Identification and characterization of GH62
bacterial α-L-arabinofuranosidase fromthermotolerant Streptomyces sp. SWU10 that
preferentially degrades branched
L-arabinofuranoses in wheat arabinoxylan
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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, Kunishige, Yuika,
Sukhumsirichart, Wasana, Sakamoto, Tatsuji
メールアドレス:所属:

URL

http://hdl.handle.net/10466/16164

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Identification and characterization of GH62 bacterial α-L-arabinofuranosidase from2

thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched L-

3arabinofuranoses in wheat arabinoxylan

45Pornpimol Phuengmaunga, Yuika Kunishigeb, Wasana Sukhumsiricharta,*, Tatsuji

6

Sakamotob,**

7
8

a

9

Sukhumvit 23, Bangkok 10110, Thailand.

Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114

10

b

11

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

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

12
13

Corresponding author:

14*Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114

15

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

166495 334.

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

18

Osaka Prefecture University, Sakai, Osaka 599-8531, Japan; Tel.: +81 72 254 9456; Fax: +81

19

72 254 9921.

20
21

E-mail addresses: pphuengmaung@gmail.com (P. Phuengmaung), swc02023@edu.osakafu-

22

u.ac.jp (Y. Kunishige), Wasanas54@gmail.com (W. Sukhumsirichart),23

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

24
25

26

Abbreviations:27
28

AcB, sodium acetate buffer; AFase, α-L-arabinofuranosidase; AXH, arabinoxylan

29arabinofuranohydrolase; GH, glycoside hydrolase; HPAEC, high-performance anion-

30

exchange chromatography; IPTG, isopropyl-β-D-thiogalactopyranoside; KPB, potassium

31

phosphate buffer; NMR, nuclear magnetic resonance; Xyl2, xylobiose; Xyl3, xylotriose;

32

Xyl4, xylotetraose.

33

2

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Abstract

35
36

We previously described thermotolerant Streptomyces sp. SWU10, which produced four

37

endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan

38

degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the39

recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of40

SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-L-

41arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified

42

in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass

43

without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity

44

at pH 5.0 and 50°C, and more than 70% of its initial enzymatic activity remained after

45

incubation at pH 4.1–10.5, while SWUAbf62A lost all activity after 1 h at 60°C.

46

SWUAbf62A activity was stimulated by Ba2+, Ca2+, and Mn2+ and decreased by Ag+, Cu2+,

47

Fe2+, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-L-arabinofuranoside48

or p-nitrophenyl-β-D-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A49

had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg,

50

and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these51

results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using 1H-

52

NMR analysis, SWUAbf62A was found to release L-arabinofuranoses singly linked to O-3 of53

wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase

54(XynSW3) and α-L-arabinofuranosidase, which releases arabinose linked to O-3 of double-

55

substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A56

is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and57

can be applied in various industrial biotechnologies.

58

3

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60

Keywords:

Arabinoxylan arabinofuranohydrolase

61

Streptomyces

62

Glycoside hydrolase family 62

63

Enzyme regioselectivity

64

Synergistic action

65

4

66

1. Introduction

67
68

α-L-Arabinofuranosyl residues are widely distributed in components of plant cell69

walls, especially pectins and hemicelluloses such as arabinogalactan, arabinan, and70

arabinoxylan [1], and exist as side chains of these polymers. Sugar beet arabinan is a71

component of pectin with L-arabinoses linked via α-1,5-glycosidic bonds and attached at72

positions C-2 and/or C-3, forming branches [2]. Arabinoxylan is the hemicellulose portion

73

containing β-1,4-xylopyranose backbones singly and/or doubly attached with α-L-arabinosyl

74

residues at C-2 and/or C-3 [3, 4]. Arabinoxylan requires the synergistic action of several

75

enzymes for complete degradation. In general, endo-xylanases (EC 3.2.1.8) and β-76

xylosidases (EC 3.2.1.37) are key enzymes that hydrolyze the backbones of xylans to77xylooligosaccharides and the oligosaccharides from their non-reducing ends to xylose

78

monomers, respectively. α-L-Arabinofuranosidases (AFases; EC 3.2.1.55), acetylxylan

79

esterases (EC 3.1.1.6), and ferulic acid esterases (EC 3.1.1.73) are also important for the80

removal of substituents of arabinoxylan. The end products of arabinoxylan degradation are D-

81

xyloses and L-arabinoses, which have biotechnological applications in medical production,82

the food industry, bioethanol production, and animal feedstock, among others [5].

83AFases specifically degrade arabinose-containing carbohydrates, which are

84

categorized into three groups based on substrate specificity according to the Beldman

85

classification [2]. The first group releases L-arabinosyl residues from arabino-

86

oligosaccharides [6]. Interestingly, the AFase PcABF43B from Penicillium chrysogenum

87

preferentially hydrolyzes arabinosidic side chains in branched arabino-oligosaccharides [7],88so PcABF43B could be classified into the subtype of this group. The second group degrades89

L-arabinosidic linkages both on arabino-oligosaccharides and branched arabinans [8]. Both of90

these AFase groups are active on p-nitrophenyl-α-L-arabinofuranoside. The last group5

91

preferentially hydrolyzes L-arabinofuranose residues at the side-chain of arabinoxylan and are

92

named arabinoxylan arabinofuranohydrolases (AXHs) [9-11]. Furthermore, the recently

93

characterized P. chrysogenum AFase PcABF43A specifically debranches L-arabinosyl side94

chains in L-arabinan, and does not fit into any of the above three AFase groups [12]. In

95

addition, the three groups are classified into different glycoside hydrolase (GH) families. The

96

first and the second groups have often been observed in the GH51 and GH54 families,97

respectively, whereas the third group has correlated with GH43 and GH62 enzymes [2].

98

AFases with different modes of action play important roles in degradation of pectins and99

hemicelluloses by synergistic actions with various enzymes, demonstrating that they are good100

candidates for biorefinery applications and other biotechnological utilizations.

101

Arabinoxylan is one of the main component of rice straw, which is an abundant102

agricultural waste in Thailand. After pretreatment, the arabinoxylan can be readily

103hydrolyzed into renewable monosaccharides by hemicellulolytic enzymes. We previously

104

identified and characterized four endo-xylanases (XynSW1 [13], XynSW2A [14], XynSW2B

105[14], and XynSW3 [15]) from thermotolerant Streptomyces sp. SWU10 (GenBank accession

106

number AB604027), suggesting the possibility that this bacterial strain produces enzymes

107

capable of complete degradation of arabinoxylan. The NCBI BLAST program

108(http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed high identity in amino acid sequences among109

the above enzymes and Streptomyces sp. CCM_MD2014 (GenBank accession number

110

CP009754). AFases are currently categorized into six families, namely, GH2, GH3, GH43,111

GH51, GH54, and GH62. Analysis of amino acid sequences using the CAZy database112(http://www.cazy.org/Glycoside-Hydrolases.html) showed that Streptomyces sp.

113

CCM_MD2014 had two GH51 and one GH62 family gene encoded putative AFase. Among

114

three genes, we chose the putative AFase gene belonging to the GH62 family (GenBank

115

accession number AIV33730.1), because GH62 AFase enzymes are known to degrade

6

116

arabinoxylan. By contrast, most of the characterized GH51 AFases show specificity for117

arabino-oligosaccharides, as mentioned above. This study deals with the cloning and118

expression of the swu62A gene from Streptomyces sp. SWU10 and biochemical

119characterization of the recombinant enzyme (SWUAbf62A). In addition, the mode of action

120of SWUAbf62A and the synergistic action of SWUAbf62A, endo-xylanase (XynSW3) [15],121and AFase towards arabinoxylan are described.

122
123

2. Materials and methods

124
125

2.1. Reagents and substrates

126
127

Escherichia coli rosetta2 (DE3) competent cells and pET22b vectors were obtained

128

from Novagen (Darmstadt, Germany). LA Taq DNA polymerase with GC buffer, the In-

129

Fusion HD Cloning Kit, and E. coli DH5α cells were purchased from Takara Bio, Inc. (Shiga,

130Japan). Endonuclease restriction enzymes were obtained from New England Biolabs, Inc.

131

(Ipswich, MA, USA). The HisTrap HP affinity column and Superdex 75 10/300 GL column

132

were purchased from GE Healthcare UK, Ltd. (Little Chalfont, Buckinghamshire, UK).

133

Sugar beet arabinan, sugar beet debranched arabinan, wheat arabinoxylan (low viscosity),134

and AFase from Bifidobacterium sp. were obtained from Megazyme International Ireland,

135

Ltd. (Wicklow, Ireland). Birch wood xylan, p-nitrophenyl-α-L-arabinofuranoside, and p-

136

nitrophenyl-β-D-xylopyranoside were purchased from Sigma-Aldrich Co. (St. Louis, MO,

137

USA). All other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka,

138

Japan) unless otherwise stated and were certified reagent grade.

139
140

2.2. Cloning and sequencing of the swu62A gene

7

141
142

The swu62A gene with the putative signal sequence removed was amplified from143

genomic DNA of Streptomyces sp. SWU10 using the following primers designed from the144

nucleotide sequence of the GH62 gene in Streptomyces sp. CCM_MD2014 (GenBank

145

accession no. AIV33730.1): GH62-pET22-Fw infusion,146

AAGGAGATATACATATGGCCGGCAGYGGCGCCCTG; GH62-pET22-Rv infusion,

147

GGTGGTGGTGCTCGAGGCGCCGCAGGGTGAGGACTCC. NdeI and XhoI restriction

148

enzyme sites (underlined) were added to the forward and reverse primers, respectively. PCR

149

was carried out as follows: one cycle at 94°C for 3 min, followed by 35 cycles at 94°C for 30

150s, at 60°C for 30 s, and at 72°C for 90 s using LA Taq DNA polymerase. Purified swu62A151

was ligated into pET22b vectors using the In-Fusion HD Cloning Kit and transformed into E.

152

coli DH5α for gene sequencing. The nucleotide sequence of swu62A was reported to the153

DDBJ/EMBL/GenBank with accession number LC340444.

154
155

2.3. Expression of the swu62A gene and purification of SWUAbf62A

156
157

E. coli rosetta2 (DE3) was transformed with the swu62A-pET22b construct for gene158

expression. For SWUAbf62A production, 10% of the culture medium was inoculated into 2 L

159

of LB broth supplemented with 50 µg/mL ampicillin and 35 µg/mL chloramphenicol and160

cultured at 37°C for 1 h. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final

161

concentration of 0.01 mM and incubated at 15°C for 4 days. Cells were harvested from162

culture broth (2 L) by centrifugation and disrupted by sonication. After centrifugation, the163

supernatant was put onto a HisTrap HP column equilibrated with a buffer containing 20 mM

164

potassium phosphate buffer (KPB; pH 6.0), 20 mM imidazole, and 500 mM NaCl. Proteins165

were eluted with a linear gradient of imidazole (160 mL, from 0 to 250 mM) in the same

8

166

buffer. Fractions containing activity were collected and loaded onto a Superdex 75 column

167equilibrated with 0.1 M NaCl in 20 mM KPB (pH 6.0). Proteins were eluted by the same

168

buffer.

169
170

2.4. Enzyme assays

171
172

A standard assay for enzyme activity was performed using wheat arabinoxylan as the173

substrate. The reaction mixture containing 195 µL of 0.1% wheat arabinoxylan in 20 mM

174

sodium acetate buffer (AcB; pH 5.0) and 5 µL of enzyme was incubated at 50°C for 30 min.

175

Released reducing sugars were quantified by the Nelson-Somogyi method [16]. One unit of176

enzyme was defined as the amount of enzyme releasing 1 µmol of L-arabinose per minute.

177

Sensitivity of the enzyme to 14 cations and EDTA was examined by running the178

standard assay conditions in the presence of 1 mM compound. Substrate specificity towards

179

polysaccharides was assessed by incubating the reaction mixture containing 195 µL of 0.1%

180

substrate in 20 mM AcB (pH 5.0) and 5 mU of enzyme at 50°C for 1 h. In these experiments,181

the released arabinose was quantified by high-performance anion-exchange chromatography

182(HPAEC) as described below.

183
184

2.5. Conditions for HPAEC

185
186

Arabinose content was measured using a Dionex DXc-500 system (Dionex Co.,187

Sunnyvale, CA, USA) with a CarboPac PA-1 column (4 × 250 mm; Dionex). Sugars were188

eluted at a flow rate of 1 mL/min with 100 mM NaOH for 5 min followed by a linear gradient

189

from 0–450 mM of sodium acetate in 100 mM NaOH for 30 min. The effluent was monitored190

with pulsed amperometric detection.

9

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192

2.6. The sensitivity of SWUAbf62A

193
194

Each buffer containing 20 mM sodium acetate/HCl (pH 2.5, 2.9, and 3.2), 20 mM

195

AcB (pH 4.1 and 5.0), 20 mM KPB (pH 6.5, 7.1, and 8.2), 20 mM sodium

196

carbonate/bicarbonate (pH 9.1 and 10.5), and 20 mM Na2HPO4-NaOH (pH 11.1 and 11.6)

197

were mixed with 0.1% wheat arabinoxylan as a substrate for determination of the optimum

198

pH by monitoring enzyme activity at 50°C for 30 min. To examine pH stability, the enzyme

199in 200 mM of each above buffer was incubated without substrate at 4°C for 16 h, and its

200

remaining activity was measured by the standard assay. The optimum temperature was

201

determined in 20 mM AcB (pH 5.0) at temperatures ranging from 20–70°C for 1 h. To

202

determine thermal stability, the enzyme was incubated at 20–70°C for 1 h, and its remaining

203

activity was measured by the standard assay.

204
205

2.7. Estimation of molecular mass and concentration of proteins

206
207

Ten percent SDS-PAGE gel was used for determination of molecular mass of proteins208

according to the method of Laemmli [17], in which proteins were visualized by Coomassie

209

Brilliant Blue R-250 staining. Protein concentration was estimated by the Coomassie-Plus

210

Bradford assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) according to the method

211

of Bradford [18] using bovine serum albumin as a standard and by measurement of

212

absorbance at 595 nm.

213
214

2.8. Nuclear magnetic resonance (NMR) spectroscopy

215

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The reaction mixture containing SWUAbf62A (20 mU) and 10 mL of 0.1% wheat

217

arabinoxylan in 20 mM AcB (pH 5.0) was incubated at 37°C for 48 h, and then boiled for 10

218

min to inactivate the enzyme. After centrifugation, the supernatant was dialyzed against

219

water to remove arabinose and lyophilized. To substitute H with D, the sample (5 mg) was

220

dissolved in D2O, lyophilized, and dissolved in 0.6 mL of D2O. 1H-NMR spectra were221

recorded using a 500 MHz spectrometer (JNM-ECZ500, JEOL, Ltd., Tokyo, Japan) in D2O

222

at 80°C as previously reported [19].

223
224

2.9. Synergistic action on wheat arabinoxylan

225
226

Enzyme activity was tested by incubating 5 mU of the enzyme with 200 µL of 0.05%

227

substrate in 20 mM KPB (pH 6.0) at 40°C for 6 h. Reducing sugars released into the reaction

228

mixture were quantified by the Nelson-Somogyi method [16].

229
230

3. Results and discussion

231
232

3.1. Cloning and identification of swu62A

233
234

The swu62A gene was cloned by PCR using genomic DNA from Streptomyces sp.

235

SWU10 and contained 1,314 bp with 68% GC content encoding a protein of 438 amino acids.

236

SWUAbf62A seems to have an N-terminal signal peptide based on the amino acid sequence237

of a putative AFase gene of Streptomyces sp. CCM_MD2014 (NCBI Reference Sequence

238

WP_061442083.1) (Fig. 1), but we PCR-amplified the region without the signal sequence in239

this study. SWUAbf62A has a calculated pI and molecular mass of 6.09 and 46,232 Da,

240

respectively. Figure 1 shows the most closely matched amino acid sequences between

11

241

SWUAbf62A and other AFases, including Streptomyces sp. CCM_MD2014 (97%: NCBI

242

Reference Sequence WP_061442083.1) [20], Streptomyces sp. M1013 (95%: NCBI

243

Reference Sequence WP_076976742.1) [21], and Streptomyces canus (95%: NCBI

244

Reference Sequence WP_059298106.1) [22], as determined by NCBI BLAST analysis

245(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The Pfam program (http://pfam.xfam.org) predicted

246

that SWUAbf62A contained a Glyco_hydro_62 (PF03664) domain at positions 1–272. One

247

glutamate and two aspartates are conserved as catalytic residues of GH62 enzymes and are248

also found in GH43 enzymes [23, 24]. Crystallization of the GH62 AFase from Streptomyces249

coelicolor (ScAbf62A) demonstrated that Asp202, Glu361, and Asp309 play the roles of a

250

general base, general acid, and pKa modulation, respectively [23]. These amino acid residues

251

are also located at the Asp165, Glu324, and Asp272 positions in SWUAbf62A (Fig. 1). In

252

addition to these three conserved amino acid residues, GH62 enzymes exhibit a 5-bladed β-

253

propeller structure similar to that of GH43 AFases [25, 26], indicating that both GH families

254

belong to the superfamily GH-F [27].

255
256

3.2. Gene expression and purification of SWUAbf62A

257
258

The pET22b expression vector was ligated with swu62A and transformed into E. coli259

rosetta2 (DE3) cells. The expression of swu62A-pET22b constructs was induced by 0.01 mM

260

IPTG, and the cells were cultured at 15°C for 4 days. The recombinant enzyme was purified

261

by two steps using HisTrap HP and Superdex 75 column chromatographies. The result of

262

each enzyme purification step is presented in Table 1; this method resulted in 32.3-fold263

purification with a final yield of 67% (1.1 mg) and a specific activity of 1.29 U/mg.

264

Recombinant SWUAbf62A was purified as a single band with an apparent molecular mass of265

49 kDa on 10% SDS-PAGE gel (Fig. 2).

12

266
267

3.3. Biochemical characterization of SWUAbf62A

268
269

SWUAbf62A presented maximal enzyme activity at 50°C and pH 5.0 when wheat

270

arabinoxylan was used as its substrate. More than 70% of its initial enzymatic activity

271

remained after incubation at pH 4–10, and 60% remained after incubation at 20–50°C (Fig. 3).

272

SWUAbf62A lost all activity after incubation at 60°C for 1 h. Other GH62 AFases show their273

best enzymatic activity at 50°C and pH 5.0, including rAXS5 from P. chrysogenum [28],274

Abf62A from Scytalidium thermophilum [29], STX-IV from Streptomyces thermoviolaceus

275[30], and X-II-A from Aspergillus sojae [31].

276

The sensitivity of SWUAbf62A to several metal ions indicated that Ba2+, Ca2+, and277

Mn2+ stimulated while Ag+, Cu2+, Fe2+, and EDTA notably inhibited its enzymatic activity.

278

Fe3+, Hg2+, and Zn2+ also reduced activity by over 30% of initial activity, whereas no other279

metals had any effect on activity (Table 2). Similarly, the GH62 AFase activity of S.

280

thermophilum (Abf62C) [29], S. thermoviolaceus OPC-520 (STX-IV) [30], S.

281

thermoviolaceus (SthAbf62A) [32], and Coprinopsis cinerea (CcAbf62A) [33] were282

enhanced by Ca2+, whereas this ion inhibited the AFase activity of S. thermophilum (Abf62A)

283[29]. On the X-ray crystallographic structure, Ca2+ was localized to the center of the 5-bladed284

β-propeller structure of SthAbf62A (S. thermoviolaceus) [32], UmAbf62A (Ustilago maydis)285[34], and PaAbf62A (Podospora anserina) [34], coordinating with histidine residues for286

structural stabilization rather than playing a role in the catalytic mechanism [32, 34]. Such287

histidines are necessary for arabinose hydrolysis by the inverting mechanism in the GH-F

288

superfamily [34]. In addition, most GH62 enzymatic activities decrease in the presence of289

Cu2+ [30, 32]. GH62 is a small family that contains only bacterial and eukaryotic AFases of

290

274 sequences, and only a small number of its AFases are characterized (5 bacterial and 17

13

291

eukaryotic AFases). Most characterizations of GH62 AFases have focused on

292

crystallographic structure, phylogenetics, and substrate specificity, whereas there have been293

few studies on the effects of metal ions, enzyme regioselectivity, and synergistic action with294

other GH enzymes.

295

Substrate specificity of SWUAbf62A on natural polymeric substrates, such as wheat296

arabinoxylan, birch wood xylan, sugar beet arabinan, and sugar beet debranched arabinan,297

were examined by analyzing the enzymatic products by HPAEC after incubation at 50°C for298

1 h (Fig. 4). The enzyme released arabinose from both wheat arabinoxylan and sugar beet299

arabinan, with specific activities of 1.29 and 0.14 U/mg, respectively. SWUAbf62A

300

presented the best Vmax and affinity for wheat arabinoxylan of 0.09 mmol/min/mg and 0.58

301

mg/mL, respectively, whereas those for sugar beet arabinan were 0.04 mmol/min/mg and302

4.00 mg/mL, respectively (Table 3). The other two substrates were resistant to the enzyme

303

under the experimental conditions. Although SWUAbf62A had a low specific activity, the304

enzyme showed good performance towards wheat arabinoxylan, similarly to other GH62

305

AFases [23, 28, 35, 36]. In addition, SWUAbf62A was not active on p-nitrophenyl-α-L-

306

arabinofuranoside or p-nitrophenyl-β-D-xylopyranoside, even in an excess enzyme reaction,

307

which confirmed that the GH62 AFases have no or low activity on these substrates [24].

308

From these results, SWUAbf62A was demonstrated to be an AFase that specifically degraded309

arabinosidic linkages in wheat arabinoxylan, i.e., SWUAbf62A was an AXH. Some GH62

310

AFases, such as ScAbf62A from S. coelicolor [23] and STX-IV from S. thermoviolaceus

311

OPC-520 [30], can release L-arabinosyl residues from branched arabinan, as SWUAbf62A

312

can.

313
314

3.4. Regioselectivity of SWUAbf62A towards wheat arabinoxylan

315

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316

AXHs are classified into three modes of action depending on arabinofuranosyl

317

substitutions. AXH-m mode enzymes are active on L-arabinofuranose residues linked to O-2

318

or O-3 of single-substituted D-xylosidic residues [9]. The AXH-d3 mode enzymes release L-

319

arabinofuranose linked to O-3 of double-substituted D-xylose residues [10, 37-39]. The last320

mode, termed AXH-md, can hydrolyze both mono- and di-substituted L-arabinofuranosyl

321side chains in arabinoxylan [11]. We investigated the mode of action of SWUAbf62A

322

towards wheat arabinoxylan by 1H-NMR analysis. There is a distinct 1H-NMR signal for the323

anomeric proton of each L-arabinofuranose residue substitution at different positions of D-

324

xylose residues. Figure 5a shows the 1H-NMR data of wheat arabinoxylan before the enzyme

325

reaction. Based on a previous report [28], the signal at 5.381 ppm represents the single L-326

arabinofuranosyl substitution at O-3 of D-xylose residues, while the signals at 5.204 and327

5.274 ppm represent doubly substituted L-arabinofuranosidic residues at the O-2 and O-3

328

positions of D-xylosidic residues, respectively. 1H-NMR analysis of SWUAbf62A activity

329

towards wheat arabinoxylan found that only the signal at 5.381 ppm decreased, while the two

330

signals at 5.204 and 5.274 ppm did not change (Fig. 5b); this indicated that SWUAbf62A acts

331

according to the AXH-m3 mode. To examine whether SWUAbf62A releases single L-332

arabinofuranosyl substituted at O-2 of D-xylose backbones, a substrate with singly substituted

333

L-arabinose residues at O-2 or O-3 of a xylosidic backbone was prepared as described334

previously [28] (Fig. 5c) using Bifidobacterium sp. AFase with AXH-d3 activity mode is335

termed as BiAFase-d3 in this paper [10]. After incubation of SWUAbf62A and the substrate,336

the signal at 5.272 ppm, which represents the mono-substituted L-arabinofuranose at O-2 on337

D-xylosidic residues, decreased (Fig. 5d). Therefore, SWUAbf62A is a GH62 family

338

bacterial AXH that acts in the AXH-m2,3 mode. Almost all characterized GH62 AFases

339

show AXH-m2,3 activity, including rAXS5 [28], SthAbf62A [32], and CcAbf62A [33].

15

340

Even though 209 bacterial sequences have been classified in the GH62 family, only

341

five bacterial AXHs, including that of Cellvibrio japonicus [40], Streptomyces342

chattanoogensis UAH23 [41], S. coelicolor A3(2) [42], Streptomyces lividans 1326 [36], and343

S. thermoviolaceus OPC-520 [30], have been characterized. The structures of two among the344

five, namely, S. coelicolor [23] and S. thermoviolaceus [32], have been clearly described,345

while regioselectivity has only been studied for SthAbf62A from S. thermoviolaceus [32].

346

Therefore, SWUAbf62A is the second GH62 bacterial enzyme whose mode of enzymatic

347

action has been studied, and is the sixth characterized GH62 bacterial enzymes.

348

Arabinoxylan degradation requires β-1,4-endo-xylanases for xylan depolymerization,349

but arabinose substitutions generally interfere with such activity [43]. Therefore, AFases are350

key enzymes for degradation of arabinoxylan before the backbone can be hydrolyzed by β-351

1,4-endo-xylanases [44-45]. We assessed the synergistic effects between SWUAbf62A,

352

BiAFase-d3, and XynSW3, an endo-xylanase, from Streptomyces sp. SWU10 [15] on353

arabinoxylan degradation. As shown in Table 4, combination of SWUAbf62A and XynSW3354

degraded arabinoxylan 4 times more compared to XynSW3 alone. Addition of BiAFase-d3 to355

the reaction mixture containing SWUAbf62A and XynSW3 further increased the release of356

reducing sugar by 2 times. These synergistic effects indicated that efficient degradation of357

arabinoxylan by an endo-xylanase requires AFases with different modes of action.

358

Subsequently, we analyzed enzymatic reaction products of wheat arabinoxylan with the359

above three enzymes alone or in combination by HPAEC. A small amount of360

oligosaccharides was detected by the action of XynSW3 alone after reacting overnight (Fig.

361

6b). Conversely, XynSW3 in combination with SWUAbf62A degraded arabinoxylan and362

released arabinose, xylobiose (Xyl2), xylotriose (Xyl3), and xylotetraose (Xyl4), along with363

unidentified oligosaccharides that seemed to be xylooligosaccharides with double-substituted

364

L-arabinofuranose residues, and were eluted at 17–18 min (Fig. 6c). When three enzymes

16

365

reacted together, most of the above unidentified oligosaccharides were degraded and a much

366

larger amount of xylooligosaccharides was accumulated than that observed as a result of the367

combinatorial effect of XynSW3 and SWUAbf62A (Fig. 6d). These results indicated that i)368

the unidentified oligosaccharides were xylooligosaccharides with double-substituted

369

arabinose, ii) the xylooligosaccharides can be efficiently obtained from wheat arabinoxylan

370

using these enzymes, and iii) the AFase reaction is the rate-limiting step in the degradation

371

process of wheat arabinoxylan [46].

372
373

3.5. Conclusions

374
375

A novel GH62 AFase from thermotolerant Streptomyces sp. SWU10, termed

376

SWUAbf62A, was identified and its biochemical properties characterized. The enzyme

377

showed optimal activity at pH 5.0 and 50°C, and specific hydrolysis of wheat arabinoxylan in378

the AXH-m2,3 activity mode. Although the specific activity of the enzyme (SWUAbf62A)

379

was rather low, SWUAbf62A acted in synergy with XynSW3 and BiAFase-d3 to digest

380

wheat arabinoxylan, indicating that SWUAbf62A can be utilized for arabinoxylan

381

degradation. Therefore, SWUAbf62A is a new candidate for hydrolysis of plant biomass in382

the agricultural industry and other biotechnological applications.

383
384

Funding385
386This research was supported by The Royal Golden Jubilee Ph.D. Program of Thailand

387

Research Fund (grant number PHD/0209/2556) and by Srinakharinwirot University (grant

388

number 366/2559). This research was performed under the Core-to-Core Program, which was

389

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

17

390

Society for the Promotion of Science. The funder had no role in the study design; collection,

391

analysis, or interpretation of data; writing of the manuscript; or in the decision to submit the392

article for publication.

393
394

Declarations of interest: none.

395
396

Acknowledgments

397
398

The authors thank Ms. Misaki Noguchi, Osaka Prefecture University, for helping with399

the NMR experiments. We would like to thank Editage (www.editage.jp) for English

400

language editing.

401
402

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403
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548

24

549

Figure legends

550
551

Fig. 1. Sequence alignment of SWUAbf62A and other GH62 AFases: Streptomyces sp.

552

CCM_MD2014 (WP_061442083.1), Streptomyces sp. M1013 (WP_076976742.1),

553

Streptomyces canus (WP_059298106.1), Streptomyces coelicolor A3(2) (CAA16189.1).

554

Identical residues are shown in white font on black background. More than three identical

555

residues are shown in black font on gray background. Three asterisks (*) denote the catalytic

556

acidic residues.

557
558

Fig. 2. SDS-PAGE gel image of each purification step of SWUAbf62A. Lane M, molecular559

marker; lane 1, cell-free extract; lane 2, HisTrap HP; lane 3, Superdex 75. The gel was

560

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

561
562

Fig. 3. Effects of temperature (a) and pH (b) on activity (dotted lines) and stability (solid563

lines) of SWUAbf62A. The experimental conditions are described in the text.

564
565

Fig. 4. HPAEC analysis of enzymatic degradation products of wheat arabinoxylan (a) and566

sugar beet arabinan (b). Enzymatic reaction mixtures of 195 µL of 0.1% substrate in 20 mM

567

sodium acetate buffer (pH 5.0) and 5 mU of SWUAbf62A were incubated at 50°C for 1 h,568

and the reaction products were analyzed using HPAEC. HPAEC conditions are described in569

the text.

570
571

Fig. 5. 1H-NMR analysis of SWUAbf62A enzymatic degradation products from reactions

572

with natural (a and b) and modified (c and d) wheat arabinoxylan. The modified wheat573

arabinoxylan was prepared by digestion with the AFase from Bifidobacterium sp. Spectra

25

574

obtained before the reaction are shown in a and c, and those obtained after in b and d.

575

Experimental conditions are described in the text.

576
577

Fig. 6. HPAEC analyses of wheat arabinoxylan hydrolyzed by XynSW3, SWUAbf62A, and578

BiAFase-d3. A total of 100 µL of 0.1% wheat arabinoxylan in 20 mM KPB (pH 6.0) was

579

incubated with the appropriate enzyme samples at 40°C for 24 h. The mixtures were boiled

580

for 5 min and subsequently, the enzymatic products were analyzed by HPAEC. a: before the581

reaction, b: digestion with XynSW3 (1 mU), c: digestion with XynSW3 (1 mU) and582

SWUAbf62A (1 mU), d: digestion with XynSW3 (1 mU), SWUAbf62A (1 mU), and583

BiAFase-d3 (1 mU).

584

26

Table. 1 Purification of SWUAbf62A from Streptomyces sp. SWU10.

Purification
step

Protein(mg)

Activity(U)

Specific
activity(U/mg)

Purification(fold)

Yield (%)

Cell-free extract
HisTrap HP

56.60
9.77

2.13
1.88

0.04
0.19

1.0
4.8

100
88

Superdex 75

1.10

1.42

1.29

32.3

67

Wheat arabinoxylan was used as the substrate for enzyme assay.

Table 2. Effect of metal ions and EDTA on enzyme activity.
CompoundRelative activity (%)

None
AgNO3
BaCl2
CaCl2
CdCl2
CoCl2
CuCl2
FeSO4
FeCl3

100
15 ± 2128 ± 0125 ± 092 ± 2100 ± 118 ± 322 ± 370 ± 2

HgCl2
KCl
MnCl2
NaCl
NiCl2
ZnSO4

59 ± 299 ± 1115 ± 1101 ± 0101 ± 172 ± 2EDTA

30 ± 1

Experiments were performed in triplicate.

Table 3. Kinetic parameters of SWUAbf62A on wheatarabinoxylan and sugar beet arabinan.
SubstrateWheatarabinoxylan
Sugar beetarabinan

Km (mg/mL)

Vmax (mmol/min/mg)

0.58

0.09

4

0.04

Table 4. Degradation of wheat arabinoxylan by a combination of XynSW3,
SWUAbf62A, and BiAFase-d3.
EnzymeReducing sugar released (µg/mL)
XynSW326.7 ± 0.8
SWUAbf62A
49.6 ± 1.6
BiAFase-d3
24.7 ± 2.8
XynSW3/SWUAbf62A
107.2 ± 2.2
XynSW3/SWUAbf62A/BiAFase-d3
205.0 ± 8.0
The experimental conditions are described in the text.
All experiments were performed in triplicate.

(kDa)
200
150
120
100
85

70
60
50
40

M

1

2

3

Fig 2

Relative and remaining activity
(%)

75

100

75

50

100

50

25

[a]

25

0
70

0
20

30
40
50
60
Temperature (°C )

0

2

4

6
pH

8

10

[b]

12

Fig 3

[a]

4
[b]

8

8

Arabinose

6

6

Arabinose

4

10

10

12 14 16 18

12 14 16 18
Retention time (min)After 1 h

Before reaction

After 1 h

Before reaction

Fig 4

[a]

[b]

5.5

5.381
5.274 5.204

ppm

5.1

[c]

[d]

5.5

5.381
5.272

ppm

5.1

Fig 5

Ara

Xyl3

Xyl4

Retention time (min)

Xyl2

[d]
[c]
[b]
[a]

Fig 6