Characterization and mechanism of action of Microbacterium imperiale glucan 1,4-α-maltotriohydrolase

Article abstract of DOI:10.1016/j.carres.2013.11.014

In this study, glucan 1,4-α-maltotriohydrolase (AMTS) from Microbacterium imperiale was purified and characterized. Hydrolysis by AMTS was affected by starch structure (e.g., amylose versus amylopectin) and hydrolysis time. During the initial phase of hydrolysis of maltooligosaccharides (G4-G7), AMTS displayed a unique transfer specificity to the transfer of maltotriosyl units. After extensive hydrolysis, maltotriose became the major end product, followed by glucose and maltose. Maltotetraose (G4) was the smallest donor in transglycosylation reactions by AMTS. This is the first study that reports transglycosylation activity of AMTS on maltooligosaccharides. The results of this study suggest that high purity maltotriose can be produced by the hydrolytic action of AMTS on starch.

Full text of DOI:10.1016/j.carres.2013.11.014

Carbohydrate Research 384 (2014) 46–50
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Characterization and mechanism of action of Microbacterium
imperiale glucan 1,4-a-maltotriohydrolase
Chunsen Wu a,b, Xing Zhou a,b, Yan Xu a, Hongyan Li a, Yaoqi Tian a,b, Xueming Xu a,b, Zhengyu Jin a,b,
⇑
a The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
b Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 August 2013
Received in revised form 16 November 2013
Accepted 20 November 2013
Available online 1 December 2013
In this study, glucan 1,4-
a
-maltotriohydrolase (AMTS) from Microbacterium imperiale was purified and
characterized. Hydrolysis by AMTS was affected by starch structure (e.g., amylose versus amylopectin)
and hydrolysis time. During the initial phase of hydrolysis of maltooligosaccharides (G4–G7), AMTS dis-
played a unique transfer specificity to the transfer of maltotriosyl units. After extensive hydrolysis, mal-
totriose became the major end product, followed by glucose and maltose. Maltotetraose (G4) was the
smallest donor in transglycosylation reactions by AMTS. This is the first study that reports transglycosy-
lation activity of AMTS on maltooligosaccharides. The results of this study suggest that high purity mal-
totriose can be produced by the hydrolytic action of AMTS on starch.
Keywords:
Glucan 1,4-a-maltotriohydrolase
Transglycosylation
Maltotriose
Ó 2013 Elsevier Ltd. All rights reserved.
Corn starch
Maltooligosaccharides
1. Introduction
ported that the starch hydrolysates derived from the enzymatic
activity of glucan 1,4-alpha-maltotriohydrolase (AMTS, EC
Maltotriose has excellent properties, for example, mild sweet-
ness, moisture preservation, low freezing-point depression, and
structural relaxation reduction, which favors glassy states and pre-
vents starch retrogradation.1–3 Owing to these characteristics, mal-
totriose can be used in the production of desserts, baked goods, and
as glucose replacement in intravenous feeding.1,4 Researchers have
recently reported that maltotriose is a nuclear localization signal
for nanoparticles and proteins and that maltotriose-modified poly
(propylene imine) dendrimers have physiological roles in cells.5,6
Moreover, modified maltotrioses can be used as novel enzyme sub-
strates7,8 and as physiological activators.9–11 However, due to its
high price, utilization of maltotriose is limited.
Maltotriose can be obtained from the enzymatic hydrolysis of
pullulan and starch and from maltose syrup purification.1,4 High
purity maltotriose can easily be produced by the hydrolytic action
of pullulanase on pullulan;4 however, pullulan is quite expensive.
The other two production methods usually result in maltotriose
of low purity. A low cost production of high purity maltotriose is
of outmost importance for large scale applications. It has been re-
3.2.1.116) consist predominantly of maltotriose.12,13 Recent chro-
matographic separation developments provide a convenient meth-
od of producing pure maltotriose from low purity syrup at a
relative low cost.14,15 From a practical viewpoint, amylolysis by
AMTS represents a potential method of producing maltotriose at
low costs and for large scale applications. This study explored
the production of maltotriose from starch using AMTS.
AMTS from Natronococcus sp. Ah-36 belongs to the family 13 of
glycoside hydrolases (http://www.cazy.org/). AMTS successively
removes maltotriose units from the non-reducing ends of polysac-
charide chains, producing maltotriose as the main end product
(http://www.brenda-enzymes.org). However, the mechanism of
action of AMTS has not been elucidated.
In this study, a commercial AMTS from Microbacterium imperiale
was purified and characterized. The objective of this study was to
assess the hydrolytic mechanism of AMTS on corn starch (CS), corn
amylose (CSAM), and corn amylopectin (CSAP). Additionally, we
assessed the hydrolytic mechanism of AMTS on maltooligosaccha-
rides, that is, maltose (G2), maltotriose (G3), maltotetraose (G4),
maltopentaose (G5), maltohexaose (G6), and maltoheptaose (G7).
2. Results and discussion
Abbreviations: AMTS, glucan 1,4-a-maltotriohydrolase; G1–G7, glucose, malt-
ose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose;
DM, degree of starch hydrolyzed into maltotriose; PM, percentage of maltotriose in
G1–G7; CS, corn starch; CSAM, corn amylose; CSAP, corn amylopectin; DP, degree of
polymerization.
2.1. Enzyme purification and characterization
To purify M. imperial AMTS, the commercial enzyme was
fractionated using a HiTrap™ desalting column. The desalted
⇑
Corresponding author. Tel./fax: +86 510 85913299.
E-mail address: jinlab2008@yahoo.com (Z. Jin).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.11.014

C. Wu et al. / Carbohydrate Research 384 (2014) 46–50
47
CSAM, 68% for CS, and 58% for CSAP. The relative high percentage
of non-reducing ends and short chains in CSAP16 may contribute
to the higher releasing rates of maltotriose during the initial phase
of hydrolysis. With extensive hydrolysis, the (1->6)-a-D-glucosidic
linkages of CSAP may hinder AMTS activity, resulting in lower mal-
totriose releasing rates.
In the starch industry, the refining stage is of outmost concern
because it is costly and time-consuming.17 One considerable chal-
lenge is to separate sugars from low molecular weight sugars with
similar chain lengths and physicochemical properties.15 Therefore,
our study evaluated the percentage of maltotriose in G1–G7 (PM)
following hydrolysis by AMTS. In general, PM values of CS, CSAM,
and CSAP were not decreased directly after hydrolysis. As a result
of changing PM values, starch hydrolysis by AMTS appears to occur
in three different stages (Fig. 3). In the first stage, PM values were
high and subsequently decreased. In the second stage, PM values
gradually decreased. In the last stage, PM values increased slightly
and subsequently remained stable. At this inflection point, DM val-
ues were 50% for CSAP, 56% for CS, and 66% for CSAM. Low molec-
ular weight dextrin molecules increased following starch
hydrolysis.18 AMTS activity on these dextrin molecules increased
with increasing hydrolysis. The increased PM values during the last
stage of hydrolysis might be attributed to the hydrolysis of low
molecular dextrins.
Therefore, there are three different steps in starch hydrolysis
by AMTS: in the first step, AMTS hydrolyzes starch molecules into
low molecular dextrins with the accumulation of maltotriose; in
the second step, AMTS simultaneously hydrolyzes the initial sub-
strate and end products (i.e., low molecular dextrins); and in the
final step, AMTS mainly hydrolyzes the remaining low molecular
sugars. In addition, PM values of CSAP had a purity of >90% before
reaching 10% DM, suggesting that pure maltotriose might be pro-
duced by a controlled partial hydrolysis of amylopectin. These
observations reveal that starch structure and hydrolysis time
determine the profiles of low molecular hydrolysates from starch
by AMTS.
Figure 1. SDS–PAGE of AMTS in 10% polyacrylamide gel. Lane 1: purified AMTS;
lane 2: commercial AMTS; lane M: molecular weight standards.
enzymatic fraction was applied to a cation-exchange chromatogra-
phy column using HiTrap™ Q fast flow, yielding a single peak
(AMTS) that corresponded to a single band in SDS-PAGE (Fig. 1).
The optimum AMTS hydrolytic conditions consisted of a tempera-
ture of 50 °C and a pH of 6.5 (Fig. 2). These results are in accordance
with those reported by other researchers.12,13
2.2. Maltotriose production from starch using AMTS
This study explored the production of maltotriose from inex-
pensive sources like CS. CSAM and CSAP were prepared to assess
the AMTS hydrolytic mechanism. Figure 3 shows distinct hydro-
lytic patterns of AMTS on CS, CSAM, and CSAP as a function of time.
Figure 3 shows that the degree of starch hydrolyzed into malto-
triose (DM) increased with hydrolysis. During the initial phase of
hydrolysis, DM of CSAP was 2.3-fold higher than that of CSAM.
However, these differences were not significant after 180 min of
hydrolysis. Subsequently, DM of CSAM was higher than that of
CS and CSAP. After extensive hydrolysis (24 h), DM was 79% for
2.3. Hydrolysis of maltooligosaccharides (G2–G7)
Extensive hydrolysis of maltooligosaccharides by AMTS was
monitored by HPAEC-PAD; the results are shown in Figure 4. G2
110
(a)
100
110
(b)
100
90
80
70
60
50
40
30
90
80
70
60
50
40
30
20
20
25
30
35
40
45
50
55
60
65
70
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
Temperature (ഒ)
Figure 2. Optimum temperature and pH of AMTS. (a): Optimum temperature; (b): optimum pH.

48
C. Wu et al. / Carbohydrate Research 384 (2014) 46–50
1
2
3
100
90
80
70
60
50
40
30
20
10
0
100
(a)
95
90
85
80
75
70
65
DM
PM
-200
0
200 400 600 800 1000 1200 1400 1600
Hydrolysis Time (min)
1 2
3
1 2
3
100
90
80
70
60
50
40
30
20
10
0
100
95
90
85
80
75
70
65
100
90
80
70
60
50
40
30
20
10
0
100
95
90
85
80
75
70
65
(b)
(c)
-200
0
200 400 600 800 1000 1200 1400 1600
Hydrolysis Time (min)
-200
0
200 400 600 800 1000 1200 1400 1600
Hydrolysis Time (min)
Figure 3. DM and PM values following starch hydrolysis by AMTS. (a): Corn starch; (b): amylose from corn starch; (c): amylopectin from corn starch. The numbers 1, 2, and 3
represent the hydrolysis stage 1, 2, and 3, respectively.
2 3 4
5
6
7
8
9 101112
13 (a)
(b)
1
1 2 3 4
5
6
7
8 9 10111213
12 h
1.5 h
10 min
5 min
2 min
0 min
(c)
(d)
12 h
1.5 h
10 min
5 min
2 min
0 min
(e)
(f)
12 h
1.5 h
10 min
5 min
2 min
0 min
0
5
10
15
20/0
5
10
15
20
Time/min
Figure 4. Maltooligosaccharides (G2–G7) hydrolyzed by AMTS. (a): Maltoheptaose (G7); (b): maltohexaose (G6); (c): maltopentaose (G5); (d): maltotetraose (G4); (e):
maltotriose (G3); (f): maltose (G2). The numbers 1–13 represent sugars with a degree of polymerization (DP) 1–13.

C. Wu et al. / Carbohydrate Research 384 (2014) 46–50
49
Figure 5. Proposed mechanism of M. imperiale glucan 1,4-
a-maltotriohydrolase on maltooligosaccharides.
and G3 were relatively stable during hydrolysis; transglycosylation
activity was observed during the hydrolysis of G4–G7. Therefore,
the smallest donor in transglycosylation reactions by AMTS was
G4. This is the first study that reports transglycosylation activity
4. Materials and methods
4.1. Materials
of AMTS. Similarly, a-amylases from Byssochlamys fulva and Ther-
Maltose, maltotriose, maltotetraose, maltopentaose, malto-
hexaose, and maltoheptaose (G2–G7) were obtained from Sigma-
Aldrich Trading Co., Ltd (Shanghai, China). Soluble starch (acid-
hydrolyzed potato starch) was supplied by Sinapharm Chemical
Reagent Co., Ltd (Shanghai, China). AMTS was purchased from
Amano Enzyme Inc. (Nagoya, Japan).
motoga matitima have the capacity to perform transglycosylation
reactions to their own hydrolysates.19,20 After 12 h of incubation
of G4–G7, different proportions of G1, G2, and G3 were produced.
Hydrolysis rate increased with increasing substrate molecular
weight as follows, G7 > G6 > G5 > G4 > G3 = G2 = 0.
During the initial phase of hydrolysis, the hydrolysis of G4 pri-
marily resulted in G1 and G3 with a small number of G7 (Fig. 4). G5
hydrolysis mainly yielded G2 and G3, followed by G8; G6 hydroly-
sis mainly yielded G3 and a small amount of G9; and G7 yielded G3
and G4 and a small amount of G10. Apart from maltotriose, AMTS
partially hydrolyzed maltooligosaccharides with DPn (n = 4, 5, 6,
and 7) into products with DPn 3. The products apart from those
with DP n 3 obtained after 5 min or 10 min of hydrolysis
(Fig. 4) might result from the hydrolysis of initial transglycosylated
products by AMTS.
This is the first study that reports that maltotriosyl transfer oc-
curs during the hydrolysis of maltooligosaccharides. To the best of
our knowledge, glucosyl and maltosyl transferases have been pre-
viously reported; however, reports on spontaneous maltotriosyl
transferases are scarce.21,22 Therefore, the maltotriosyl transfer
activity of AMTS can be used to produce special transglycosylated
products.
Based on these results, the proposed mechanism of action of
AMTS on maltooligosaccharides is showed in Figure 5. At the
beginning, hydrolysis and maltotriosyl transfer reactions occur
simultaneously. Following extensive hydrolysis, maltotriose is
the major product with different proportions of glucose, maltose,
and other by-products. This mechanism is important to understand
the hydrolytic behavior of AMTS on CS. The different PM values
from the hydrolysis of CS, CSAP, and CSAM could partially be
attributed to the hydrolytic action of AMTS on different low molec-
ular sugars released from starch.
4.2. Enzyme purification and characterization
AMTS was partially purified using the ÄKTA purifier™ 10 sys-
tem (GE Healthcare, Uppsala, Sweden) equipped with a UV detec-
tor and a fraction collector. HiTrap™ desalting and HiTrap™ Q FF
columns (GE Healthcare, Uppsala, Sweden) were used for purifica-
tion. Fractions of high AMTS activity were pooled. Protein purity
was assessed by sodium dodecylsulfate–polyacrylamide gel elec-
trophoresis (SDS–PAGE). Protein concentration was determined
by the Coomassie brilliant blue method with bovine serum albu-
min as the standard.23
To identify the optimum pH of AMTS, enzymatic assays were
performed in 1% soluble starch dissolved in 0.1 M phosphate buffer
at different pH values (pH 5.0–8.5). The optimum temperature of
AMTS was determined by incubating AMTS in 1% soluble starch
at different temperatures (30–65 °C). Samples were periodically re-
moved; the ratio (in percentage) between the enzyme activity at a
certain pH or temperature and the corresponding optimum activity
was calculated.
AMTS activity was determined by quantifying the amount of
reducing sugars24 released from 1% soluble starch in 0.1 M phos-
phate buffer (pH 6.5) at 50 °C. Maltotriose (0–1
as a standard. The results were expressed as
equivalents released per min. One enzymatic unit (U) was defined
l
mol/mL) was used
lmol of maltotriose
as the amount of enzyme required to release 1.0
ose equivalents per min.
lmol of maltotri-
4.3. Enzymatic hydrolysis of starch into maltotriose
3. Conclusions
4.3.1. CS fractionation
This study described the production of maltotriose from starch
hydrolysis by AMTS. Maltotriose production by AMTS depends on
the specific starch structure (e.g., amylose versus amylopectin)
and hydrolysis time. A specific transglycosylation activity by AMTS
was elucidated. AMTS preferably transfers maltotriosyl units dur-
ing the initial phase of hydrolysis. The smallest donor of transgly-
cosylation reactions by AMTS was maltotetraose (G4).
CSAM and CSAP were separated from CS according to the meth-
od reported by Takeda et al.25,26 CS was defatted three times with
90% dimethyl sulfoxide and precipitated with 6 volumes of etha-
nol. The resulting residue was vacuum-dried at 40 °C for 24 h.
The dried residue was re-dissolved and precipitated with n-butyl
alcohol at 4 °C for 24 h. Following centrifugation, CSAM and CSAP

50
C. Wu et al. / Carbohydrate Research 384 (2014) 46–50
were separated from each other. Crude CSAM was repeatedly pre-
cipitated in saturated n-butyl alcohol solution until the purity was
>98%. For CSAP preparation, the supernatant was further precipi-
tated with n-butyl alcohol to remove amylose. The precipitate
was discarded. Ethanol was added to the supernatant and stored
overnight at 4 °C. This procedure was repeated several times. Sub-
sequently, CSAM and CSAP were washed in ethanol and vacuum
dried for purification purposes.
coupled with a GP40 gradient pump and an electrochemical detec-
tor. Bound material was eluted with 0.15 M NaOH and a sodium
gradient at 1 mL/min.28,29
4.5. Statistical analyses
Data were analyzed using PASW Statistics 18 (SPSS Inc., Chi-
cago, USA). All results were reported as mean SD by single-tree
analysis in triplicate.
4.3.2. Preparation of starch dispersions
Aqueous starch dispersions (1%, w/v) were prepared according
to the protocol reported by Yoo and Jane.27 CS, CSAM, and CSAP
were dispersed in DMSO (95%, w/v) under constant stirring at
100 °C for 60 min. The dispersions were further stirred for 24 h at
30 °C. Ethanol was added to precipitate starch, which was sus-
pended in acetone. Following centrifugation, the starch pellet
was vacuum dried, dispersed in boiling 0.1 M phosphate buffer
(pH 6.5), and stirred for 60 min.
Acknowledgement
This study was financially supported by the Natural Science
Foundation of China (Nos. 31230057 and 31301505).
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DM ¼
ꢀ 100
Search content ðmg; dry basisÞ
From the peak areas corresponding to the digestion of different
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equation,
Peak areas of maltotriose
Total peak areas of low molecular sugars ðG1—G7Þ
PM ¼
ꢀ 100:
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4.4. Hydrolysis of maltooligosaccharides (G2–G7)
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(100 ll, 10 U). The hydrolysates obtained after 2 min, 5 min,
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