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Expression and characterization of levansucrase from Clostridium acetobutylicum Song Gao, Xianghui Qi, Darren J. Hart, Herui Gao, and Yingfeng An J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05165 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017
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Journal of Agricultural and Food Chemistry
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Expression and characterization of levansucrase from
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Clostridium acetobutylicum
3
Song Gao a,b§, Xianghui Qi c§, Darren J. Hart d, Herui Gao a, Yingfeng An a §
§
*
4 5 6 7
a
College of Biosciences and Biotechnology, Shenyang Agricultural University,
Shenyang 110161, China b
College of Food Science, Shenyang Agricultural University, Shenyang
8
110161, China
9
c
School of Food and Biological Engineering, Jiangsu University, Zhenjiang
10
212000, China
11
d
12
Grenoble 38044, France
Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes,
13 14 15 16 17 18
* Corresponding author: Yingfeng An
19
Email:
[email protected] 20
Tel: +86-24-88487163.
Fax: +86-24-88487163
21 22
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Abstract
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The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase
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was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287
26
bp and encodes 428 amino acid residues, which could convert 150 mmol/L
27
sucrose to levan with the liberation of glucose. The optimum pH and
28
temperature of this enzyme for levan formation were pH 6 and 60 ℃ ,
29
respectively. Levansucrase activity of Ca-SacB was completely abolished by 5
30
mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were
31
calculated to be 64 mmol/L and 190 µmol/min/mg, respectively. Interestingly,
32
Ca-SacB
33
fructooligosaccharide was identified in the product, indicating that Ca-SacB
34
may be valuable for industrial production of levan. In addition, Ca-SacB is the
35
first characterized levansucrase isolated from an anaerobic bacterium, which
36
should be valuable for exploring new enzyme resources and deepening the
37
understanding of the catalytic mechanisms of levansucrases.
was
found
to
have
high
product
specificity
38 39
Key words:
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Ca-SacB; Clostridium acetobutylicum; levan; levansucrase
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Introduction
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Levansucrase (EC 2.4.1.10), one of the fructosyltransferases, belongs to
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glycoside hydrolase family 68 (GH68) 1 and catalyzes the production of levan,
48
composed of β-(2-6)-linked fructose residues.2 Levan has varieties of
49
applications in the fields of foods, cosmetics, and pharmaceuticals.3,4
50
Levansucrases are produced by various microorganisms belonging to the
51
genera Bacillus, Acetobacter, Lactobacillus, Geobacillus, Leuconostoc,
52
Zymomonas, Pseudomonas, etc.5 Levansucrase activity is involved in varieties
53
of processes including survival of bacteria in soil (e.g., B. subtilis), symbiosis
54
(e.g., Paenibacillus polymyxa) and phytopathogenesis (e.g., Pseudomonas
55
and Erwinia species) of plant interactive bacteria.6 Levansucrases catalyze at
56
least three different reactions: polymerization of fructose derived from sucrose,
57
hydrolysis of sucrose and hydrolysis of levan. 7
58
The 3D structures
of levansucrases 10
from Bacillus subtilis,8 B.
59
megaterium,9 Lactobacillus johnsonii
60
resolved. These levansucrases share a β-propeller fold consisting of five
61
antiparallel β-strands and a central negatively charged cavity, which are also
62
the significant characteristics of members of GH68.9
and Erwinia amylovora
11
were
63
Until now, all the reported levansucrases are from aerobic bacteria and
64
microaerobes, but no levansucrases from anaerobic bacteria have been
65
characterized. Therefore, isolating and characterizing of levansucrases from
66
anaerobic bacteria should be valuable for exploring new enzyme resources 3
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and deepening the understanding of their catalytic mechanisms. C.
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acetobutylicum is a very important anaerobic bacterium which can be used for
69
producing acetone, ethanol, and butanol from starch. Although levansucrase
70
Ca-SacB from C. acetobutylicum can be predicted by BLAST, no detailed
71
information about this enzyme has been reported. In the present study, we
72
report the first-time molecular cloning and expression of C. acetobutylicum
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levansucrase (Ca-SacB) in E. coli. This result will give information for better
74
understanding of the catalytic strategies of Ca-SacB and laying the
75
foundations for industrial applications of this enzyme for the production of
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levan.
77 78
Materials and methods
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2.1. Strains, plasmids and media
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C. acetobutylicum was anaerobically cultured in medium containing 3%
81
(w/v) glucose, 0.5% (w/v) yeast extract, 0.07% (w/v) (NH4)2HPO4, 0.2% (w/v)
82
CaCO3, pH7.0. E. coli JM109 strain (Promega, USA) was used for molecular
83
cloning and propagation of the plasmids, and E. coli BL21(DE3) strain was
84
used for expression of the recombinant levansucrase. LB agar plates
85
supplemented with 5% (w/v) sucrose, 50 mg/L kanamycin, and 0.1 mmol/L
86
isopropyl-β-d-thiogalactopyranoside (IPTG) were used for the identification of
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the levansucrase phenotype.
88
2.2. Cloning, expression and characterization of Ca-SacB 4
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Genomic DNA was isolated from C. acetobutylicum and used as template
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for PCRs. Ca-SacB gene was amplified by PCR using primers: sacB-For
91
(5’-CTAGG ACGTC GTTGA AAACA AGAAA AACTT ATAAA ATGAT ATCTT
92
CGC-3’, Aat II underlined) and sacB-Rev1 (5’-TACCA CTAGT ATGTG
93
CAGGC GTAAC TACTC CTTCC CCAAG-3’, Spe I underlined). The PCR
94
products were cloned into the corresponding restriction enzyme sites of
95
pETM11 to give pET-Ca-SacB. pET-Ca-SacB was transformed into E. coli
96
BL21(DE3).
97
supplemented with 50 mg/L kanamycin. DNA sequencing was carried out by
98
GENEWIZ Company in China. BLAST program was used for sequence
99
homology searches of GenBank (NCBI, Bethesda, MD, USA).
The transformants
were
replicated
on
LB
agar
plates
100
For protein expression and purification, the strain of E. coli BL21(DE3)
101
transformed with pET-Ca-SacB was cultured in TB media and protein
102
expression was induced by 0.1 mmol/L IPTG. The cells were pelleted by
103
centrifugation, resuspended in 50 mmol/L sodium phosphate buffer (pH 5.9),
104
and then disrupted by sonication. The expressed protein was purified using
105
Ni-NTA agarose (Qiagen, Germany) chromatography. The lysate was
106
incubated with Ni-NTA slurry at 4℃ for 10 min followed by loading to a column.
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The sample was washed three times with washing buffer (20 mmol/L imidazole,
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300 mmol/L NaCl, 50 mmol/L NaH2PO4, pH 8.0) and Ca-SacB protein was
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then eluted with 0.5 ml of elution buffer (250 mmol/L imidazole, 300 mmol/L
110
NaCl, 50 mmol/L NaH2PO4, pH 5.9). To determine kinetic parameters, sucrose 5
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hydrolysis was analyzed in a reaction containing an appropriate amount of
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sucrose (10-1000mmol/L) and purified enzyme. The reactions were incubated
113
at 50℃, and the glucose content was determined by Glucose Assay Kit (HuiLi
114
Biotech Co., China).
115
2.3. Isolation and component analysis of fructan
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Purified Ca-SacB was added to 50 mmol/L sodium phosphate buffer (pH
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5.9) containing 10% (w/v) sucrose. The reaction mixture was incubated at 20℃
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for 72 h. The soluble section of the products catalyzed by Ca-SacB was filtered
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through 0.22-µm Millipore filters and analyzed by high-performance liquid
120
chromatography (HPLC). HPLC analysis was carried out on a Waters 1525
121
HPLC system (Milford, MA, USA) using a Waters Symmetry C18 column (250
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mm × 4.5 mm). The standards contain fructose (F), glucose (G), sucrose (GF),
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1-kestose (GF2), nystose (GF3), and fructofuranosyl-nystose (GF4) (Meiji
124
Seika Kaisha Ltd). Degassed 70% acetonitrile at 1.0 mL/min was used as
125
mobile phase. The eluate was monitored with a 2414 Refractive Index
126
Detector.
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Then
13
C-NMR spectrometry was used to analyze linkage type of the
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fructan. An equal volume of ethanol was added to the reaction mixture,
129
followed by incubation at 4℃ for 12 h to allow the precipitation of fructan.
130
Fructan was recovered by centrifugation (200,000×g) and resuspended in
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water. The fructan pellet was washed twice by precipitations as described
132
above, and then the pellet was dehydrated by lyophilization. Then 6
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C-NMR
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spectrometry was run at 125 MHz on AMX-500 (Bruker, Germany).
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Assignment of peaks was based on the report of Shimamura et al.12
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2.4. Effect of temperature, pH, metal ions and chemicals
136
Effects of temperatures between 20 and 80℃ on stability and activity of
137
the Ca-SacB were studied. Thermostability was determined by incubating the
138
enzyme (1133 U/mg) for 30 min at a designated temperature, where 1 U was
139
defined as the amount of enzyme required to release 1 µmol of glucose per
140
minute under standard conditions. After incubation, the residual enzyme
141
activity was assayed under the standard reaction condition at 50℃. The effect
142
of pH on enzyme activity was assayed by varying pH between 3.0 and 8.0.
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McIlvaine buffer (prepared by mixing 0.1 mol/L Na2HPO4 and 0.1 mol/L citric
144
acid) and borax buffer (prepared by mixing 0.05 mol/L borax and 0.2 mol/L
145
boric acid) were used for pH 3.0-7.0 and pH 8.0-9.0, respectively. Then
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Ca-SacB was incubated at the indicated pH for 30 min at 50℃, and at each pH
147
the activity prior to incubation was used as positive control to determine pH
148
stability via assaying the residual activity after incubation. The effect of various
149
metal ions [CuCl2, AlCl3, Hg(NO3)2, MnCl2, MgSO4, KCl, LiCl, NaCl, ZnSO4,
150
CaCl2, BaCl2, NiSO4, CoCl2, SnCl2, RbCl, AgNO3, and FeSO4] and chelating
151
agents (EDTA, Urea and SDS) on levansucrase activity were studied by
152
incubating Ca-SacB solution with the respective chemicals for 30 min under
153
optimized conditions of 50℃ and pH5.9.
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3. Results and discussion 7
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3.1. Cloning, expression and characterization of Ca-SacB
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The Ca-SacB gene was composed of 1287 bp nucleotides encoding 428
157
amino acid residues. The deduced amino acid sequence of Ca-SacB gene
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was compared with some reported levansucrases from other microorganisms.
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It showed identity with amino acid sequences of levansucrases from
160
Brevibacillus formosus (53%),13 Streptomyces olindensis (44%),14 Rahnella
161
aquatilis (37%),15 Zymomonas mobilis (36%),16 Pseudomonas syringae
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(36%),17 B. subtilis (29%).1 Ca-SacB originates from a gram-positive strain,
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and its gene sequence was most homologous to those of other gram-positive
164
strains, such as B. formosus and S. olindensis.
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Sequence alignment of Ca-SacB and levansucrase from B. subtilis
166
(Bs-SacB) based on structural superimposition was generated by ESPript 3.0
167
(Fig. 1). Secondary structure elements were labelled using the structure of
168
Bs-SacB as template. The conserved regions are suggested to be important
169
for the activities, e.g., sucrose hydrolysis and transfer of fructose to the proper
170
acceptors.18,19 There are totally seven conserved regions in the reported
171
levansucrases from gram-positive strains,20 six of which (i.e., II to VII) are
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conserved in Ca-SacB. In addition, three crucial amino acid residues (i.e., D in
173
conserved region II, D in region IV, and E in region V) that function together at
174
the center of the active site (i.e., catalytic triad) in reported levansucrases
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were also conserved in Ca-SacB.
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21
To further understand the structure and functions of Ca-SacB, a protein 8
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model was built by SWISS-MODEL using the crystal structure of Bacillus
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subtilis levansucrase (PDB ID: 1oyg) as template (Fig. 2). According to the
179
model, Ca-SacB has the typical structure of β-propeller fold consisting of five
180
blades with antiparallel β-strands (Fig. 2-a), and the β-propeller of each
181
structure forms a central negatively charged cavity, which is essential for
182
activity (Fig. 2-a, b). Although the protein sequences of CA-SacB and Bs-SacB
183
have a low identity (29%), the alignment of the model of CA-SacB and the
184
solved Bs-SacB structure shows that their structures might have high identity
185
(Fig. 2-c).
186
Based on Bs-SacB structure1 and alignment of amino acid sequence of
187
Bs-SacB (Fig 1 and Fig 2-d), we could predict three fully conserved active site
188
amino acid residues (D71, D222 and E306). The D71 (corresponding to D86 in
189
Bs-SacB) and E306 (corresponding to E342 in Bs-SacB) might form the pair of
190
essential catalytic side chains, whereas D222 (corresponding to D247 in
191
Bs-SacB) might interact with hydroxyls of the fructosyl unit of substrate, and
192
form strong hydrogen bonds. E306 might be part of a complex network of
193
interactions that includes R221 (corresponding to R246 in Bs-SacB), H324
194
(corresponding to Arg360 in Bs-SacB) and Y371 (corresponding to Y411 in
195
Bs-SacB), and Q304 (corresponding to E340 in Bs-SacB). Although some of
196
the amino acid residues mentioned above are not fully conserved, their side
197
chains or properties are similar to that of Bs-SacB.
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In order to characterize the enzymatic properties of Ca-SacB, His-tagged 9
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Ca-SacB was purified by Ni–NTA chromatography. The purified levansucrase
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from E. coli lysate showed specific activity of 1133 U/mg. The Km of Ca-SacB
201
was 64 mmol/L sucrose and the Vmax was 190 µmol/min/mg. The Km value of
202
this enzyme was similar to the Km of levansucrase from R. aquatilis JCM-1683
203
(50 mmol/L).22 However, levansucrases from Z. mobilis
204
were reported to have Km of higher values: 160 and 122 mmol/L, respectively.
205
3.2. Analysis of sugar components
23
and P. syringae
24
206
The transfructosylation reactions and levan formation by Ca-SacB were
207
assayed in a standard reaction containing sucrose. As a result, Ca-SacB
208
expressed in E. coli could catalyze the production of turbid levan (Fig. 3-a).
209
The soluble section of the products catalyzed by Ca-SacB was analyzed by
210
HPLC. As a result, nearly no fructooligosaccharide was identified from this
211
section, indicating that levan was the only product of transfructosylation by
212
Ca-SacB (Fig. 3-b). According to the HPLC diagram, about 61% sucrose has
213
been converted into levan and glucose during the reaction.
214
The linkage type of the polymer was analyzed by
13
C-NMR spectrometry
215
(Fig 4). Assignment of peaks was based on the report of Shimamura et al.12
216
The results indicate that the polymer is levan of β-2, 6-fructan. In this study,
217
only the linkage type of the insoluble polymer with high molecular mass has be
218
analyzed by 13C-NMR spectrometry, because the soluble sucrose and
219
glucose in the reaction mixture have been eliminated by centrifugation.
220
3.3. Effect of temperature, pH, metal ions and chemicals 10
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As shown in Fig. 5, the optimum temperature of Ca-SacB was 60℃. The
222
activity was greatly reduced at temperatures below 30℃ or above 70℃ (Fig.
223
5-a). At temperatures higher than 70 ℃ , this enzyme was inactive. The
224
thermostability decreased sharply above a 70℃ threshold temperature. The
225
optimum pH of Ca-SacB was found to be 6.0 (Fig. 5-b). The activity was
226
greatly reduced at pH below 4.0 or above pH 7.0.
227
The effect of metal ions and other reagents on levansucrase activity of
228
Ca-SacB was determined by incubating Ca-SacB in the presence of reagents
229
at 50℃ for 30 min. The residual activity was assayed by the standard method.
230
As a result, the activity was strongly inhibited by CuCl2, Hg(NO3)2, AgNO3 and
231
SDS; while SnCl2 and MnCl2 increased levansucrase activity by 43% and 28%,
232
respectively (Table 1). SDS is a commonly used protein-denaturing agent in
233
biology laboratories. The levansucrases from A. diazotrophicus,6 Bacillus sp.
234
TH4-2,20 and Leuconostoc mesenteroides
235
inactivated by Hg2+ and Ag+. Recently, Mn2+ has also been found to have
236
positive effect on the activity of levansucrase from B. subtilis, which was
237
postulated to be associated with the folding cofactor effect of this metal.25
238
Interestingly, in this study SnCl2 was found to have the strongest activation
239
effect on Ca-SacB, but it has never been reported to have similar effect on
240
other levansucrases.
7
were also strongly inhibited or
241
To sum up, in the present study, we first describe the cloning,
242
heterologous expression and characterization of levansucrase gene Ca-SacB 11
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from C. acetobutylicum, which should lay the foundation for further
244
modification of this enzyme for more efficient production of fructan. Further
245
studies aimed at better understanding the catalysis of transfructosylation by
246
Ca-SacB is now in progress.
247
Acknowledgments
248 249
The authors would like to thank Sergi Castellano and Promdonkoy Patcharee for helpful discussions and review of this manuscript.
250 251
§The authors Song Gao and Xianghui Qi contributed equally to this
252
work.
253 254
Funding Sources
255
This work was supported by National Natural Science Foundations of
256
China (grant numbers 31100045, 31270114, 31571806), Program for Liaoning
257
Excellent Talents in University (grant number LR2014018), and Liaoning
258
BaiQianWan Talents Program (grant number 2015-40).
259 260 261 262 263 264 265
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(19) Song, K.B.; Joo, H.K.; Rhee, S.K. Nucleotide sequence of
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levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988). Biochim.
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Biophys. Acta. 1993,1173,320−324.
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(20) Seo, J.W.; Song, K.B.; Jang, K.H.; Kim, C.H.; Jung, B.H.; Rhee, S.K.
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Molecular cloning of a gene encoding the thermoactive levansucrase from
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Rrahnella
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Eescherichia coli. J. Biotechnol. 2000,81,63−72.
aquatilis
and
its
growth
phase-dependent
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expression
in
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(21) Verhaest, M.; Van den Ende, W.; Roy, K.L.; De Ranter, C.J.; Laere,
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A.V.; Rabijns, A. X-ray diffraction structure of a plant glycosyl hydrolase family
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32 protein: fructan 1-exohydrolase IIa of Cichorium intybus. Plant J.
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2005,41,400−411.
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(22) Hernandez, L.; Arrieta, J.; Menendez, C.; Vazquez, R., Coego, A.;
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Suarez, V.; Selman, G.; Petit-Glatron, M.F.; Chambert, R. Isolation and
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enzymic properties of levansucrase secreted by Acetobacter diazotrophicus
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SRT4,
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1995,309,113−118.
a
bacterium
associated
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cane.
Biochem.
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(23) Yanase, H.; Iwata, M.; Nakahigashi, R.; Kita, K.; Kato, N.; Tonomura,
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K. Purification, crystallization and properties of the extracellular levansucrase
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from Zymomonas mobilis, Biosci. Biotechnol. Biochem. 1992,56,1335−1336.
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(24) Hettwer, U.; Gross, M.; Rudolph, K. Purification and characterization
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of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola.
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J. Bacteriol. 1995,177,2834−2839.
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(25) Artur, S.; Kamila, G.; Małgorzata, G. Synthesis of ß-(2-6)-linked
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fructan with a partially purified levansucrase from Bacillus subtilis. J. Mol. Catal.
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B-Enzym. 2016,131,1−9.
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Fig. 1 Ca-SacB and Bs-SacB sequence alignment based on structural
355
superimposition. Secondary structure elements α-helices and β-strands are
356
indicated by squiggles and arrows, respectively. The α-helices (labelled α) and
357
β-strands (labelled β) are consecutively numbered. The regions considered as
358
important for activity are underlined and consecutively numbered from I to VII.
359
The catalytic triad at the expected center of the active site (i.e., D in conserved
360
region II, D in region IV, and E in region V) are marked with triangles.
361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 17
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Fig. 2 Analysis of protein model of CA-SacB. (a) shows model of Ca-SacB.
392
α-helices are shown by solid lines in red, and β-strands in blue; (b) shows
393
surface of protein model of Ca-SacB. The central negatively charged cavity in
394
red shows the center of the active site; (c) shows alignment of Bs-SacB
395
structure (in green) and the model of CA-SacB (in blue); (d) shows alignment
396
of some important amino acid residues Bs-SacB structure (in blue) and in the
397
model of CA-SacB (in green). Numbering of amino acid residues is based on
398
Bs-SacB, with the numbering of CA-SacB in parentheses.
399 400 401 402 403 404 405 406 407 408 409 410 411 412
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Fig. 3 Production of turbid levan introduced by Ca-SacB in the presence
414
of sucrose and analysis of the soluble section by HPLC. (a) shows the
415
production of turbid levan by transfructosylation activity of Ca-SacB. 1 and 2
416
refer to reactions be associated with E. coli BL21(DE3) harboring plasmid
417
pET-Ca-sacB and pETM11, respectively; (b) shows HPLC chromatogram of
418
the soluble section of the products catalyzed by Ca-SacB. G, GF, GF2, GF3
419
and
420
fructofuranosyl-nystose, respectively
GF4
refer
to
glucose,
sucrose,
1-kestose,
421 422 423 424 425 426 427 428 429 430 431 432 433 434
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nystose
and
Journal of Agricultural and Food Chemistry
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Fig. 4. Analysis of components of sugar synthesized using purified
436
Ca-SacB by
437
synthesized using Ca-SacB; (b) shows chemical shifts for C-NMR spectra of
438
levan and polymer synthesized using Ca-SacB.
13
C-NMR spectra. (a)
13
C-NMR spectra shows polymer
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Fig. 5. Effect of temperature (a) and pH (b) on activity and stability of
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Ca-SacB. The activity (shown as solid circles) and stability (shown as solid
459
squares) were measured using 5% (w/v) sucrose as substrate. Error bars
460
represent means ± standard deviations (n=3).
461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 21
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Table 1 Effect of metal ions and detergents (5mmol/L) on levansucrase activity Compound Relative activity(%) Control (without any metal ion) 100 AgNO3 2 Hg(NO3)2 2 55 AlCl3 BaCl2 107 FeSO4 50 LiCl 104 NaCl 100 NiSO4 109 SnCl2 143 RbCl 99 KCl 118 ZnSO4 41 CoCl2 104 CuCl2 5 CaCl2 113 128 MnCl2 MgSO4 115 EDTA 36 Urea 96 SDS 3
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Journal of Agricultural and Food Chemistry
Fig. 1 Ca-SacB and Bs-SacB sequence alignment based on structural superimposition. Secondary-structural elements α-helices and β-strands are indicated by squiggles and arrows, respectively. The α-helices (labelled α) and β-strands (labelled β) are consecutively numbered. The regions considered as important for activity are underlined and consecutively numbered from I to VII. The catalytic triad at the expected center of the active site (i.e., D in conserved region II, D in region IV, and E in region V) are marked with triangles.
99x108mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Fig. 2 Analysis of protein model of CA-SacB. (a) shows model of Ca-SacB. α-helices are shown by solid lines in red, and β-strands in blue; (b) shows surface of protein model of Ca-SacB. The central negatively charged cavity in red shows the center of the active site; (c) shows alignment of Bs-SacB structure (in green) and the model of CA-SacB (in blue); (d) shows alignment of some important amino acid residues Bs-SacB structure (in blue) and in the model of CA-SacB (in green). Numbering of amino acid residues is based on Bs-SacB, with the numbering of CA-SacB in parentheses.
85x80mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Fig. 3 Production of turbid levan introduced by Ca-SacB in the presence of sucrose and analysis of the soluble section by HPLC. (a) shows the production of turbid levan by transfructosylation activity of Ca-SacB. 1 and 2 refer to reactions be associated with E. coli BL21(DE3) harboring plasmid pET-Ca-sacB and pETM11, respectively; (b) shows HPLC chromatogram of the soluble section of the products catalyzed by Ca-SacB. G, GF, GF2, GF3 and GF4 refer to glucose, sucrose, 1-kestose, nystose and fructofuranosyl-nystose, respectively
33x13mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Fig. 4. Analysis of components of sugar synthesized using purified Ca-SacB by 13C-NMR spectra. (a) 13CNMR spectra shows polymer synthesized using Ca-SacB; (b) shows chemical shifts for C-NMR spectra of levan and polymer synthesized using Ca-SacB. 57x38mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Fig. 5. Effect of temperature (a) and pH (b) on activity and stability of Ca-SacB. The activity (shown as solid circles) and stability (shown as solid squares) were measured using 5% (w/v) sucrose as substrate. Error bars represent means ± standard deviations (n=3).
82x80mm (300 x 300 DPI)
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TOC Graphic 44x24mm (300 x 300 DPI)
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