Subscriber access provided by LAURENTIAN UNIV
Article
Synthesis of isomalto-oligosaccharides by Pichia pastoris displaying the Aspergillus niger #-glucosidase Nannan Zhao, Yanshan Xu, Kuang Wang, and Suiping Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04140 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 35
Journal of Agricultural and Food Chemistry
Synthesis of isomalto-oligosaccharides by Pichia pastoris displaying the Aspergillus niger α-glucosidase Nannan Zhao1, Yanshan Xu1, Kuang Wang, Suiping Zheng*
1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China 2. Guangdong research center of Industrial enzyme and Green manufacturing technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, P. R. China
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 35
1
ABSTRACT
2
We explored the ability of an Aspergillus niger α-glucosidase displayed on P. pastoris
3
to act as a whole-cell biocatalyst (Pp-ANGL-GCW61) system to synthesize
4
isomalto-oligosaccharides (IMOs). IMOs are a mixture that includes isomaltose (IG2),
5
panose (P) and isomaltotriose (IG3). In this study, the IMOs were synthesized by a
6
hydrolysis-transglycosylation reaction in an aqueous system of maltose. In a 2-mL
7
reaction system, the IMOs were synthesized with a conversion rate of approximately
8
49% in 2 h when 30% maltose was utilized under optimal conditions by
9
Pp-ANGL-GCW61. Additionally, 0.5-L reaction system was conducted in a 2-L
10
stirred reactor with a conversion rate of approximately 44% in 2 h. Moreover, the
11
conversion rate was relatively stable after the whole-cell catalyst was reused three
12
times. In conclusion, Pp-ANGL-GCW61 has a high reaction efficiency and
13
operational stability, which makes it a powerful biocatalyst available for industrial
14
scale synthesis.
15
Aspergillus
niger
α-glucosidase,
16
Keywords:
17
Transglycosylation, IMOs, Scale synthesis
Yeast
2
ACS Paragon Plus Environment
whole-cell
catalyst,
Page 3 of 35
Journal of Agricultural and Food Chemistry
18
INTRODUCTION
19
The isomalto-oligosaccharides (IMOs) are a group of glucans that consist of glucosyl
20
saccharide units linked by α-1, 6-glycosidic linkages. Its main functional components
21
include isomaltose (IG2), panose (P) and isomaltotriose (IG3). The most direct
22
physiological function of IMOs is to promote the growth of probiotics. On this basis,
23
its indirect effects include regulating intestinal flora, inhibiting the growth of
24
pathogenic bacteria, improving constipation1-3, and increasing the synthesis of
25
essential vitamins and mineral absorption4. Wang Yu et al. found that IMOs can
26
regulate lipid metabolism2. In addition, IMOs have approximately 45-50% of the
27
sweetness of sucrose as well as having lower calories. Thus, it is an ideal sugar
28
substitute for a person with diabetes. IMOs have already been used in
29
pharmaceuticals2, 5 and food products6-7.
30
The production and application of IMOs first originated in Japan in 1985.
31
Commercial Isomalt-500 is an enzymatic product that contains approximately 50%
32
IMOs8. During the industrial scale production of IMOs, starch is first liquefied with a
33
thermostable bacterial α-amylase (e.g., Termamyl SC, Novozymes) to produce limit
34
dextrins, which are further saccharified by fungal α-amylase (e.g., Fungamyl 800L,
35
Novozymes) and transglucosylated using α-glucosidase (e.g., Transglucosidase L,
36
Amano Enzymes Inc, Japan) in a separate process to produce IMOs9.
37
α-Glucosidase (EC 3.2.1.20), a well-known hydrolyzing enzyme, can be used to carry
38
out the transglucosylation of maltose10. It is the key to producing IMOs. The
39
hydrolyzing reaction of α-glucosidase releases α-D-glucose from the non-reducing 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
40
end of oligosaccharides. The transfer occurs most frequently at the 6-OH group of the
41
non-reducing glucose unit, producing isomaltose from D-glucose or panose from
42
maltose11.
43
Many α-glucosidases from different sources have been found, and their characteristics
44
were determined12-15. The α-glucosidase from Aspergillus niger (ANGL) has a high
45
level of transglycosylation activity, even at quite low substrate concentrations16. The
46
mutagenesis, immobilization and heterologous expression of the α-glucosidase from
47
Aspergillus niger became research hotspots. The complete amino acid sequence of the
48
α–glucosidase from Aspergillus niger has been reported in 199217. Until now, ANGL
49
has been expressed in and secreted from E. coli, A. nidulans and P. pastoris18-20.
50
Fermentation, to produce a large amount of stable, high-quality α–glucosidase, is
51
extremely important. At the present, the most mature Aspergillus niger technology is
52
in Amano Enzymes Inc, and the α–glucosidase IMOs products account for almost all
53
imports from Japan into China. However, the Amano enzymes are free enzymes, and
54
because of their import and difficulty in recycling, the cost of IMOs production is
55
high in China.
56
Whole-cell catalysts are a kind of unique immobilized technology that do not need the
57
complicated free enzyme purification process. It can be recycled and reused by
58
centrifugation, which reduces the free protein contamination in the process of product
59
collection and purification. Moreover, it can also be used in fluidized beds to develop
60
the continuous production of lots of IMOs. The approach of displaying an enzyme
61
protein on the P. pastoris cell surface by using the GPI-modified cell-wall protein 4
ACS Paragon Plus Environment
Page 4 of 35
Page 5 of 35
Journal of Agricultural and Food Chemistry
62
(GCW) has been successful in our laboratory21. It could be a cost-effective
63
bioconversion process that could be applied in manufacturing with a simpler
64
preparation.
65
In this study, we constructed successfully a recombinant P. pastoris strain with ANGL
66
immobilized on the cell surface by GCW61. We explored the ability of the
67
ANGL-displaying P. pastoris whole-cell biocatalyst (Pp-ANGL) to synthesize IMOs
68
using maltose. We mainly focused on the synthesis of isomaltose (IG2), panose (P)
69
and isomaltotriose (IG3). Reaction parameters such as pH and temperature were
70
optimized, and the effect of the concentrations of the whole-cell biocatalyst and
71
substrate were determined. Subsequently, scaled-up reactions were performed in a 2-L
72
stirred reactor under optimized conditions. We also tested the the reusability of
73
Pp-ANGL-GCW61. In this paper, we report the first, synthesis of IMOs using the
74
whole-cell biotransformation approach by cell surface displaying the α-glucosidase
75
from Aspergillus niger.
76
MATERIALS AND METHODS
77
Strains and growth conditions
78
E. coli was used as the host strain for plasmid storage and amplification. P. pastoris
79
GS115 was used for cell surface display. The recombinant Dalbergia cochinchinensis
80
Pierre β-glucosidase (DCBGL) surface-displaying plasmid pKDCBGL-GCWn
81
(n=12,19,21,49,61)was previously constructed by Guo et al.22 in our laboratory. On
82
this basis, the recombinant plasmid pKANGL-GCW61 and ANGL-displaying P.
83
pastoris strain Pp-ANGL-GCW61 were constructed for this study. DNA sequences 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 35
84
encoding mature ANGL (NCBI accession No. XP_001402053.1) were synthetized after
85
codon usage optimization by Genrey (Shanghai, China) and maintained in
86
Escherichia coli Top10 cells.
87
E. coli was cultivated on LB plates or in LB medium (1% NaCl, 0.5% yeast extract,
88
and 1% tryptone, plates also contain 2% agar) containing 50 µg/mL kanamycin or 25
89
µg/mL zeocin at 37 °C and 250 revolutions per minute (rpm). Yeast strains were
90
grown on either MD plates (2% glucose, 1.34% yeast nitrogen base, and 2% agar) or
91
in BMGY/BMMY media (1.34% yeast nitrogen base, 1% yeast extract, 2% peptone,
92
100 mM potassium phosphate (pH 6.0), and 1% glycerol or 2% methanol). P. pastoris
93
GS115 strain was cultured at 30 °C and 250 rpm. P. pastoris GS115 transformed with
94
pPIC9K (GS115/9K) was used as a negative control.
95
Standard chemicals including isomaltose, panose and isomaltotriose were purchased
96
from Sigma-Aldrich (St. Louis, USA).
97
Construction
98
recombinant yeast strain GS115/ pPIC9K-ANGL-GCW61
99
The target gene encodes the mature ANGL was amplified from the synthetized
of
the
pPIC9K-ANGL-GCW61
expression
plasmid
and
100
template by PCR, with the upstream 5’-
101
CCGGAATTCATGTACCCATACGATGTTCCAGATTACGCTTCTACAACTGCACC
102
AA -3’ and downstream 5’-
103
CGACGCGTCCATTCCAAAACCCAGT -3’ primers, while an HA peptide tag was
104
added at the N-terminus for further study. The PCR product and the plasmid
105
pKDCBGL-GCW61 were both digested with EcoRI and MluI, gel-purified and then 6
ACS Paragon Plus Environment
Page 7 of 35
Journal of Agricultural and Food Chemistry
106
ligated to form the recombinant plasmid pPIC9K-ANGL-GCW6122.
107
The recombinant plasmid pPIC9k-ANGL-GCW61 was linearized with Kpn2I and
108
integrated into the host strain P. pastoris GS115 by electroporation in homologous
109
recombination method23. The transformants were selected by incubation at 30 °C for 3
110
days on MD plates. The confirmed transformants were precultured first in 10 mL
111
BMGY medium at 30 °C and 200 rpm. After 24 h, the cells were collected by
112
centrifugation at 6000 rpm for 5 min and then resuspended in 25 mL BMMY medium
113
containing 2% (v/v) methanol to make sure that the initial OD600 was controlled to
114
be 1. To maintain the induced expression of the fusion proteins, 500 µL of methanol
115
was added to the culture every 24 h throughout the induction phase.
116
Hydrolysis activity of the ANGL-displaying P. pastoris cell
117
α-Glucosidase activity was determined by release of p-nitrophenol from
118
4-nitrophenyl-α-D-glucopyranoside (pNPG) solution. Briefly, the hydrolysis activity
119
was measured in a 500-µL reaction at 55°C for 10 min with 5 mM pNPG in 0.04 M
120
Britton-Robinson buffer (pH 5.0)24. The 500-µL reaction included the recombinant P.
121
pastoris cells collected from 250 µL fermentation broth, which washed and
122
resuspended with 250 µL Britton-Robinson buffer (pH 5.0). The reaction was
123
terminated and colored by adding 500 µL of 2 M sodium carbonate. Then, the
124
p-nitrophenol released in the reaction was determined by measuring its absorbance at
125
405 nm22. One unit of hydrolysis activity was defined as the amount of biocatalyst
126
required to release 1 µmol of p-nitrophenol from pNPG per min at 55 °C. Using this
127
method,
the
hydrolysis
activity
of
the
yeast
7
ACS Paragon Plus Environment
whole-cell
biocatalyst
Journal of Agricultural and Food Chemistry
128
(Pp-ANGL-GCW61) was 2.88 U/ (g dry cell).
129
Preparation of the ANGL-displaying P. pastoris whole-cell biocatalyst
130
After 5 days of methanol induction, the recombinant P. pastoris cells were collected,
131
washed, and then resuspended in a 0.04 M Britton-Robinson buffer (pH 4.0) with 0.2
132
M trehalose added as a protectant for freeze-drying25. Freeze-dried ANGL-displaying
133
P. pastoris whole-cell biocatalysts (Pp-ANGL-GCW61) were used for subsequent
134
experiments.
135
The synthetic ability of Pp-ANGL-GCW61 was analyzed with maltose as the
136
substrate26. To this end, 0.1 g of the whole-cell biocatalyst was added to 2 mL of 30%
137
(w/v) maltose dissolved in 0.04 M Britton-Robinson buffer (pH 4.0) in a 10-mL
138
stoppered glass Erlenmeyer flask. The mixture was incubated on a rotary shaker at
139
55 °C and 200 rpm for 1 h. One unit of synthesis activity corresponded to the amount
140
of biocatalyst that caused 1 µmol of maltose into IMOs (IMOs=IG2+P+IG3) per min
141
at 55 °C. A control reaction was performed using the same procedure and the control
142
whole cells but without the whole-cell biocatalyst. Using this method, the synthetic
143
ability of Pp-ANGL-GCW61 was 54.76 U/ (g dry cell).
144
Optimization of synthesis of IMOs by Pp-ANGL-GCW61
145
The IMOs synthesis by transglycosylation was carried out in 10 mL Erlenmeyer shake
146
flasks containing 2 mL of reactant. The flasks were placed in a shaking incubator at
147
200 rpm and at an appropriate temperature. The pH, temperature, and whole-cell
148
biocatalyst and substrate concentrations were optimized through univariate
149
optimization experiments. 8
ACS Paragon Plus Environment
Page 8 of 35
Page 9 of 35
Journal of Agricultural and Food Chemistry
150
The effects of pH were examined at 55 °C and 200 rpm using 62.5 mg/mL
151
Pp-ANGL-GCW61, 300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer.
152
When a single variable was changed, all of the other variables were kept constant. The
153
pH value was set at 3.0, 4.0, 5.0,6.0 or 7.0. The effect of temperature was also
154
examined by performing the reactions at 40, 45, 50, 55 and 60 °C with 50 mg/mL
155
Pp-ANGL-GCW61, 300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer
156
(pH 4.0) at 200 rpm for 4 h. After this reaction, the whole-cell biocatalysts were kept
157
warm for 15 h at the selected pH or temperature before the tolerance of
158
Pp-ANGL-GCW61 was tested.
159
The effect of the substrate concentration on the reaction was investigated by using 200,
160
300, 400 and 500 mg/mL concentrations, while the Pp-ANGL-GCW61 concentration
161
was maintained at 50 mg/mL. The Pp-ANGL-GCW61 concentration was set at 25,
162
37.5, 50, 62.5 and 75 mg/mL, while the substrate concentration was maintained at 300
163
mg/mL.
164
To study the stability of the whole-cell catalyst under the optimal reaction conditions,
165
the Pp-ANGL-GCW61 was reused three times to investigate any changes in catalytic
166
ability. The Pp-ANGL-GCW61 was collected by centrifugation after the reaction.
167
Next, the cells were washed with 0.04 M Britton-Robinson buffer three times. After
168
centrifugation and freeze-drying, the Pp-ANGL-GCW61 was used for the next
169
reaction. Moreover, the reuse experiments were performed with a 10 mL reaction
170
system in the 50-mL Erlenmeyer shake flasks.
171
At predetermined time intervals, 50 µL of reaction liquid was removed from the 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
172
reaction mixture and centrifuged at 14,000 rpm for 1 min. Three parallel samples were
173
removed for each reaction.
174
In the present study, the conversion rate was referred to as the molar conversion,
175
which is equal to the number of moles of maltose that were completely transformed
176
into product divided by the number of moles of substrate.
177
Synthesis of IMOs and reuse of Pp-ANGL-GCW61 in a 2-L stirred reactor
178
To reduce the cost, a large quantity of Pp-ANGL-GCW61 fermentation broth of 30 L
179
fed-batch fermentation was spray-dried directly without washing after 5 days of
180
methanol induction. This whole-cell catalyst was prepared to be used in large-scale
181
reactions.
182
A 0.5-L reaction system was conducted in a 2-L stirred reactor. The reaction was
183
carried out under optimized conditions: 62.5 mg/mL Pp-ANGL-GCW61, 300 mg/mL
184
maltose, and 0.04 M Britton-Robinson buffer (pH 4.0) at 55°C and 200 rpm. Aliquots
185
of the reaction liquid were removed from the reaction mixture at predetermined time
186
intervals. In addition, the Pp-ANGL-GCW61 was reused three times to investigate
187
any changes in catalytic ability over time. The Pp-ANGL-GCW61 was reused as
188
described above.
189
HPLC analysis
190
The quantitative analyses of maltose (M), isomaltose (IG2), panose (P) and
191
isomaltotriose (IG3) were performed using an HPLC (Waters model 2695) equipped
192
with an evaporative light-scattering detector (Waters model 2424 ELSD) and a Waters
193
SPHERISORB NH2 column (particle size, 5 µm; dimensions, 250 mm × 4.6 mm). 10
ACS Paragon Plus Environment
Page 10 of 35
Page 11 of 35
Journal of Agricultural and Food Chemistry
194
The reaction liquid was centrifuged at 13,000 rpm for 2 min. The supernatant (20 µL)
195
was collected, diluted 50-fold with water to a total volume of 1 mL and filtered
196
through a cellulose nitrate membrane (0.25 µm). Next, the samples were characterized
197
by HPLC. A standard calibration curve was prepared using IG2, P and IG3 standards
198
that were purchased from Sigma–Aldrich. The analysis was carried out at a column
199
temperature of 30 °C using an acetonitrile-water (80:20, v/v HPLC GRADE) solvent
200
as the mobile phase, at a flow rate of 1.0 mL/min. Using the ELSD detector, the drift
201
tube was set at 65 °C using a nebulizer gas as the carrier gas at a flow rate of 30 psi.
202
RESULTS AND DISCUSSION
203
Display of ANGL on the yeast cell surface
204
The constructed plasmid pPIC9K-ANGL-GCW61 is shown in Figure 1a. Gel
205
electrophoresis of an EcoRI/MluI digestion of the recombinant vector revealed that
206
this plasmid was constructed successfully. The isolated transformants were cultured in
207
BMGY/BMMY medium. A growth curve of the transformants is shown in Figure 1b.
208
As seen from the growth curves, the growth of the recombinant yeast strain is
209
consistent with the growth of GS115/9K, showing that the expression of the fusion
210
protein has no obvious influence on the growth of this strain. With the increase of the
211
induction time, the enzyme activity and OD600 increased substantially, reaching the
212
highest levels (Figure 1c).
213
According
214
transglycosylation activity of the Aspergillus niger α-glucosidase that was obtained
215
via secretory expression in Aspergillus niger or other microorganisms. This report is
to
previous
studies,
researchers
studied
11
ACS Paragon Plus Environment
the
hydrolysis
and
Journal of Agricultural and Food Chemistry
216
the first in which the Aspergillus niger α-glucosidase was displayed on the yeast cell
217
surface with a higher activity, and there are little activity existed in the free-form
218
(Figure 1d). As a cost-effective whole-cell catalyst, its preparation and storage were
219
convenient. Moreover, its operation stability was suitable for large-scale reactions.
220
Effect of various reaction parameters on the IMOs yield by Pp-ANGL-GCW61
221
The products were quantified using HPLC. Glucose (G), maltose (M), isomaltose
222
(IG2), panose (P) and isomaltotriose (IG3) were well separated by a Waters amino
223
propyl column (2). The retention times were 5.820 min for G, 7.575 min for M, 8.430
224
for IG2, 11.142 min for P and12.607 min for IG3. The conversion rate was greatly
225
affected by the reaction conditions, such as the pH, temperature, whole-cell
226
biocatalyst concentration, substrate concentration and the properties of the enzyme.
227
Effect of pH on IMOs synthesis
228
A tiny deviation from the optimal pH can change the ionization of groups in the
229
enzyme active site and reduce the activity of the enzyme. In addition, large deviations,
230
which disturb many non-covalent bonds that maintain the enzyme’s three-dimensional
231
structure, cause the enzyme to denature. As shown in Figure 3a, the effect of pH was
232
obvious. The highest molar conversion was reached at pH 4.0 and reaction duration
233
for 2 h. The initial synthesis activity was increased when the pH value decreased. The
234
molar conversion of all reaction mixtures was approximately 30% under different pH
235
conditions when the reaction was performed for 4 h.
236
The pH-activity curves of IMOs synthesis for the Amano enzyme are shown in Figure
237
3b. The optimal pH of the activity of Pp-ANGL-GCW61 is from 3.0~4.0, while the 12
ACS Paragon Plus Environment
Page 12 of 35
Page 13 of 35
Journal of Agricultural and Food Chemistry
238
optimal pH is from 5.0~6.0 for transglucosidase L (Amano Enzymes Inc, Japan).
239
These changes in the optimal pH range have rarely been reported by other
240
researchers18-20. The essential ionizable groups of the α-glucosidase are two kinds of
241
carboxyl groups: one is charged, and the other is a protonated 27. One possible reason
242
for this behavior was that the GPI-modified cell-wall protein (GCW), which was
243
co-expressed with ANGL, influenced the three-dimensional structure and the charge
244
of the enzyme. We also studied the relationship between pH and stability of
245
Pp-ANGL-GCW61. The activity was still high when the pH value was 3 and 4. We
246
chose pH 4.0 as the optimal pH of the transglucosylation reaction.
247
Effect of temperature on IMOs synthesis
248
The influence of temperature on the enzymatic reaction includes two aspects; on the
249
one hand, when the temperature rises, the reaction rate is accelerated, as in a general
250
chemical reaction. On the other hand, the enzyme gradually degrades as the
251
temperature rises, thus slowing the enzyme’s reaction by reducing the activity of the
252
enzyme. Just below the optimal temperature, the former effect is dominant; but above
253
the optimal temperature, the latter effect is dominant, resulting in the loss of enzyme
254
activity and a decrease in the reaction rate.
255
As shown in Figure 4a, the Pp-ANGL-GCW61 activity gradually increases with an
256
increase in temperature; the high temperature resulted in a high initial rate because the
257
high temperature accelerated molecular diffusion and increased the solubility of the
258
substrate. When the temperature reached 60 °C, the molar conversion was highest.
259
When the temperature continued to increase, the enzyme would be inactivated 13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
260
irreversibly, so the molar conversion sharply reduced. At 50, 55 and 60 °C, the molar
261
conversion was higher and above 35%. Its properties are similar to the properties of
262
the transglucosidase L (Amano Enzymes Inc, Japan)16.
263
The 50 mg/mL whole-cell biocatalyst was kept warm for 15 h to test its thermal
264
tolerance at 50, 55 and 60 °C. As shown in Figure 4b, the Pp-ANGL-GCW61 has
265
good thermal stability at 50 and 55 °C, and the molar conversion still can reach
266
approximately 25% after the heat treatment, although the conversion only reaches 10%
267
at 60°C. Therefore, 55 °C was considered to be the most suitable temperature for
268
further optimization research and biocatalyst reuse.
269
Effect of the Pp-ANGL-GCW61 concentration on IMOs synthesis
270
As the Pp-ANGL-GCW61 concentration increases, the initial reaction rate increases.
271
The larger the dose of enzyme was added, the shorter reaction time was required to
272
get to the molar conversion peak. As shown in Figure 5a, when 75 mg/mL
273
Pp-ANGL-GCW61 was added, the conversion rate peaked at 32% in 3 h of reaction.
274
When the added amount of catalyst was 25 mg/mL, the maximum conversion rate
275
(30%) required the reaction to continue for 9 h. Optimizing the enzyme dose is
276
important to reduce the reaction time, as well as to control costs.
277
The results showed that the ultimate conversion rate that the different enzyme doses
278
achieved was consistent, approximately 30%, when the reaction continues for a
279
sufficient period. When the enzyme concentration reaches a certain value, restricted
280
by the amount of substrate, the enzymatic reaction rate will not increase. When 62.5
281
and 75 mg/mL Pp-ANGL-GCW61 were added, the conversion rate curves were 14
ACS Paragon Plus Environment
Page 14 of 35
Page 15 of 35
Journal of Agricultural and Food Chemistry
282
similar (Figure 5a). Therefore, considering the reaction time and enzyme cost, 62.5
283
mg/mL Pp-ANGL-GCW61 was found to be the optimal enzyme concentration.
284
Effect of maltose concentration on IMOs synthesis
285
ANGL has exhibited typical substrate inhibition kinetics10. The rate increased linearly
286
with increasing substrate concentration (up to 100 mg/mL maltose) and then exhibited
287
a lower, incrementally changing slope in a non-linear phase between 100 and 200
288
mg/mL of substrate. Once the substrate concentration increased above 200 mg/mL,
289
the reaction velocity entered a non-linear deceleration phase, indicating strong
290
substrate inhibition by the maltose10.
291
As shown in Figure 5b , when the maltose concentration increased from 200 mg/mL,
292
the IMOs concentrations showed a corresponding increase, in contrast, the
293
conversation rates decreased. The reaction mixtures containing 200, 300, 400 and 500
294
mg/mL maltose reached the highest conversion rate (approximately 35%) in 3, 5, 7
295
and 9 h, respectively. After enough reaction time, the reaction system is a mixture of
296
IMOs, glucose and small amounts of maltose. Since the maximum conversion rates of
297
the reaction system of different substrate concentrations are similar, when the initial
298
substrate concentration is higher, more maltose is not converted into IMOs.
299
Considering the reaction time and substrate cost, 300 mg/mL maltose was used for
300
further research
301
Reuse of Pp-ANGL-GCW61 in 10-mL and 50-mL Erlenmeyer shake flasks under
302
optimum conditions
303
After single-factor optimization of the factors influencing IMOs synthesis by 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
the
optimum
reaction
conditions
304
Pp-ANGL-GCW61,
305
Pp-ANGL-GCW61, 300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer
306
(pH 4.0) at 55 °C and 200 rpm for 4 h.
307
In the initial stage of the reaction, a mass of maltose was hydrolyzed into glucose.
308
Because of the high concentrations of maltose in the mixture, free glucose residues are
309
preferred to participate in the synthesis reaction of maltose to panose; As shown in
310
Figure 6a, the synthesis rate of panose was highest28. When the reaction reached a
311
certain time, the maltose concentration was greatly reduced, and the panose
312
concentration increased; the effect of panose hydrolysis was remarkable, as shown in
313
the conversion rate drop. The glucose content in the reaction system continued to
314
increase. The reaction reached equilibrium when the glucose content was high enough
315
to inhibit the hydrolysis activity of Pp-ANGL-GCW61.
316
To study the reuse ability of the whole-cell catalyst, the Pp-ANGL-GCW61 was
317
reused three times firstly in the 2-mL reaction system. Batches 1- 3 indicated that
318
Pp-ANGL-GCW61 was reused once, twice and three times. As shown in Figure 7a,
319
the conversion rate in the 2-mL reaction system achieved approximately 35% in 4 h
320
when Pp-ANGL-GCW61 was reused once. There are two reasons: the loss of added
321
enzyme and the loss of enzyme activity, which decreased the conversion rate. Then
322
we enlarged the reaction system to 10-mL, while the conversion rate was a little
323
higher in 10-mL reaction system, as shown in Figure 7b. The conversion rate of
324
batches 2, 3 still reached more than 30% in 5 h both in 2-mL and 10-mL reaction
325
system. Besides, the curves of batches 1, 2 and 3 were similar, indicating that 16
ACS Paragon Plus Environment
were
Page 16 of 35
62.5
mg/mL
Page 17 of 35
Journal of Agricultural and Food Chemistry
326
Pp-ANGL-GCW61 was stable and reusable in a 2-mL and 10-mL reaction system.
327
Synthesis of IMOs in a 2-L stirred reactor and the reuse of Pp-ANGL-GCW61
328
IMOs were synthesized in 500-mL reaction system under the optimized conditions in
329
a 2-L stirred reactor. Batches 1- 3 indicated that Pp-ANGL-GCW61 was reused once,
330
twice and three times, respectively. Compared with the 2-mL and 10-mL reaction
331
system, the initial conversion rate of the 500-mL reaction system was a little decrease
332
in the reuse study (Table 1). The reaction in the small shake flask system may have
333
been more uniform than that in the stirred reactor. Similar to the 2-mL and 10-mL
334
reaction system, batch 1 achieved the highest conversion rate in 4 h, and the
335
conversion rate was still increasing after reacting for 5 h in batches 2 and 3 (Figure
336
7c).
337
In general, Pp-ANGL-GCW61, which has a high operational stability and the
338
advantages of being inexpensive and conveniently prepared, is very suitable for the
339
large-scale synthesis of IMOs through the transglycosylation reaction. This study
340
provided a reference for enzymatic industrial applications.
341
In conclusions, the P. pastoris strain that displayed the Aspergillus niger
342
α-glucosidase was successfully constructed. The hydrolysis and synthetic activities of
343
Pp-ANGL-GCW61 were 2.88 U/(g dry cell) and 54.76 U/(g dry cell), respectively.
344
The ANGL-displaying P. pastoris whole-cell biocatalyst (Pp-ANGL-GCW61) was
345
prepared by vacuum lyophilization. After a single-factor optimization test, we
346
determined that the optimum reaction conditions are 62.5 mg/mL Pp-ANGL-GCW61,
347
300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer (pH 4.0) at 55 °C and 17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 35
348
200 rpm for 4 h. In a 2-mL reaction system, IMOs were synthesized with
349
approximately
350
Pp-ANGL-GCW61.
351
The whole-cell biocatalyst was reused in a 500-mL reaction system under the
352
optimized conditions in a 2-L stirred reactor. Furthermore, the reusability of the
353
Pp-ANGL-GCW61 indicated that Pp-ANGL-GCW61 was suitable for large-scale
354
synthesis because of its high stability, low cost and moderate conversation rate.
355
Because of its cost-effectiveness, high stability and catalytic activity, the whole-cell
356
catalyst Pp-ANGL-GCW61 provides a reference for the enzymatic synthesis of IMOS
357
on an industrial scale.
358
Abbreviations Used
359
GPI,
360
pNPG, 4-nitrophenyl-α-D-glucopyranoside; GCW61, GPI-modified cell wall protein
361
from Pichia pastoris; M, maltose; IMOs, isomalto-oligosaccharides; IG2, isomaltose;
362
P,
363
ACKNOWLEDGMENT
364
All the authors are thankful for the financial support of the Science and Technology
365
Planning Project of Guangzhou City (No.201607010307), the National Natural
366
Science Foundation of China (No.31671840), the Recruitment Program of Leading
367
Talents in Innovation and Entrepreneurship of Guangzhou (LCY201322) to Suiping
368
Zheng.
369
NOTES
49%
conversion
in
2
h
under
optimum
conditions
by
glycosylphosphatidylinositol; ANGL, the Aspergillus niger α-glucosidase;
panose; IG3, isomaltotriose; rpm, revolutions per minute.
18
ACS Paragon Plus Environment
Page 19 of 35
Journal of Agricultural and Food Chemistry
370
The authors declare no competing financial interest.
371
References
372
1.
373
Isomaltooligosaccharides for Increasing Fecal Bifidobacteria. Agricultural and Biological
374
Chemistry 1991, 55 (8), 2157-2159.
375
2.
376
of isomalto-oligosaccharides improved colonic microflora profile, bowel function, and blood
377
cholesterol levels in constipated elderly people—A placebo-controlled, diet-controlled trial.
378
Nutrition 2011, 27 (4), 445-450.
379
3.
380
from resistant starch in a pig model. Journal of the Science of Food and Agriculture 1998, 77 (1),
381
71-80.
382
4.
383
enhance the mineral absorption and counteract the adverse effects of phytic acid in mice. Nutrition
384
2010, 26 (3), 305-311.
385
5.
386
Functions and Indicators of Nutritional Status in Constipated Elderly Men. Journal of the
387
American College of Nutrition 2001, 20 (1), 44-49.
388
6.
389
Characteristics of Sponge Cake. Cereal chemistry. 2008, 85 (4), 515-521.
390
7.
391
isomalto-oligosaccharides on broiler performance and intestinal microflora. (0032-5791 (Print)).
Kohmoto,
T.;
Fukui,
F.;
Takaku,
H.;
Mitsuoka,
T.,
Dose-response
Test
of
Yen, C.-H.; Tseng, Y.-H.; Kuo, Y.-W.; Lee, M.-C.; Chen, H.-L., Long-term supplementation
Martin, L. J. M.; Dumon, H. J. W.; Champ, M. M. J., Production of short-chain fatty acids
Wang, Y.; Zeng, T.; Wang, S.-e.; Wang, W.; Wang, Q.; Yu, H.-X., Fructo-oligosaccharides
Chen, H.-L.; Lu, Y.-H.; Lin, J., Jr.; Ko, L.-Y., Effects of Isomalto-Oligosaccharides on Bowel
Lee, C. C.; Wang, H. F.; Lin, S. D., Effect of Isomaltooligosaccharide Syrup on Quality
Zhang, W. F.; Li Df Fau - Lu, W. Q.; Lu Wq Fau - Yi, G. F.; Yi, G. F., Effects of
19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
392
8.
Kanno, T., Some functional properties of so-called isomalto-oligosaccharides and their
393
applications to food industry. Journal of Applied Glycoscience 1990, 37 (2), 87-97.
394
9.
395
simultaneous saccharification and transglucosylation from starch and sustainable sources. Process
396
Biochemistry 2016, 51 (10), 1464-1471.
397
10. Basu, A.; Mutturi, S.; Prapulla, S. G., Modeling of enzymatic production of
398
isomaltooligosaccharides: a mechanistic approach. Catal. Sci. Technol. 2015, 5 (5), 2945-2958.
399
11. Goffin, D.; Delzenne, N.; Blecker, C.; Hanon, E.; Deroanne, C.; Paquot, M., Will
400
Isomalto-Oligosaccharides, a Well-Established Functional Food in Asia, Break through the
401
European and American Market? The Status of Knowledge on these Prebiotics. Critical Reviews
402
in Food Science and Nutrition 2011, 51 (5), 394-409.
403
12. Giuliano, M.; Schiraldi, C.; Marotta, M. R.; Hugenholtz, J.; De Rosa, M., Expression of
404
Sulfolobus solfataricus alpha-glucosidase in Lactococcus lactis. Applied microbiology and
405
biotechnology 2004, 64 (6), 829-32.
406
13. Yamamoto, T.; Unno, T.; Watanabe, Y.; Yamamoto, M.; Okuyama, M.; Mori, H.; Chiba, S.;
407
Kimura, A., Purification and characterization of Acremonium implicatum alpha-glucosidase having
408
regioselectivity for alpha-1,3-glucosidic linkage. Biochimica et biophysica acta 2004, 1700 (2),
409
189-98.
410
14. Nakai, H.; Ito, T.; Hayashi, M.; Kamiya, K.; Yamamoto, T.; Matsubara, K.; Kim, Y. M.;
411
Jintanart, W.; Okuyama, M.; Mori, H.; Chiba, S.; Sano, Y.; Kimura, A., Multiple forms of
412
alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare). Biochimie 2007, 89 (1), 49-62.
413
15. Ojha, S.; Mishra, S.; Chand, S., Production of isomalto-oligosaccharides by cell bound
Basu, A.; Mutturi, S.; Prapulla, S. G., Production of isomaltooligosaccharides (IMO) using
20
ACS Paragon Plus Environment
Page 20 of 35
Page 21 of 35
Journal of Agricultural and Food Chemistry
414
α-glucosidase of Microbacterium sp. LWT - Food Science and Technology 2015, 60 (1), 486-494.
415
16. Kita, A.; Matsui, H.; Somoto, A.; Kimura, A.; Takata, M.; Chiba, S., Substrate Specificity and
416
Subsite Affinities of Crystalline α-Glucosidase from Aspergillus niger. Agricultural and
417
Biological Chemistry 1991, 55 (9), 2327-2335.
418
17. Kimura, A.; Takata, M.; Sakai, O.; Matsui, H.; Takai, N.; Takayanagi, T.; Nishimura, I.;
419
Uozumi, T.; Chiba, S., Complete Amino Acid Sequence of Crystalline (α–Glucosidase from
420
Aspergillus niger. Bioscience, Biotechnology, and Biochemistry 1992, 56 (8), 1368-1370.
421
18. Nakamura, A.; Nishimura, I.; Yokoyama, A.; Lee, D.-G.; Hidaka, M.; Masaki, H.; Kimura, A.;
422
Chiba, S.; Takeshi, U., Cloning and sequencing of an α-glucosidase gene from Aspergillus niger
423
and its expression in A. nidulans. Journal of Biotechnology 1997, 53 (1), 75-84.
424
19. Chen, D.-L.; Tong, X.; Chen, S.-W.; Chen, S.; Wu, D.; Fang, S.-G.; Wu, J.; Chen, J.,
425
Heterologous expression and biochemical characterization of alpha-glucosidase from Aspergillus
426
niger by Pichia pastroris. J Agric Food Chem 2010, 58 (8), 4819-4824.
427
20. Ogawa, M. N. U., Fujisawa, Kanagawa (Japan). Coll. of Bioresource Sciences); Nishio, T.;
428
Minoura, K.; Uozumi, T.; Wada, M.; Hashimoto, N.; Kawachi, R.; Oku, T., Recombinant
429
alpha-glucosidase from Aspergillus niger. overexpression by Emericella nidulans, purification and
430
characterization. jan2006, v. 53.
431
21. Zhang, L.; Liang, S.; Zhou, X.; Jin, Z.; Jiang, F.; Han, S.; Zheng, S.; Lin, Y., Screening for
432
glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant
433
expression on the cell surface. Applied and environmental microbiology 2013, 79 (18), 5519-26.
434
22. Sambrook, J.; Fritsch, E. F.; Maniatis, T., Molecular cloning: a laboratory manual. CSH:
435
1989; p 895–909. 21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
436
23. Güldener, U.; Heck, S.; Fielder, T.; Beinhauer, J.; Hegemann, J. H., A new efficient gene
437
disruption cassette for repeated use in budding yeast. Nucleic Acids Research 1996, 24 (13), 2519.
438
24. Hartner, F. S.; Ruth, C.; Langenegger, D.; Johnson, S. N.; Hyka, P.; Lincereghino, G. P.;
439
Lincereghino, J.; Kovar, K.; Cregg, J. M.; Glieder, A., Promoter library designed for fine-tuned
440
gene expression in Pichia pastoris. Nucleic Acids Research 2008, 36 (12), e76.
441
25. Li, C.; Lin, Y.; Zheng, X.; Pang, N.; Liao, X.; Liu, X.; Huang, Y.; Liang, S., Combined
442
strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris. Bmc
443
Biotechnology 2015, 15 (1), 1-11.
444
26. Cregg, J. M.; Tolstorukov, I.; Kusari, A.; Sunga, J.; Madden, K.; Chappell, T., Expression in
445
the yeast Pichia pastoris. Methods in Enzymology 2009, 463, 169.
446
27. Waterham, H. R.; Digan, M. E.; Koutz, P. J.; Lair, S. V.; Cregg, J. M., Isolation of the Pichia
447
pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.
448
Gene 1997, 186 (1), 37.
449
28. Cregg, J. M.; Barringer, K. J.; Hessler, A. Y.; Madden, K. R., Pichia pastoris as a host system
450
for transformations. Mol Cell Biol 5: 3376-3385. Molecular & Cellular Biology 1986, 5 (12),
451
3376-85.
452
29. Cheng, L.; Ying, L.; Huang, Y.; Liu, X.; Liang, S., Citrobacter amalonaticus Phytase on the
453
Cell Surface of Pichia pastoris Exhibits High pH Stability as a Promising Potential Feed
454
Supplement. Plos One 2014, 9 (12), e114728.
455
30. Nordén, K.; Agemark, M.; Danielson, J. Å.; Alexandersson, E.; Kjellbom, P.; Johanson, U.,
456
Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris.
457
Bmc Biotechnology 2011, 11 (11), 566-570. 22
ACS Paragon Plus Environment
Page 22 of 35
Page 23 of 35
Journal of Agricultural and Food Chemistry
458
31. Zhao, X.; Xie, W.; Lin, Y.; Lin, X.; Zheng, S.; Han, S., Combined strategies for improving the
459
heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia
460
pastoris. Process Biochemistry 2013, 48 (9), 1317-1323.
461
32. Hohenblum, H.; Gasser, B.; Maurer, M.; Borth, N.; Mattanovich, D., Effects of gene dosage,
462
promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris.
463
Biotechnology and bioengineering 2004, 85 (4), 367.
464
33. Hou, J.; Tyo, K.; Liu, Z.; Petranovic, D.; Nielsen, J., Engineering of vesicle trafficking
465
improves heterologous protein secretion in Saccharomyces cerevisiae. Metabolic Engineering
466
2012, 14 (2), 120-127.
467
34. Vogl, T.; Hartner, F. S.; Glieder, A., New opportunities by synthetic biology for
468
biopharmaceutical production in Pichia pastoris. Current Opinion in Biotechnology 2013, 24 (6),
469
1094-1101.
470
35. Cregg, J. M.; Cereghino, J. L.; Shi, J.; Higgins, D. R., Recombinant protein expression in
471
Pichia pastoris. Molecular Biotechnology 2000, 16 (1), 23-52.
472
36. Cereghino, J. L.; Cregg, J. M., Heterologous protein expression in the methylotrophic yeast
473
Pichia pastoris. Fems Microbiology Reviews 2000, 24 (1), 45-66.
474
37. Lin, C. G.; Lin, C. J.; Sunga, A. J.; Johnson, M. A.; Lim, M.; Gleeson, M. A.; Cregg, J. M.,
475
New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation
476
of Pichia pastoris. Gene 2001, 263 (1-2), 159.
477
38. Nett, J. H.; Gerngross, T. U., Cloning and disruption of the PpURA5 gene and construction of
478
a set of integration vectors for the stable genetic modification of Pichia pastoris. 2003, 20 (15),
479
1279-1290. 23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
480
39. Nett, J. H.; Hodel, N.; Rausch, S.; Wildt, S., Cloning and disruption of the Pichia pastoris
481
ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers. Yeast
482
2005, 22 (4), 295–304.
483
40. Thor, D.; Xiong, S.; Orazem, C. C.; Kwan, A. C.; Cregg, J. M.; Lincereghino, J.;
484
Lincereghino, G. P., Cloning and characterization of the Pichia pastoris MET2 gene as a
485
selectable marker. Fems Yeast Research 2005, 5 (10), 935–942.
486
41. Kimura, M.; Kamakura, T.; Tao, Q. Z.; Kaneko, I.; Yamaguchi, I., Cloning of the blasticidin S
487
deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for
488
Schizosaccharomyces pombe and Pyricularia oryzae. Molecular Genetics and Genomics 1994,
489
242 (2), 121-129.
490
42. Scorer, C. A.; Clare, J. J.; Mccombie, W. R.; Romanos, M. A.; Sreekrishna, K., Rapid
491
selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign
492
gene expression. Bio/technolgy 1994, 12 (2), 181-184.
493
43. Yang, J.; Jiang, W.; Yang, S., mazF as a counter‐selectable marker for unmarked genetic
494
modification of Pichia pastoris. Fems Yeast Research 2009, 9 (4), 600.
495
44. Gasser, B.; Dragosits, M.; Mattanovich, D., Engineering of biotin-prototrophy in Pichia
496
pastoris for robust production processes. Metabolic Engineering 2010, 12 (6), 573-580.
497
45. Williams, K. E.; Jiang, J.; Ju, J.; Olsen, D. R., Novel strategies for increased copy number
498
and expression of recombinant human gelatin in Pichia pastoris with two antibiotic markers.
499
Enzyme & Microbial Technology 2008, 43 (1), 31-34.
500
46. Mellitzer, A.; Glieder, A.; Weis, R.; Reisinger, C.; Flicker, K., Sensitive high-throughput
501
screening for the detection of reducing sugars. Biotechnology Journal 2012, 7 (1), 155-62. 24
ACS Paragon Plus Environment
Page 24 of 35
Page 25 of 35
Journal of Agricultural and Food Chemistry
502
47. HansMarx; AstridMecklenbräuker; BrigitteGasser; MichaelSauer; DiethardMattanovich,
503
Directed gene copy number amplification in Pichia pastoris by vector integration into the
504
ribosomal DNA locus. FEMS Yeast Research 2009, 9 (8), 1260-70.
505
48. Lin, X. Q.; Han, S. Y.; Zhang, N.; Hu, H.; Zheng, S. P.; Ye, Y. R.; Lin, Y., Bleach boosting
506
effect of xylanase A from Bacillus halodurans C-125 in ECF bleaching of wheat straw pulp.
507
Enzyme & Microbial Technology 2013, 52 (2), 91-98.
508
49. Idiris, A.; Tohda, H.; Kumagai, H.; Takegawa, K., Engineering of protein secretion in yeast:
509
strategies and impact on protein production. Applied Microbiology and Biotechnology 2010, 86 (2),
510
403-417.
511
50. Whyteside, G.; Alcocer, M. J.; Kumita, J. R.; Dobson, C. M.; Lazarou, M.; Pleass, R. J.;
512
Archer, D. B., Native-state stability determines the extent of degradation relative to secretion of
513
protein variants from Pichia pastoris. Plos One 2011, 6 (7), e22692.
514
51. Cudna, R. E.; Dickson, A. J., Endoplasmic reticulum signaling as a determinant of
515
recombinant protein expression. Biotechnology & Bioengineering 2003, 81 (1), 56–65.
516
52. Shusta, E. V.; Raines, R. T.; Plückthun, A.; Wittrup, K. D., Increasing the secretory capacity
517
of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nature
518
Biotechnology 1998, 16 (8), 773-7.
519
53. Ruddock, L., Method for producing natively folded proteins in a prokaryotic host. EP: 2016.
520
54. Delic, M.; Göngrich, R.; Mattanovich, D.; Gasser, B., Engineering of protein folding and
521
secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins.
522
Antioxidants & Redox Signaling 2014, 21 (3), 414.
523
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
524
Table titles
525
Table 1 Initial conversion rate of Pp-ANGL-GCW61 in reuse study in 0.5 h.
526 527
Figure captions
528
Figure 1. (a) Construction of the pPIC9K-ANGL-GCW61 plasmid used for
529
displaying ANGL on the yeast cell surface. (b) Growth-related curves of P. pastoris
530
GS115/ANGL–GCW61 and GS115/9K in 250-mL shake flasks. (c) Fermentation time
531
curves of P. pastoris GS115/ANGL–GCW61 and GS115/9K in 250-mL shake flasks.
532
(d) Determination of Pp-ANGL-GCW61 fermentations upermatant hydrolytic activity.
533
Error bars represent standard deviations, and three replicates were performed.
534 535
Figure 2. HPLC-ELSD analyses of reaction products from maltose by enzymatic
536
activity of Pp-ANGL-GCW61; G, glucose; M, maltose; IG2, isomaltose; P, panose ;
537
IG3, isomaltotriose.
538 539
Figure 3. (a) Effect of pH on IMOs synthesis by Pp-ANGL-GCW61, (b) pH-activity
540
curves of Amano and Pp-ANGL-GCW61 for IMOs synthesis, and (c) pH-stability
541
relationship of Pp-ANGL-GCW61 for IMOs synthesis. Error bars represent standard
542
deviations, and three replicates were performed.
543
544
Figure 4. (a) Effect of temperature on IMOs synthesis by Pp-ANGL-GCW61 (b)
545
Temperature-stability relationship of Pp-ANGL-GCW61 for IMOs synthesis. Error 26
ACS Paragon Plus Environment
Page 26 of 35
Page 27 of 35
Journal of Agricultural and Food Chemistry
546
bars represent standard deviations, and three replicates were performed.
547
548
Figure 5. (a) Effect of the whole-cell biocatalyst Pp-ANGL-GCW61 concentration on
549
IMOs synthesis. (b) Effect of the maltose concentration on IMOs synthesis. Error bars
550
represent standard deviations, and three replicates were performed.
551
Figure 6. Production of IMOs under optimum conditions. Error bars represent
552
standard deviations, and three replicates were performed.
553
554
Figure 7. (a) Reuse of Pp-ANGL-GCW61 in the 2-mL reaction system. (b) Reuse of
555
Pp-ANGL-GCW61 in the 10-mL reaction system. (c) Reuse of Pp-ANGL-GCW61 in
556
the 500-mL reaction system. Error bars represent standard deviations, and three
557
replicates were performed.
558
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 28 of 35
Tables Table 1 times reused
2-mL system
10-mL system
500-mL system
batch 1
9.84%
9.97%
9.68%
batch 2
9.26%
9.67%
7.42%
batch 3
6.74%
8.94%
5.40%
28
ACS Paragon Plus Environment
Page 29 of 35
Journal of Agricultural and Food Chemistry
Figures Figure 1
29
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 2
30
ACS Paragon Plus Environment
Page 30 of 35
Page 31 of 35
Journal of Agricultural and Food Chemistry
Figure 3
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 4
32
ACS Paragon Plus Environment
Page 32 of 35
Page 33 of 35
Journal of Agricultural and Food Chemistry
Figure 5
Figure 6
33
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 7
34
ACS Paragon Plus Environment
Page 34 of 35
Page 35 of 35
Journal of Agricultural and Food Chemistry
For Table of Contents Only
35
ACS Paragon Plus Environment