Enhanced Purification Efficiency and Thermal Tolerance of

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Enhanced Purification Efficiency and Thermal Tolerance of T. aotearoense #-Xylosidase through Aggregation Triggered by Short Peptides Tianwang Xu, Xiongliang Huang, Zhe Li, Carol Sze Ki Lin, and Shuang Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00551 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Journal of Agricultural and Food Chemistry

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Enhanced Purification Efficiency and Thermal

2

Tolerance of T. aotearoense β-Xylosidase through

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Aggregation Triggered by Short Peptides

4

Tianwang Xu†1, Xiongliang Huang†1 and Zhe Li†, Carol Sze Ki Lin‡, Shuang Li†,*

5 6

†

7

Biology and Biological Engineering, South China University of Technology,

8

Guangzhou, China

9

‡

10

Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of

School of Energy and Environment, City University of Hong Kong, Tat Chee

Avenue, Kowloon, Hong Kong

11 12

1

T. X. and X. H. contributed equally to this work.

13 14

**Corresponding author (Tel: +86-20-39380601. E-mail: [email protected])

1

ACS Paragon Plus Environment


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ABSTRACT

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To simplify purification and to improve heat tolerance of a thermostable β-xylosidase

17

(ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated

18

as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form.

19

However, ThXylC-ELK assembled into aggregates with 98.6% of total β-xylosidase

20

activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles

21

were collected with 92.57% activity recovery and 95% purity, respectively.

22

Meanwhile, the wild type ThXylC recovery yield was less than 55% after heat

23

inactivation, affinity and desalting chromatography followed by HRV 3C protease

24

cleavage purification. Catalytic efficiency (Kcat/Km) was increased from 21.31 mM-1s-1

25

for ThXylC to 32.19 mM-1s-1 for ThXylC-ELK accompanied with the a little increase

26

of Km value. Heat tolerance of ThXylC-ELK at high temperatures was also increased.

27

The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C.

28

Released reducing sugars were raised 1.3-fold during sugarcane bagasse hydrolysis

29

when ThXylC-ELK was supplemented into the combination of XynA∆SLH and

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Cellic CTec2.

31 32

Keywords: β-Xylosidase, self-assembly amphipathic peptide, active aggregates,

33

purification, heat tolerance

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INTRODUCTION

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Second-generation biofuels from lignocellulosic biomass are considered one of

36

the most promising energy. Enzymatic hydrolysis of the lignocellulosic biomass into

37

fermentable sugars played a significant role for the process to be economically

38

feasible because of the recalcitrance of feedstock.1-3 Within this context, the

39

exploitation of functional enzyme characteristics and effective production process

40

received extensive attention.4 Presently, a number of approaches have been proposed

41

to achieve the goals, including the site-directed mutagenesis and directed evolution. 5,

42

6

43

formulation and immobilization are also applied to improve the catalytic performance

44

of the target enzymes.7, 8

Moreover, downstream processes in enzyme manufacturing, i.e. purification,

45

Through the combination of directed evolution and site-directed mutagenesis,

46

Zhang and coworkers obtained a xylanase mutant which showed the optimal

47

temperature increased from 77 oC to 87 oC and displayed 90% increase in catalytic

48

efficiency (kcat/Km).9 The thermal stability parameter of a β-xylosidase XylBH43 was

49

enhanced by 8.8 oC by DNA shuffling and saturation mutagenesis.10 Although these

50

approaches achieved tremendous success, there are still several drawbacks and

51

challenges to be overcome,11 such as efficient high-throughput screening technique,

52

knowledge on the relationship between enzyme function and structure, etc.

53

Earlier research showed that attachment of some peptides to the N or C terminus

54

of an enzyme could enhance its catalysis performance11 and thermostability.12 Fusion

55

of a short self-assembling amphipathic peptide (SAP) to the N terminus of

56

lipoxygenase resulted in about 4.5-fold enhancement of thermostability at 50 °C.

57

By genetic fusion, a small 27-residue β-propeller like segment was linked to HIV1

58

envelope glycoprotein13 and short collagen14 to improve their thermodynamic stability. 3


12


59

Meanwhile, stimulus-responsive polymers, elastin-like peptides (ELPs) were used to

60

purify target proteins by Inverse Transition Cycling (ITC) triggered by temperature or

61

salt without chromatography.15-17 Wu et al reported ELK16 (LELELKLK)2 was able

62

to induce the cytoplasmic inclusion bodies formation in Escherichia coli (E. coli)

63

when attached to the carboxyl termini of the model protein.18 In our previous study, an

64

amphipathic octadecapeptide (18A) was fused to the C terminus of a nitrilase, the

65

formed active aggregates of Nit-SEA possessed higher thermal stability than the

66

native nitrilase.19

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The thermoacidophilic bacterial strain Thermoanaerobacterium aotearoense

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SCUT27 has been reported to be an excellent utilizer of xylan.20 A glycoside

69

hydrolase family 10 (GH10) xylanase from it was also cloned and characterized by

70

our group.21 In this work, a β-xylosidase from T. aotearoense P8G3#4 was cloned and

71

characterized. The more important objective of this work was to develop an efficient

72

and convenient method to simplify enzyme purification steps with satisfactory activity

73

recovery and to improve the catalytic performance at higher temperature of this

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β-xylosidase based on aggregation triggered by a SAP.

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MATERIALS AND METHODS

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Strains, media, plasmids and reagents

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T. aotearoense P8G3#4 (CGMCC No. 9000) was isolated from a hot spring and

78

cultured in the modified MTC medium.20 E. coli DH5α (Invitrogen, San Diego, CA,

79

USA) and BL21(DE3) (Novagen, Wisconsin, WI, USA) were used as DNA

80

manipulation and expression host, respectively. E. coli cells were cultured in

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Luria-Bertani (LB) medium supplemented with 50 µg·mL-1 of kanamycin if necessary.

82

The plasmid pET-30a(+) was obtained from Novagen (Wisconsin, USA). Human

4


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Rhinovirus (HRV) 3C Protease for fusion tag cleavage was from TaKaRa (Dalian,

84

China). p-Nitrophenyl β-D-xylopyranoside (pNPX) was purchased from Tokyo

85

Chemical Industry Co. Ltd., (TCI, Tokyo, Japan). Xylooligosaccharides (xylobiose,

86

xylotriose, xylotetraose and xylopentaose) were bought from Megazyme (Bray,

87

Ireland).

88

Plasmid construction

89

DNA was manipulated according to standard protocols. High-fidelity DNA

90

polymerase PrimerSTAR was from TaKaRa (Dalian, China). Restriction enzymes and

91

T4 DNA ligase were obtained from Fermentas (Thermo Scientific., Waltham, USA).

92

The gene xylC, encoding the β-xylosidase, was amplified from the genomic

93

DNA

94

5′-TCGGCTCATATGGAATACCATGTGGCTAAAA-3′ and reverse primer (xylC-R)

95

5′-TAGCAACTCGAGAGAAGAGCCCCAAACTTTTATGTAATTATTTCCT-3′

96

(NdeI and XhoI are underlined). Purified PCR product was digested and introduced

97

into the NdeI and XhoI sites of pET-30a(+) to yield the pET30a-xylC.

98

of

T.

aotearoense

P8G3#4

with

the

forward

primer

(xylC-F)

PCR amplification was carried out with primer pairs of xylC-F and xylC-HRV

99

(5′-CCGCTCGAGGGGTCCCTGAAAGAGGACTTCAAGCCAAACTTTTATGT

100

AATTATTTCC-3', the HRV 3C protease recognition sites were in bold) using

101

pET30a-xylC as template. Amplified product was subcloned into pET-30a(+) to obtain

102

pET30a-xylC-HRV,

103

(LeuGluValLeuPheGlnGlyPro) was introduced between the xylC and His-tag.

104

in

which

the

protelytic

cleavage

site

Using pET30a-xylC as template, PCR products amplified through primer pairs of

105

xylC-F

and

xylC-R2

106

(5′-TCGTTCTCGAGTCATTTCAGCTTTAATTCTAATTCCAGTTTTAACTTCAGT

107

TCAAGTTCCAGCAGAAGAGCCCCAAACTTTTATG-3') were also inserted into 5



108

the NdeI and XhoI sites of pET-30a(+) to construct pET-xylC-ELK. The ELK16

109

peptide (LELELKLKLELELKLK) was fused to the C-terminus of XylC through this

110

manipulation. The 6×His tag was omitted by the addition of stop codon (TGA)

111

between the ELK16 and His-tag coding sequences.

112

Expression and purification

113

The recombinant E. coli BL21(DE3) cells containing pET30a-xylC-HRV or

114

pET-xylC-ELK cells were cultured at 37 oC for about 2 h to the early exponential

115

phase (OD600 = 0.5-0.6). Isopropyl β-D-1-galactoside (IPTG) was added to a final

116

concentration of 0.1 mM to induce the β-xylosidase expression. After incubation at 30

117

o

118

for 30 min.

C for 24 h, the recombinant E. coli cells were harvested by centrifugation at 4300 g

119

To purify ThXylC-HRV-His, the cell pellets were resuspended in a lysis buffer

120

(20 mM sodium phosphate, 0.5 M NaCl, 50 mM imidazole, pH 7.4) and subjected to a

121

brief sonication (4 s each with 4 s interval for 50 times) in an ice-water bath. The

122

supernatants of cell lysates were collected by centrifugation at 15,422 g for 30 min

123

and incubated at 65 oC for 30 min to inactivate unstable proteins. After centrifugation,

124

the soluble fractions were passed through a 0.22 µm filter and applied to the standard

125

immobilized metal affinity chromatography (IMAC) purification through a HiTrap™

126

Chelating HP column (GE Healthcare, Piscataway, NJ, USA), which was

127

chelated with nickel (Ni2+) ions. Aliquots containing β-xylosidase activity were

128

pooled and loaded onto a HiPrep™ 26/10 desalting column (GE Healthcare) and

129

eluted for buffer exchange. The final collected ThXylC-HRV-His was stored in 100

130

mM phosphate buffer (pH 6.5) at 4 °C.

131

For His-tag cleavage, 16 mg ThXylC-HRV-His, 200 µL HRV 3C protease (200

132

U), 1 mL 10×HRV 3C Protease Cleavage Buffer and sterile distilled water were 6


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combined to make a 10 mL total reaction volume. The reaction mixture were

134

incubated at 4 oC for 16 h and then loaded onto HiTrap™ Chelating HP column

135

following the standard protocol described previously. The reaction mixture,

136

flow-through and eluted fractions were all analyzed by sodium dodecyl

137

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

138

For the insoluble ThXylC-ELK purification, cell pellets were gathered and lysed

139

by centrifugation and sonication as described before. Then the cell debris containing

140

ThXylC-ELK was collected and washed twice with the same volume of wash buffer

141

(50 mM sodium phosphate, 50 mM NaCl, 0.8% Triton X-100 (v/v), pH 7.5). When

142

incubated on ice for 20 min, the membrane proteins were re-solubilized by Triton

143

X-100 and removed by centrifugation. After centrifugation, the supernatants were

144

discarded, and the enzyme aggregates, ThXylC-ELK, were collected and resuspended

145

with Tris-Cl (50 mM, pH7.0) for further characterization.

146

Enzyme characterization

147

Protein purity and molecular mass were estimated by SDS-PAGE in a 10%

148

separating gel and stained with Coomassie Brilliant Blue R-250 (Sangon). Protein

149

concentration was determined by a BCA Protein Assay Kit (Sangon, Shanghai, China)

150

using bovine serum albumin (BSA, Sangon) as the standard. The enzymatic activity

151

distributions were determined by measurements of the corresponding β-xylosidase

152

activity for the soluble and insoluble fractions of cell lysates. The whole enzyme

153

activity was the sum of soluble and insoluble parts.

154

The β-xylosidase activity was determined by measuring the amount of

155

p-nitrophenol released from the substrate pNPX. The reaction mixture consisting of

156

170 µL 0.1 M phosphate buffer (pH 6.5) and 20 µL 40 mM pNPX was incubated at 65

157

o

C for 1 min, followed by the addition of 10 µL enzyme solution. After incubation at 7



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65 oC for 5 min, the reaction was stopped by the addition of 600 µL 1 M Na2CO3 and

159

put into an ice-water bath for 2 min. The produced p-nitrophenol was measured at 405

160

nm as literature reported.

161

β-xylosidase required to release 1 µmol of p-nitrophenol per minute under the reaction

162

conditions. Each assay was performed in triplicate.

22, 23

One unit activity was defined as the amount of

163

The optimum pH was determined by measuring enzyme activity under various

164

pH values at 65 oC. The pH was adjusted by the addition of sodium acetate buffer (pH

165

4.0 to 5.5) or potassium phosphate buffer (pH 5.5 to 8.0) in the range from 4.0 to 8.0.

166

The optimum temperature was investigated by measuring the enzyme activity at

167

different temperatures ranging from 45 to 90 oC in 0.1 M potassium phosphate buffer

168

(pH 6.5). Thermostability assays were carried out by measuring the residual

169

β-xylosidase activities after pre-incubation at 65 and 70 oC for different hours.

170

The Michaelis constant (Km), maximum activity (Vmax) and catalytic efficiency

171

(Kcat/Km) values were determined by measuring the initial rates at various pNPX

172

concentrations ranging from 0.1 to 1.0 mM under optimal reaction conditions. The

173

kinetic parameters were calculated by nonlinear regression using the GraphPad

174

Software

175

Michaelis-Menten equation in the panel of Enzyme kinetics - Substrate v Velocity was

176

chosen. 24

177

Enzyme synergism analysis

program

(GraphPad

Software,

Inc.

CA,

USA),

in

which

the

178

The synergistic actions of the recombinant xylanase A (XynA∆SLH, purified by

179

our group previously21) and β-xylosidase on the beechwood xylan (Sigma-Aldrich)

180

hydrolysis were investigated. The hydrolysis experiments were carried out at 55 oC in

181

0.1 M phosphate buffer, pH 6.5, containing 1.5 mg·mL-1 of substrate and different

182

amounts of enzymes (0.5 U of XynA∆SLH with or without 1.0 U of ThXylC-ELK). A 8


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solution of xylobiose (X2), xylotriose (X3), xylotetraose (X4) or xylopentaose (X5)

184

(Megazyme, Bray, Ireland) in the concentration of 1 mg·mL-1 for each mixed with 0.5

185

U of ThXylC-ELK was incubated at 65 °C for 3 h in 50 mM phosphate buffer (pH

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6.5). The hydrolyzed xylooligosaccharides and xylan were centrifuged and the

187

supernatants were analyzed on silica gel TLC plates (CF254, 100 × 100 mm, Merck).

188

The developing solvent was the mixture of chloroform/acetic acid/water (3:6:1, by

189

vol.). The plates were sprayed with a mixture of ethanol and concentrated sulfuric

190

acid (95:5, v/v) and then incubated at 105 oC for 15 min for color development. The

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standard mixture containing 1 mg⋅mL-1 of xylose, X2, X3, X4 and X5 was spotted to

192

determine the xylooligosaccharide identity.

193

Sugarcane bagasse was acquired from Guangzhou Sugar Cane Industry Research

194

Institute (Guangzhou, China). In preliminary processing, the air-dried sugarcane

195

bagasse was milled to pass through with 0.3 mm sieve.19 0.2 g of pretreated sugarcane

196

bagasse was hydrolyzed in 20 mL of 0.1 M Bis-Tris-HCl (pH 6.5) containing (a) 10 U

197

of XynA∆SLH, (b) 10 U of XynA∆SLH and 20 U of ThXylC-ELK, (c) 10 U of

198

XynA∆SLH and 0.2 filter paper unit (FPU) of Cellic CTec2 (Novozyme, Denmark),

199

or (d) 10 U of XynA∆SLH, 20 U of ThXylC-ELK and 0.2 FPU of Cellic CTec2.

200

Samples were taken at different time intervals to determine the released reducing

201

sugars, which were measured by the standard dinitrosalicylic acid (DNS)

202

colorimetric method using xylose as standard.25

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RESULTS

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Overexpression and purification of recombinant enzymes

205

Heterologous expression of recombinant β-xylosidases in E. coli BL21(DE3)

206

was confirmed by SDS-PAGE and the catalytic activity against pNPX. The

207

ThXylC-HRV-His (attached with a 6×His-tag, 74.66 kD) was predominantly 9



208

expressed as soluble protein (Figure 1a). The β-xylosidase activity in the supernatants

209

of cell lysates was detected as 39.02 U⋅mL-1, which accounts for about 98.6% of the

210

total activity. To purify the ThXylC-HRV-His, the supernatants of cell lysates were

211

subjected to heat treatment to inactivate thermally unstable proteins with the protein

212

recovery yield of 86.45%. After three-step purification (heat-treatment, affinity

213

purification and desalting), highly enriched preparations of the recombinant

214

ThXylC-HRV-His were obtained with an enzyme recovery of 62.50% and 9.78-fold

215

purification (Figure 1a and Table 1). After incubation with HRV 3C protease at 4 oC

216

for 16 h, cleavage reaction solution containing the ThXylC-HRV-His showed two

217

major protein bands of ThXylC (~ 73.44kD) and HRV 3C protease (~22 kD, Figure

218

1b lane 1). The target ThXylC flew through and the terminal His-tag and HRV 3C

219

protease remained on resin when IMAC Ni-charged resin is used.

220

On the contrary, almost no β-xylosidase activity (1.5 U⋅mL-1) was measured in

221

the supernatant fractions of cell lysate for E. coli BL21(DE3)/pET-xylC-ELK.

222

However, about 96.2% of the whole activity (37.65 U⋅mL-1) were accumulated in the

223

insoluble fractions. Judged by SDS-PAGE, the fusion ThXylC-ELK was found to be

224

predominantly in the insoluble fractions (Figure 1c). Furthermore, the final E. coli cell

225

densities (OD600) for ThXylC-HRV-His and ThXylC-ELK expression were 2.78±0.21

226

and 3.28±0.18, respectively.

227

The aggregates containing the β-xylosidase activity (ThXylC-ELK) were

228

purified by buffer washing and centrifugation with almost 95% of purity estimated

229

from the SDS-PAGE and 92.57% enzyme activity recovery. In addition, the specific

230

activity of ThXylC-ELK (108.19±1.74 U⋅mg-1) was increased about 1.53-fold

231

compared with the purified ThXylC (71.04±1.39 U⋅mg-1) (Table 1).

10


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Biochemical characterization of recombinant ThXylC and ThXylC-ELK

233

Characteristics of ThXylC are almost the same as those of ThXylC-HRV-His

234

(Figure 2 and Table 2), thus only ThXylC will be discussed hereafter when compared

235

with ThXylC-ELK.

236

The soluble ThXylC and the aggregates of ThXylC-ELK both showed relatively

237

high activities in the range of pH 6.0 - 7.0 with the optimum activity at pH 6.5 in 0.1

238

M KH2PO4-K2HPO4 buffer (Figure 2a). The catalytic activity for ThXylC-ELK

239

exhibited higher activity in the acidic pH range when compared with ThXylC. For

240

example, the activity of ThXylC-ELK was about 60% at pH 5.5 relative to the activity

241

at pH 6.5, whereas it was only 16% for ThXylC under the same conditions.

242

In terms of the effect of temperature on β-xylosidase activity, the optimal

243

temperature of the purified ThXylC was found to be 65 °C (Figure 2b). It is

244

interesting to find that the optimal temperature for ThXylC-ELK was increased to

245

70 °C. When reaction temperature was set at 75 °C, the catalytic activity of

246

ThXylC-ELK still remained about 90% relative to that at 70 °C. The ThXylC retained

247

27.6% of the initial activity after an incubation of 48 h at 65 °C, and lost most of its

248

catalytic ability after an incubation at 70 °C for 6 h, with obvious white sediments at

249

the bottom of the reaction tube (Figure 2c). However, the residual activity of

250

ThXylC-ELK retained more than 50% after the incubation at 65 °C for 48 h. Even

251

incubated at 70 °C for 48 h, it still preserved about 26.6% of its initial activity. The

252

calculated half-lives for ThXylC and ThXylC-ELK at 65 °C were 8.9 h and 54.8 h,

253

respectively.

254

To understand substrate affinity and catalytic efficiency, kinetic parameters of the

255

purified ThXylC using pNPX as substrate were determined by the Michaelis-Menten

256

analysis. The values of Km and Vmax of recombinant ThXylC were 20.37 mM and 11



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257

434.10 U⋅mg-1, respectively. And the catalytic efficiency (kcat/Km) of the enzyme was

258

determined as 21.31 mM-1s-1. The purified ThXylC exhibited unusual values of Km

259

and Vmax compared with other β-xylosidases (Table 2), implying the relative lower

260

affinity for the artificial substrate of pNPX. However, the Km, Vmax and kcat/Km of

261

ThXylC-ELK were measured as 27.67 mM, 890.7 U⋅mg-1 and 32.19 mM-1s-1,

262

respectively.

263

Synergistic action of ThXylC and XynA∆SLH

264

To evaluate the potential of ThXylC-ELK in practical applications, the action

265

mode of ThXylC-ELK was analyzed using different xylooligosaccharides as

266

substrates and assayed on silica gel plates. As shown in Figure 3a, the ThXylC-ELK

267

completely degrades X2, X3, X4 and X5 into xylose when incubated at 65°C for 3 h.

268

In addition, we also investigated the efficiency of combined action of previously

269

reported endo-xylanase, XynA∆SLH21 and ThXylC on beechwood xylan (Figure 3b).

270

Being consistent with our previous investigation,

271

beechwood xylan only by XynA∆SLH released xylobiose and presumed

272

methylglucuronoxylotriose (MeGlcAXyl3). The MeGlcAxyl3 might be the aldouronic

273

acid liberated from methylglucuronoxylan by xylanases.

274

ThXylC-ELK could not hydrolyze MeGlcAXyl3 neither.

21

the depolymerization of

26, 27

However, the

275

After 60 h hydrolysis, the reducing sugar catalyzed only by XynA∆SLH was

276

recorded as 0.065 mmol⋅g-1 sugarcane bagasse (Figure 4). When the reaction was

277

supplemented with the purified ThXylC-ELK, the produced sugar was increased to

278

0.233 mmol⋅g-1 sugarcane bagasse. And the similar increase of final hydrolysis

279

product was observed from 0.366 mmol⋅g-1 catalyzed by the combination of

280

XynA∆SLH with purchased cellulase (Cellic CTec2, from Novozyme) to 0.460

12


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281

mmol⋅g-1 supplemented with ThXylC-ELK.

282

DISCUSSION

283

In this study, we cloned the xylC gene, encoding β-xylosidase in T. aotearoense

284

P8G3#4. The native β-xylosidase, designated as ThXylC was expressed primarily as a

285

soluble protein (Figure 1a and 1b). Through overexpression and affinity purification,

286

the ThXylC was obtained from the soluble fractions of the recombinant E. coli

287

BL21(DE3) cells. When the ELK16 short peptide was introduced to the C-terminus of

288

ThXylC, the recombinant protein, ThXylC-ELK, was found to be predominantly in

289

insoluble fraction with relative high β-xylosidase activity (Figure 1c). Differing from

290

the previous study,18 the introduction of the ELK16 peptide to the C terminus of

291

ThXylC did not disturb the cell growth. After three-step purification and HRV 3C

292

protease cleavage, the final activity recovery yield of ThXylC was about 54.37%.

293

However, the particles of ThXylC-ELK could be easily collected only by buffer

294

washing and centrifugation with outstanding high activity recovery rate (92.57%) and

295

purity (~95%) (Table 1 and Figure 1c). Using the thermally driven, phase transition

296

property of the elastin-like peptides (ELPs), it allows the target protein fused with

297

ELPs to be isolated from cell contaminants by the phase change from soluble

298

monomers to insoluble aggregates, which was termed Inverse Transition Cycling

299

(ITC).16, 17 Both the ELK16 mediated purification method and the ITC protocol do not

300

require the use of chromatography, and are all cost effective and easy to scale up. It

301

should be noted that the ITC method needs 3-5 rounds of “hot-spin” and “cold-spin”

302

to attain the desired purity, which is dependent on the protein.16 However, the

303

ELK16-fusion protein was expressed as insoluble aggregates with good catalytic

304

activity, and two-time buffer wash with one centrifugation are enough to obtain the

305

desired purity. 13



306

The recombinant ThXylC exhibited the highest activity at 65 °C. It is exciting to

307

find that when the ELK16 peptide was attached to the terminus of ThXylC, relative

308

activities at the pH values investigated and the heat tolerance were both improved

309

(Figure 2 and Table 2). For example, the optimal temperature of fusion ThXylC-ELK

310

was increased by 5°C compared to that of ThXylC. And the half-life at 65 °C was also

311

elongated from 8.9 h for ThXylC to 54.8 h for ThXylC-ELK. The vast enhancement

312

of thermostability should be attributed to the aggregate formation.19 According to the

313

previous studies, thermal stability and specific activity of lipoxygenase was enhanced

314

by fusing with SAP,12 and the operational stability of nitrilase from Alcaligenes

315

faecalis JM3 was also dramatically improved by the introduction of the 18A peptide.19

316

However to our best knowledge, this is the first report that the terminal SAP

317

attachment to an enzyme could increase its optimal temperature.

318

Another attractive aspect is that the catalytic efficiency (kcat/Km) of ThXylC-ELK

319

was increased 1.51-fold compared with that of ThXylC (Table 2). In our previous

320

result,19 the Km value was increased from 25.6 mM to 33.0 mM because of the

321

attachment of 18A peptide at the nitrilase C-terminus. Similarly in this study, the

322

introduction of ELK16 to the ThXylC enhanced the Km by about 35%. It was reported

323

that the less hydrophobic peptide EAK16 originated from Zuotin protein, could

324

spontaneously form a β-sheet structure.28 In contrast, the 18A peptide would

325

self-assemble into coiled-coil structure in aqueous solution.29 However, the

326

differences of the secondary structure for ELK16 and 18A did not cause changes in

327

trend of Km values between the native and fusion enzymes.

328

The synergy in the degradation of beechwood xylan by XynA∆SLH and

329

ThXylC-ELK offered the possibility to degrade lignocellulosic biomass for industrial

330

applications. The amount of released reducing sugars is one of the most important 14


Page 14 of 32

Page 15 of 32


331

aspects in the lignocellulosic biomass hydrolysis. The results indicate that the

332

recombinant ThXylC-ELK could degrade short xylooligosaccharides to xylose.

333

However, it could not attack a site containing glucuronic acid as a substituted residue

334

deduced from the fact that ThXylC-ELK did not degrade MeGlcAXyl3.

335

The XynA∆SLH is an endoxylanase that randomly cleaves the internal linkages

336

in xylan. And β-xylosidase cleaves the nonreducing termini of xylobiose and

337

xylooligosaccharide segments into xylose. Complete degradation of xylan requires the

338

synergistic action of various enzymes.30 The sugarcane bagasse hydrolysis showed

339

that ThXylC-ELK supplementation in the reaction resulted in the enhanced

340

production of reducing sugar production (Figure 4). The final concentration of

341

reducing sugar catalyzed by the additional ThXylC-ELK was improved by 3.6-fold

342

and 1.3-fold relative to the counterparts catalyzed by the XynA∆SLH alone and the

343

combination of XynA∆SLH with Cellic CTec2, respectively. The results indicate that

344

the supplementation of ThXylC-ELK is beneficial to releasing the polysaccharide

345

releasing in the lignocellulosic biomass degradation.

346

In this study, introduction of a short ELK16 peptide to the β-xylosidase

347

C-terminus gave a facile protein purification scheme with high activity recovery and

348

purity. Taking into account its inherent good heat tolerance of ThXylC, 5 °C increase

349

of optimal temperature and significant improvement of thermostability were still

350

achieved by simple attachment of an ELK16 peptide at its C-terminus. When used in

351

sugarcane bagasse hydrolysis, approximately 25.7% increase in the production of

352

reducing sugars was observed in the Cellic CTec2 and XynA∆SLH supplemented

353

with ThXylC-ELK compared to that without ThXylC-ELK.

354 355

ACKNOWLEDGEMENTS 15



356

This work was financial supported in part by the National Natural Science Foundations

357

of China (U1701243), the Project on the Integration of Industry, Education and

358

Research of Guangzhou, China (201704020183), the Science and Technology Planning

359

Project of Guangdong Province, China (2017A010105019), and the Fundamental

360

Research Funds for the Central Universities, SCUT (2015ZZ108).

361

REFERENCES

362

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stable trimers formed by human immunodeficiency virus type 1 envelope

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self-assembled into insoluble active xylanase particles. J. Biotechnol. 2014, 177,

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Elastin-like polypeptides: a strategic fusion partner for biologics. Biotechnol. Bioeng.

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2016, 113, 1617-1627.

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(17) Hassouneh, W.; Christensen, T.; Chilkoti, A., Elastin-like polypeptides as a

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purification tag for recombinant proteins. Curr. Protoc. Protein Sci. 2010, Chapter 6,

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terminally attached self-assembling peptide ELK16 in Escherichia coli. Microb. Cell

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Thermoanaerobacterium strain. Bioresource Technol. 2010, 101, 8718-8724.

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aotearoense SCUT27 in Bacillus subtilis. J. Agr. Food Chem. 2015, 63, 6430-6439.

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xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11

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xylanase from Humicola insolens and their synergy in the degradation of xylan. Food

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Chem. 2014, 148, 381-387.

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(23) Bhalla, A.; Bischoff, K. M.; Sani, R. K., Highly thermostable GH39 β-xylosidase

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from a Geobacillus sp strain WSUCF1. BMC Biotechnol. 2014, 14, 963.

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(24) Zorn, H.; Li, Q. X., Trends in Food Enzymology. J. Agr. Food Chem. 2017, 65,

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4-5.

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(25) Miller, G. L., Use of dinitrosalicylic acid reagent for determination of ruducing

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sugar. Anal. Chem. 1959, 31, 426-428.

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(26) Gallardo, Ó.; Pastor, F. I.; Polaina, J.; Diaz, P.; Lysek, R.; Vogel, P.; Isorna, P.;

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González, B.; Sanz-Aparicio, J., Structural insights into the specificity of Xyn10B

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from Paenibacillus barcinonensis and its improved stability by forced protein

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xylanase

from

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by

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ldh-deleted

Thermoanaerobacterium


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evolution. J. Biol. Chem. 2010, 285, 2721-33.

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(27) Rhee, M. S.; Sawhney, N.; Kim, Y. S.; Rhee, H. J.; Hurlbert, J. C.; St. John, F. J.;

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Nong, G.; Rice, J. D.; Preston, J. F., GH115 α-glucuronidase and GH11 xylanase from

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Paenibacillus sp. JDR-2: potential roles in processing glucuronoxylans. Appl.

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Microbiol. Biotechnol. 2017, 101, 1465-1476.

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(28) Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A., Zuotin, a putative

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Z-DNA binding protein in Saccharomyces cerevisiae. EMBO. J. 1992, 11, 3787-3796.

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(29) Anantharamaiah, G. M.; Jones, J. I.; Brouillette, C. G.; Schmidt, C. F.; Chung, B.

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H.; Hughes, T. A.; Bhown, A. S.; Segrest, J. P., Studies of synthetic peptide analogs of

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the amphipathic helix - structure of complexes with dimyristoyl phosphatidylcholine.

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J. Biol. Chem. 1985, 260, 248-255.

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(30) Matsuzawa,

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characterization of a GH43 family β-xylosidase/α-arabinofuranosidase from a

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compost microbial metagenome. Appl. Microbiol. Biotechnol. 2015, 99, 8943-8954.

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(31) Falck, P.; Linares-Pasten, J. A.; Adlercreutz, P.; Karlsson, E. N., Characterization

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of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic

472

Weissella sp. strain 92. Glycobiology 2016, 26, 193-202.

473

(32) Wongwisansri, S.; Promdonkoy, P.; Matetaviparee, P.; Roongsawang, N.;

474

Eurwilaichitr, L.; Tanapongpipat, S., High-level production of thermotolerant

475

β-xylosidase of Aspergillus sp BCC125 in Pichia pastoris: Characterization and its

476

application in ethanol production. Bioresource Technol. 2013, 132, 410-413.

T.; Kaneko, S.; Yaoi, K.,

Screening,

477

20


identification,

and

Page 21 of 32


478

FIGURE CAPTIONS

479

Figure 1. SDS-PAGE analysis of the recombinant β-xylosidase. (a) Expression and

480

purification of ThXylC-HRV-His. M, protein marker; 1, supernatants of cell lysates

481

after sonication; 2, heat-treated crude extracts; 3, flow-through fractions loading on

482

HiTrap

483

ThXylC-HRV-His after desalting; (b) Purification of ThXylC from ThXylC-HRV-His

484

by HRV 3C protease cleavage. M, protein marker; 1, reaction solution after cleavage

485

with HRV 3C protease at 4 oC for 16 h; 2, flow-through fractions containing ThXylC

486

loading

487

ThXylC-HRV-His and HRV 3C protease. (c) Expression and purification of

488

ThXylC-ELK16. M, protein marker; 1, supernatants of lysates after sonication; 2,

489

insoluble pellets of the lysates; 3, cell pellets recovered by two-step wash. 19

column;

on

4,

HiTrap

eluent

column;

possessing

3,

β-xylosidase

elution

fractions

activity;

including

5,

purified

un-cleaved

490 491

Figure 2. pH and temperature profiles on ThXylC, ThXylC-HRV-His and

492

ThXylC-ELK. (a) Effect of pH on enzyme activity measured at 65 °C using sodium

493

acetate buffer (pH 4.0 to 5.5) and potassium phosphate buffer (pH 5.5 to 8.0). (b)

494

Effect of temperature on enzyme activity measured at pH 6.5 in potassium phosphate

495

buffer, pH 6.5. (c) Thermostability incubated at 65 and 70 °C in potassium phosphate

496

buffer (pH 6.5) for various periods of time. Samples were withdrawn at each time

497

point and assayed at optimal conditions. Error bars represent standard deviation of

498

triplicate assays.

499 500

Figure 3. Thin-layer chromatography (TLC) of sugars produced during hydrolysis. (a)

501

Xylooligosaccharide hydrolysis by the recombinant ThXylC-ELK. Lane M, mixture

502

of xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4) and xylopentaose 21



Page 22 of 32

503

(X5). Lanes 2, 3, 4 and 5 represent standard samples of X2, X3, X4 and X5,

504

respectively. Lanes 0 represent the degradation products of X2, X3, X4 and X5 by the

505

recombinant ThXylC-ELK. Xylooligosaccharides at concentrations of 1 mg·mL-1

506

were incubated with 0.5 U of purified ThXylC-ELK for 3 h at 65 °C in 80 mM

507

phosphate buffer, pH 6.5. (b) Xylan hydrolysis products. Beechwood xylan (1.5

508

mg⋅mL-1) was hydrolyzed for 24 h at 65 °C in 100 µL of 80 mM phosphate buffer, pH

509

6.5. Lane 1, Xylan was hydrolyzed only by 0.5 U of recombinant XynA∆SLH

510

Lane 2, Xylan was degraded by the combination of 0.5 U XynA∆SLH and 1.0 U

511

ThXylC-ELK.

21

.

512 513

Figure 4. Synergistic hydrolysis of sugarcane bagasse by combinations of

514

ThXylC-ELK, XynA∆SLH and Cellic CTec2. ■, hydrolyzed by XynA∆SLH only;

515

●, hydrolyzed by XynA∆SLH and ThXylC-ELK; ▲, hydrolyzed by XynA∆SLH

516

and Cellic CTec2; ▼, hydrolyzed by the combination of XynA∆SLH, ThXylC-ELK

517

and Cellic CTec2. Different enzyme combinations were added into the 20 mL reaction

518

mixture containing 0.2 g of sugarcane bagasse in 0.1 M Bis-Tris-HCl buffer system,

519

pH 6.5. The amounts of added enzymes are 10 U, 20 U and 0.2 FPU for XynA∆SLH,

520

ThXylC-ELK and Cellic Ctec2, respectively.

22


Page 23 of 32


TABLES Table 1 Purification of different forms of β-xylosidase a. Specific Enzymes

Fraction

Total Protein (mg)

Total Activity (U)

Activity

Purification Fold

% Yield

(U·mg-1)

ThXylC-HRV-His Crude extracts

ThXylC b

250.00

1808.40

7.23

1.00

100. 00

Heat treatments

122.31

1563.45

12.78

1.77

86.45

Ni-affinity

17.09

1281.13

74.96

10.36

70.84

Desalting

15.98

1130.26

70.71

9.78

62.50

13.93

983.22

71.04

9.76

54.37

Crude extracts

235.15

1739.30

7.40

1.00

100

Two-step buffer washing

14.88

1610.07

108.19

14.62

92.57

HRV 3C protease cleavage and IMAC purification

ThXylC-ELK

a

The β-xylosidase activity was determined using pNPX as substrate at 65 °C, pH 6.5. Each value is the mean of three preparations. Standard

deviation was less than 5% and has been omitted. 23



b

ThXylC was purified from ThXylC-HRV-His by HRV 3C protease cleavage.

24


Page 24 of 32

Page 25 of 32


Table 2 Comparison of the Enzymatic Properties of Thermostable β-xylosidase

ThXylC-HRV-Hi Name

WSUCF1

Xyl43A

Xyl43B

WXyn43

β-xylosidase

ThXylC

ThXylC-ELK s

Geobacill us

sp Humicola

Humicola

Weissella

sp. Aspergillus

sp. T. aotearoense T.

aotearoense T. aotearoense

insolens

insolens

strain 92

BCC125

P8G3#4

P8G3#4

P8G3#4

GH39

GH3

GH43

GH43

GH3

GH120

GH120

GH120

133

11.6

1.7

11.2

156

71.04

70.71

108.19

6.5

6.0

7.0

6.0-6.5

4.0-5.0

6.5

6.5

6.5

70

60

50

55

60

65

65

70

3.5 h at 60°C a

8.9 h at 65°C

8.7 h at 65°C

54.8 h at 65°C

Source strain WSUCF1 GH family

Specific activity (U·mg-1) Optimum pH Optimum Temperature (°C)

Thermostability

9

d

at 2.8 h at 50°C a

34 h at 50°C a 4 min at 55°C

25



Page 26 of 32

70°C

Km (mM)

2.38

2.51

1.29

7.4

1.7

20.37

20.62

27.67

Vmax (U·mg-1)

147

37.33

2.18

N.A.b

211.5

434.10

437.50

890.70

Kcat/Km (mM-1s-1 )

-

-

-

34.9

198.8

21.31

21.22

32.19

Reference

23

22

22

31

32

This study

This study

This study

a

, calculated from the reported data

b

, N.A. not analyzed

26


Page 27 of 32


Figure 1

28



a 120 100

ThXylC ThXylC-HRV-His ThXylC-ELK

80 60 40 20 0 4.0

5.0

6.0

7.0

8.0

pH

b 120 100

ThXylC ThXylC-HRV-His ThXylC-ELK

80 60 40 20 0 40

50

60

70

80

90

Temperature (°C)

c 120

ThXylC, 65°C ThXylC-HRV-His, 65°C ThXylC-ELK, 65°C ThXylC, 70°C ThXylC-HRV-His, 70°C ThXylC-ELK, 70°C

100 80 60 40 20 0 0

10

20

30

40

Time (h)

Figure 2

29


50

Page 28 of 32

Page 29 of 32


Figure 3

30



Figure 4

31


Page 30 of 32

Page 31 of 32


For Table of Contents Only

32


Affinity & desalting chromatography protease cleavage


>95% purity ~50% recovery Lower cataly�c efficiency Poorer heat tolerance

Soluble expression

Enzyme expression

Cell lysis

Centrifugation Two-time buffer wash

Ac�ve aggregates

ThXylC

ThXylC-ELK

different contaminates


>95% purity >90% recovery Facile scheme Higher cataly�c efficiency Be�er heat tolerance

Page 32 of 32

Enhanced Purification Efficiency and Thermal Tolerance of

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