Identification and characterization of the first
β-1,3-D-xylosidase from a gram-positivebacterium, Streptomyces sp. SWU10
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言語: English
出版者:
公開日: 2018-12-12
キーワード (Ja):
キーワード (En):作成者: Phuengmaung, Pornpimol, Fujiwara, Daisuke,
Sukhumsirichart, Wasana, Sakamoto, Tatsuji
メールアドレス:所属:

URL

http://hdl.handle.net/10466/16165

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Identification and characterization of the first β-1,3-D-xylosidase from a gram-positive

2bacterium, Streptomyces sp. SWU10

3
4

Pornpimol Phuengmaunga, Daisuke Fujiwarab, Wasana Sukhumsiricharta,*, Tatsuji

5

Sakamotoc,**

6
7

a

8

Sukhumvit 23, Bangkok 10110, Thailand.

9bDepartment of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114

Department of Biological Science, Graduate School of Science, Osaka Prefecture

10

University, Sakai, Osaka 599-8531, Japan.

11

c

12

Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.

Division of Applied Life Sciences, Graduate School of Life and Environmental

13
14

Corresponding author:

15*Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University.

16

114 Sukhumvit 23, Wattana district, Bangkok 10110, Thailand; Tel.: +66 2 2602 122;

17

Fax: +66 2 6495 334.

18**Division of Applied Life Sciences, Graduate School of Life and Environmental

19

Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan; Tel.: +81 72 254

209456; Fax: +81 72 254 9921.

21
22

E-mail addresses: pphuengmaung@gmail.com (P. Phuengmaung),

23

fujiwara@b.s.osakafu-u.ac.jp (D. Fujiwara), wasanas54@gmail.com (W.

24

Sukhumsirichart), sakamoto@biochem.osakafu-u.ac.jp (T. Sakamoto).

1

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Abbreviations:26

GH, glycoside hydrolase; HPAEC, high-performance anion-exchange chromatography;

27

KPB, potassium phosphate buffer; MALDI-TOF MS, matrix-assisted laser desorption

28

ionization-time of flight mass spectrometry; pNPA, p-nitrophenyl-α-L-

29arabinofuranoside; pNPX, p-nitrophenyl-β-D-xylopyranoside; XOS,

30

xylooligosaccharide; Xyl, xylose; Xyl2, xylobiose; Xyl3, xylotriose; Xyl4, xylotetraose.

31

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Abstract: In previous reports, we characterized four endo-xylanases produced by

33

Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To

34

obtain a set of enzymes to achieve complete xylan degradation, a β-D-xylosidase gene35

was cloned and expressed in Escherichia coli, and the recombinant protein, named

36

rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not

37

contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A

38

was revealed to contain a Glyco_hydro_43 domain and possess the three conserved

39

amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass

40

of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60

41

kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40 °C. The

42

enzyme was stable up to 40 °C over a wide pH range (3.1 to 8.9). rSWU43A activity

43

was enhanced by Fe2+ and Mn2+ and inhibited by various metals (Ag+, Cd2+, Co2+, Cu2+,44

Hg2+, Ni2+, and Zn2+), D-xylose, and L-arabinose. rSWU43A showed activity on p-

45

nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside substrates,46

with specific activities of 0.09 and 0.06 U/mg, respectively, but not on any xylosidic or

47

arabinosidic polymers. rSWU43A efficiently degraded β-1,3-xylooligosaccharides to48

produce xylose but showed little activity towards β-1,4-xylobiose, with specific49

activities of 1.33 and 0.003 U/mg, respectively. These results demonstrate that50

SWU43A is a β-1,3-D-xylosidase (EC 3.2.1.72), which to date has only been described3

51

in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first52

β-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a

53

specific tool for the structural elucidation and production of xylose from β-1,3-xylan in54

seaweed cell walls.

55
56

Keywords:

Streptomyces57

β-1,3-D-Xylosidase

58

Cloning59

Glycoside hydrolase family 43

60

Seaweed

61

4

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1. Introduction

63
64

Several biotechnological applications of Streptomyces have been reported, such65

as the production of hemicellulolytic enzymes that are tolerant to wide pH and66

temperature ranges and have unique biochemical characterizations [1]. Therefore,

67

Streptomyces species have been extensively studied for industrial applications in food68

and beverage industries, pharmaceuticals, and the pulp and paper industry, as well as for69

medical trials.

70

Xylans are important hemicellulosic polysaccharides that have diverse chemical

71

structures. In the cell wall of terrestrial plants, the backbone is composed of β-1,4-xylan,

72

which is sometimes substituted with arabinose, glucuronic acid, and acetic acid73

depending on the plant source. Their chemical structures that include arabinoxylan,

74

glucuronoarabinoxylan, and acetylated glucuronoxylan, and their degrading-enzymes

75

that include β-1,4-endo-xylanases (EC 3.2.1.8), β-xylosidases (EC 3.2.1.37), α-L-

76

arabinofuranosidases (EC 3.2.1.55), α-D-glucuronidases (EC 3.2.1.139), and77

acetylxylan esterases (EC 3.1.1.6), have been well studied thus far [2, 3]. In contrast, β-78

1,3-xylan is only found as the main cell wall component of red algae of Bangia sp. [4]79and Porphyra sp. [5], and green algae of Caulerpa sp. and Bryopsis sp. [5]. The β-1,3-80xylan structure of a marine green algae, Caulerpa lentillifera, was studied and its

81monosaccharides consist of xylose and glucose with contents of 97.42% and 2.58%,

82

respectively [6]. This structure was also found in algae species including Caulerpa

83brachypus [7, 8], Caulerpa filiformis [9], and Caulerpa racemosa var. laetevirens [10].

84In addition, a small amount of galactose is also presented in C. lentillifera. [11].

85

Because almost all of the β-1,3-xylan structural elements are β-1,3-glycosidic linkages

5

86of D-xyloses, endo-β-1,3-xylanases (EC 3.2.1.32) and β-1,3-xylosidase (EC 3.2.1.72)

87

are two focal enzymes for β-1,3-xylan degradation. To date, six endo-β-1,3-xylanases

88

from Alcaligenes sp. XY-234 [12], Aspergillus terreus A-07 [13], Pseudomonas sp. PT-89

5 [14], Thermotoga neapolitana DSM 4359 [15], Vibrio sp. AX-4 [16, 17], and Vibrio

90

sp. XY-214 [18, 19], and one β-1,3-xylosidase (XloA) from Vibrio sp. XY-214 [20]91

have been studied with regard to purification and characterization, whereas the other92

enzymes such as glucosidases and galactosidases for β-1,3-xylan degradation have not93

been studied yet.

94

We have previously isolated Streptomyces sp. SWU10 (GenBank accession no.

95

AB604027) from rice straw and characterized four endo-β-1,4-xylanases (XynSW1 [21],96

XynSW2A [22], XynSW2B [22], and XynSW3 [23]), indicating that this strain has a97

potent ability to degrade xylans. Therefore, we tried to determine the genes encoding β-

98xylosidases from Streptomyces sp. SWU10 to obtain a set of enzymes capable of99

completely degrading xylans. The amino acid sequences of the above endo-xylanases

100

show the highest similarities to those from Streptomyces sp. CCM_MD2014 (GenBank

101

accession no. CP009754), as determined using the NCBI BLAST program

102(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Because the genomic sequence of Streptomyces

103

sp. CCM_MD2014 is available [24], the sequence was used to clone the β-xylosidase

104

genes from Streptomyces sp. SWU10. β-Xylosidases are classified into glycoside105

hydrolase (GH) families 1, 3, 30, 39, 43, 51, 52, 54, 116, and 120 in the CAZy database106(http://www.cazy.org/Glycoside-Hydrolases.html). Streptomyces sp. CCM_MD2014

107

possesses five genes for GH1, eight for GH3, one for GH30, four for GH43, and two for

108

GH51 (http://www.cazy.org/b3848.html). These GH families include various enzymes

109

with different catalytic activities, and not just β-xylosidases. Thus, the deduced amino

6

110

acid sequences of these 20 genes were analyzed using the NCBI BLAST program,111

indicating that the 16 proteins of GH1, GH3, GH30, and GH51 were anticipated not to112

encode β-xylosidase. Among the four proteins belonging to GH43, we selected a113

putative β-xylosidase (accession no. AIV32177.1) that showed high identity to amino

114

acid sequences of β-xylosidase from Streptomyces sp. Here, we report the cloning and115

overexpression of the β-xylosidase gene (swu43A) of Streptomyces sp. SWU10 in116

Escherichia coli, and the biochemical characterization of the recombinant enzyme

117(rSWU43A).

118
119

2. Materials and methods

120
121

2.1. Reagents

122
123

Tks Gflex DNA polymerase and E. coli DH5α cells were obtained from Takara

124

Bio (Shiga, Japan). Restriction endonucleases were purchased from New England

125

Biolabs (Ipswich, MA, USA). E. coli BL21 (DE3) cells and the pET22b vector were126

obtained from Novagen (Darmstadt, Germany). The HisTrap HP affinity

127

chromatography column and Superdex 200 Increase 10/300 GL column were purchased

128

from GE Healthcare UK (Little Chalfont, Buckinghamshire, UK). All other chemicals

129

were obtained from Wako Pure Chemical Industries (Osaka, Japan) unless otherwise

130

stated and were of certified reagent grade.

131
132

2.2. Gene amplification

133

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The full-length of the swu43A gene was amplified from the genomic DNA of135

Streptomyces sp. SWU10 using two primers, SWU43AFw

136(AAGGAGATATACATATGGTGCGCATGTACGACA) and SWU43ARv

137(GGTGGTGGTGCTCGAGGAGCCGCATACCTTCCT), designed from the nucleotide

138

sequence of the GH43 gene (GenBank accession no. AIV32177.1) of Streptomyces sp.

139

CCM_MD2014. These primers contain NdeI and XhoI recognition sites (underlined),140

respectively. PCR was performed using Tks Gflex DNA polymerase for 35 cycles at

141

98 °C for 10 s, 63 °C for 15 s, and 68 °C for 90 s. The nucleotide sequence of swu43A142

was determined and submitted to the DDBJ/EMBL/GenBank and is available under the143

accession number LC322286.

144
145

2.3. Gene cloning in an expression vector and synthesis of rSWU43A

146
147

The purified PCR fragment was ligated into the pET22b vector using the In-

148

fusion HD Cloning Kit (Takara Bio) and transformed into E. coli DH5α and BL21

149

(DE3) competent cells for gene cloning and expression, respectively. For rSWU43A

150

production, 10% of an overnight culture of bacterial transformants containing the151

swu43A-pET22b construct was inoculated into 100 mL of LB medium with 50 µg/mL

152

ampicillin and cultured at 37 °C for 1 h. Next, 0.01 mM isopropyl-β-153

thiogalactopyranoside was added, and incubation was continued at 15 °C for 4 days.

154

Cells obtained from 100 mL of the culture broth were harvested by centrifugation,

155

resuspended in 20 mM potassium phosphate buffer (KPB; pH 8.0), and lysed by156

sonication in ice. Next, the cell-free extract was centrifuged and applied onto a HisTrap

157

HP column equilibrated with 20 mM imidazole and 500 mM NaCl in 20 mM KPB (pH

8

158

8.0). Bound proteins were eluted using a linear gradient of imidazole (60 mL, from 0 to

159

500 mM) in the same buffer.

160
161

2.4. Substrates

162
163

α-1,5-L-Arabinobiose, sugar beet arabinan, sugar beet debranched arabinan, and164

wheat arabinoxylan (low viscosity) were obtained from Megazyme International Ireland

165(Wicklow, Ireland). Oat spelt xylan, birch wood xylan, p-nitrophenyl-α-L-

166

arabinofuranoside (pNPA), and p-nitrophenyl-β-D-xylopyranoside (pNPX) were167

purchased from Sigma-Aldrich (St. Louis, MO, USA). β-1,4-Xylobiose (β-1,4-Xyl2)

168

was kindly provided by Dr. S. Kitamura (Osaka Prefecture University, Japan).

169

β-1,3-Xylooligosaccharides (XOSs) were prepared by mild acid hydrolysis of β-170

1,3-xylan isolated from the green alga Caulerpa lentillifera with H2SO4 (pH 2.4) as

171

described previously [12]. The hydrolysates were applied onto an activated charcoal

172

column (1.5 × 10 cm) equilibrated with water. β-1,3-Xyl2 and the mixture of Xyl2,

173xylotriose (Xyl3), and xylotetraose (Xyl4) were eluted with 30 % and 60 % ethanol,

174

respectively.

175
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2.5. Enzymatic assays

177
178

Standard assays for enzymatic activity of β-D-xylosidase and α-L-

179

arabinofuranosidase were performed using the chromogenic substrates pNPX and pNPA,

180

respectively, at a concentration of 0.02 % in 20 mM KPB (pH 6.0). After incubation at

181

45 °C for 10 min, 0.2 M Na2CO3 was added to stop the reaction, and the absorbance at

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182

420 nm was determined using a spectrophotometer. An enzyme unit is defined as the183

amount of enzyme producing 1 µmol of pNP per minute.

184

The β-1,3- or 1,4-xylosidase activity was determined using 0.02% β-1,3-Xyl2 or

185

β-1,4-Xyl2 in 20 mM KPB (pH 6.0) as the substrate. After 30 min of incubation at

186

45 °C, the enzymatic mixture was boiled at 100°C to stop the reaction and analyzed by187

high-performance anion-exchange chromatography (HPAEC) under the conditions

188

described below. One unit of enzyme is defined as the amount of enzyme producing 1

189µmol of D-Xyl in 1 minute.

190

The effect of metal ions on enzyme activity was assayed in the presence of 1

191

mM Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, K+, Mn2+, Na+, Ni2+, or Zn2+. In addition,192

the effect of EDTA, an ion chelator, was examined. Tolerance of enzyme activity to D-

193

Xyl and L-arabinose in concentrations ranging from 0.5 to 100 mM was assessed using194

the standard assay method.

195

Polysaccharide degradation activity was measured by incubating rSWU43A (5196mU) with 0.2 mL of 0.1 % substrate in 20 mM KPB (pH 6.0) at 45 °C for 16 h. The

197

reaction products were analyzed using HPAEC.

198
199

2.6. HPAEC analysis

200
201

HPAEC was performed using a Dionex DXc-500 system (Dionex, Sunnyvale,

202

CA, USA) with a CarboPac PA-1 column (4 × 250 mm; Dionex). Sugars were eluted at203

a flow rate of 1 mL/min with 100 mM NaOH for 5 min, followed by a linear gradient204

from 0 to 450 mM sodium acetate in 100 mM NaOH for 30 min. The effluent was

205

monitored with pulsed amperometric detection.

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207

2.7. Effect of pH and temperature on enzymatic activity and stability

208
209

Optimum pH was determined by monitoring enzyme activity in buffer pH

210

ranging from 1.8 to 11.6 at 45 °C for 10 min using pNPX as a substrate. Various buffers

211

were used: 20 mM glycine-HCl (pH 1.8, 2.5, and 3.1), 20 mM acetate buffer (pH 4.1

212

and 5.1), 20 mM KPB (pH 6.5, 7.1, and 8.2), 20 mM glycine-NaOH (pH 8.9 and 9.9),213

and 20 mM Na2HPO4-NaOH (pH 11.1 and 11.6). pH stability was determined by214

incubating the enzyme in 200 mM of the above buffers at 4 °C for 16 h and measuring

215

the remaining enzymatic activity with the standard assay. The optimum temperature216

was determined in 20 mM KPB (pH 6.0) in a temperature range of 20 °C to 70 °C for217

10 min, and the thermal stability was determined by pre-incubating the enzyme without

218

substrate in 200 mM KPB (pH 6.0) in a temperature range of 20 °C to 70 °C for 1 h.

219

The remaining enzymatic activity was assayed as described above.

220
221

2.8. Concentration and molecular mass of proteins

222
223

Protein concentration was determined using the Coomassie-Plus-The Better

224

Bradford Assay Kit (Pierce Biotechnology, Rockford, IL, USA) using bovine serum

225

albumin as a standard, and the absorbance at 595 nm was determined using a Multiskan

226

GO Microplate spectrophotometer (Thermo Scientific). The molecular mass of the227

proteins was estimated using 10 % gel SDS-PAGE according to Laemmli [25]. The gel

228

was stained with Coomassie Brilliant Blue R-250 for protein visualization.

229

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2.9. Matrix-assisted laser desorption ionization-time of flight mass spectrometry

231
232

The β-1,3-XOSs were mixed with the matrix solution prepared by dissolving 10233

mg of 2,5-dihydroxybenzoic acid in 1 mL of 20 % ethanol and were analyzed using an

234

Autoflex II matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)

235

mass spectrometer (Bruker Daltonics, Bremen, Germany) as previously reported [26].

236
237

3. Results and discussion

238
239

3.1. swu43A gene cloning and nucleotide sequence analysis

240
241

The swu43A gene was PCR-amplified from the genomic DNA of Streptomyces242

sp. SWU10 using primers SWU43AFw and SWU43ARv, generating a 1,566 bp

243

fragment with 71 % GC content and encoding a 522-amino acid protein. No N-terminal

244

signal peptide was found in the SWU43A protein sequence using the SignalP prediction

245

program (http://www.cbs.dtu.dk/services/SignalP), indicating that it is an intracellular246

enzyme. These results are consistent with most reports of bacterial xylosidases [27, 28].

247

SWU43A has a calculated molecular mass and isoelectric point of 56,835 and 5.49,

248

respectively. The deduced amino acid sequence of SWU43A is highly similar to those249

of an undefined protein (GenBank accession no. AIV32177.1: 99 %) of Streptomyces sp.

250

CCM_MD2014 [24], GH family 43 protein (GenBank accession no. WP_087792600.1:

25195 %) of Streptomyces sp. CS159 [29], and xylosidase/arabinofuranosidase (GenBank

252

accession no. EHN79169.1: 95 %) of Streptomyces coelicoflavus ZG0656 [30] (Fig. 1).

253

SWU43A was predicted to contain a Glyco_hydro_43 (PF04616) domain at positions 4

12

254

to 282, as determined by Pfam program analysis (http://pfam.xfam.org). The catalytic

255

residues are perfectly conserved in the GH43 proteins, consisting of one glutamic acid256

and two aspartic acids [31]. On the basis of X-ray crystallographic studies with the257

GH43 β-xylosidase (XynB3) of Geobacillus stearothermophilus T-6, Asp15, Glu187,

258

and Asp128 act as the general base, general acid, and modulator of the pKa, respectively

259[32]. These amino acid residues are also conserved in SWU43A: Asp15, Glu174, and260

Asp123 (Fig. 1). β-1,3-xylosidase has only been reported for XloA from Vibrio sp. XY-

261

214 and belongs to GH43. The catalytic residues are also conserved at Asp16, Glu189,

262

and Asp130 in XloA [20] (Fig. 1).

263
264

3.2. swu43A expression in E. coli and purification of the recombinant enzyme

265

To obtain a recombinant protein, a DNA fragment containing the swu43A gene266

was ligated into the pET22b vector and transformed into E. coli BL21 (DE3) cells. The

267

recombinant fusion protein was designed to contain a histidine tag at the C-terminal of268

SWU43A. rSWU43A was purified from cell-free extracts in one step using a HisTrap

269

HP column, which led to a 2.7-fold enrichment with a final yield of 57 % (13.8 mg) and270

specific activity of 0.09 U/mg when 0.02 % pNPX was used as the substrate (Table 1).

271

rSWU43A was also active on pNPA with a specific activity of 0.06 U/mg.

272
273

3.3. General characterization of rSWU43A

274
275

Determination of the molecular mass of purified rSWU43A using SDS-PAGE

276

showed a strong signal corresponding to a 60 kDa protein (Fig. 2), which was in good277

agreement with the calculated molecular mass of SWU43A (56,835). In addition, gel

13

278

filtration chromatography on a Superdex 200 Increase 10/300 GL column showed the279

native rSWU43A enzyme molecular mass to be 55 kDa, which indicated that rSWU43A

280

is a monomeric protein (data not shown). Similarly, a bifunctional281

arabinofuranosidase/xylosidase (deAX) from a compost starter mixture has been

282

described to be a monomer [27], whereas XynB3 of G. stearothermophilus [32] and283

XloA of Vibrio sp. XY-214 [20] were reported to be tetramers.

284

rSWU43A showed its optimal activity at pH 6.5 and 40 °C (Fig. 3). The

285

enzymatic activity remained above 75 % of initial levels after incubation in a pH range286

of 3.1 to 8.9 in 200 mM of various buffers at 4 °C for 16 h. rSWU43A was stable at287

temperatures ranging from 20 °C to 40 °C (Fig. 3). The GH43 xylosidases of288Enterobacter sp. [33], Lactobacillus brevis [34], Lactobacillus rossiae DSM 15814T

289[35], and Penicillium purpurogenum [36] have an optimum pH and temperature of290

approximately pH 6 and 40 °C, respectively, while the Selenomonas ruminantium

291

enzyme has an optimal temperature of 40 °C but a lower optimum pH of 5.3 [37].

292rSWU43A activity was significantly enhanced by addition of Fe2+ and Mn2+. In

293

contrast, Ag+, Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+ led to reduced activity using both294

pNPX and pNPA as substrates, whereas Ca2+ inhibited only arabinofuranosidase295

activity. K+, Na+, and EDTA did not affect enzymatic activity (Table 2). Activation and296

inhibition of bacterial β-xylosidases that belong to GH43 occur when different metal

297

cations are exogenously added (Table 3). Similar to rSWU43A, β-xylosidase activities298

are often activated in the presence of Mn2+ [39, 41−45, 47, 48]. However, Mn2+ has

299

been demonstrated to be irrelevant to the structural or the catalytic role in GH43

300

enzymes [42]. Most enzymes are inhibited by various kinds of divalent cations and by

301

Ag+. Some GH43 β-xylosidases can be activated by Ca2+, such as CoXyl43A from a

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302

compost microbial metagenome [39], RS223-BX from metagenomic library [42], and303

β-xylosidase from Paecilomyces thermophila [44]. Ca2+ has been demonstrated to304

tightly bind to some GH43 enzymes by crystal structure analysis [49]. The presence of305

Ca2+ has an important role in the structure and/or the catalytic mechanism of enzymes

306[50, 51] such as RS223-BX, which is the first GH43 β-xylosidase in which Ca2+ was at307

the back of the active site [52]. Neither Ca2+ nor EDTA affected the enzyme activity of308

rSWU43A (Table 2), suggesting that the structure of rSWU43A may be different from309

those of the above-described Ca2+-dependent enzymes.

310

Evaluation of the tolerance of rSWU43A to Xyl and arabinose at 0.5 to 100 mM

311

revealed that enzymatic activity retained 41 % and 46 % of the initial levels when in the312

presence of 100 mM D-Xyl and L-arabinose, respectively. These results are similar to313

those described for the GH43 β-xylosidase of Paecilomyces thermophila [44]. Kinetic

314

parameters of rSWU43A were calculated using standard Michaelis-Menten kinetics

315with various concentrations of pNPX as a substrate at 40 °C for 10 min, and Km and316

Vmax corresponded to 0.108 mM and 0.088 µmol/min/mg, respectively.

317
318

3.4. Substrate specificity of rSWU43A

319
320

The substrate specificity of the recombinant enzyme was evaluated. Polymeric321

substrates such as sugar beet arabinan, sugar beet debranched arabinan, oat spelt xylan,322

birch wood xylan, and wheat arabinoxylan were resistant to the action of rSWU43A,

323

even after a prolonged 16 h reaction.

324
325

To evaluate the preference of rSWU43A for β-1,3-XOSs and β-1,4-XOSs, theformer was prepared by mild acid hydrolysis of β-1,3-xylan [11]. The acid hydrolysates

15

326

were applied to an activated charcoal column and separated into two fractions (1 and 2)

327

that were eluted with 30 % and 60 % ethanol, respectively. HPAEC analyses of328

fractions 1 and 2 revealed major peaks eluted at around 13 min and 16 min, respectively

329(Fig. 4a and 4b). Fraction 2 was then analyzed by MALDI-TOF MS, which exhibited

330

major peaks at m/z 305.3, 437.2, and 569.2 (Fig. 4c). These observed compounds

331

corresponded to sodiated molecules [M + Na]+. Thus, fraction 2 was shown to contain332

β-1,3-Xyl2, β-1,3-Xyl3, and β-1,3-Xyl4. Comparison of the HPAEC chromatograms

333

shown in Figures 4a and 4b indicated that fraction 1 was mostly composed of β-1,3-334

Xyl2. These fractions were used for enzymatic assays. Figures 5a and 5b show HPAEC

335

chromatograms of reaction products of fraction 1 (β-1,3-Xyl2) and β-1,4-Xyl2 with

336

rSWU43A. The enzyme completely degraded β-1,3-Xyl2 to Xyl in 1 h, whereas a small

337

amount of Xyl was released even after incubation for 3 h when β-1,4-Xyl2 was used as

338

the substrate. The specific activities of rSWU43A towards β-1,3-Xyl2 and β-1,4-Xyl2339

were 1.33 and 0.003 U/mg, respectively. The activity of the enzyme on β-1,3-Xyl2 was340

15 times higher than that on pNPX. Furthermore, rSWU43A was able to degrade β-1,3-341

Xyl3 and β-1,3-Xyl4 to produce Xyl in a 1 h reaction (Fig. 5c). In contrast, α-1,5-L-

342

arabinobiose was not degraded even in the presence of an excess of the rSWU43A

343

enzyme. These results demonstrated that β-1,3-XOSs are the most preferred substrates344

for rSWU43A and that the enzyme can be deduced to be β-1,3-xylosidase.

345

Many microorganisms produce β-1,4-xylosidases, which have been extensively

346

studied, whereas β-1,3-xylosidase has only been reported in the marine bacterium

347

Vibrio sp. strain XY-214 (XloA) [20]. Although both SWU43A and XloA belong to

348

GH43, they showed only 35 % identity in a 442-amino acid overlap. XloA releases a

16

349

small amount of Xyl from β-1,4-XOSs, while completely hydrolyzing β-1,3-XOSs.

350

However, it is not active on pNPA, in contrast to rSWU43A, indicating that the351

enzymes have a somewhat different mechanism for substrate recognition.

352

Vibrio sp. XY-214 possesses not only the β-1,3-xylosidase gene but also the353

endo-β-1,3-xylanase gene [18, 19] to achieve efficient degradation of β-1,3-xylan. To

354date, all of the characterized bacterial endo-β-1,3-xylanases are classified into GH26355

[15]. However, no GH26 gene was found in the genome of Streptomyces sp.

356

CCM_MD2014, indicating that Streptomyces sp. SWU10 does not possess an endo-β-357

1,3-xylanase gene. Although it remains possible that the Streptomyces sp. SWU10 strain

358

has an endo-β-1,3-xylanase gene belonging to other GH families, it is unclear how the359

Streptomyces sp. SWU10 strain degrades β-1,3-xylan without an endo-β-1,3-xylanase.

360

Streptomyces are one of the best-known microorganisms with potential abilities for361

adaptation to very different environments. Some Streptomyces found in marine

362

environments were related to terrestrial Streptomyces according to a 16S rRNA gene363

analysis [53]. Although marine Streptomyces have been suggested to be related to364

terrestrial Streptomyces, some genes are only found in marine or terrestrial

365

Streptomyces, indicating that evolution has occurred [53]. Although Streptomyces sp.

366

SWU10 was isolated from rice straw and is not a marine bacterium, the fact that this367

strain possesses the β-1,3-xylosidase gene suggests that Streptomyces sp. SWU10 is the368intermediate in the evolution from terrestrial to marine environments. Although369

Streptomyces sp. SWU10 is a thermotolerant microorganism, the temperature profile of370

the enzyme was ordinary. Thus, thermotolerance may not be required in the371

environment in which SWU43A is active.

372

17

373

3.5. Conclusions

374
375

We have identified the first xylosidase from the Streptomyces sp. SWU10 strain.

376

Interestingly, rSWU43A revealed unique characteristics including a preference for β-377

1,3-XOSs over β-1,4-XOSs and also activity toward both the synthetic pNPX and

378

pNPA substrates. Only one other β-1,3-xylosidase (XloA) has been reported to date379[20]. Although both SWU43A and XloA belong to the GH43 family and show three380

conserved catalytic residues, their amino acid identity is only 35 %. In addition, XloA

381

does not show activity for pNPA, in contrast to SWU43A. Consequently, SWU43A of

382

Streptomyces sp. SWU10 has different properties from XloA and is the first β-1,3-383

xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool384

for the structural elucidation and production of xylose from β-1,3-xylan in seaweed cell

385

walls.

386
387

Funding388
389This work was supported by grants from The Royal Golden Jubilee Ph.D.

390

Program of Thailand Research Fund [PHD/0209/2556] and Srinakharinwirot University

391[366/2559]. This work was performed under the Core-to-Core Program, which was

392

financially supported by the National Research Council of Thailand (NRCT) and Japan

393

Society for the Promotion of Science (JSPS). The funding agencies played no role in the394

study design; collection, analysis, and interpretation of data; writing of the manuscript;

395

or in the decision to submit the article for publication.

396

18

397

Declaration of conflicts of interest

398
399

The authors declare that they have no conflicts of interest.

400
401

Acknowledgments

402
403

The authors thank Shinichi Kitamura, Osaka Prefecture University, for404

providing β-1,4-Xyl2. We would like to thank Editage (http://www.editage.jp) for

405

English language editing.

406
407

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26

585

Figure legends

586
587

Fig. 1. Amino acid sequence alignment of SWU43A and other GH43 enzymes.

588

Streptomyces sp. CCM_MD 2014: GH protein (AIV32177.1), Streptomyces sp. CS159:

589

GH family 43 protein (WP_087792600.1), Streptomyces coelicoflavus ZG0656:590

xylosidase/arabinofuranosidase (EHN79169.1), Geobacillus stearothermophilus

591(XynB3): GH43 β-xylosidase (AAT98625.1), Vibrio sp. XY-214 (XloA): β-1,3-592

xylosidase (BAF98235.1). Identical residues are shown in white font on a black593

background. More than four identical residues are shown in black font on a gray594

background. Catalytic residues are indicated with asterisks (*).

595
596

Fig. 2. SDS-PAGE of rSWU43A. Lane 1, protein markers; lane 2, cell extracts of597

Escherichia coli harboring swu43A-pET22b; lane 3, rSWU43A. Proteins were598

visualized with Coomassie Brilliant Blue R-250 staining.

599
600

Fig. 3. Effects of pH (a) and temperature (b) on the activity (solid lines) and stability

601(dotted lines) of rSWU43A. Experimental conditions are described in the text.

602
603

Fig. 4. High-performance anion-exchange chromatography analyses of fractions 1 (a)604

and 2 (b) and matrix-assisted laser desorption ionization-time of flight mass605

spectrometry analysis of fraction 2 (c). Experimental conditions are described in the text.

606
607

Fig. 5. High-performance anion-exchange chromatography (HPAEC) analysis of β-1,3-608

Xyl2 (a), β-1,4-Xyl2 (b), and β-1,3-xylooligosaccharides (c) reaction products with

27

609

rSWU43A. Reaction mixtures containing 0.1 mL of 4 µg/mL substrate in 20 mM

610

potassium phosphate buffer (pH 6.0) and 5 mU of rSWU43A were incubated at 45 °C

611

for the time periods shown, followed by analysis of the reaction products by HPAEC.

612

HPAEC conditions are described in the text.

28

Table 1. Purification of rSWU43A enzyme.
Purification step
Cell-free extract
HisTrap HP

Protein (mg)
65.0
13.8

Activity (U)
2.15
1.22

Enzyme activity was assayed using pNPX as the substrate.

Specific activity (U/mg)
0.033
0.088

Purification (fold)1
2.7

Yield (%)
100
57

Table 2. Effect of metal ions and EDTA on enzyme activity.
Relative activity (%)
pNP-xylopyranoside
pNP-arabinofuranoside
None
100
100
15 ± 019 ± 0AgNO3103 ± 365 ± 1
CaCl2
22 ± 030 ± 1
CdCl2
51 ± 023 ± 3CoCl2
32 ± 119 ± 2CuCl2139 ± 1113 ± 2FeSO4
16 ± 217 ± 3HgCl2
KCl
98 ± 083 ± 3154 ± 1216 ± 0
MnCl2
NaCl
102 ± 284 ± 140 ± 118 ± 0NiCl2
43 ± 126 ± 0ZnSO4EDTA
97 ± 398 ± 2* Experiments were performed in triplicate.
CompoundCa2+, Mn2+

Ag+, Cu2+, Ni2+, Zn2+

Cu2+, Fe2+, Zn2+
Ag+, Hg2+

Ag+, Zn2+

Inhibition

[43]

[42]

[41]

[39]
[40]

[38]

Reference

Table 3. Effect of metal cations on β-xylosidase and xylosidase/arabinofuranosidase activity in the literature.

Mn2+

Cu2+, Zn2+

[44]

Xylosidase/arabinofuranosidase (CoXyl43A) A compost microbial metagenome
β-Xylosidase
Geobacillus stearothermophilus T-6
Xylosidase/arabinofuranosidase (xyl43A and
Humicola insolens
xyl43B)
Xylosidase/arabinofuranosidase (RS223-BX) Metagenomic library

Activation

Ca2+, Co2+, Fe2+, Mn2+, Ni2+

Ca2+, Ni2+, Zn2+

Resource

Co2+, Mn2+

Ag+, Co2+, Cu2+, Na+, Zn2+

EnzymeA mixed population of microorganisms was isolated
from rumen fluid
Ca2+, Fe2+, Mn2+

Bifidobacterium animalis subsp. lactis BB-12

Xylosidase/arabinofuranosidase (RUM630BX)
Paecilomyces thermophila[45]

Xylosidase/arabinofuranosidase (BXA43)

β-Xylosidase

Cu2+, Fe2+, EDTA

[46]

Ca2+, Mn2+, Ni2+

Co2+, Cu2+, Fe2+, Mn2+, Ni2+, Zn2+

[47]

Paenibacillus woosongensis

Co2+, Cu2+, Ni2+

[48]

Xylosidase/arabinofuranosidase (XylC)

Ca2+, Mn2+

Cu2+, Hg2+, Zn2+

Phanerochaete chrysosporium

Co2+, Ca2+, Mn2+

Xylosidase/arabinofuranosidase

Talaromyces thermophiles

Xylosidase/arabinofuranosidase (XylM1989) Synthetic metagenomics approach
β-Xylosidase

Table 4. rSWU43A tolerance to xylose and arabinose.
SubstratepNP-xylopyranoside

Concentration of sugar

None
0.5 mM of xylose
1 mM of xylose
10 mM of xylose
50 mM of xylose
100 mM of xylose
pNP-arabinofuranosideNone
0.5 mM of arabinose
1 mM of arabinose
10 mM of arabinose
50 mM of arabinose
100 mM of arabinose* Experiments were performed in triplicate.

Relative activity (%)
100
86 ± 166 ± 060 ± 053 ± 141 ± 2100
100 ± 176 ± 073 ± 151 ± 146 ± 1

*

*

*

Fig 1

(kDa)

85
70
60
50
40

1

2

3

Fig 2

Relative and remaining activity (%)

(a)

100
75

100
75

12

50

10

50

8

25

6

25

4

0

2

0
0

pH

10

(b)

20

30

40

50

Temperature (°C )Fig 3

6070

β-1,3-Xyl3β-1,3-Xyl4

12
16
18
14
Retention time (min)

20

400

500

600 (m/z)

569.233(c)

300

305.323

(b)

(a)

10

β-1,3-Xyl2437.150

Fig 4

6

Xyl

8

(a)

1h

20

Before reaction

10 12 14 16 18
Retention time (min)β-1,3-Xyl2

6

Xyl

8

(b)

3h

20

Before reaction

10 12 14 16 18
Retention time (min)β-1,4-Xyl2

6

8

(c)

β-1,3-Xyl2β-1,3-Xyl3β-1,3-Xyl4

10 12 14 16 18
Retention time (min)Before reaction

Xyl

1h

20

Fig 5