- Email: [email protected]
Mechanism study of Tween 80 enhancing the pullulan production by Aureobasidium pullulans
Accepted Manuscript Title: Mechanism study of Tween 80 enhancing the pullulan production by Aureobasidium pullulans Author: Long Sheng Guilan Zhu Qunyi Tong PII: DOI: Reference:
S0144-8617(13)00413-X http://dx.doi.org/doi:10.1016/j.carbpol.2013.04.058 CARP 7676
To appear in: Received date: Revised date: Accepted date:
15-3-2013 12-4-2013 15-4-2013
Please cite this article as: Sheng, L., Zhu, G., & Tong, Q., Mechanism study of Tween 80 enhancing the pullulan production by Aureobasidium pullulans, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.04.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mechanism study of Tween 80 enhancing the pullulan production by
2
Aureobasidium pullulans
3
Long Shenga, Guilan Zhub, Qunyi Tonga,*
ip t
1
4
The State Key Laboratory of Food Science and Technology, School of Food
cr
6
a
Science and Technology, Jiangnan University, Wuxi 214122, China
us
5
7 b
Department of Life Science, Hefei Normal University, Hefei 230061, China
an
8 9
*Corresponding author:
11
Qunyi Tong
12
Tel:/Fax: +86 510 85919170
13
E-mail address: [email protected]
15 16 17 18
d
te
Ac ce p
14
M
10
19 20 21 22
1
Page 1 of 17
23
Abstract The influences of Tween 80 on the pullulan production, cell growth,
25
glucosyltransferase activity and fatty acid composition of the cells were studied.
26
The addition of Tween 80 to the culture medium significantly enhanced the
27
pullulan
28
Glucosyltransferase activity involved in pullulan synthesis was remarkable
29
promoted
30
unsaturated/saturated fatty acids contained in the cells was significantly higher
31
in 0.5% Tween 80 in comparison with the absence of Tween 80. Results
32
indicated that the incease of pullulan yield brought by Tween 80 was highly
33
correlated with glucosyltransferase activity and fatty acid composition. Tween
34
80 was not degraded to serve as extra carbon source.
35
Keywords
36
Pullulan; Aureobasidium pullulans; Tween 80; Glucosyltransferase; Fatty acids
had
no
impact
to
biomass
accumulation.
an
increase
of
Tween
80
content.
The
ratio
of
te
d
M
an
with
us
cr
but
Ac ce p
37
production,
ip t
24
38
Abbreviations
39
A. pullulans, Aureobasidium pullulans; UDP-glucose, uridine diphosphate
40
glucose; PDA, potato dextrose agar; DCW, dry cell weight
41 42
1. Introduction
43
Pullulan, an exocellular homopolysaccharide, is produced by the strains of
44
Aureobasidium pullulans. It is mainly made of maltotriose repeating units joined
2
Page 2 of 17
by (1 → 6)-α linkages (Pan et al., 2013). It has a number of remarkable
46
properties, such as non toxicity, low viscosity, high plasticity and good
47
film-forming. This polysaccharide is widely used in food and pharmaceutical
48
industries (Cheng et al., 2011).
ip t
45
Pullulan production was under complex metabolic and environmental
50
control. It was reported that pullulan chains originated from UDP-glucose
51
(Shingel, 2004). Cately and McDowell (1982) had proposed the following order
52
of the biochemical events preceding pullulan formation. The first stage was
53
UDPG-mediated attachment of a D-glucose residue to the lipid molecule (LPh)
54
with a phosphoester bridge. A further transfer of the D-glucose residue from
55
UDP-glucose gave lipid-linked isomaltose. In the next step, isomaltose
56
participated in the reaction with lipid-linked glucose to yield an isopanosyl
57
residue. Further, isopanosyl residues were polymerized into the pullulan chain
58
(Daran et al., 1997). Therefore, glucosyltransferase might play an important role
59
in biosynthesis of pullulan with high yield.
us
an
M
d
te
Ac ce p
60
cr
49
Tween 80 was a non-ionic surfactant and known as polyethylene glycol
61
sorbitan monooleate. It had been reported that Tween 80 had the capability to
62
enhance glucosyltransferase synthesis in Streptococcus mutants (Tomita et al.,
63
1998) and Leuconostoc dextranicum (Majumder & Goyal, 2008). Tween 80 was
64
also found to stimulate the production of pullulan in A. pullulans, although there
65
was inadequate information available for explaining this phenomenon (West &
66
Reed-Hamer,
1995).
It
had
been
confirmed
that
the
activity
of
3
Page 3 of 17
glucosyltransferase was highly correlated with the amount of pullulan produced
68
and the carbon source used (Duan et al., 2008). However, the relationships
69
among Tween 80 concentration, glucosyltransferase activity and pullulan
70
production have not been studied.
ip t
67
The objectives of this paper were to investigate the effect of Tween 80 on
72
the pullulan production, biomass accumulation and glucosyltransferase activity
73
of A. pullulans CGMCC1234. The effect of Tween 80 on the fatty acid
74
composition of the cells was also evaluated.
an
us
cr
71
75
2. Materials and methods
77
2.1. Organisms and inoculum preparation
M
76
A. pullulans CGMCC1234 was maintained on potato dextrose agar (PDA)
79
at 4 °C and subcultured every 2 weeks. The seed culture medium contained
80
50.0 g sucrose, 1.5 g yeast extract, 4.0 g K2HPO4, 0.8 g (NH4)2SO4, 0.2 g
81
MgSO4, and 2.0 g NaCl per liter of deionized water. The pH was adjusted to 6.5,
82
and the medium was autoclaved at 121 °C for 15 min.
84
te
Ac ce p
83
d
78
2.2. Fermentative production of pullulan
85
Cultures were grown on PDA at 28 °C for 4 days and then transferred to a
86
250-ml flask that contained 50 ml of the seed culture medium and subsequently
87
incubated at 28 °C for 2 days with shaking at 200 rpm. The pullulan production
88
was carried out in a 5 l stirred tank fermentor (KF-5l, KoBioTech
4
Page 4 of 17
Bio-engineering Equipment Co., Ltd.) with a working volume of 3 l of the
90
production medium containing (g/l) 80.0 g sucrose, 0.9 g yeast extract, 6.0 g
91
K2HPO4, 0.6 g (NH4)2SO4, 0.5 g MgSO4, 4.0 g NaCl and 0-10.0 g Tween 80.
92
Fermentor was consisted of a glass vessel with stainless-steel endplates and
93
three equally spaced vertical baffles. Agitation was provided by a six-flat-blade
94
impeller (diameter 4 cm) located 3 cm above the bottom of the vessel.
95
Temperature, pH, dissolved oxygen, agitation speed and aeration rate in the
96
vessel could be monitored and controlled automatically. The fermentor with 3 l
97
of the production medium was sterilized at 121 °C for 15 min. After cooling, the
98
medium was inoculated with 150 ml of the seed culture. The pullulan production
99
was carried out at constant temperature of 28 °C, aeration rate of 3 l/min and
100
agitation speed of 300 rpm. The pH was controlled at 6.5 by feeding with either
101
2 M NaOH or 2 M HCl.
103 104
cr
us
an
M
d
te
Ac ce p
102
ip t
89
2.3. Isolation of exocellular polysaccharide The fermentation broth was appropriately diluted with distilled water and
105
heated at 80 °C in water bath for 30 min. The heated culture was centrifuged at
106
10,000 g for 20 min to remove the microorganisms and other precipitates. The
107
supernatant was mixed with two volumes of cold 95% (v/v) ethanol and held at
108
4 °C for 24 h to precipitate the pullulan completely. The precipitate was
109
separated by centrifugation at 15,000g for 20 min, rewashed with ethanol,
110
recovered by filtration and dried at 80 °C to a constant weight. The biomass
5
Page 5 of 17
pellet from the initial centrifugation was washed three times with distilled water
112
by centrifugation at 10,000g for 5 min and dried at 80 °C until the dry cell weight
113
(DCW) was constant. DCW and pullulan content were expressed as g/l.
ip t
111
114
2.4. Preparation of cell-free extract
cr
115
The cells in 5.0 ml of the culture were collected by centrifugation at 10000 g
117
for 20min at 4 °C. The cell pellet was washed three times with ice-cold distilled
118
water by centrifugation at 10,000g for 5 min. The pellet was suspended in 1.0
119
ml of ice-cold 1.0 M Tris-HCl (pH 7.6) to make a thick paste. The paste was
120
homegenized in a Homegenizer (DY89-I, Xinzhi, Zhejiang, China) for 1h on the
121
ice bath. The cell debris was removed by centrifugation at 14,000 g for 30 min
122
at 4 °C. The supernatant (the cell-free extract) was stored at -80 °C and used as
123
a source of enzymes. Protein concentration in the cell-free extract was
124
determined by the method of Bradford with bovine serum albumin as standard
125
(Bradford, 1976).
127 128
an
M
d
te
Ac ce p
126
us
116
2.5. Enzymes assays
Glucosyltransferase activity was determined according to methods
129
described by Duan et al (2008). A suitably diluted enzyme preparation (0.2 ml)
130
was incubated with pre-equilibrated p-nitrophenyl α-D-glucopyranoside (0.2 ml,
131
10 mM) in 0.1 M sodium acetate buffer (pH 4.0) for 5 min at 40 °C. The reaction
132
was terminated by the addition of an aqueous solution of Trizma base (3.0 ml,
6
Page 6 of 17
2.0% w/v), and the absorbance at 410 nm was measured. Enzyme activity was
134
expressed in terms of μM of p-nitrophenol released/min and calculated by
135
reference to a standard curve (the standard curve was made by measuring
136
OD410nm values of the solutions with different concentrations of p-nitrophenol).
137
The mixture with the diluted enzyme preparation that had been heated at 100
138
°C for 5 min was used as the control.
us
cr
ip t
133
139
2.6. Lipid analysis
an
140
Cells harvested at the late-logarithmic phase were washed three times with
142
the distilled water by centrifugation at 10,000g for 5 min. Total lipids extracted
143
from the freeze-dried cells by the method of Bligh and Dyer (1959) were
144
methylated based on the report of Ahn et al (2012). Fatty acids methyl esters
145
were analyzed by gas chromatography as reported previously (Ahn et al.,
146
2012).
d
te
Ac ce p
147
M
141
148
3. Results and discussion
149
3.1. Effect of the concentration of Tween 80 on pullulan fermentation
150
Influence of various concentrations of Tween 80 on pullulan production and
151
cell growth by A. pullulans CGMCC1234 are shown in Fig. 1. The addition of
152
Tween 80 to the nutrient medium significantly increased the pullulan production.
153
The pullulan production increased as the Tween 80 concentration in the
154
medium
increased.
Certain
experimental
results
evidenced
that
7
Page 7 of 17
glucose-containing lipid intermediates played a crucial role in pullulan
156
biosynthesis (Shingel, 2004). The lipid was used as a carrier in which pullulan
157
was taken to the outside of the plasmalemma. The pullulan arranged in a
158
network covering outer surface of the cells. Tween 80 was a well known
159
industrial surfactant, therefore it was assumed that its presence in the media
160
would affect the homogeneity of the broth and facilitate the nutrient and oxygen
161
transfer to the microorganism. However, the addition of Tween 80 to the nutrient
162
medium had no effect on biomass production, indicating that Tween 80 was not
163
degraded to serve as extra carbon source.
an
us
cr
ip t
155
165
M
164
3.2. Effect of the concentration of Tween 80 on glucosyltransferase production Effect of different concentrations of Tween 80 on glucosyltransferase
167
activity are shown in Fig. 2. Glucosyltransferase activity was remarkably
168
promoted with an increase of Tween 80 content. Furthermore, when Tween 80
169
was added to the cell-free extract, no increase in the glucosyltransferase
170
activity was observed (date not shown). As a result of this, Tween 80 did not
171
affect the activity of glucosyltransferase by converting the enzyme from an
172
inactive form to an active form and thus the increased glucosyltransferase
173
activity might be due to the improvement of glucosyltransferase production in
174
presence of Tween 80. Similar results that Tween 80 stimulated the production
175
of glucosyltransferase and had no direct activating effect on enzyme activity in
176
Streptococcus mutants was reported (Umesaki et al., 1977).
Ac ce p
te
d
166
8
Page 8 of 17
177 178
3.3. Effect of Tween 80 on fatty acid compositions Table 1 shows the effect of Tween 80 on the fatty acid composition of the
180
cells grown to the late-logarithmic phase. Enhancement of extracellular pullulan
181
production and intracellular glucosyltransferase activity by Tween 80 was
182
associated with changes in fatty acid composition in the cells. The major fatty
183
acids contained in the cells were palmitic acid (C16:0), palmitoleic acid (C16:1),
184
stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2). In the
185
presence of Tween 80, the ratio of unsaturated/saturated fatty acids increased
186
from 2.34 to 3.90. The increase in the unsaturated degrees by Tween 80 was
187
mainly due to the result that oleic acid increased with a decrease of palmitic
188
acid. The interaction of Tween 80 with cells affected the permeability of the cell
189
membrane, which might enhance to uptake the essential components including
190
vitamins from surroundings (Taoka et al., 2011) and to release of pullulan into
191
the extracellular fluid.
193 194
cr
us
an
M
d
te
Ac ce p
192
ip t
179
4. Conclusions
The addition of Tween 80 to the culture medium enhanced pullulan
195
production, but had no effect on biomass production. The mechanism by which
196
Tween 80 enhanced the production of pullulan was associated with the
197
glucosyltransferase activity and fatty acid composition.
198
9
Page 9 of 17
199
References Ahn, J. W., Hwangbo, K., Lee, S. Y., Choi, H. G., Park, Y., Liu, J. R., & Jeong, W.
201
J. (2012). A new Arctic Chlorella species for biodiesel production.
202
Bioresource Technology, 125, 340-343.
ip t
200
cr
203
Blight, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and
205
purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917.
us
204
an
206
Bradford, M. M. (1976). A rapid and sensitive method for quantitation of
208
microgram quantities of protein utilizing the principle of protein-dye binding.
209
Analytical Biochemistry, 72, 248-253.
M
207
te
d
210
Cately, B. J., & McDowell, W. (1982). Lipid-linked saccharides formed during
212
pullulan biosynthesis in Aureobasidium pullulans. Carbohydrate Research,
213 214
Ac ce p
211
103, 65-75.
215
Cheng, K. C., Demirci, A., & Catchmark, J. M. (2011). Pullulan: biosynthesis,
216
production, and applications. Applied Microbiology and Biotechnology, 92,
217
29-44.
218 219
Daran, J. M., Bell, W., & Francois, J. (1997). Physiological and morphological
220
effects of genetic alterations leading to a reduced synthesis of UDP-glucose
10
Page 10 of 17
221
in Saccharomyces cerevisiae. FEMS Microbiology Letters, 153, 89-96.
222
Duan, X. H., Chi, Z. M., Wang, L., & Wang, X. H. (2008). Influence of different
224
sugars on pullulan production and activities of α-phosphoglucose mutase,
225
UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan
226
synthesis in Aureobasidium pullulans Y68. Carbohydrate Polymers, 73,
227
587-593.
us
cr
ip t
223
an
228
Majumder, A., & Goyal, A. (2008). Enhanced production of exocellular
230
glucansucrase from Leuconostoc dextranicum NRRL B-1146 using response
231
surface method. Bioresource Technology, 99, 3685-3691.
M
229
te
d
232
Pan, S. K., Yao, D. R., Chen, J., & Wu, S. J. (2013). Influence of controlled pH
234
on the activity of UDPG-pyrophosphorylase in Aureobasidium pullulans.
235 236
Ac ce p
233
Carbohydrate Polymers, 92, 629-632.
237
Shingel, K. I. (2004). Current knowledge on biosynthesis, biological activity, and
238
chemical modification of the exopolysaccharide, pullulan. Carbohydrate
239
Research, 339, 447-460.
240 241
Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., & Hayashi, M.
242
(2011). Effect of Tween 80 on the growth, lipid accumulation and fatty acid
11
Page 11 of 17
243
composition of Thraustochytrium aureum ATCC 34304. Journal of Bioscience
244
and Bioengineering, 111, 420-424.
245
Tomita, Y., Watanabe, T., Takeuchi, T., Nanbu, A., Shinozaki, N., Ikemi, T., &
247
Fukushima, K. (1998). Effects of surfactants on glucosyltransferase
248
production and in vitro sucrose-dependent colonization by Streptococcus
249
mutants. Archives of Oral Biology, 43, 735-740.
an
250
us
cr
ip t
246
Umesaki, Y., Kawai, Y. & Mutai, M. (1977). Effect of Tween 80 on
252
glucosyltransferase production in Streptococcus mutants. Applied and
253
Environment Microbiology, 34, 115-119.
M
251
257
te
256
West, T. P., & Reed-Hamer, B. (1995). Effect of oils and surfactants on pullulan production relative to nitrogen source. Microbios, 83, 249-59.
Ac ce p
255
d
254
258
Figure captions
259
Fig. 1. Effect of Tween 80 concentration on pullulan production and cell growth
260
by Aureobasidium pullulans CGMCC1234. Values are given as means ±
261
standard deviation (n=3). Bars denoted with different capital letters are
262
significantly different (P＜0.05).
263 264
Fig. 2. Effect of Tween 80 concentration on glucosyltrasferase activity. Values
12
Page 12 of 17
are given as means ± standard deviation (n=3). Bars denoted with different
266
capital letters are significantly different (P＜0.05).
Ac ce p
te
d
M
an
us
cr
ip t
265
13
Page 13 of 17
Highlights (for review)
The addition of Tween 80 significantly enhanced pullulan production. Tween 80 had no impact to biomass accumulation. Glucosyltransferase activity was promoted by Tween 80.
ip t
The ratio of unsaturated/saturated fatty acids in the cells increased
Ac
ce pt
ed
M
an
us
cr
with Tween 80.
Page 14 of 17
Table(s)
Table 1 Fatty acid composition of A. pullulans cells grown with and without Tween 80 Fatty acids
% Total fatty acids 0.5% Tween 80
C16:0
23.7 ± 1.2
16.0 ± 0.4*
C16:1
1.3 ± 0.1
4.3 ± 0.2*
C18:0
6.2 ± 0.3
4.4 ± 0.1*
C18:1
34.8 ± 0.9
55.7 ± 1.0*
34.0 ± 0.7
19.6 ± 0.5*
2.34
3.90
C18:2
cr
unsaturated/saturated
a
ip t
Control
Values are given as means ± standard deviation (n=3). Values denoted with an asterisk
ce pt
ed
M
an
Ratio of unsaturated/saturated = (C16:1 + C18:1 + C18:2)/(C16:0 + C18:0).
Ac
a
us
have significant difference between control and treatment group (P＜0.05).
13
Page 15 of 17
Figure(s)
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 1
14
Page 16 of 17
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 2
15
Page 17 of 17
S0144-8617(13)00413-X http://dx.doi.org/doi:10.1016/j.carbpol.2013.04.058 CARP 7676
To appear in: Received date: Revised date: Accepted date:
15-3-2013 12-4-2013 15-4-2013
Please cite this article as: Sheng, L., Zhu, G., & Tong, Q., Mechanism study of Tween 80 enhancing the pullulan production by Aureobasidium pullulans, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.04.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mechanism study of Tween 80 enhancing the pullulan production by
2
Aureobasidium pullulans
3
Long Shenga, Guilan Zhub, Qunyi Tonga,*
ip t
1
4
The State Key Laboratory of Food Science and Technology, School of Food
cr
6
a
Science and Technology, Jiangnan University, Wuxi 214122, China
us
5
7 b
Department of Life Science, Hefei Normal University, Hefei 230061, China
an
8 9
*Corresponding author:
11
Qunyi Tong
12
Tel:/Fax: +86 510 85919170
13
E-mail address: [email protected]
15 16 17 18
d
te
Ac ce p
14
M
10
19 20 21 22
1
Page 1 of 17
23
Abstract The influences of Tween 80 on the pullulan production, cell growth,
25
glucosyltransferase activity and fatty acid composition of the cells were studied.
26
The addition of Tween 80 to the culture medium significantly enhanced the
27
pullulan
28
Glucosyltransferase activity involved in pullulan synthesis was remarkable
29
promoted
30
unsaturated/saturated fatty acids contained in the cells was significantly higher
31
in 0.5% Tween 80 in comparison with the absence of Tween 80. Results
32
indicated that the incease of pullulan yield brought by Tween 80 was highly
33
correlated with glucosyltransferase activity and fatty acid composition. Tween
34
80 was not degraded to serve as extra carbon source.
35
Keywords
36
Pullulan; Aureobasidium pullulans; Tween 80; Glucosyltransferase; Fatty acids
had
no
impact
to
biomass
accumulation.
an
increase
of
Tween
80
content.
The
ratio
of
te
d
M
an
with
us
cr
but
Ac ce p
37
production,
ip t
24
38
Abbreviations
39
A. pullulans, Aureobasidium pullulans; UDP-glucose, uridine diphosphate
40
glucose; PDA, potato dextrose agar; DCW, dry cell weight
41 42
1. Introduction
43
Pullulan, an exocellular homopolysaccharide, is produced by the strains of
44
Aureobasidium pullulans. It is mainly made of maltotriose repeating units joined
2
Page 2 of 17
by (1 → 6)-α linkages (Pan et al., 2013). It has a number of remarkable
46
properties, such as non toxicity, low viscosity, high plasticity and good
47
film-forming. This polysaccharide is widely used in food and pharmaceutical
48
industries (Cheng et al., 2011).
ip t
45
Pullulan production was under complex metabolic and environmental
50
control. It was reported that pullulan chains originated from UDP-glucose
51
(Shingel, 2004). Cately and McDowell (1982) had proposed the following order
52
of the biochemical events preceding pullulan formation. The first stage was
53
UDPG-mediated attachment of a D-glucose residue to the lipid molecule (LPh)
54
with a phosphoester bridge. A further transfer of the D-glucose residue from
55
UDP-glucose gave lipid-linked isomaltose. In the next step, isomaltose
56
participated in the reaction with lipid-linked glucose to yield an isopanosyl
57
residue. Further, isopanosyl residues were polymerized into the pullulan chain
58
(Daran et al., 1997). Therefore, glucosyltransferase might play an important role
59
in biosynthesis of pullulan with high yield.
us
an
M
d
te
Ac ce p
60
cr
49
Tween 80 was a non-ionic surfactant and known as polyethylene glycol
61
sorbitan monooleate. It had been reported that Tween 80 had the capability to
62
enhance glucosyltransferase synthesis in Streptococcus mutants (Tomita et al.,
63
1998) and Leuconostoc dextranicum (Majumder & Goyal, 2008). Tween 80 was
64
also found to stimulate the production of pullulan in A. pullulans, although there
65
was inadequate information available for explaining this phenomenon (West &
66
Reed-Hamer,
1995).
It
had
been
confirmed
that
the
activity
of
3
Page 3 of 17
glucosyltransferase was highly correlated with the amount of pullulan produced
68
and the carbon source used (Duan et al., 2008). However, the relationships
69
among Tween 80 concentration, glucosyltransferase activity and pullulan
70
production have not been studied.
ip t
67
The objectives of this paper were to investigate the effect of Tween 80 on
72
the pullulan production, biomass accumulation and glucosyltransferase activity
73
of A. pullulans CGMCC1234. The effect of Tween 80 on the fatty acid
74
composition of the cells was also evaluated.
an
us
cr
71
75
2. Materials and methods
77
2.1. Organisms and inoculum preparation
M
76
A. pullulans CGMCC1234 was maintained on potato dextrose agar (PDA)
79
at 4 °C and subcultured every 2 weeks. The seed culture medium contained
80
50.0 g sucrose, 1.5 g yeast extract, 4.0 g K2HPO4, 0.8 g (NH4)2SO4, 0.2 g
81
MgSO4, and 2.0 g NaCl per liter of deionized water. The pH was adjusted to 6.5,
82
and the medium was autoclaved at 121 °C for 15 min.
84
te
Ac ce p
83
d
78
2.2. Fermentative production of pullulan
85
Cultures were grown on PDA at 28 °C for 4 days and then transferred to a
86
250-ml flask that contained 50 ml of the seed culture medium and subsequently
87
incubated at 28 °C for 2 days with shaking at 200 rpm. The pullulan production
88
was carried out in a 5 l stirred tank fermentor (KF-5l, KoBioTech
4
Page 4 of 17
Bio-engineering Equipment Co., Ltd.) with a working volume of 3 l of the
90
production medium containing (g/l) 80.0 g sucrose, 0.9 g yeast extract, 6.0 g
91
K2HPO4, 0.6 g (NH4)2SO4, 0.5 g MgSO4, 4.0 g NaCl and 0-10.0 g Tween 80.
92
Fermentor was consisted of a glass vessel with stainless-steel endplates and
93
three equally spaced vertical baffles. Agitation was provided by a six-flat-blade
94
impeller (diameter 4 cm) located 3 cm above the bottom of the vessel.
95
Temperature, pH, dissolved oxygen, agitation speed and aeration rate in the
96
vessel could be monitored and controlled automatically. The fermentor with 3 l
97
of the production medium was sterilized at 121 °C for 15 min. After cooling, the
98
medium was inoculated with 150 ml of the seed culture. The pullulan production
99
was carried out at constant temperature of 28 °C, aeration rate of 3 l/min and
100
agitation speed of 300 rpm. The pH was controlled at 6.5 by feeding with either
101
2 M NaOH or 2 M HCl.
103 104
cr
us
an
M
d
te
Ac ce p
102
ip t
89
2.3. Isolation of exocellular polysaccharide The fermentation broth was appropriately diluted with distilled water and
105
heated at 80 °C in water bath for 30 min. The heated culture was centrifuged at
106
10,000 g for 20 min to remove the microorganisms and other precipitates. The
107
supernatant was mixed with two volumes of cold 95% (v/v) ethanol and held at
108
4 °C for 24 h to precipitate the pullulan completely. The precipitate was
109
separated by centrifugation at 15,000g for 20 min, rewashed with ethanol,
110
recovered by filtration and dried at 80 °C to a constant weight. The biomass
5
Page 5 of 17
pellet from the initial centrifugation was washed three times with distilled water
112
by centrifugation at 10,000g for 5 min and dried at 80 °C until the dry cell weight
113
(DCW) was constant. DCW and pullulan content were expressed as g/l.
ip t
111
114
2.4. Preparation of cell-free extract
cr
115
The cells in 5.0 ml of the culture were collected by centrifugation at 10000 g
117
for 20min at 4 °C. The cell pellet was washed three times with ice-cold distilled
118
water by centrifugation at 10,000g for 5 min. The pellet was suspended in 1.0
119
ml of ice-cold 1.0 M Tris-HCl (pH 7.6) to make a thick paste. The paste was
120
homegenized in a Homegenizer (DY89-I, Xinzhi, Zhejiang, China) for 1h on the
121
ice bath. The cell debris was removed by centrifugation at 14,000 g for 30 min
122
at 4 °C. The supernatant (the cell-free extract) was stored at -80 °C and used as
123
a source of enzymes. Protein concentration in the cell-free extract was
124
determined by the method of Bradford with bovine serum albumin as standard
125
(Bradford, 1976).
127 128
an
M
d
te
Ac ce p
126
us
116
2.5. Enzymes assays
Glucosyltransferase activity was determined according to methods
129
described by Duan et al (2008). A suitably diluted enzyme preparation (0.2 ml)
130
was incubated with pre-equilibrated p-nitrophenyl α-D-glucopyranoside (0.2 ml,
131
10 mM) in 0.1 M sodium acetate buffer (pH 4.0) for 5 min at 40 °C. The reaction
132
was terminated by the addition of an aqueous solution of Trizma base (3.0 ml,
6
Page 6 of 17
2.0% w/v), and the absorbance at 410 nm was measured. Enzyme activity was
134
expressed in terms of μM of p-nitrophenol released/min and calculated by
135
reference to a standard curve (the standard curve was made by measuring
136
OD410nm values of the solutions with different concentrations of p-nitrophenol).
137
The mixture with the diluted enzyme preparation that had been heated at 100
138
°C for 5 min was used as the control.
us
cr
ip t
133
139
2.6. Lipid analysis
an
140
Cells harvested at the late-logarithmic phase were washed three times with
142
the distilled water by centrifugation at 10,000g for 5 min. Total lipids extracted
143
from the freeze-dried cells by the method of Bligh and Dyer (1959) were
144
methylated based on the report of Ahn et al (2012). Fatty acids methyl esters
145
were analyzed by gas chromatography as reported previously (Ahn et al.,
146
2012).
d
te
Ac ce p
147
M
141
148
3. Results and discussion
149
3.1. Effect of the concentration of Tween 80 on pullulan fermentation
150
Influence of various concentrations of Tween 80 on pullulan production and
151
cell growth by A. pullulans CGMCC1234 are shown in Fig. 1. The addition of
152
Tween 80 to the nutrient medium significantly increased the pullulan production.
153
The pullulan production increased as the Tween 80 concentration in the
154
medium
increased.
Certain
experimental
results
evidenced
that
7
Page 7 of 17
glucose-containing lipid intermediates played a crucial role in pullulan
156
biosynthesis (Shingel, 2004). The lipid was used as a carrier in which pullulan
157
was taken to the outside of the plasmalemma. The pullulan arranged in a
158
network covering outer surface of the cells. Tween 80 was a well known
159
industrial surfactant, therefore it was assumed that its presence in the media
160
would affect the homogeneity of the broth and facilitate the nutrient and oxygen
161
transfer to the microorganism. However, the addition of Tween 80 to the nutrient
162
medium had no effect on biomass production, indicating that Tween 80 was not
163
degraded to serve as extra carbon source.
an
us
cr
ip t
155
165
M
164
3.2. Effect of the concentration of Tween 80 on glucosyltransferase production Effect of different concentrations of Tween 80 on glucosyltransferase
167
activity are shown in Fig. 2. Glucosyltransferase activity was remarkably
168
promoted with an increase of Tween 80 content. Furthermore, when Tween 80
169
was added to the cell-free extract, no increase in the glucosyltransferase
170
activity was observed (date not shown). As a result of this, Tween 80 did not
171
affect the activity of glucosyltransferase by converting the enzyme from an
172
inactive form to an active form and thus the increased glucosyltransferase
173
activity might be due to the improvement of glucosyltransferase production in
174
presence of Tween 80. Similar results that Tween 80 stimulated the production
175
of glucosyltransferase and had no direct activating effect on enzyme activity in
176
Streptococcus mutants was reported (Umesaki et al., 1977).
Ac ce p
te
d
166
8
Page 8 of 17
177 178
3.3. Effect of Tween 80 on fatty acid compositions Table 1 shows the effect of Tween 80 on the fatty acid composition of the
180
cells grown to the late-logarithmic phase. Enhancement of extracellular pullulan
181
production and intracellular glucosyltransferase activity by Tween 80 was
182
associated with changes in fatty acid composition in the cells. The major fatty
183
acids contained in the cells were palmitic acid (C16:0), palmitoleic acid (C16:1),
184
stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2). In the
185
presence of Tween 80, the ratio of unsaturated/saturated fatty acids increased
186
from 2.34 to 3.90. The increase in the unsaturated degrees by Tween 80 was
187
mainly due to the result that oleic acid increased with a decrease of palmitic
188
acid. The interaction of Tween 80 with cells affected the permeability of the cell
189
membrane, which might enhance to uptake the essential components including
190
vitamins from surroundings (Taoka et al., 2011) and to release of pullulan into
191
the extracellular fluid.
193 194
cr
us
an
M
d
te
Ac ce p
192
ip t
179
4. Conclusions
The addition of Tween 80 to the culture medium enhanced pullulan
195
production, but had no effect on biomass production. The mechanism by which
196
Tween 80 enhanced the production of pullulan was associated with the
197
glucosyltransferase activity and fatty acid composition.
198
9
Page 9 of 17
199
References Ahn, J. W., Hwangbo, K., Lee, S. Y., Choi, H. G., Park, Y., Liu, J. R., & Jeong, W.
201
J. (2012). A new Arctic Chlorella species for biodiesel production.
202
Bioresource Technology, 125, 340-343.
ip t
200
cr
203
Blight, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and
205
purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917.
us
204
an
206
Bradford, M. M. (1976). A rapid and sensitive method for quantitation of
208
microgram quantities of protein utilizing the principle of protein-dye binding.
209
Analytical Biochemistry, 72, 248-253.
M
207
te
d
210
Cately, B. J., & McDowell, W. (1982). Lipid-linked saccharides formed during
212
pullulan biosynthesis in Aureobasidium pullulans. Carbohydrate Research,
213 214
Ac ce p
211
103, 65-75.
215
Cheng, K. C., Demirci, A., & Catchmark, J. M. (2011). Pullulan: biosynthesis,
216
production, and applications. Applied Microbiology and Biotechnology, 92,
217
29-44.
218 219
Daran, J. M., Bell, W., & Francois, J. (1997). Physiological and morphological
220
effects of genetic alterations leading to a reduced synthesis of UDP-glucose
10
Page 10 of 17
221
in Saccharomyces cerevisiae. FEMS Microbiology Letters, 153, 89-96.
222
Duan, X. H., Chi, Z. M., Wang, L., & Wang, X. H. (2008). Influence of different
224
sugars on pullulan production and activities of α-phosphoglucose mutase,
225
UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan
226
synthesis in Aureobasidium pullulans Y68. Carbohydrate Polymers, 73,
227
587-593.
us
cr
ip t
223
an
228
Majumder, A., & Goyal, A. (2008). Enhanced production of exocellular
230
glucansucrase from Leuconostoc dextranicum NRRL B-1146 using response
231
surface method. Bioresource Technology, 99, 3685-3691.
M
229
te
d
232
Pan, S. K., Yao, D. R., Chen, J., & Wu, S. J. (2013). Influence of controlled pH
234
on the activity of UDPG-pyrophosphorylase in Aureobasidium pullulans.
235 236
Ac ce p
233
Carbohydrate Polymers, 92, 629-632.
237
Shingel, K. I. (2004). Current knowledge on biosynthesis, biological activity, and
238
chemical modification of the exopolysaccharide, pullulan. Carbohydrate
239
Research, 339, 447-460.
240 241
Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., & Hayashi, M.
242
(2011). Effect of Tween 80 on the growth, lipid accumulation and fatty acid
11
Page 11 of 17
243
composition of Thraustochytrium aureum ATCC 34304. Journal of Bioscience
244
and Bioengineering, 111, 420-424.
245
Tomita, Y., Watanabe, T., Takeuchi, T., Nanbu, A., Shinozaki, N., Ikemi, T., &
247
Fukushima, K. (1998). Effects of surfactants on glucosyltransferase
248
production and in vitro sucrose-dependent colonization by Streptococcus
249
mutants. Archives of Oral Biology, 43, 735-740.
an
250
us
cr
ip t
246
Umesaki, Y., Kawai, Y. & Mutai, M. (1977). Effect of Tween 80 on
252
glucosyltransferase production in Streptococcus mutants. Applied and
253
Environment Microbiology, 34, 115-119.
M
251
257
te
256
West, T. P., & Reed-Hamer, B. (1995). Effect of oils and surfactants on pullulan production relative to nitrogen source. Microbios, 83, 249-59.
Ac ce p
255
d
254
258
Figure captions
259
Fig. 1. Effect of Tween 80 concentration on pullulan production and cell growth
260
by Aureobasidium pullulans CGMCC1234. Values are given as means ±
261
standard deviation (n=3). Bars denoted with different capital letters are
262
significantly different (P＜0.05).
263 264
Fig. 2. Effect of Tween 80 concentration on glucosyltrasferase activity. Values
12
Page 12 of 17
are given as means ± standard deviation (n=3). Bars denoted with different
266
capital letters are significantly different (P＜0.05).
Ac ce p
te
d
M
an
us
cr
ip t
265
13
Page 13 of 17
Highlights (for review)
The addition of Tween 80 significantly enhanced pullulan production. Tween 80 had no impact to biomass accumulation. Glucosyltransferase activity was promoted by Tween 80.
ip t
The ratio of unsaturated/saturated fatty acids in the cells increased
Ac
ce pt
ed
M
an
us
cr
with Tween 80.
Page 14 of 17
Table(s)
Table 1 Fatty acid composition of A. pullulans cells grown with and without Tween 80 Fatty acids
% Total fatty acids 0.5% Tween 80
C16:0
23.7 ± 1.2
16.0 ± 0.4*
C16:1
1.3 ± 0.1
4.3 ± 0.2*
C18:0
6.2 ± 0.3
4.4 ± 0.1*
C18:1
34.8 ± 0.9
55.7 ± 1.0*
34.0 ± 0.7
19.6 ± 0.5*
2.34
3.90
C18:2
cr
unsaturated/saturated
a
ip t
Control
Values are given as means ± standard deviation (n=3). Values denoted with an asterisk
ce pt
ed
M
an
Ratio of unsaturated/saturated = (C16:1 + C18:1 + C18:2)/(C16:0 + C18:0).
Ac
a
us
have significant difference between control and treatment group (P＜0.05).
13
Page 15 of 17
Figure(s)
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 1
14
Page 16 of 17
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 2
15
Page 17 of 17