Accepted Manuscript

Title: The GH67 ␣-glucuronidase of Paenibacillus

curdlanolyticus B-6 removes hexenuronic acid groups and
facilitates biodegradation of the model xylooligosaccharide
hexenuronosyl xylotriose

Author: Krisna Septiningrum Hiroshi Ohi Rattiya Waeonukul

Patthra Pason Chakrit Tachaapaikoon Khanok
Ratanakhanokchai Junjarus Sermsathanaswadi Lan Deng
Panida Prawitwong Akihiko Kosugi

PII: S0141-0229(15)00007-1
DOI: http://dx.doi.org/doi:10.1016/j.enzmictec.2015.01.006
Reference: EMT 8723

To appear in: Enzyme and Microbial Technology

Received date: 4-9-2014

Revised date: 25-11-2014
Accepted date: 19-1-2015

Please cite this article as: Septiningrum K, Ohi H, Waeonukul R, Pason P,

Tachaapaikoon C, Ratanakhanokchai K, Sermsathanaswadi J, Deng L, Prawitwong
P, Kosugi A, The GH67 rmalpha-glucuronidase of Paenibacillus curdlanolyticus
B-6 removes hexenuronic acid groups and facilitates biodegradation of the model
xylooligosaccharide hexenuronosyl xylotriose, Enzyme and Microbial Technology
(2015), http://dx.doi.org/10.1016/j.enzmictec.2015.01.006

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1  HIGHLIGHTS

2  · GH67 α-glucuronidase (AguA) from P. curdlanolyticus was characterized.

3  · AguA releases xylotriose and hexenuronic acid from hexenuronosyl xylotriose

t
4  (ΔX3).

ip
5  · ΔX3 was degraded by intracellular α-glucuronidase and β-xylosidase activities.

cr
6  · GH67 AguA is applicable enzymes which remove HexA from ΔX3.

us
7 

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p te
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7  The GH67 α-glucuronidase of Paenibacillus curdlanolyticus B-6 removes

8  hexenuronic acid groups and facilitates biodegradation of the model

9  xylooligosaccharide hexenuronosyl xylotriose

10 

t
ip
11 

12  Krisna Septiningruma,b, Hiroshi Ohia, Rattiya Waeonukulc, Patthra Pasonc, Chakrit

cr
13  Tachaapaikoonc, Khanok Ratanakhanokchaid, Junjarus Sermsathanaswadie, Lan Dengb,

us
14  Panida Prawitwongb, Akihiko Kosugia,b*

15 

an
a
16  Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1

17  Tennodai, Tsukuba, Ibaraki 305-8572, Japan

M
b
18  Biological Resources and Post-harvest Division, Japan International Research Center

19  for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
d

c
20  Pilot Plant Development and Training Institute, King Mongkut’s University of
te

21  Technology Thonburi, Bangkuntien, Bangkok 10150, Thailand

p

d
22  School of Bioresources and Technology, King Mongkut’s University of Technology
ce

23  Thonburi, Bangkuntien, Bangkok 10150, Thailand

e
24  Department of Chemical Technology, Faculty of Science and Technology, Suan Dusit
Ac

25  Rajabhat University, 295 Rajasrima Road, Dusit, Bangkok 10300, Thailand

26 

27  Running title: Release of HexA from ΔX3 by α-glucuronidase

28  * Corresponding author

29  E-mail address: akosugi@affrc.go.jp

30  Tel./Fax: +81-29-838-6623

31 

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31  ABSTRACT

32  4-O-Methylglucuronic acid (MeGlcA) side groups attached to the xylan backbone
33  through α-1,2 linkages are converted to hexenuronic acid (HexA) during alkaline
34  pulping. α-Glucuronidase (EC 3.2.1.131) hydrolyzes 1,2-linked MeGlcA from

t
35  xylooligosaccharides. To determine whether α-glucuronidase can also hydrolyze HexA-

ip
36  decorated xylooligosaccharides, a gene encoding α-glucuronidase (AguA) was cloned
37  from Paenibacillus curdlanolyticus B-6. The purified protein degraded hexenuronosyl

cr
38  xylotriose (ΔX3), a model substrate prepared from kraft pulp. AguA released xylotriose
39  and HexA from ΔX3, but the Vmax and kcat values for ΔX3 were lower than those for

us
40  MeGlcA, indicating that HexA side groups may affect the hydrolytic activity. To
41  explore the potential for biological bleaching, ΔX3 degradation was performed using

an
42  intracellular extract from P. curdlanolyticus B-6. The intracellular extract, with
43  synergistic α-glucuronidase and β-xylosidase activities, degraded ΔX3 to xylose and
44  HexA. These results indicate that α-glucuronidase can be used to remove HexA from
M
45  ΔX3 derived from pulp, reducing the need for chemical treatments in the pulping
46  process.
d

47 
te

48  Keywords: Hexenuronic acid; α-Glucuronidase; GH67; Hexenuronosyl xylotriose;

49  Paenibacillus curdlanolyticus. 
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50  1. Introduction

51  During alkaline pulping of wood chips, 4-O-methyl-D-glucuronic acid (MeGlcA)

52  groups attached exclusively at α-1,2 linkages to the xylan backbone are about 75–90%

t
53  degraded. The residual MeGlcA groups are converted to unsaturated 4-deoxy-β-L-threo-

ip
54  hex-4-enopyranosyluronic acid (hexenuronic acid or HexA) groups by β-elimination of

cr
55  methanol directly or via the intermediate product 4-O-methyliduronic acid [1, 2] (Fig.

56  1). HexA is a major uronic acid substituent of both industrial kraft pulp and some

us
57  unconventional alkali-based organosolv pulps, accounting for 83–88% of total uronic

58  acids [3]. The rate of formation of HexA depends on operating variables of the kraft

59 

an
process, including cooking time [4], temperature [5], and effective alkali concentration

[6, 7]. HexA groups are significant because of their role in the bleaching process and
M
60 

61  their influence on the final pulp properties. HexA contributes to kappa number [8],

increases the consumption of bleaching agents [9], retains metal ions, increases
d
62 

brightness reversion [10], and contributes to the formation of oxalic acid. There have
te

63 

64  been attempts to reduce the HexA content of pulp by inserting acid stages between
p

65  cooking and bleaching [9], using an electrophilic oxidant such as elemental chlorine or
ce

66  chlorine dioxide, or using ozonation or a peracid treatment [11]. Enzymatic methods

67  have also shown promise. Specifically, the use of xylanases and laccases in pulp-
Ac

68  bleaching sequences has proved effective for removing HexA [12-14], indicating that

69  enzymatic bleaching is a promising technology for the pulp and paper industry.

70  However, these enzymes have low efficiency: only about 30% of HexA was removed

71  from eucalyptus pulp treated with laccase and xylanase [14]. Furthermore, the addition

72  of hydroxybenzotriazole is necessary for the removal of HexA by xylanases and

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73  laccases [12]. Therefore, more efficient and direct enzymatic bleaching technologies are

74  required for the pulp and paper industry.

75  α-Glucuronidase (EC 3.2.1.139) hydrolyzes the α-1,2 glycosidic bond between α-D-

t
76  glucuronic acid (GlcA) or its 4-O-methyl ether (MeGlcA) and D-xylose residues of

ip
77  xylooligosaccharides (aldouronic acids). The majority of α-glucuronidases that

cr
78  hydrolyze these bonds are located in glycoside hydrolase family-67 (GH67) [15, 16]. 

79  These enzymes, however, remove uronic acid only from glucuronoxylooligosaccharides

us
80  and not from glucuronoxylan. α-Glucuronidase might be applicable for the direct

81  removal of HexA groups from xylooligosaccharides because of the similar structures of

82 

an
HexA and MeGlcA (Fig. 1). It was recently reported that a xylanolytic Paenibacillus

strain could degrade hexenuronosyl xylotriose (ΔX3) using a combination of

M
83 

84  intracellular and extracellular enzymes such as xylanases and β-xylosidase; however, it

is still unclear how these enzymes cooperate to degrade ΔX3 [17]. In particular, there
d
85 

are no reports on whether α-glucuronidase can remove HexA from ΔX3. Therefore, it is
te

86 

87  important to characterize the α-glucuronidase from Paenibacillus strains that degrade
p

88  ΔX3. To determine whether α-glucuronidase can remove HexA, we prepared ΔX3 from
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89  alkaline and enzymatically treated eucalyptus kraft pulp for use as a model substrate.

90  Here we report the direct removal of HexA from ΔX3 using the GH67 α-glucuronidase
Ac

91  from Paenibacillus curdlanolyticus B-6. This organism has been well characterized for

92  xylanolytic enzymes and can efficiently degrade xylan polymers substituted to varying

93  degrees with GlcA, MeGlcA, acetyl, feruloyl, or p-coumaroyl side-chain groups

94  through a multienzyme complex of cellulases and hemicellulases [18, 19]. α-

95  Glucuronidase from P. curdlanolyticus B-6 may improve the brightness stability of pulp

96  through enzymatic removal of HexA.

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97 

98  2. Materials and Methods

99  2.1. Bacterial strains and plasmids

t
ip
100  Paenibacillus curdlanolyticus B-6 has been deposited with the BIOTEC Culture

Collection of the National Center for Genetic Engineering and Biotechnology, Thailand

cr
101 

102  (BCC No. 11175). P. curdlanolyticus B-6 was grown on Berg’s mineral salt medium,

us
103  pH 7.0 [18, 20], containing 2 g of NaNO3, 0.5 g of K2HPO4, 0.2 g of MgSO4·7H2O,

104  0.02 g of MnSO4·H2O, 0.02 g of FeSO4·7H2O, and 0.02 g of CaCl2·2H2O and

an
105  supplemented with 5 g of birchwood xylan (Sigma-Aldrich, St. Louis, MO, USA) per

106  liter of distilled water. Chemicals were purchased from Wako Pure Chemical Industries
M
107  (Osaka, Japan). Escherichia coli strains JM109, HST 08, and BL21(DE3) (Takara Bio,

108  Shiga, Japan) and plasmids pTAC-1 (DynaExpress TA PCR Cloning Kit, Tokyo, Japan)
d

109  and pET22b (Merck KGaA, Darmstadt, Germany) were used for cloning and expression.
te

110  E. coli cells were grown at 37°C in Luria–Bertani (LB) medium containing ampicillin
p

111  (100 μg/mL).

ce

112 
Ac

113  2.2. Cloning and sequencing of the α-glucuronidase gene from P. curdlanolyticus B-6

114  Genomic DNA was prepared by phenol/chloroform extraction [19] and plasmid DNA

115  was prepared using a QIAprep Spin Miniprep Kit in accordance with the manufacturer’s

116  protocol (Qiagen, Frederick, MD, USA). The oligonucleotide primers used in this

117  research are listed in Table 1. After comparing the amino acid sequences of more than

118  five GH67 α-glucuronidases from Paenibacillus, we designed two degenerate primers

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119  (Table 1) based on two highly-conserved amino acid sequences (consensus sequences of

120  DGSIERGYAG and SGKTVIQHIY). To amplify the corresponding region from P.

121  curdlanolyticus B-6 genomic DNA, polymerase chain reaction (PCR) was performed

122  with Ex Taq polymerase (Takara Bio) under standard conditions according to the

t
ip
123  manufacturer’s instructions. The purified PCR products were ligated with pTAC-1 and

124  both strands were sequenced using M13 primers. Homology analysis through BLAST

cr
125  confirmed that the amplified region was a fragment of an α-glucuronidase gene. Four

us
126  gene-specific primers (Table 1) were designed to perform genome-walking PCR using

127  the Universal GenomeWalker 2.0 kit (Takara Bio) according to the manufacturer’s

an
128  instructions. The purified products of the nested PCR were cloned into pTAC-1 and

129  sequenced. Sequence assembly was performed with GENETYX ver.12 software
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130  (GENETYX CORPORATION, Tokyo, Japan). Nucleotide and amino acid sequences

131  were analyzed with the blastn and blastp programs, respectively
d

132  (https://blast.ncbi.nlm.nih.gov/Blast). The GH67 α-glucuronidase gene from P.

te

133  curdlanolyticus B-6 was named aguA.

p

134 
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135  2.3. Construction of the AguA expression vector

Ac

136  Specific primers (Table 1) were designed to amplify the open reading frame of the aguA

137  gene and to provide restriction sites for generating fragments for ligation and cloning. A

138  2,070-bp fragment of the aguA gene was produced by PCR using Ex Taq polymerase

139  (Takara Bio) under the following conditions: one cycle of 1 min at 98°C; 30 cycles of

140  10 s at 98°C, 1 min at 60°C, and extension for 2 min at 68°C; and an additional

141  extension for 10 min at 72°C. The PCR products were purified by agarose gel

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142  electrophoresis, digested with NcoI plus XhoI, and cloned into the vector pET22b

143  digested with NcoI and XhoI.

144 

t
2.4. Production and purification of recombinant AguA

ip
145 

For production and characterization of α-glucuronidase, pET22b containing the aguA

cr
146 

147  gene was transformed into E. coli BL21. Transformants were selected on LB plates

us
148  containing ampicillin (100 μg/mL). E. coli BL21 containing the aguA expression vector

149  was grown at 37°C in 300 mL of LB medium supplemented with ampicillin (100

an
150  μg/mL) until the absorbance at 600 nm reached 0.6–0.8. Protein expression was carried

151  out at 30°C with the addition of 1 mM isopropyl-β-D-thiogalactopyranoside to the

M
152  culture medium. After cultivation for 3 h, E. coli cells were harvested by centrifugation

153  (8,500 × g, 10 min, 4°C) and frozen at −80°C for 24 h. The frozen cell pellet was
d

154  resuspended in 50 mM sodium phosphate buffer (pH 7.0), and recombinant proteins
te

155  were released by sonication. Cell-free extracts were separated into lysates and cell
p

156  debris by centrifugation (8,500 × g, 10 min, 4°C). The recombinant protein was purified
ce

157  with the Profinia Affinity Chromatography Protein Purification System using a Bio-

158  Scale Mini Profinity IMAC cartridge and a Bio-Gel P6 desalting cartridge in
Ac

159  accordance with the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA,

160  USA). Protein concentrations were determined using the Coomassie (Bradford) protein

161  assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin

162  as the standard. The homogeneity of the purified protein was analyzed by sodium

163  dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was

164  performed on 5 to 20% gradient polyacrylamide gels (ATTO, Tokyo, Japan) according

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165  to the manufacturer’s instructions. Samples for SDS-PAGE were boiled for 10 min in

166  sample buffer containing dithiothreitol (Sigma-Aldrich). After electrophoresis, gels

167  were stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories). Molecular

168  mass standards were from Bio-Rad Laboratories.

t
ip
169 

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170  2.5. Preparation of hexenuronosyl xylotriose (ΔX3) from eucalypt kraft pulp

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171  Hexenuronosyl xylotriose (ΔX3) was prepared from alkaline- and enzyme-treated

172  eucalypt (Eucalyptus globulus L.) kraft pulp [17]. Hardwood oxygen-delignified kraft

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173  pulp (LOKP) (2 kg, obtained from Hokuetsu Kishu Paper Co., Ltd., Niigata, Japan) was

174  soaked in 15% (w/v) NaOH (Wako Pure Chemical Industries) for 24 h at 25°C. The
M
175  pulp residue was filtered and washed with water, and the filtrate was collected for

176  further treatment as described below. After the filtrate had been neutralized with
d

177  sulfuric acid, the xylan was precipitated (approximately 87 g) from the kraft pulp and
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178  separated by centrifugation (8,500 × g, 30 min, 25°C), washed with distilled water, and
p

179  dried under vacuum at room temperature. The modified xylan was hydrolyzed for 72 h
ce

180  at 45°C and pH 4.5 (50 mM sodium acetate buffer) with xylanase (Shearzyme;

181  Novozymes A/S, Bagsvaerd, Denmark) (544 U/g sediment) and cellulase (Onozuka R-
Ac

182  10; Wako Pure Chemical Industries) (74 U/g sediment). Enzymatic hydrolysates were

183  boiled for 10 min. Clear supernatants containing xylooligosaccharides were obtained by

184  centrifugation at 8,500 × g for 30 min and applied to a packed column of activated

185  carbon (Wako Pure Chemical Industries). The active carbon was washed with 12 L of

186  distilled water and eluted with 9 L of 40% (v/v) ethanol. The eluted mixture was

187  concentrated to 15 mL on a rotary evaporator. The mixture contained approximately 5.8

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188  g of ΔX3. To avoid contamination with xylooligosaccharides such as xylotriose, the

189  mixture containing ΔX3 was incubated with 10 units of β-xylosidase from Bacillus

190  pumilus (Megazyme, Bray, Ireland) for 3 h at 35°C. The mixture was boiled for 10 min,

191  and a clear supernatant containing ΔX3 was eventually obtained by centrifugation

t
ip
192  (16,400 × g, 15 min, 4ºC). The concentration of ΔX3 was measured using high-

193  performance anion-exchange chromatography with pulsed amperometric detection 

cr
194  (HPAEC-PAD) as previously described [17]. Eventually, 5.1 g of purified ΔX3 without

us
195  xylooligosaccharides was obtained from 2 kg of LOKP.

an
196 

197  2.6. Enzyme activities

M
198  α-Glucuronidase activity was measured by determining the amount of glucuronic acid

199  liberated from aldouronic acid (Megazyme) using a colorimetric assay [21]. The
d

200  incubation mixture for the α-glucuronidase assay (total volume, 0.2 mL) contained 0.16
te

201  mL of substrate (2 mg of aldobiouronic, aldotriouronic, aldotetraouronic, and

p

202  aldopentaouronic acids [10:60:20:10] in 0.1 M sodium acetate buffer [pH 6.0]) and 0.04
ce

203  mL of the enzyme solution to be assayed. The reaction was started by addition of the

204  enzyme. After 30 min of incubation at 40°C, the reaction was stopped by boiling the
Ac

205  samples for 4 min. Next, 0.6 mL of copper reagent, prepared as described by Milner and

206  Avigad [21], was added to each tube, and then the samples were boiled for 10 min and

207  cooled on ice. Subsequently, 0.4 mL of arsenomolybdate reagent [22] was added. The

208  samples were mixed gently, 0.8 mL of H2O was added, and the absorbance at 620 nm

209  was measured against H2O. Controls were prepared by boiling a complete assay mixture

210  at time zero, before incubation at 40°C. A substrate control was made by adding water

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211  instead of enzyme solution. A standard curve was prepared using D-glucuronic acid

212  (Sigma-Aldrich). One α-glucuronidase unit is the amount of enzyme liberating 1 μmol

213  of glucuronic acid per minute under standard assay conditions. The activity of α-

214  glucuronidase towards birchwood xylan and MeGlcA (Sigma-Aldrich) was measured

t
ip
215  using the colorimetric assay after incubation at 40°C for 15 h [23]. The activity towards

216  p-nitrophenyl-β-D-glucopyranoside (Sigma-Aldrich) was determined by measuring the

cr
217  absorbance of p-nitrophenol at 400 nm. The optimal pH was determined with

us
218  aldouronic acid in the following buffers at 40°C: 0.1 M sodium acetate buffer, pH 4.0 to

219  6.0; 0.1 M phosphate buffer, pH 6.0 to 8.0; and 0.1 M Tris-HCl buffer, pH 8.0 to 9.0.

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220  To determine pH stability, the enzyme was preincubated without substrate in buffers of

221  different pHs for 3 h at 37°C, and then the α-glucuronidase activity was measured at
M
222  40°C for 10 min (pH 6.0). The optimal temperature was determined at pH 6.0 (50 mM

223  sodium acetate buffer) from 30°C to 60°C. Thermostability was monitored by
d

224  preincubating the enzyme without substrate in sodium acetate buffer (pH 6.0) for 3 h at
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225  30–60°C and then assaying residual enzyme activity under standard assay conditions.
p

226  Various metal ions at a concentration of 1 mM were added to the standard assay in 100
ce

227  mM sodium acetate buffer (pH 6.0 at 40°C). Blanks with inactive enzyme were

228  included to verify that the observed activity was not due to the colorimetric assay
Ac

229  reagents. Xylanase activity was measured by determining the amount of reducing sugars

230  released from birchwood xylan (Sigma-Aldrich) [18]. Released reducing sugars were

231  quantified by the Somogyi–Nelson method using xylose as a standard [23]. One unit of

232  xylanase activity was defined as the amount of enzyme that liberated 1 μmol of

233  reducing sugar in 1 min under the assay conditions. β-Glucosidase and β-xylosidase

234  activities were based on measurement of p-nitrophenol release from p-nitrophenyl β-D-

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235  glucoside and p-nitrophenyl β-D-xyloside (both from Sigma-Aldrich), respectively [18]. 

236  One unit of enzyme released 1 µmol equivalent of p-nitrophenol per minute.

237 

t
2.7. Hydrolytic activity of AguA against ΔX3

ip
238 

Hydrolytic activity against ΔX3 was measured by directly determining the amount of

cr
239 

240  xylotriose liberated from ΔX3 by HPAEC-PAD using a DX-500 series chromatograph

us
241  equipped with a PAD II pulsed amperometric detector (Dionex, Sunnyvale, CA, USA).

242  AguA (50–60 µg/mL) was incubated with the prepared ΔX3 (4.7–6.0 mM) in 100 mM

an
243  sodium acetate buffer (pH 6.0) at 40°C for 3 h, and then the reaction mixture was boiled

244  for 10 min and cooled on ice. The xylooligosaccharides were analyzed by HPAEC-PAD
M
245  with a CarboPac PA-100 column (250 mm × 4 mm) and a PA-100 guard column (25

246  mm × 3 mm) at 30°C. Gradient elution was performed with 100 mM NaOH and 100
d

247  mM NaOH/1 M sodium acetate at a flow rate of 1 mL/min. Authentic ΔX3, provided by
te

248  Dr. Shigeki Yoshida (University of Tsukuba, Ibaraki, Japan), was used as a standard.
p

249  The α-glucuronidase from Geobacillus stearothermophilus was purchased from

ce

250  Megazyme International (Bray, Ireland). D-Xylose, 1,4-β-D-xylobiose, 1,4-β-D-

251  xylotriose, and 1,4-β-D-xylotetraose (Megazyme International) were used as external

Ac

252  standards. The specificity of AguA for ΔX3 was determined by incubating purified

253  enzyme (1 μg/μL) with 100 nmol of substrate in 100 μL of reaction buffer (50 mM

254  sodium acetate, pH 6.0) at 40°C for approximately 16 h. The digested products (20 μL

255  of a complete digest containing 20 nmol equivalents of product) were spotted on a thin-

256  layer chromatography plate (10 by 10 cm; thickness, 0.25 mm; Silica Gel 60; Merck

257  KGaA) along with 20 nmol of xylose or xylooligosaccharides (xylobiose, xylotriose,

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258  and xylotetraose) and HexA. The plate was developed with n-butanol–acetic acid–water

259  (1:1.5:1.5, v/v) for 1.5 h, air-dried for 10 min, and sprayed with 1% (w/v) thiobarbituric

260  acid in methanol. The stained plate was baked in an oven at 110°C for 10 min for

261  visualization (pink color) of HexA [17].

t
ip
262 

cr
263  2.8. Biological degradation of ΔX3 using P. curdlanolyticus B-6

us
264  P. curdlanolyticus B-6 was grown for 48 h at 37°C in 300 mL of Berg’s mineral salt

265  medium supplemented with 0.5% (w/v) birchwood xylan as the sole carbon source. P.

an
266  curdlanolyticus B-6 cells were harvested by centrifugation (8,500 ×g, 10 min, 4°C) and

267  frozen at −80°C for 24 h. The culture broth was used as the extracellular fraction. The
M
268  frozen cell pellet was washed with 50 mM sodium acetate buffer (pH 6.0) and

269  resuspended in 10 mL of the same buffer. Cell-free extract was prepared by sonication
d

270  and separated into lysate and cell debris by centrifugation (10,810 ×g, 10 min, 4°C).
te

271  The lysate was used as the intracellular fraction. Protein concentrations were determined
p

272  using the Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific) with
ce

273  bovine serum albumin as the standard. The protein concentrations of the extracellular

274  and intracellular fractions were estimated as 0.17 mg/mL and 2.7 mg/mL, respectively.
Ac

275 

276  2.8. Nucleotide sequence accession number

277  The GenBank accession number for aguA from P. curdlanolyticus B-6 is KM278173.

278 

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279  3. Results and Discussion

280  3.1. Characterization of AguA from P. curdlanolyticus B-6

281  According to the Carbohydrate-Active enZYmes (CAZy) Database, α-glucuronidases

t
are found in three glycoside hydrolase families (GH4, GH67, and GH115). The majority

ip
282 

283  of bacterial and fungal α-glucuronidases are classified within GH67. To determine

cr
284  whether α-glucuronidases can remove HexA side groups from xylooligosaccharides, a

us
285  GH67 α-glucuronidase gene was cloned from P. curdlanolyticus B-6. An α-

286  glucuronidase gene fragment (1,400 bp) was amplified by PCR using degenerate

an
287  primers based on two highly conserved amino acid sequences (DGSIERGYAG and

288  SGKTVIQHIY) (Table 1). The 5′ and 3′ flanking regions were amplified by PCR-based
M
289  genome walking and assembled with the known partial sequence. The full-length α-

290  glucuronidase gene, aguA, contains 2,070 bp and encodes a protein of 690 amino acids
d

291  with a calculated molecular mass of 77.3 kDa. The conserved amino acid sequences,
te

292  DGSIERGYAG and SGKTVIQHIY, are located at amino acid residues 167–176 and

293  613–622, respectively. The deduced amino acid sequence encoded by aguA showed
p

294  homology with putative GH67 α-glucuronidases from Paenibacillus mucilaginosus

ce

295  (69% identity, WP_014369631), Paenibacillus sp. oral taxon 786 (68% identity,
Ac

296  WP_009223235), Cohnella thermotolerans (67% identity, WP_027092093),

297  Paenibacillus barengoltzii (67% identity, WP_016312234), Paenibacillus sp. JDR-2

298  (62% identity, WP_015842945), and Paenibacillus polymyxa (60% identity,

299  WP_025719411).

300  To characterize the enzymatic properties of AguA, recombinant AguA was

301  expressed in E. coli BL21, purified to electrophoretic homogeneity using immobilized-

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302  metal affinity chromatography (Fig. 2), and assayed for activity with aldouronic acids

303  and other substrates. AguA showed highly specific activity for MeGlcA side groups of

304  aldouronic acids, while no activity was observed with birchwood xylan, 4-O-methyl-D-

305  glucurono-D-xylan, or p-nitrophenyl-β-D-glucopyranoside when assayed at 40°C (pH

t
ip
306  6.0). The optimal pH for α-glucuronidase activity of AguA was 6.0, and the enzyme

307  was stable in the range of pH 4.0–7.0. The temperature for maximum activity was found

cr
308  to be 40°C at pH 6.0. These enzymatic properties and the narrow substrate specificity

us
309  indicate that AguA, like other known GH67 α-glucuronidases, removes uronic acid

310  from the nonreducing end of glucuronoxylooligosaccharides but does not attack

an
311  glucuronoxylan [16, 24-26]. To measure the substrate affinity and catalytic efficiency of

312  AguA toward aldouronic acids, kinetic parameters were determined by Michaelis–
M
313  Menten analysis. Initial reaction rates were determined from the kinetic curves of

314  reactions containing different concentrations of aldouronic acid. The Km and Vmax values
d

315  for AguA were estimated to be 1.67 ± 0.1 mM and 838 ± 0.5 μmol/(min·mg protein),
te

316  respectively, corresponding to a kcat of 1081.8/s. The effect of various metal ions on α-
p

317  glucuronidase activity was also evaluated. Although it seems unlikely that AguA
ce

318  requires a specific metal ion for its catalytic activity, 1 mM Co2+, Fe3+, Cu2+, and Ni2+

319  enhanced AguA activity by 173%, 244%, 194%, and 119%, respectively. In contrast,
Ac

320  Mg2+, Mn2+, Ca2+, Zn2+, and K+ caused losses of activity of 88%, 78%, 47%, 71%, and

321  70%, respectively. These metal ions are known to affect the activity of GH67 α-

322  glucuronidases and have considerable affinity for functional groups such as thiols,

323  imidazoles, and amines [25]. Thus, it is likely that these metal ions affect the active site

324  and structure of AguA.

325 

15
 
Page 15 of 33
  
 

326  3.2. Removal of hexenuronic acid (HexA) groups from hexenuronosyl xylotriose (ΔX3)

327  by AguA

328  To determine whether GH67 α-glucuronidase can remove HexA from hexenuronosyl

t
329  xylooligosaccharides, hexenuronosyl xylotriose (ΔX3) was prepared from eucalyptus

ip
330  kraft pulp and used as a model substrate. The fractionation was carried out as described

cr
331  by Winyasuk et al. [17] (Fig. 3a). After treatment with commercial xylanases and

332  cellulases, HPAEC-PAD analysis showed that the fraction of ΔX3 containing

us
333  xylooligosaccharides still contained xylotriose (Fig. 3b). To clearly observe the release

334  of xylotriose from ΔX3 by AguA, the ΔX3 fraction containing xylotriose was treated

335 

an
with commercial β-xylosidase. Free xylotriose in this fraction was converted to xylose,

but the ΔX3 concentration was not affected (Fig 3b), indicating that β-xylosidase cannot
M
336 

337  degrade xylotriose decorated with HexA.

d

338  HPAEC-PAD analysis showed that xylotriose was liberated when AguA was
te

339  incubated with ΔX3, and the ΔX3 peak decreased with the appearance of xylotriose (Fig.

340  4a). To confirm whether the peak observed following incubation with AguA was
p

341  xylotriose, β-xylosidase was added to the reaction (Fig. 4a). The xylotriose was
ce

342  completely converted to xylose by the β-xylosidase. These results clearly indicated that
Ac

343  xylotriose was liberated from ΔX3 by AguA, which can hydrolyze the α-1,2 glycosidic

344  bond even when the side group is HexA instead of MeGlcA (Fig. 4a). The Km for ΔX3

345  was estimated to be 4.6 ± 0.5 mM. The affinity of AguA for ΔX3 was only slightly

346  lower than that for MeGlcA-substituted aldouronic acids. However, Vmax for the release

347  of xylotriose from ΔX3 was 5.0 ± 0.6 µmol xylotriose/(min·mg protein), and the kcat

348  was estimated to be 6.4/s. These results indicate that although the affinity of AguA for

349  ΔX3 was not different from that for MeGlcA-substituted aldouronic acids, the velocity

16
 
Page 16 of 33
  
 

350  and efficiency of AguA were significantly affected by HexA side groups. Recently, the

351  three-dimensional structure and biochemical properties of a GH67 α-glucuronidase

352  (GlcA67A) from Pseudomonas cellulosa (Cellvibrio japonicus) were reported [15].

353  GlcA67A (GenBank accession No. CP000934) has an amino acid similarity of 46%

t
ip
354  with AguA. The bacterial α-glucuronidases belonging to GH67 catalyze bond cleavage

355  by an SN2-like single displacement mechanism in which water attacks the anomeric

cr
356  carbon of the uronic acid, concomitant with protonation of the glycosidic oxygen and

us
357  departure of the leaving group, leading to an inversion of the aromatic configuration and

358  generation of the β-anomer of MeGlcA [16, 27]. Co-crystallization of GlcA67A with

an
359  glucuronic acid revealed a binding site within a deep, partially hydrophobic pocket in

360  the enzyme surface [15]. The hydrophobic pocket provides exquisite recognition
M
361  elements for the carboxylate moiety of glucuronic acid and may accommodate a 4-O-

362  methyl substituent through surrounding hydrophobic amino acid residues such as
d

363  Val210 and Trp160 [15]. Thus, if the 4-O-methyl substituent is lost by β-elimination of
te

364  methanol during alkaline pulping, the binding of the substrate in the hydrophobic pocket
p

365  might be unstable.

ce

366  Thin-layer chromatography (TLC) was also carried out to confirm the release of

367  HexA in reactions with or without AguA. Deoxy sugars such as HexA yield a red spot
Ac

368  on TLC plates developed with thiobarbituric acid, but no color is observed for ΔX3 [17,

369  28]. Although the reaction of ΔX3 with boiled AguA did not show a red spot on a TLC

370  plate sprayed with thiobarbituric acid, the reaction of ΔX3 with native AguA did show a

371  red spot (Fig. 4b). These results strongly support the detection of xylotriose in the

372  reaction with AguA by HPAEC-PAD analysis. To determine whether other bacterial α-

373  glucuronidases belonging to GH67 can release HexA from ΔX3, we performed the

17
 
Page 17 of 33
  
 

374  hydrolysis tests using the well-characterized GH67 α-glucuronidase from G.

375  stearothermophilus [25, 29, 30]. Because this α-glucuronidase is commercially

376  available as a high-purity recombinant enzyme from Megazyme International, it might

377  give more reproducible results than non-commercial preparations. Complete three-

t
ip
378  dimensional structures are available for only two GH67 enzymes [30]: GlcA67A from

379  C. japonicus and the α-glucuronidase from G. stearothermophilus [29]. The overall

cr
380  structures of these two enzymes appear to be quite similar [30]. The α-glucuronidase

us
381  from G. stearothermophilus also hydrolyzed ΔX3 to xylotriose and HexA, but it had a

382  lower Vmax (2.7 µmol xylotriose/(min·mg protein)) than AguA. These results suggest

an
383  that GH67 α-glucuronidases may be useful for the specific removal of HexA in kraft

384  pulping processes.

M
385 
d

386  3.3. Possibility of biological degradation using P. curdlanolyticus B-6

te

387  An attempt to directly remove HexA from ΔX3 using intracellular enzymes from
p

388  Paenibacillus sp. strain 07-G-dH has been reported [17]. The absence of an N-terminal
ce

389  signal sequence indicates that AguA of P. curdlanolyticus B-6 might be an intracellular

390  enzyme. The majority of α-glucuronidases belonging to GH67 have been reported to be
Ac

391  either membrane-bound or intracellular enzymes [16, 24-26, 31]. To characterize the

392  enzymatic properties of P. curdlanolyticus B-6 in relation to ΔX3 degradation,

393  extracellular and intracellular fractions were prepared from cells cultured with

394  birchwood xylan as the sole carbon source. α-Glucuronidase and β-xylosidase activities

395  in the intracellular fraction extracted from P. curdlanolyticus B-6 cells were

396  considerably higher than those in the culture supernatant (Table 2). These results

18
 
Page 18 of 33
  
 

397  indicate that the GH67 α-glucuronidase of P. curdlanolyticus B-6 may be an

398  intracellular enzyme [18]. To explore the possibility of direct biological bleaching using

399  α-glucuronidase, the ability of the intracellular fraction to degrade ΔX3 was assessed.

400  Although only a small amount of xylotriose was released when the intracellular fraction

t
ip
401  was incubated with ΔX3, an increase in the amount of xylose occurred in connection

402  with a decrease in the amount of ΔX3 (Fig. 5). In contrast, when the extracellular

cr
403  fraction (culture broth) was incubated with ΔX3, no increase in xylose or xylotriose

us
404  concentration or decrease in ΔX3 concentration was observed (data not shown). These

405  results may be understood as the complete degradation of ΔX3 by the synergistic

an
406  activity of intracellular α-glucuronidase and β-xylosidase. The reaction catalyzed by

407  AguA—the removal of HexA from ΔX3—may be the rate-limiting step in hydrolysis of
M
408  ΔX3. The released xylotriose should be quickly converted to xylose monomers by β-

409  xylosidase. The pattern of these end products is similar to that observed when
d

410  intracellular enzymes from Paenibacillus sp. strain 07-G-dH [17] were tested for
te

411  degradation of ΔX3.

p

412 
ce

413  4. CONCLUSIONS
Ac

414  The presence of HexA negatively affects pulps by decreasing brightness. To determine

415  whether HexA-decorated xylooligosaccharides could be hydrolyzed by α-glucuronidase,

416  AguA, a GH67 α-glucuronidase from P. curdlanolyticus B-6, was characterized for its

417  ability to degrade ΔX3 as a model substrate. When ΔX3 was incubated with AguA,

418  xylotriose and HexA were released and accumulated in the reaction mixture. ΔX3 can

419  be directly degraded by the α-glucuronidase activity of intracellular extract from P.

19
 
Page 19 of 33
  
 

420  curdlanolyticus B-6. These findings provide new insight into the direct biological

421  degradation of ΔX3 by α-glucuronidase and may help reduce the consumption of active

422  bleaching chemicals. This is the first report of direct degradation of ΔX3 by a GH67 α-

423  glucuronidase.

t
ip
424 

cr
425  ACKNOWLEDGEMENTS

us
426  We would like to thank Dr. Shigeki Yoshida (University of Tsukuba, Ibaraki, Japan) for

427  providing authentic ΔX3. K. Septiningrum was supported by a Japan Educational

an
428  Exchanges and Services Sponsored Scholarship (JEES) (2012–2013) from Mitsubishi

429  Cooperation.
M
430 
d

431  REFERENCES
te

432  [1] Johansson MH, Samuelson O. Epimerization and degradation of 2-Ο-(4-Ο-methyl-

p

433  α-D-glucopyranosyluronic acid)-D-xylitol in alkaline medium. Carbohydr Res

ce

434  1977;54:295-9.

435  [2] Šimkovic I, Ebringerová A, Hirsch J, Königstein J. Alkaline degradation of model

Ac

436  compounds related to (4-Ο-methyl-D-glucurono)-D-xylan. Carbohydr Res

437  1986;152:131-6.

438  [3] Shatalov A, Pereira H. Uronic (hexenuronic) acid profile of ethanol–alkali

439  delignification of giant reed Arundo donax L. Cellulose 2004;11:109-17.

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440  [4] Daniel AID, Neto CP, Evtuguin DV, Silvestre AJD. Hexenuronic acid contents of

441  Eucalyptus globulus kraft pulps: variation with pulping conditions and effect on ECF

442  bleachability. Tappi J 2003;2:3-8.

443  [5] Buchert J, Tenkanen M, Viikari L, Teleman A, Harjunpaa V, Vuorinen T. Effect of

t
ip
444  cooking and bleaching on the structure of xylan in conventional pine kraft pulp. Tappi J

445  1995;78:125-30.

cr
446  [6] Simão JPF, Egas APV, Baptista CMSG, Carvalho MG, Castro JAAM. Evolution of

us
447  methylglucuronic and hexenuronic acid contents of Eucalyptus globulus pulp during

448  kraft delignification. Ind Eng Chem Res 2005;44:2990-6.

an
449  [7] Takahashi S, Nakagawa-izumi A, Ohi H. Differential behavior between acacia and

450  Japanese larch woods in the formation and decomposition of hexenuronic acid during
M
451  alkaline cooking. J Wood Sci 2011;57:27-33.

452  [8] Li J, Gellerstedt G. On the structural significance of the kappa number measurement.
d

453  Nord Pulp Paper Res J 1988;13:153-8.

te

454  [9] Vuorinen T, Fagerström P, Buchert J, Tenkanen M, Teleman A. Selective hydrolysis

p

455  of hexenuronic acid groups and its application in ECF and TCF bleaching of kraft pulps.
ce

456  J Pulp Pap Sci 1999;25:155-62.

457  [10] Granstrom A, Eriksson T, Gellerstedt G, Roost C, Larsson P. Variables affecting

Ac

458  the thermal yellowing of TCF-bleached birch kraft pulps. Nord Pulp Paper Res J

459  2001;16:18-23.

460  [11] Cadena EM, Vidal T, Torres AL. Influence of the hexenuronic acid content on

461  refining and ageing in eucalyptus TCF pulp. Bioresour Technol 2010;101:3554-60.

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462  [12] Valls C, Colom JF, Baffert C, Gimbert I, Roncero MB, Sigoillot J-C. Comparing

463  the efficiency of the laccase–NHA and laccase–HBT systems in eucalyptus pulp

464  bleaching. Biochem Eng J 2010;49:401-7.

465  [13] Valls C, Roncero MB. Using both xylanase and laccase enzymes for pulp

t
ip
466  bleaching. Bioresour Technol 2009;100:2032-9.

467  [14] Valls C, Vidal T, Roncero MB. The role of xylanases and laccases on hexenuronic

cr
468  acid and lignin removal. Process Biochem 2010;45:425-30.

us
469  [15] Nurizzo D, Nagy T, Gilbert HJ, Davies GJ. The structural basis for catalysis and

470  specificity of the Pseudomonas cellulosa α-glucuronidase, GlcA67A. Structure

an
471  2002;10:547-56.

472  [16] Nagy T, Nurizzo D, Davies GJ, Biely P, Lakey JH, Bolam DN, et al. The α-
M
473  glucuronidase, GlcA67A, of Cellvibrio japonicus utilizes the carboxylate and methyl

474  groups of aldobiouronic acid as important substrate recognition determinants. J Biol

d

475  Chem 2003;278:20286-92.

te

476  [17] Winyasuk W, Gomi S, Shimokawa T, Hishiyama S, Satake T, Yoshida S.

p

477  Screening of enzyme system for specific degradation of hexenuronosyl-xylotriose. Int J

ce

478  Agric Technol 2012;8:103-16.

479  [18] Pason P, Kyu KL, Ratanakhanokchai K. Paenibacillus curdlanolyticus strain B-6
Ac

480  xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides. Appl

481  Environ Microbiol 2006;72:2483-90.

482  [19] Pason P, Kosugi A, Waeonukul R, Tachaapaikoon C, Ratanakhanokchai K, Arai T,

483  et al. Purification and characterization of a multienzyme complex produced by

484  Paenibacillus curdlanolyticus B-6. Appl Microbiol Biotechnol 2010;85:573-80.

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485  [20] Berg B, Hofstan BV, Petterson B. Growth and cellulase formation by Cellvibrio

486  fulvus. J Appl Bacteriol 1972;35:201-14.

487  [21] Milner Y, Avigad G. A copper reagent for the determination of hexuronic acids and

488  certain ketohexoses. Carbohydr Res 1967;4:359-61.

t
ip
489  [22] Nelson N. A photometric adaptation of the Somogyi method for determination of

490  glucose. J Biol Chem 1944;153:375-80.

cr
491  [23] Wood TM, Bhat KM. Methods for measuring cellulase activities. In: Willis A.

us
492  Wood STK, editor. Methods Enzymol: Academic Press; 1988. p. 87-112.

493  [24] Bronnenmeier K, Meissner H, Stocker S, Staudenbauer WL. α-D-Glucuronidases

an
494  from the xylanolytic thermophiles Clostridium stercorarium and

495  Thermoanaerobacterium saccharolyticum. Microbiology 1995;141:2033-40.

M
496  [25] Zaide G, Shallom D, Shulami S, Zolotnitsky G, Golan G, Baasov T, et al.

497  Biochemical characterization and identification of catalytic residues in α-glucuronidase

d

498  from Bacillus stearothermophilus T-6. Eur J Biochem 2001;268:3006-16.

te

499  [26] Nagy T, Emami K, Fontes CMGA, Ferreira LMA, Humphry DR, Gilbert HJ. The
p

500  membrane-bound α-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-Ο-

ce

501  methyl-D-glucuronoxylooligosaccharides but not 4-Ο-methyl-D-glucuronoxylan. J

502  Bacteriol 2002;184:4925-9.

Ac

503  [27] Biely P, de Vries RP, Vršanská M, Visser J. Inverting character of α-glucuronidase

504  A from Aspergillus tubingensis. Biochim Biophys Acta 2000;1474:360-4.

505  [28] Warren L. Thiobarbituric acid spray reagent for deoxy sugars and sialic acids.

506  Nature 1960;186:237.

507  [29] Golan G, Shallom D, Teplitsky A, Zaide G, Shulami S, Baasov T, et al. Crystal

508  structures of Geobacillus stearothermophilus α-glucuronidase complexed with its

23
 
Page 23 of 33
  
 

509  substrate and products: MECHANISTIC IMPLICATIONS. J Biol Chem

510  2004;279:3014-24.

511  [30] Shallom D, Golan G, Shoham G, Shoham Y. Effect of dimer dissociation on

512  activity and thermostability of the α-glucuronidase from Geobacillus

t
ip
513  stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside

514  hydrolases. J Bacteriol 2004;186:6928-37.

cr
515  [31] de Vries RP, Poulsen CH, Madrid S, Visser J. aguA, the gene encoding an

us
516  extracellular α-glucuronidase from Aspergillus tubingensis, is specifically induced on

517  xylose and not on glucuronic acid. J Bacteriol 1998;180:243-9.

518 

an
M
d
p te
ce
Ac

24
 
Page 24 of 33
  
 

518  Figure Captions

519  Fig. 1. Structures of 4-O-methyl-glucuronoxylan and hexenuronosyl xylan.

520 

t
ip
521  Fig. 2. SDS-PAGE analysis of AguA purification steps.

cr
522  Recombinant protein was produced by E. coli harboring an expression plasmid. Lane M,

523  standard protein molecular mass markers; lane 1, crude lysate; lane 2, flow-through;

us
524  lane 3, wash; lane 4, eluate (purified protein). Lane 4 contains 3.5 μg (10 μL) of protein.

an
525 

526  Fig. 3. Scheme for the fractionation of ΔX3 (a) and its treatment with β-xylosidase (b).
M
527  (a) A total of 5.1 g of purified ΔX3 was obtained from 2 kg of hardwood oxygen-
d

528  delignified kraft pulp (LOKP). (b) HPAEC-PAD chromatograms for the fractionation of
te

529  ΔX3 before (top) and after (bottom) treatment with β-xylosidase. The PAD response

530  (nA) and elution time (min) are shown on the y- and x-axis, respectively. The dotted
p

531  lines in the chromatograms show the concentration of eluent.

ce

532 
Ac

533  Fig. 4. HPAEC-PAD (a) and TLC (b) analyses of xylotriose and HexA released from

534  ΔX3 by AguA.

535  (a) Three HPAEC-PAD chromatograms show analysis of reaction mixtures of ΔX3

536  without AguA (top), with AguA (middle), and following addition of β-xylosidase after

537  hydrolysis with AguA (bottom). The PAD response (nA) and elution time (min) are

538  shown on the y- and x-axis, respectively. The dotted lines in the chromatograms show

25
 
Page 25 of 33
  
 

539  the concentration of eluent. (b) HexA was detected on TLC plates sprayed with

540  thiobarbituric acid solution.

541 

t
Fig. 5. Biological degradation of ΔX3 using extract from P. curdlanolyticus B-6.

ip
542 

Biological degradation of ΔX3 was carried out using intracellular extract from xylan-

cr
543 

544  grown P. curdlanolyticus B-6 cells. Concentrations of ΔX3 (triangles), xylotriose

us
545  (diamonds), and xylose (squares) were measured by HPAEC-PAD analysis. The

546  concentration of released HexA (circles) was estimated from the decrease in the

an
547  concentration of ΔX3. M
548 
d
p te
ce
Ac

26
 
Page 26 of 33
  
 

548  TABLE 1 Oligonucleotide primers used for cloning of aguA

Primer Sequence (5′→3′)

Degenerate primersa

t
AguMF1 GAY GGN WSN ATH GAR MGN GGN TAY GCN GGN

ip
AguMR1 RTC RTA DAT RTG YTG DAT NAC NGT YTT NCC NSW

cr
Gene-specific primers

us
GWR1 CAGACGCGCATAATCTTCGATCCGGCC

GWR2 AGGCTGGCCGTCATGACCGAACACACG

an
GWF1 GGACCTAACGCAGCTAAGTTCAATTCG

GWF2 CTCGAAGGGCTGATCGAGCCAGAGACA
M
Expression primersb

AguA_pET22F CATG CCATG GGCGAAAGAACGAAACGCAGC

d
te

AguA_pET22R GGGCTCGAGATAAATTTTCCGGCCGTGATC

a
549  The two degenerate primers were designed based on highly conserved amino acid
p

550  sequences (DGSIERGYAG and SGKTVIQHIY) in bacterial α-glucuronidases. D

ce

551  represents G or A or T; H represents A or C or T; M represents A or C; N is any base; R

552  represents G or A; W represents A or T; and Y represents T or C.
Ac

b
553  NcoI and XhoI restriction sites are underlined.

554 

27
 
Page 27 of 33
  
 

554 

555  TABLE 2 Enzymatic properties of extracellular and intracellular fractions from P.

556  curdlanolyticus B-6

Specific activity (U/mg protein)

t
ip
Enzymatic activity Extracellular fractiona Intracellular fraction

α-Glucuronidaseb 0.0027±0.0002 0.038±0.001

cr
β-Xylosidase 0.0031±0.003 0.040±0.005

us
β-Glucosidase 0.0014±0.0008 0.007±0.0009

an
Xylanase 5.51±1.5 0.73±0.08

557  Values are means of triplicate experiments ± standard deviation.

M
a
558  Culture broth from a xylan-grown culture was used directly as the enzyme preparation
559  for each assay.
d

b
560  Aldouronic acid was used as substrate.
te

561 
p
ce
Ac

28
 
Page 28 of 33
Figure(s)

Figure 1. Septiningrum et al.

t
ip
4-O-methyl-glucuronoxylan

cr
us
α-glucuronidase (GH4, GH67, GH115)

an
M
ed

Hexenuronosyl xylan
pt
ce
Ac

Page 29 of 33
 t
ip
cr
Figure 2. Septiningrum et al.

us
an
kDa M 1 2 3 4
150

100
M
80

50
ed

37
pt

25
ce
Ac

Page 30 of 33
Figure 3. Septiningrum et al.

a.
LOKP

t
(2 kg)

ip
- Treatment by alkaline solution (15% NaOH)

cr
Supernatant
Sediment (87 g)

us
- Treatment by xylanase (Shearzyme) and cellulase (Onozuka R-10)
- Removal of monosaccharides by active charcoal

an
ΔX3 containing
xylooligosaccharides
(5.8 g)
M
- Treatment by ß-xylosidase

ΔX3
(5.1 g)
ed

b.
pt

ΔX3
ce

Xylotriose
Xylose
Ac

ΔX3
Xylose

Page 31 of 33
Figure 4. Septiningrum et al.

a. ΔX3

t
Xylose

ip
cr
us
Xylotriose ΔX3

an
Xylose
M
ΔX3
Xylose
ed
pt
ce

b.
Ac

HexA

ΔX3 ΔX3
hydrolysate with
AguA Page 32 of 33
Figure 5. Septiningrum et al.

t
ip
cr
us
an
M
6.0 16.0

Xylose concentration (mM)

ΔX3, HexA, and xylotriose

5.0
concentrations (mM)

ed

12.0
4.0

3.0 8.0
pt

2.0
4.0
ce

1.0

0.0 0.0
Ac

0 1 2 3 4 5 6

Hydrolysis time (h)

Page 33 of 33