Enhancing the Thermostability of Feruloyl Esterase EstF27 by Directed

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Enhancing the thermostability of feruloyl esterase EstF27 by directed evolution and the underlying structural basis Lichuang Cao, Ran Chen, Wei Xie, and Yuhuan Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03424 • Publication Date (Web): 02 Sep 2015 Downloaded from http://pubs.acs.org on September 6, 2015

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Journal of Agricultural and Food Chemistry

Enhancing the thermostability of feruloyl esterase EstF27 by directed evolution and the underlying structural basis

Li-chuang Cao§, 1, 3, Ran Chen §, 1, 2, Wei Xie*, 1, 2, Yu-huan, Liu*, 1, 3

1

School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China

2

State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen

University, Guangzhou 510275, P. R. China 3

South China Sea Bio-Resource Exploitation and Utilization Collaborative

Innovation Center, Sun Yat-sen University, Guangzhou 510275, P. R. China

§

These authors contributed equally to this work.

*Correspondence should be addressed to: Yu-huan Liu: [email protected] And Wei Xie: [email protected]

Tel: 86-20-84113712 Fax: 86-20-84036215

1

ACS Paragon Plus Environment


1

Abstract

2

To improve the thermostability of EstF27, two rounds of random mutagenesis were

3

performed. A thermostable mutant M6 with six amino acids substitutions was

4

obtained. The half-life of M6 at 55 °C is 1, 680 h while that of EstF27 is 0.5 h. The

5

Kcat/Km value of M6 is 1.9-fold higher than that of EstF27. The ferulic acid released

6

from destarched wheat bran by EstF27 and M6 at their respective optimal temperature

7

was 223.2 ± 6.8 and 464.8 ± 11.9 µΜ, respectively. To further understand the

8

structural basis of the enhanced thermostability, the crystal structure of M6 is

9

determined at 2.0 Å. Structural analysis shows that a new disulfide bond and

10

hydrophobic interactions formed by the mutations may play an important role in

11

stabilizing the protein. This study not only provides us with a robust catalyst, but also

12

enriches our knowledge about the structure-function relationship of feruloyl esterase.

13 14

Keywords: Feruloyl esterase, Directed evolution, Thermostability, Wheat bran,

15

Crystal structure, X-ray crystallography

16 17 18 19 20 21 22 23 24 2


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Introduction Ferulic acid (FA), also named 4-hydroxy-3-methoxycinnamic acid, has attracted

27

increasing interest in recent years as an effective natural antioxidant with broad

28

potential applications in the food and pharmaceutical industries.1-3 It is abundant

29

present in the cell walls of cereals, such as wheat bran (0.5%, w/w),4 sugar beet pulp

30

(0.8%, w/w)5 and maize bran (3.0%, w/w).6 The release of FA through enzymatic

31

routine needs the action of feruloyl esterases in synergy with xylanases. Xylanases

32

cleave the xylan main chain to expose feluloyl ester bonds, and feruloyl esterases

33

release ferulic acid from feruloylated oligosaccharides, hemicellulose or pectin.6, 7

34

Feruloyl esterase (EC 3.1.1.73) is also known as ferulic acid esterase (FAE) and

35

belongs to a subclass of carboxylesterase (EC 3.1.1).7 The enzyme not only can

36

hydrolyze the ester bond of ferulic acid (FA) or diferulic acid (diFA) esterified to

37

arabinoxylans (AXs) and certain pectins present in plant cell, but also synthesize

38

novel bioactive components through a transesterification reaction.7-9 FAEs can be

39

divided into four types (type A through D), based on their primary sequence

40

homology and substrate specificities towards the methyl esters of caffeic (CA), ferulic

41

(FA), sinapic (SA) and p-coumaric acid (p-CA).10 Currently, FAEs have wide

42

applications in food, agricultural and pharmaceutical industries. In addition to

43

releasing FA from agro-industrial waste materials,11-13 they can also be used to disrupt

44

and loosen the cell wall structure in pulp and paper industry,14, 15 improve the sugar

45

yield of lignocellulosic materials,16, 17 increase the digestibility and the calorific value

46

of complex plant cell walls in ruminants,18, 19 and expand the application potential of

47

hydroxycinnamic acids (ferulic, p-cumatic, caffeic, sinapinic) in health and

48

pharmaceutical industries via esterification.20-22

49

Excellent thermostability is an attractive property of catalysts that is of interest 3



50

because it decreases the enzyme load, allows long-term storage, increases the

51

operational flexibility and thus reduces costs.23 An effective method to enhance the

52

thermostability of proteins is directed evolution, which mimics the natural evolution

53

of enzymes in a laboratory. This approach has been successfully applied to a broad

54

range of enzymes including esterase,24 β-Lactamase,25 phytase 26 and xylanase.27

55

However, there has been only one report on improving the thermostability of FAE

56

using directed evolution up to date, which identified twelve amino acid substitutions

57

that are beneficial to the thermostability of the FAE from A. niger CIB 423.1 by

58

screening a random mutagenesis library.28 Because different biochemical reactions

59

require FAEs with respective properties, and the employment and development of

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FAEs in these reactions ultimately depends on the thorough understanding of the

61

fundamentals of their structure-function relationship,8, 29, 30 more studies on this aspect

62

are still needed.

63

In previous work, a novel feruloyl esterase EstF27 was isolated from a

64

metagenomic library of Turpan Depression and characterized.31. When mixed with

65

xylanase, it released FA efficiently from destarched wheat bran (DSWB). The main

66

drawback of EstF27 is the poor thermostability at temperatures higher than 50 °C. To

67

improve its thermostability, two rounds of random mutagenesis were performed in

68

this study. The enzymatic properties of the thermostable mutant M6 were studied and

69

compared to that of the wild type (WT). Their synergistic effects with xylanase on

70

releasing ferulic acid from DSWB were also investigated. To further elucidate the

71

structural basis of the thermostability enhancement, we tried to solve the structures of

72

EstF27 protein and M6 using X-ray crystallography. The crystal of the WT was

73

obtained from a condition containing PEG 8000. The unit cell was relatively large and

74

there was likely to be ten protein subunits present in the asymmetric unit.32 Molecular 4


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replacement using various structures available was not successful. Consequently, we

76

solved the structure of M6 using experimental phasing with CsCl as the anomalous

77

scatterer. The aim of this study was to improve the thermostability of EstF27 by

78

directed evolution and also provide useful information about the structure-function

79

relationship.

80 81

Materials and Methods

82

Materials

83

E. coli DH5α and pUC19 (TaKaRa, Dalian, China) were used for the construction

84

of the random mutagenesis libraries. The E. coli strain BL21 (DE3) and the pET-28a

85

(+) plasmid (Novagen, Madison, WI, USA) was used for protein expression.

86

Restriction endonucleases, DNA polymerase and T4 DNA ligase were purchased from

87

Thermo Fisher Scientific (Hudson, NH, USA). Ferulic acid (FA) was purchased from

88

Sigma-Aldrich (St. Louis, MO). 4-Nitro-phenyl ferulate (pNPF) procured from the

89

Slovak Academy of Sciences (Bratislava, Slovakia). All other chemicals and reagents

90

were of analytical grade and purchased from commercial sources, unless indicated

91

otherwise.

92 93 94

Construction and screening of random mutagenesis library The plasmid pUC19-estF27 (GenBank accession number: HQ241478) was used as

95

the template for the first round of mutagenesis and gave rise to a stable mutant with

96

four mutations (named M4, T27I/S84L/V161I/G243C). Then the plasmid pUC19-M4

97

was further used as the template for the second round of mutagenesis. The random

98

mutagenesis was performed with GeneMorph II Random Mutagenesis Kit (Stratagene,

99

La Jolla, CA, USA) following the manufacture’s protocol with the primers 5’5



100

TATAACAGCTATGACCATGATTACGCCAAGCTTATG-3’ (forward) and

101

5’-GAATT CGAGCTCGGTACCCGGGGATCC-3’ (reverse). The error-prone PCR

102

(epPCR) product was recovered and digested with BamH I and Hind III, and then

103

ligated into pUC19, which had been previously digested with the same restriction

104

enzymes. The ligation product was transformed into E. coli DH5α via electroporation

105

and the transformants were cultured on LB-agar plates containing 100 µg/mL

106

ampicillin, 0.1% (v/v) tributyrin, 0.3% (w/v) polyvinyl alcohol, 80 µg/mL rhodamine

107

B and 0.1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 37 °C overnight.

108

Single colonies that formed a transparent zone were picked, transferred on duplicate

109

LB-agar plates containing 100 µg/mL ampicillin and 0.02 mM IPTG, and were

110

cultivated at 37 °C for 48 h. One plate was incubated for 50 min at 60 °C for the first

111

round of mutagenesis or 25 min at 85 °C for the second round of mutagenesis. After

112

cooling to room temperature, about 5 mL mixture containing 0.3 mg/mL 1-naphthyl

113

acetate and 1.3 mg/mL Fast Blue B Salt was added onto the plate. Positive colonies

114

were confirmed by the brown halos formed around the colonies. The plasmid of the

115

transformants expressing the mutants with improved thermostability was subsequently

116

extracted for sequence analysis.

117 118 119

Site-directed mutagenesis In vitro site-directed mutagenesis was performed with the TaKaRa MutanBEST Kit

120

(TaKaRa, Dalian, China) following the instructions of the manufacturer by using the

121

plasmid pET-28a (+) as the template. The primers used were listed in SI Table S1.

122

The correctness of the mutants was confirmed by sequencing.

123 124

Overexpression and purification of the target proteins 6


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The full-length EstF27 gene was PCR-amplified using the primers

126

5’-GATAGGGCCATATGACCCCCGAATTGCGCGCCAA-3’ and

127

5’-TTGCACTTCTCGAGTCACCGCGTACCCTGCGCAC-3’. After double digestion

128

by the XhoI and NdeI restriction enzymes, the digested PCR product was ligated into a

129

modified pET28a (+) vector in which the thrombin site in this vector (recognition

130

peptide LVPRGS) was replaced by a Tobacco etch virus (TEV) protease cleavage site

131

(recognition peptide ENLYFQG). The plasmids encoding the target proteins contain a

132

6×histidine tag at the N-terminus, and were transformed into E. coli BL21 (DE3) for

133

overexpression.

134

The cells carrying pET28a/EstF27 plasmids were grown at 37 °C and 190 rpm

135

overnight in LB medium containing 30 µg/mL kanamycin. 500 mL fresh culture

136

medium was then inoculated with 5 mL overnight culture. When absorbance at 600

137

nm (OD600) reached 0.6-0.8, 1 mM IPTG was added to the culture and the cells were

138

subsequently induced overnight at 37 °C. Then the cells were harvested by

139

centrifugation at 5, 000g for 15 min and resuspended in pre-chilled

140

nickel-nitrilotriacetic acid (Ni-NTA) buffer A containing 40 mM Tris-HCl (pH 8.0),

141

250 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF) and

142

1 mM β-mercaptoethanol. After sonication, the cell lysate was cleared by

143

centrifugation at 14, 000g for 1 h at 4 °C. The resulting supernatant was mixed with

144

Ni-NTA (Qiagen) resin at 4 °C for 1 h. The bound protein was washed with 10

145

column volumes of buffer A. The target protein was then eluted with Ni-NTA buffer B

146

containing 40 mM Tris-HCl (pH 8.0), 250 mM NaCl, 250 mM imidazole, 1 mM

147

PMSF and 1 mM β-mercaptoethanol. The protein was pooled, concentrated and

148

loaded onto a Superdex 200 column (GE healthcare) and eluted with a buffer

149

containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT. The eluted 7



150

protein displayed a symmetric peak on the Superdex 200 column and was

151

concentrated to 15 mg/mL before being flash frozen and stored at -80 °C for further

152

crystallization experiments.

153

The recombinant proteins used for activity analysis were purified according to the

154

same method without the addition of PMSF in Ni-NTA buffers. The enzymes in buffer

155

B were further dialyzed three times in phosphate buffer (100 mM, pH 7.0), and then

156

stored at 4 °C for further study.

157

The molecular mass of the recombinant protein was determined by using sodium

158

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with suitable size of

159

protein markers (Thermo Fisher Scientific, Waltham, MA, USA) as standards. The

160

protein concentration was determined by using the CoomassiePlusTM (Bradford)

161

Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the product

162

manual.

163 164

Determination of thermostability and kinetic parameters

165

The optimal pHs and optimal temperatures of EstF27 and the mutants were

166

determined by using pNPF as substrate. The concentration of pNP released was

167

determined by measuring the absorbance of the solution at 405 nm. The reaction

168

mixture contained 10 µL diluted enzyme solution and 490 µL substrate solution. The

169

substrate solution was prepared according to Mastihuba et al.33 The pH buffers

170

included 100 mM citric acid-sodium citrate buffer (pH 5.0-6.0), phosphate buffer (pH

171

6.0-8.0), Tris-HCl buffer (pH 8.0-9.0) and glycine-NaOH buffer (pH 9.0-10.0). The

172

optimal temperature was determined by measuring the initial reaction rates in the

173

temperature range of 30 °C-70 °C in phosphate buffer (100 mM, pH 7.0).

174

Two parameters were measured to evaluate the thermostability of EstF27 and the 8


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mutants. One is the half-life (t1/2), which is defined as the incubation time needed to

176

inactivate 50% of the initial enzyme activity. The purified enzymes (0.1 mg/mL) were

177

incubated at 55, 65 or 70 °C in 100 mM phosphate buffer (pH 7.0). Samples were

178

collected at various time intervals, cooled in ice and assayed at 40 °C using pNPF as

179

substrate. The other one is the average size of the protein particles (Z-average),

180

measured by Dynamic Light Scattering. The measurements were carried out with a

181

photogoniometer as the plate reader (Wyatt Technology, Goleta, CA, USA).34 The WT

182

protein and different mutants (1 mg/mL, 100 mM phosphate buffer, pH 7.0) were

183

centrifuged at 14, 000g for 10 min, and the supernatant was transferred to the

184

384-well plates. Mineral oil was added on top to prevent evaporation during the

185

heating process. The plate was subjected to a 2, 000g centrifugation for 2 min to

186

remove air bubbles before readout in the temperature-controlled DynaPro plate reader.

187

The temperature range was set to 25 °C to 75 °C, and the temperature-rising rate was

188

0.35 °C/min. Each sample was run in triplicates and each well was measured 5 times,

189

with 5 s acquisition time.

190

The storage stability of EstF27 and the mutant M6 was determined at 25 °C in 100

191

mM phosphate buffer (pH 7.0). After different storage times (0-150 days), the residual

192

activities were tested. The initial activity of the enzymes was defined as 100%,

193

respectively.

194

The kinetic parameters (Kcat and Km) of EstF27 and the mutant M6 were determined

195

by assaying the activity in 100 mM phosphate buffer (pH 7.0) at 40 and 65 °C with

196

seven different concentrations (0.5-5.0× Km) of pNPF as substrate. The kinetic data

197

were fitted to Lineweaver-Burk curve using the SigmaPlot software (Systat Software,

198

Chicago, IL, USA).

199 9



200 201

Release of FA from DSWB The wild type EstF27 and the thermostable mutant M6 were each applied to

202

degrade destarched wheat bran (DSWB) synergistically with xylanase from

203

Thermomyces lanuginosus (Sigma-Aldrich, St. Louis, MO). The reactions were

204

performed at 40 and 65 °C in 1 mL phosphate buffer (100 mM, pH 7.0) containing 50

205

mg DSWB. The enzyme loads were 10 U/mL for xylanase and 0.5 mg/mL for EstF27

206

and M6, respectively. Samples were collected at different time intervals, boiled for 5

207

min and filtered. The concentration of FA released was determined by

208

High-Performance Liquid Chromatography (HPLC). A C18 reverse-phase column

209

(8.0×300 mm, 5 µm, ODS, Japan) was used for the HPLC analysis. A mixture of

210

methanol: HPLC-grade water containing 1% (w/v) acetic acid (30: 70, v/v) was used

211

as the mobile phase at a flow rate of 1.0 mL/min at 30 °C. The injection volume was

212

10 µL and the absorbance at 280 nm and 320 nm was recorded. The wheat bran was

213

destarched based on the method described by Topakas et al.35 The total FA content in

214

the DSWB was determined according to the method of Garcia et al.36

215 216 217

Crystallization and data collection The mutant M6 was crystallized using the sitting-drop vapor-diffusion method in

218

96-well plates. The commercial screens Index, Crystal Screen and a homemade

219

PEG/ammonium sulfate-based screen, were used for initial crystallization screening.

220

Crystals of M6 grew to optimal sizes in 5-7 d at 25 °C in 1.2 M ammonium sulfate,

221

100 mM HEPES (pH 7.5). A single crystal was flash-frozen in liquid nitrogen using

222

20% glycerol (v/v) plus the crystallization condition as a cryoprotectant.

223

A native X-ray diffraction dataset (a total of 214 frames) were collected at 100 K

224

using an in-house Oxford Diffraction Xcalibur Nova diffractometer operating at 50 10


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kV and 0.8 mA. The rotation was 0.75° per frame and the crystal-to-detector distance

226

was 60 mm. The exposure time was 30 s, and the data were recorded with a 165 mm

227

Onyx CCD detector. In order to determine the phase, derivative crystals were obtained

228

by soaking the native crystals in the above cryoprotectant with 1 M CsCl for 15 min.

229

The soaked crystals had no apparent decay and a dataset of 180 frames was collected

230

afterward, with a rotation of 1° per frame. The crystal-to-detector distance was 65 mm

231

and the exposure time was 90 s.

232 233 234 235

Accession Numbers The atomic coordinates and structure factors have been deposited in the Protein Data Bank with the accession code 4ZRS.

236 237

Results and Discussion

238

Screening for mutants with improved thermostability

239

Two epPCR libraries were successfully constructed for screening mutants with

240

improved thermostability, which contained about 30, 000 and 25, 000 clones,

241

respectively. Twenty clones of each library were randomly picked and sequenced to

242

estimate the diversity of the library. Results showed that the error rates of the libraries

243

were 0.95 and 1.2 nucleotide changes/kb, respectively. About 45% of the clones were

244

identified to be active by the transparent zone formed around the colonies. Then they

245

were transformed to duplicate LB-agar plates for further screening the mutants with

246

enhanced thermostability.

247

To avoid significant changes of the protein expression on the transformed plates, a

248

low induction concentration of IPTG (0.02 mM) was applied (SI Figure S1). After a

249

heat treatment for 50 min at 60 °C, the WT completely lost activity (SI Figure S2A). 11



250

Four positive clones were identified out of nearly 13, 500 clones after screening the

251

first library. Sequence analysis identified that the mutations were T27I, S84L, G243C

252

and V161I/G243C, respectively. The individual contribution of each mutation to the

253

thermostabilization was subsequently determined (Table 1). The combination of four

254

such mutations resulted in the mutant M4 with a half-life of 4 h at 65 °C. To further

255

improve the thermostability, a second random library was constructed on the basis of

256

M4. The screening was performed after the heat treatment for 25 min at 85 °C (SI

257

Figure S2B). Two more mutants, H195L and A259V were identified. Combination of

258

all the beneficial mutations gave rise to the mutant M6.

259 260 261

Enzymatic characterization The optimal temperature (Topt) of EstF27 is 40 °C (Figure 1A). The mutations

262

increase the optimal temperatures to 60 °C (M4) and 65 °C (M6), which are 20 °C and

263

25 °C higher than that of the WT, respectively (Figure 1A). Meanwhile, the optimal

264

pHs of the recombinant protein shift 0.5~1.0 unit towards alkaline direction by the

265

mutations (Figure 1B). The half-life of EstF27 at 55 °C is 0.5 h (Table 1), different

266

from that in the previous report, in which EstF27 remained only 25% activity after

267

incubation for 10 min at 55 °C.31 The discrepancy may be due to the effects of the

268

fusion tags present in the different expression vectors. Four mutations (T27I, S84L,

269

V161I and G243C) were identified after the first round of screening, resulting in

270

3~30-fold improvement of the half-life (Table 1). Combination of the four

271

thermostabilizing mutations results in the mutant M4 with half-life of 70 h at 55 °C,

272

which is about 140-fold improvement over that of the WT. The combination of

273

another two mutations (H195L and A259V) obtained from the second random library

274

further extends the half-life from 70 h (M4) to 1, 680 h (M6). 12


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Dynamic light scattering (DLS) is a useful instrument at monitoring the presence of

276

aggregated protein as has been used to monitor change in dimension of the protein

277

during denaturation and renaturation.37, 38 The effects of temperature on the protein

278

particle sizes measured by DLS are shown in Figure 2. The temperatures that

279

significantly changed the protein particle size of M4 and M6 are 63 °C and 68 °C,

280

which are 7 °C and 12 °C higher than that of the WT (56 °C), respectively. These

281

critical temperatures at which the protein particle sizes change dramatically are an

282

indication of formation of aggregates in solution, which may lead to further

283

denaturation.39 The increase in these temperatures suggests improvement in enzymatic

284

thermostability, consistent with the trend observed by the half-life measurements.

285

The storage stability of EstF27 and the mutant M6 was also determined. After

286

stored at 25 °C for 150 days, the activity of M6 was almost constant (100.5 ± 2.1%),

287

whereas that of the WT was only 21.2 ± 2.7% (Table 2). This result again showed the

288

better thermostability of mutant M6 than WT.

289

The measured kinetic parameters of EstF27 and the mutant M6 are listed in Table 3.

290

The catalytic efficiency of the protein was increased by the mutations. The Kcat/Km

291

value of M6 at its Topt of 65 °C is about 1.9-fold higher that of WT at its Topt of 40 °C.

292

Previous studies on directed evolution of enzymes have suggested that increased

293

thermostability of enzymes may come at the cost of their activities at low

294

temperature.40, 41 However, the mutations identified in this work significantly

295

enhanced the thermostability of EstF27 while maintaining its activity at 40 °C. A

296

reasonable explanation is that only the clones that showed transparent zones at 37 °C

297

were picked for further screening and the positive clones were identified at room

298

temperature after heat treatment. Thus both properties of thermostability and activity

299

at low temperature were constrained during the directed evolution process.24 13



300 301

Releasing FA from DSWB

302

Wheat bran is one of the major by-products of the milling industries and a good

303

source of FA.4, 42 FAEs usually work synergistically with xylanases to degrade such

304

natural substrates. To investigate the effects of enhanced thermostability on the

305

hydrolysis of destarched wheat bran (DSWB), such reactions were performed at both

306

40 °C and 65 °C. The total FA present in DSWB was determined to be 0.49% (w/w).

307

No FA was detected when only xylanase was added (Figure 3, SI Figure S4). During a

308

hydrolysis of 10 h at 40 °C, the FA released by EstF27 and M6 was 223.2 ± 6.8 and

309

273.3 ± 8.1 µΜ, representing 17.7% and 21.6% of the total FA. At 65 °C, the FA

310

released by M6 in 10 h was 464.8 ± 11.9 µΜ, representing 36.8% of the total FA.

311

Whereas EstF27 appeared to have completely lost its activity quickly as little FA was

312

detected (Figure 3). M6 released 2-fold more ferulic acid at its optimal temperature

313

(Topt) of 65 °C than EstF27 at its Topt of 40 °C. These results show the advantage of

314

the thermostable mutant M6 over EstF27 in releasing ferulic acid at high temperature.

315 316 317

Structure of the mutant M6 and comparison to other structural homologs To further understand the structural basis of the enhanced thermostability, we seek

318

to solve the crystal structure of the thermostable mutant M6. EstF27 belongs to a

319

distant family of carboxylesterase and its closest orthologs in sequence is a

320

phenmedipham hydrolase from Altererythrobacter atlanticus (AKH43926), which

321

shares 72% sequence identity with EstF27. The molecular replacement with several

322

homologous structures as the search probes using the native data did not yield a

323

plausible solution. In order to solve the structure, experimental phasing was pursued

324

using CsCl as the anomalous scatterer. Cesium has a f” of 7.9 e- at 1.54 Å (the copper 14


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325

Kα wavelength), which is higher than that of iodine (6.8 e-). The crystals did not show

326

apparent morphology changes after 15 min-CsCl soaking and a complete SAD dataset

327

was collected at this wavelength to a resolution 2.4 Å at the home X-ray source.

328

Surprisingly, cell contents analyses indicated that the asymmetric unit contains only

329

one molecule, in contrast to the original two in the native dataset. Therefore, the

330

quick-soaking procedure still induced significant changes in the unit cells.

331

Experimental phasing by Phaser generated a high-quality map, in which seven cesium

332

sites were found. Of these, one cesium ion is bound to the dimer interface and most

333

likely disrupts the association of the two monomers (data not shown). The Cs-bound

334

derivative structure was built using the autobuild option of Phenix followed by

335

manual rebuilding. The structure of the mutant M6 was subsequently solved at a

336

resolution of 2.0 Å using molecular replacement with the SAD solution as the search

337

probe.

338

The crystals of the mutant M6 belong to an orthorhombic system with the space

339

group P21, and each asymmetric unit contains two monomers with 54.6% solvent

340

content. Both chains are completely intact from G-1 to R290 with no internal

341

disorders and of excellent electron density (SI Table S2). The spherical protein

342

displays the α/β hydrolase fold, featuring a central eight-stranded mixed β-sheet

343

sandwiched by five helices on each side (Figure 4A). Among these, only the β2 strand

344

is antiparallel to the other strands while the remainder strands are all parallel to each

345

other.

346

A DALI search shows that the closest structural homologs for this protein are a

347

putative carboxylesterase from Lactobacillus plantarum (PDB code 3BJR) and a

348

cloned carboxylesterase EstE1from a metagenomic library (PDB code 2C7B).43 The

349

basic α/β hydrolase fold is conserved as indicated by structure overlay. The r.m.s. 15



350

deviation values are 2.6 Å over 223 Cα atoms and 3.2 Å over 248 Cα atoms between

351

the M6 structure and the above two structures respectively (Figure 4B). While the

352

central 8-stranded sheet structures align well, noticeable local structural differences

353

are also observed. For example, as shown in Figure S5, when compared to EstE1, the

354

N-terminus of EstF27 resolved more residues and forms two helices instead of a

355

single helix; in addition, regions between β6 and β7 display substantial

356

rearrangements: the β6-α6 loop shorter and forms only one helix (α6), as compared to

357

the two helices in EstE1, and the Cα trace of the α6-α7 helices in EstF27 has also

358

significant shifts. These structural changes may be related to the substrate specificity

359

that each enzyme catalyzes.

360

Additionally, most of the essential residues are conserved as demonstrated by the

361

multiple sequence alignment (SI Figure S6). A catalytic triad formed by Ser, His, and

362

Asp/Glu residues has been reported to be critical to the activity of α/β hydrolases.44, 45

363

At the active site, the catalytic triad Ser151-His263-Asp230 is well conserved,

364

suggesting that these proteins share a common catalytic mechanism. The OD1 atom of

365

the carboxylate group in Asp 230 forms a hydrogen bond with the ND1 atom of the

366

His263 imidazole ring and constitutes part of the proton-shuffling network.

367

Interestingly, we found that extra density was connected to the side chain of S151.

368

After careful inspection, a sulfoamyl moiety was built into the density successfully (SI

369

Figure S7). The most possible scenario was that S151 acts as a nucleophile and

370

attacks PMSF (intended for protease inhibition) during the course of protein

371

purification, leading to the covalent linkage. Additionally, the four-residue motif

372

73HGGG76, a signature of the hormone-sensitive esterase/lipase (HSL) family,46, 47 is

373

located in a loop, flanking the other side of the S151 nucleophile.

374

EstF27 exists as a dimer in solution as indicated by size-exclusion chromatography 16


Page 16 of 46

Page 17 of 46


375

studies, and this observation is consistent with the fact of two monomers being in the

376

asymmetric unit. However, we have encountered problems in assigning the dimer

377

interface. In the current structure, the two subunits dimerizes through a C-terminal

378

helix α8 (dimer type I). Alternatively, could provide intermolecular contacts and

379

results in another type of dimer via interactions using α2 and α6 (dimer type II) (SI

380

Figure S8). Interestingly, both types of dimers are reasonable in terms of dimer

381

interface as well as free energy according to the PISA server: the buried surface is 2,

382

570 and 2, 380 Å2, and the ∆Gint is -16.8 and -19.7 kcal/mol respectively. To find out

383

the physiological dimer interface, we examined the structure and discovered that

384

C243, a residue at the dimer interface, forms a disulfide bond with its counterpart

385

from the other subunit in the electron density map (Figure 5A). The S-S bond

386

formation is supported by the extra band at ~70 kDa on a SDS-PAGE gel under

387

non-reducing environment. Upon reduction, this band disappears and the

388

monomeric-form band appears (Figure 5B), indicating the interface as shown in the

389

crystal structure is the genuine biological dimerization mode. We also tested the

390

mutant C243G, which reverts the cysteine residue back to the original glycine and the

391

extra band was not observed, further strengthening our argument (Figure 5B).

392

Coincidentally, C243 is also one of the resulting mutants obtained from our

393

first-round mutagenesis screen and the disulfide bridge enhances the stability of the

394

dimer.

395

Another interesting discovery is that K164-G172, a fragment connecting α5 and β6,

396

adopts different path in each subunit (SI Figure S9). This region is at least 16 Å from

397

the nucleophile S151, and the cause of different conformation and its biological

398

significance is unclear.

399

17



400

Rationalization of the mutations

401

We generated a series of mutants through random mutagenesis that greatly promote

402

the thermostability of EstF27. We subsequently determined the crystal structure of the

403

most stable mutant M6 at high resolution and the crystal structure reveals that G243C,

404

a mutant at the dimer interface, forms a disulfide bridge with C243’ and presumably

405

stabilizes the dimer through the S-S covalent bond. Examination of the other mutant

406

sites on the structure suggests that most of the mutations tend to increase the

407

hydrophobicity of the protein and several of them besides C243, are located at the

408

dimer interface. For an example, the mutations T27I, H195L, S84L all change polar

409

residues to hydrophobic residues while the mutations V161I and A259V tend to

410

increase the hydrophobicity of EstF27. Hydrophobic interactions may play a very

411

important role in forming the inner core of the enzyme and these mutations turn out to

412

boost the thermostability. Specifically, T27I may form hydrophobic interactions with

413

L5 (the closest distance between the two residues is 4.94 Å), L9 (4.0 Å) and L12 (4.3

414

Å) of α1, which is close to the dimer interface (Figure 6A). Similarly A259V may

415

promote the stability of the dimer interface by increasing the hydrophobic interactions

416

α10’ from the other monomer (Figure 6B). Byun et al.43 discovered that the

417

thermostability of EstE1, an EstF27 homolog, correlates with its ability to form a

418

dimer. In contrast, disruption of the hydrophobic interactions between the two

419

monomers destabilizes its stability, which supports our current data. Furthermore,

420

after mutation, L195 is in a hydrophobic environment formed by Y199, F77, W206

421

and I183. The closest distances between the δC atoms of L195 and the aromatic rings

422

of the former three residues of are 3.7-3.9 Å. Similarly, L84 is in close range from

423

F89 and Y90. I161 is also in a hydrophobic pocket, and within 5.0 Å, it is surrounded

424

by residues L222, L215, I220, I157, I179 and I174 (SI Figure S10). These findings

425

provide an explanation for contribution of the P195L, S84L, and V161I mutations. 18


Page 18 of 46

Page 19 of 46


426

One must realize that the crystal structure could not provide a perfect explanation for

427

the role of every mutant, because the crystal structure only reflects a static state of the

428

mutant M6 in the crystal lattice. The thermostability evaluation, performed at a

429

temperature as high as 70 oC (Table 1), is a combined result of both thermodynamic as

430

well as dynamic properties of each mutant. Ideally, the crystal structure of each

431

mutant and detailed solution studies using NMR etc., will greatly help to elucidate of

432

the structural basis of the thermostability and enzymatic behavior behind.

433

In conclusion, the thermostability of EstF27 was significantly enhanced by the

434

substitutions of six amino acids. The mutations also enhanced the storage stability and

435

improved the catalysis efficiency. The thermostable mutant M6 released 2-fold more

436

ferulic acid at its optimal temperature (Topt) of 65 °C than the wild type at its Topt of

437

40 °C in a 10 h hydrolysis of destarched wheat bran. The crystal structure of M6 was

438

determined at 2.0 Å and structural analysis showed that a new disulfide bond, as well

439

as hydrophobic interactions formed by the mutations may play an important role in

440

stabilizing the protein. These results may provide useful information for the further

441

engineering of EstF27 in subsequent study.

442 443 444 445 446 447 448 449 450 19



451

Abbreviations Used

452

E. coli, Escherichia coli; CIAP, calf intestine alkaline phosphatase; IPTG,

453

isopropyl-β-D-1-thiogalactopyranoside; DSWB, destarched wheat bran; SDS-PAGE,

454

sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pNPF, 4-nitro-phenyl

455

ferulate; PMSF, phenylmethylsulfonyl fluoride; HPLC, High-Performance Liquid

456

Chromatography

457 458

Acknowledgements

459

This research was supported by National Sciences Foundation of China (31100579),

460

Guangdong Innovative Research Team Program (2011Y038), National Natural

461

Science Foundation of China (31170117), Science & Technology Projects of

462

Guangdong Provincial Oceanic and Fishery Bureau (A201301C04), National Marine

463

Research Special Funds for Public Welfare Projects of China (201205020), Major

464

Science and Technology Projects of Guangdong Province, China (2011A080403006).

465 466

Author contributions

467

W. X. and Y-H, L. designed research and wrote the paper; L-C, C. and R. C.

468

performed the experiments. All authors have read and approved the final manuscript.

469 470

Additional Information

471

Competing financial interests: The authors declare no competing financial interests.

472

473

474 20


Page 20 of 46

Page 21 of 46


475

Supporting Information

476

Optimization of the IPTG concentration used in the epPCR libraries screening;

477

Determination of the heat treatment condition of library screening for mutants with

478

enhanced thermostability; SDS-PAGE analysis of the recombinant EstF27 and the

479

mutants; High-Performance Liquid Chromatography (HPLC) analysis of the

480

concentration of the ferulic acid released from DSWB ; The comparison of structures

481

of EstF27 (A) and EstE1 (B); Multiple sequence alignment of EstF27,

482

carboxylesterase from Lactobacillus plantarum (PDB code 3BJR), and

483

carboxylesterase from a metagenomic library (PDB code 2C7B); The electron map

484

around the modified active-site residue S151; The two possible dimerization modes in

485

the asymmetric unit, with the interfacial surface circled by the ovals; Structure overlay

486

of the two monomers of the EstF27 homodimer; The local environment of three

487

mutated residues L195 (A), L84 (B) and I161(C); Primers used to construct the

488

mutants of EstF27 and Data collection and refinement statistics. This material is

489

available free of charge via the Internet at http://pubs.acs.org.

21



References 1. Kroon, P. A.; Williamson, G. Hydroxycinnamates in plants and food: current and future perspectives. J. Sci. Food Agric. 1999, 79, 355-361. 2. Ou, S.; Kwok, K.-C. Ferulic acid: pharmaceutical functions, preparation and applications in foods. J. Sci. Food Agric. 2004, 84, 1261-1269. 3. Mancuso, C.; Santangelo, R. Ferulic acid: pharmacological and toxicological aspects. Food Chem. Toxicol. 2014, 65, 185-95. 4. Lequart, C.; Nuzillard, J.-M.; Kurek, B.; Debeire, P. Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides. Carbohydr. Res. 1999, 319, 102-111. 5. Micard, V.; Renard, C. M. G. C.; Colquhoun, I. J.; Thibault, J.-F. End-products of enzymic saccharification of beet pulp, with a special attention to feruloylated oligosaccharides. Carbohydr. Polym. 1997, 32, 283-292. 6. Saulnier, L.; Thibault, J. F. Ferulic acid and diferulic acids as components of sugar-beet pectins and maize bran heteroxylans. J. Sci. Food Agric. 1999, 79, 396-402. 7. Williamson, G.; Kroon, P. A.; Faulds, C. B. Hairy plant polysaccharides: a close shave with microbial esterases. Microbiology 1998, 144, 2011-2023. 8. Topakas, E.; Vafiadi, C.; Christakopoulos, P. Microbial production, characterization and applications of feruloyl esterases. Process Biochem. 2007, 42, 497-509. 9. Koseki, T.; Fushinobu, S.; Ardiansyah; Shirakawa, H.; Komai, M. Occurrence, properties, and applications of feruloyl esterases. Appl. Microbiol. Biotechnol. 2009, 84, 803-10. 10. Crepin, V. F.; Faulds, C. B.; Connerton, I. F. Functional classification of the microbial feruloyl esterases. Appl. Microbiol. Biotechnol. 2004, 63, 647-52. 22


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11. Benoit, I.; Navarro, D.; Marnet, N.; Rakotomanomana, N.; Lesage-Meessen, L.; Sigoillot, J. C.; Asther, M.; Asther, M. Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products. Carbohydr. Res. 2006, 341, 1820-7. 12. Rakotoarivonina, H.; Hermant, B.; Chabbert, B.; Touzel, J. P.; Remond, C. A thermostable feruloyl-esterase from the hemicellulolytic bacterium Thermobacillus xylanilyticus releases phenolic acids from non-pretreated plant cell walls. Appl. Microbiol. Biotechnol. 2011, 90, 541-52. 13. Abokitse, K.; Wu, M.; Bergeron, H.; Grosse, S.; Lau, P. C. Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran. Appl. Microbiol. Biotechnol. 2010, 87, 195-203. 14. Record, E.; Asther, M.; Sigoillot, C.; Pages, S.; Punt, P. J.; Delattre, M.; Haon, M.; van den Hondel, C. A. M.J. J.; Sigoillot, J. C.; Lesage-Meessen, L.; Asther, M. Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application. Appl. Microbiol. Biotechnol. 2003, 62, 349-55. 15. Tapin, S.; Sigoillot, J-C.; Asther, M.; Petit-Conil, M. Feruloyl Esterase Utilization for Simultaneous Processing of Nonwood Plants into Phenolic Compounds and Pulp Fibers. J. Agric. Food Chem. 2006, 54, 3697-3703. 16. Tabka, M. G.; Herpoël-Gimbert, I.; Monod, F.; Asther, M.; Sigoillot, J. C. Enzymatic saccharification of wheat straw for bioethanol production by a combined cellulase xylanase and feruloyl esterase treatment. Enzyme Microb. Technol. 2006, 39: 897-902. 17. Yu, P.-Q.; Mckinnon, J. J.; Maenz, D. D.; Olkowski, A. A.; Racz, V. J.; Christensen, D. A. Enzymic Release of Reducing Sugars from Oat Hulls by as Influenced by Aspergillus Ferulic Acid Esterase and Trichoderma Xylanase. J. Agric. 23



Food Chem. 2003, 51, 218-223. 18. Yu, P.; McKinnon, J.; Christensen, D. Improving the nutritional value of oat hulls for ruminant animals with pretreatment of a multienzyme cocktail: in vitro studies. J. Anim. Sci. 2005, 83, 1133-1141. 19. Chen, J.; Fales, S.L.; Varga, G.A.; Royse, D.J. Biodegradation of cell wall components of maize stover colonized by white-rot fungi and resulting impact on in-vitro digestibility. J. Agric. Food Chem. 1995, 68, 91-98. 20. Topakas, E.; Vafiadi, C.; Stamatis, H.; Christakopoulos, P. Sporotrichum thermophile type C feruloyl esterase (StFaeC): purification, characterization, and its use for phenolic acid (sugar) ester synthesis. Enzyme Microb. Technol. 2005, 36, 729-736. 21. Mastihubová, M.; Mastihuba, V.; Bilaničová, D.; Boreková, M. Commercial enzyme preparations catalyse feruloylation of glycosides. J. Mol. Catal. B: Enzym. 2006, 38: 54-57. 22. Kikugawa, M.; Tsuchiyama, M.; Kai, K.; Sakamoto, T. Synthesis of highly water-soluble feruloyl diglycerols by esterification of an Aspergillus niger feruloyl esterase. Appl. Microbiol. Biotechnol. 2012, 95, 615-22. 23. Haki, G. Developments in industrially important thermostable enzymes: a review. Bioresour Technol. 2003, 89, 17-34. 24. Giver, L.; Gershenson, A.; Freskgard, P.-O.; Arnold, F. H. Directed evolution of a thermostable esterase. Proc. Natl. Acad. Sci. 1998, 95, 12809-12813. 25. Hecky, J.; Müller, K. M. Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of β-lactamase. Biochemistry 2005, 44, 12640-12654. 26. Kim, M. S.; Lei, X. G. Enhancing thermostability of Escherichia coli phytase 24


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AppA2 by error-prone PCR. Appl. Microbiol. Biotechnol. 2008, 79, 69-75. 27. Ruller, R.; Deliberto, L.; Ferreira, T. L.; Ward, R. J. Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes. Proteins: Struct. Funct. Bioinf. 2008, 70, 1280-1293. 28. Zhang, S. B.; Pei, X. Q.; Wu, Z. L. Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger. Bioresour. Technol. 2012, 117, 140-147. 29. Wong, D. W. S. Feruloyl Esterase. Appl. Biochem. Biotech. 2006, 133, 87-112. 30. Fazary, A. E.; Ju, Y.- H. Feruloyl Esterases as Biotechnological Tools: Current and Future Perspectives. Acta Bioch. Bioph. Sin. 2007, 39, 811-828. 31. Sang, S. L.; Li, G.; Hu, X. P.; Liu, Y. H. Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library. J. Mol. Microbiol. Biotechnol. 2011, 20, 196-203. 32. Chen, S. K.; Wang, K.; Liu, Y. H.; Hu, X. P. Crystallization and preliminary X-ray analysis of a novel halotolerant feruloyl esterase identified from a soil metagenomic library. Acta crystallographica. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 767-70. 33. Mastihuba, V. r.; Kremnický, L. r.; Mastihubová, M.; Willett, J.; Côté, G. L. A spectrophotometric assay for feruloyl esterases. Anal. Biochem. 2002, 309, 96-101. 34. Leiros, H. K.; Flydal, M. I.; Martinez, A. Structural and thermodynamic insight into phenylalanine hydroxylase from the human pathogen Legionella pneumophila. FEBS Open Bio. 2013, 3, 370-8. 35. Topakas, E.; Stamatis, H.; Biely, P.; Kekos, D.; Macris, B. J.; Christakopoulos, P. Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water–organic solvent mixtures. J. 25



Biotechnol. 2003, 102, 33-44. 36. Mukherjee, G.; Singh, R. K.; Mitra, A.; Sen, S. K. Ferulic acid esterase production by Streptomyces sp. Bioresour. Technol. 2007, 98, 211-213. 37. Nicoli, D.; Benedek, G. Study of thermal denaturation of lysozyme and other globular proteins by light-scattering spectroscopy. Biopolymers 1976, 15, 2421-2437. 38. Gast, K.; Damaschun, H.; Misselwitz, R.; Müller-Frohne, M.; Zirwer, D.; Damaschun, G. Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate. Eur. Biophys. J. 1994, 23, 297-305. 39. Morris, A. M.; Watzky, M. A.; Finke, R. G. Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature. Biochim. Biophys. Acta 2009, 1794, 375-97. 40. Shoichet, B. K.; Baase, W. A.; Kuroki, R.; Matthews, B. W. A relationship between protein stability and protein function. Proc. Natl. Acad. Sci. 1995, 92, 452-456. 41. Cherry, J. R.; Lamsa, M. H.; Schneider, P.; Vind, J.; Svendsen, A.; Jones, A.; Pedersen, A. H. Directed evolution of a fungal peroxidase. Nat. Biotechnol. 1999, 17, 379-384. 42. Hegde, S.; Muralikrishna, G. Isolation and partial characterization of alkaline feruloyl esterases from Aspergillus niger CFR 1105 grown on wheat bran. World J. Microbiol. Biotechnol. 2009, 25, 1963-1969. 43. Byun, J. S.; Rhee, J. K.; Kim, N. D.; Yoon, J.; Kim, D. U.; Koh, E.; Oh, J. W.; Cho, H. S. Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties. BMC Struct. Biol. 2007, 7, 47. 26


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44. Heikinheimo,P.; Goldman, A.; Jeffries, C.; Ollis, D. L. Of barn owls and bankers: a lush variety of α/β hydrolases. Structure 1999, 7, R141-R146. 45. Wei, Y.; Contreras, J. A.; Sheffield, P.; Osterlund, T.; Derewenda, U.; Kneusel, R. E.; Matern, U.; Holm, C.; Derewenda, Z. S. Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase. Nat. Struct. Mol. Biol. 1999, 6, 340-345. 46. De Simone, G.; Menchise, V.; Manco, G.; Mandrich, L.; Sorrentino, N.; Lang, D.; Rossi, M.; Pedone, C. The Crystal Structure of a Hyper-thermophilic Carboxylesterase from the Archaeon Archaeoglobus fulgidus. J. Mol. Biol. 2001, 314, 507-518. 47. Lenfant, N.; Hotelier, T.; Velluet, E.; Bourne, Y.; Marchot, P.; Chatonnet, A. ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions. Nucleic Acids Res. 2013, 41, D423-D429.

27



Figure Legends Figure 1. Effects of temperature (A) and pH (B) on the initial reaction rates of EstF27 (■), M4 (○) and M6 (▲). Data points are the average of triplicate measurements, and error bars represent standard deviation.

Figure 2. The effects of temperature on the protein particles sizes of EstF27 (A), mutants M4 (B) and M6 (C).

Figure 3. Time course of ferulic acid production from DSWB at 40 oC and 65 oC. Xylanase (♦) was used alone as a control. Data points are the average of triplicate measurements, and error bars represent standard deviation.

Figure 4. The overall structure of EstF27 and comparison with orthologs. (A) The two subunits are colored in cyan and in green. The major secondary elements and the N, C termini are labeled. The intersubunit C243-C243’ disulfide bond is shown in ball-and-stick model and circled. The modified S151 on each subunit is also circled. (B) Structure overlay of Cα traces of EstF27 (green) and a cloned carboxylesterase from a metagenomic library (PDB code 2C7B). The catalytic triad is shown as ball-and-stick model.

Figure 5. The structural and biochemical characterization of the G243 mutation. (A) The electron map around the C243-C243’ disulfide bond. The map is contoured at 1σ and the Cα trace of each subunit is shown. (B) SDS-PAGE analysis of the molecular 28


Page 28 of 46

Page 29 of 46


weight of mutant M6 and mutant M6-C243G. Lane M, standard protein molecular mass marker (Thermo Fisher Scientific, sizes in kilodaltons are indicated on the left); Line 1 to Line 3, the purified recombinant protein of M6 which was untreated (Line 1) and treated by 2 mM oxidized glutathione (GSSG) (Line 2) or 5 mM DTT (Line 3); Line 4 to Line 6, the purified recombinant protein of M6-C243G which was untreated (Line 4) and treated by 2 mM oxidized glutathione (GSSG) (Line 5) or 5 mM DTT (Line 6).

Figure 6. The details of the hydrophobic interaction. (A) The local environment of I27, which could possibly form hydrophobic interactions with L5, L9 and L12 residues from α1 (close to the interface). (B) The local environment of V259, which could possibly form hydrophobic interactions with α10’ from the other subunit. The residues of interests are shown as ball-and-stick model and the major secondary elements are labeled.

29



Table 1 Half-lives of EstF27and the mutants. Enzymes EstF27 EstF27-T27I EstF27-S84L EstF27-V161I EstF27-G243C M4 M4-H195L M4-A259V M4-H195L/A259V (M6)

T1/2 at 55 oC (h) 0.5a 30 6 1.5 45 70 430 330 1, 680

T1/2 at 65 oC (h) ND ND ND ND ND 4 30 20 140

T1/2 at 70 oC (h) ND ND ND ND ND ND ND ND 3

a. which was different from that in the previous report,31 because of the fusion tag was different; ND, not determined.

30


Page 30 of 46

Page 31 of 46


Table 2 The storage stability of EstF27 and the mutant M6 at 25 °C. Storage time

Residual activity (%)

(day)

EstF27

M6

0

100.0 ± 1.1

100.0 ± 2.3

10

92.5 ± 1.6

100.0 ± 0.3

20

87.6 ±3.5

99.8 ± 1.1

50

78.5 ± 2.8

102.2 ± 1.6

80

56.2 ± 2.4

100.8 ± 2.5

100

40.6 ± 1.1

99.5 ± 1.7

150

21.2 ± 2.7

100.5 ± 2.1

31



Page 32 of 46

Table 3 Kinetic parameters of EstF27 and the mutant M6 Temperature

Enzymes

Km (mM)

Kcat (s-1)

(oC) 40

65

Kcat/Km(s-1

Reference

mM-1) EstF27

0.5±0.04

19.7±3

39.4

31

M6

0.36±0.06

18.85±0.94

52.36

This study

EstF27

ND

ND

ND

This study

M6

0.42±0.07

32.16±1.62

76.56

This study

ND, not determined.

32


Page 33 of 46


Figure 1.

33



Figure 2.

34


Page 34 of 46

Page 35 of 46


Figure 3.

35



Figure 4.

36


Page 36 of 46

Page 37 of 46


Figure 5.

37



Figure 6.

38


Page 38 of 46

Page 39 of 46


Graphical Table of Contents

39



Figure 1. Effects of temperature (A) and pH (B) on the initial reaction rates of EstF27 (■), M4 (○) and M6 (▲). Data points are the average of triplicate measurements, and error bars represent standard deviation. 101x40mm (300 x 300 DPI)


Page 40 of 46

Page 41 of 46


Figure 2. The effects of temperature on the protein particles sizes of EstF27 (A), mutants M4 (B) and M6 (C). 48x68mm (300 x 300 DPI)



Figure 3. Time course of ferulic acid production from DSWB at 40 °C and 65 °C. Xylanase (♦) was used alone as a control. Data points are the average of triplicate measurements, and error bars represent standard deviation. 127x98mm (300 x 300 DPI)


Page 42 of 46

Page 43 of 46


Figure 4. The overall structure of EstF27 and comparison with orthologs. (A) The two subunits are colored in cyan and in green. The major secondary elements and the N, C termini are labeled. The intersubunit C243C243’ disulfide bond is shown in ball-and-stick model and circled. The modified S151 on each subunit is also circled. (B) Structure overlay of Cα traces of EstF27 (green) and a cloned carboxylesterase from a metagenomic library (PDB code 2C7B). The catalytic triad is shown as ball-and-stick model. 59x76mm (300 x 300 DPI)



Figure 5. The structural and biochemical characterization of the G243 mutation. (A) The electron map around the C243-C243’ disulfide bond. The map is contoured at 1σ and the Cα trace of each subunit is shown. (B) SDS-PAGE analysis of the molecular weight of mutant M6 and mutant M6-C243G. Lane M, standard protein molecular mass marker (Thermo Fisher Scientific, sizes in kilodaltons are indicated on the left); Line 1 to Line 3, the purified recombinant protein of M6 which was untreated (Line 1) and treated by 2 mM oxidized glutathione (GSSG) (Line 2) or 5 mM DTT (Line 3); Line 4 to Line 6, the purified recombinant protein of M6-C243G which was untreated (Line 4) and treated by 2 mM oxidized glutathione (GSSG) (Line 5) or 5 mM DTT (Line 6). 50x60mm (300 x 300 DPI)


Page 44 of 46

Page 45 of 46


Figure 6. The details of the hydrophobic interaction. (A) The local environment of I27, which could possibly form hydrophobic interactions with L5, L9 and L12 residues from α1 (close to the interface). (B) The local environment of V259, which could possibly form hydrophobic interactions with α10’ from the other subunit. The residues of interests are shown as ball-and-stick model and the major secondary elements are labeled. 59x80mm (300 x 300 DPI)



Graphical Table of Contents 82x34mm (300 x 300 DPI)


Page 46 of 46

Enhancing the Thermostability of Feruloyl Esterase EstF27 by Directed

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