains of endoglucanase A (CenA) of Cellulomonas 502

fimi alters its conformation and catalytic activity. J Biol Chem 266:11335-40. 503

45. Shoseyov, O., Z. Shani, and I. Levy. 2006. Carbohydrate binding modules: 504

biochemical properties and novel applications. Microbiol Mol Biol Rev 505

70:283-95. 506

46. Tomme, P., A. Boraston, B. McLean, J. Kormos, A. L. Creagh, K. Sturch, 507

N. R. Gilkes, C. A. Haynes, R. A. Warren, and D. G. Kilburn. 1998. 508

Characterization and affinity applications of cellulose-binding domains. J 509

Chromatogr B Biomed Sci Appl 715:283-96. 510

47. Tomme, P., D. P. Driver, E. A. Amandoron, R. C. Miller, Jr., R. Antony, J. 511

Warren, and D. G. Kilburn. 1995. Comparison of a fungal (family I) and 512

bacterial (family II) cellulose-binding domain. J Bacteriol 177:4356-63. 513

48. Walton, T. J., and P. E. Kolattukudy. 1972. Determination of the structures 514

of cutin monomers by a novel depolymerization procedure and combined gas 515

chromatography and mass spectrometry. Biochemistry 11:1885-96. 516

49. Wong, W. K., B. Gerhard, Z. M. Guo, D. G. Kilburn, A. J. Warren, and R. 517

C. Miller, Jr. 1986. Characterization and structure of an endoglucanase gene 518


.asm.org/

Dow

nloaded from

http://aem.asm.org/

25

cenA of Cellulomonas fimi. Gene 44:315-24. 519

50. Zhang, S., G. Lao, and D. B. Wilson. 1995. Characterization of a 520

Thermomonospora fusca exocellulase. Biochemistry 34:3386-95. 521


.asm.org/

Dow

nloaded from

http://aem.asm.org/

26

Figure Legends 522

523

Figure 1. Construction of cutinase-CBM fusion proteins. (A) T. fusca cutinase was 524

joined with the CBM from T. fusca Cel6A using overlapping extension PCR. The 525

fusion of T. fusca cutinase with CBM from C. fimi CenA followed the similar strategy. 526

(B) Amino acid composition of the fusion proteins. 527

528

Figure 2. SDS-PAGE analysis of the expression and purification of cutinase-CBMs. 529

(A) cutinase-CBMCel6A. lane M, molecular mass markers; lane 1, culture supernatant 530

of cutinase-CBMCel6A; lane 2, purified cutinase-CBMCel6A. (B) cutinase-CBMCenA. 531

lane M, molecular mass markers; lane 1, culture supernatant of cutinase-CBMCenA; 532

lane 2, purified cutinase-CBMCenA. 533

534

Figure 3. Scouring effects of cutinase-CBMs and cutinase on cotton fiber. (A) 535

Adsorption on cotton fiber. Purified cutinase or cutinase-CBM fusion protein was 536

incubated in 20 mM Tris-HCl buffer (pH 8.0, 25 °C). without pectinase, with 537

pectinase. (B) Analysis of products released from cotton fiber. 50µM purified cutinase 538

or cutinase-CBM fusion protein was incubated in 20 mM Tris-HCl buffer (pH 8.0, 50 539

°C) within/without pectinase，and the amount of released fatty acids was measured by 540

titration with 0.02 N NaOH. ◆ cutinase-CBMCel6A alone, ◇ cutinase-CBMCel6A and 541

pectinase, ▲ cutinase-CBMCenA alone, △ cutinase-CBMCenA and pectinase, ■ cutinase 542

alone, □ cutinase and pectinase.543


.asm.org/

Dow

nloaded from

http://aem.asm.org/

27

544

545

Table 1. Primers used in the construction of the fusion genes 546

P1 : 5'-GGAATACCATATGTCCATGGCCAACCCCTACGAGCGCGG-3' (NcoI)

P2 : 5'-CGCGGCGATCGCCATGAACGGGCAGGTGGA-3'

P3 : 5'-TCCACCTGCCCGTTCATGGCGATCGCCGCG-3'

P4 : 5'-CATCTCGAGAGAATTCGGGCAGGTAAGGGTCGGAACAG-3' (EcoR1)

P2': 5'-GCGGCAGCCGGGAGCGGGAGGCGGCGTGGG-3'

P3': 5'-CCCACGCCGCCTCCCGCTCCCGGCTGCCGC-3'

P4': 5'-CATCTCGAGAGAATTCGGGGTGCCCGTGCAGGTGGTGC-3' (EcoR1)

Restriction enzyme sites are in bold and italicized. 15 base pairs of the overlapping regions are 547

underlined.548


.asm.org/

Dow

nloaded from

http://aem.asm.org/

28

549

Table 2. Monomeric products released from cutin hydrolysis by cutinase-CBMs and 550

cutinase. 551

Cutin hydrolysis by cutinase-CBM and cutinase was carried out in 25 mM potassium phosphate 552

buffer (pH 8.0) at 50 °C, for 18 h. Each value represents the mean of three independent 553

measurements, and the variation about the mean is below 5%. 554

555

Cutin hydrolysis products cutinase

hydrolysis

Area (%)

cutinase-CBMCel6A

hydrolysis

Area (%)

cutinase-CBMCenA

hydrolysis

Area (%)

Hexadecanoic acid 27.6 30.6 31.4

Octadecenoic acid 26.7 25.2 27.5

9-Octadecenoic acid 0.50 0.43 0.36

9,12-Octadecadienoic acid 1.08 1.02 1.01

16-Hydroxyhexadecanoic acid 0.54 0.54 0.58

18-Hydroxyoctadeca-9-enoic acid 1.01 0.87 0.91

18-Hydroxyoctadeca-9,12-dienoic acid 1.12 1.76 1.70

9,10,18-Trihydroxyoctadecanoic acid 0.47 0.47 0.51


.asm.org/

Dow

nloaded from

http://aem.asm.org/

29

556

557

558

Table 3. Kinetic parameters of cutinase-CBMs and cutinase 559

Kinetic parameters cutinase cutinase-CBMCel6A cutinase-CBMCenA

KmpNPB

(µM) 640 ± 40 620 ± 40 620 ± 30

Kcat(s-1

) 220 ± 10 200 ± 10 180 ± 10

Kcat/Km(s-1

) 0.34 0.32 0.29

560


.asm.org/

Dow

nloaded from

http://aem.asm.org/

30

561

cutinase-CBM /pET-20b

4.9kb

pelB

cutinase

NcoI

EcoR1His tagT7 terminator

f1 origin

Amp

ColE1 pBR322 origin

Linker

CBMCel6A

Cel6A

cutinase

P1

P2

cutinase

P1

P2

CD Linker CBMCel6A

P3

P4

Plasmid pET20b-Tfu_0883 Thermobifida fusca total DNA

cutinase

P1

Linker CBMCel6A

P4

PCR PCR

Overlapping PCR

pET20b(+) -NcoI/EcoRI

-Ligation

(A)

Linker CBMCel6Acutinase Linker CBMCel6Acutinasecutinase

(cutinase-CBMCel6A) ANPY…EVEEYRSTCP--------MAIA…PTPTPPP---------GSSG…NSNSVPTLTC

cutinase: 1~260aa Linker of CBMCel6A: 261-288aa CBMCel6A: 289~387aa

(cutinase-CBMCenA) ANPY…EVEEYRSTCP--------MAIA…PTPTPPP---------APGC…SLNGTTCTGT

cutinase: 1~260aa Linker of CBMCel6A: 261-288aa CBMCenA: 289~395aa

(B)

(cutinase-CBMCel6A) ANPY…EVEEYRSTCP--------MAIA…PTPTPPP---------GSSG…NSNSVPTLTC

cutinase: 1~260aa Linker of CBMCel6A: 261-288aa CBMCel6A: 289~387aa

(cutinase-CBMCenA) ANPY…EVEEYRSTCP--------MAIA…PTPTPPP---------APGC…SLNGTTCTGT

cutinase: 1~260aa Linker of CBMCel6A: 261-288aa CBMCenA: 289~395aa

(B)

Figure 1. Construction of cutinase-CBM fusion proteins.


.asm.org/

Dow

nloaded from

http://aem.asm.org/

31

562

563

564

Figure 2. SDS-PAGE analysis of the expression and purification of cutinase-CBMs.565


.asm.org/

Dow

nloaded from

http://aem.asm.org/

32

566

567

Figure 3. Scouring effects of cutinase-CBMs and cutinase on cotton fiber. 568

569

(A)

0

10

20

30

40

50

60 without pectinase

with pectinase

Bin

din

g r

atio

(%

)

cutinase cutinase-CBMCel6A

cutinase-CBMCenA

(B)

0 2 4 6 8 10 12 14 16 180

20

40

60

80

100

120

cutinase cutinase+pectinase

cutinase-CBMCel6A

cutinase-CBMCel6A

+pectinase

cutinase-CBMCenA

cutinase-CBMCenA

+pectinase

Fre

e f

atty

aci

ds

(µm

ol)

Time (h)


.asm.org/

Dow

nloaded from

http://aem.asm.org/

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2010, p. 7896 Vol. 76, No. 230099-2240/10/$12.00 doi:10.1128/AEM.02348-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

ERRATUM

Characterization of Thermobifida fusca Cutinase–Carbohydrate-Binding ModuleFusion Proteins and Their Potential Application in Bioscouring

Yao Zhang,1,2 Sheng Chen,1,2 Meng Xu,1,2 Artur Cavaco-Paulo,3 Jing Wu,1,2* and Jian Chen1,2*State Key Laboratory of Food Science and Technology1 and School of Biotechnology and Key Laboratory of

Industrial Biotechnology,2 Ministry of Education, Jiangnan University, Wuxi 214122, China, andDepartment of Textile Engineering, University of Minho, 4800 Guimaraes, Portugal3

Volume 76, no. 20, pages 6870–6876, 2010. Page 6870: The article byline should read as shown above.

7896

characterization of thermobifida fusca cutinase-cbm...

Documents