Cloning, expression, properties, and functional amino acid residues of new trehalose synthase from Thermomonospora curvata DSM 43183

Article abstract of DOI:10.1016/j.molcatb.2013.01.014

A new trehalose synthase (TreS) gene from Thermomonospora curvata DSM 43183 was cloned and expressed in Escherichia coli XL10-Gold. The purified recombinant enzyme (TreS-T.C) could catalyze the reversible interconversion of maltose and trehalose of sucrose into trehalulose without other disaccharides including isomaltulose at an optimum temperature of 35 °C and a pH of 6.5. The K_m of TreS-T.C for maltose (96 mM) was lower than those for trehalose (198 mM) and sucrose (164 mM), suggesting that maltose is the optimum substrate. The maximum trehalose and trehalulose yields were 70% and >80%, respectively. Active TreS-T.C is a trimer comprising three identical 60 kDa subunits. Homology modeling analysis revealed that TreS-T.C had a GH13-typical (β/α)₈ barrel catalytic domain. Two sites, one determining substrate specificity (L116) and the other affecting product formation (E330), were found near the active center by homology modeling combined with site-directed mutagenesis. TreS-T.C may be used effectively as a potential biocatalyst for the production of trehalose and trehalulose from maltose and sucrose in a one-step reaction, respectively. This study also provides a feasible and effective method for studying functional amino acid residues around TreS without performing crystal structure analysis and high-throughput screening.

Full text of DOI:10.1016/j.molcatb.2013.01.014

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Cloning, expression, properties, and functional amino acid residues of new
trehalose synthase from Thermomonospora curvata DSM 43183
Jiayuan Lianga,c, Ribo Huanga,b,c, Ying Huanga,c, Xiaobo Wanga,c, Liqin Dua,c, Yutuo Weia,c,∗
a
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, China
National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, 98 Daling Road, Nanning, Guangxi 530007, China
College of Life Science & Technology, Guangxi University, Nanning 530004, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2012
Received in revised form 15 January 2013
Accepted 15 January 2013
Available online 23 January 2013
A new trehalose synthase (TreS) gene from Thermomonospora curvata DSM 43183 was cloned and
expressed in Escherichia coli XL10-Gold. The purified recombinant enzyme (TreS-T.C) could catalyze the
reversible interconversion of maltose and trehalose of sucrose into trehalulose without other disaccha-
◦
rides including isomaltulose at an optimum temperature of 35 C and a pH of 6.5. The Km of TreS-T.C for
maltose (96 mM) was lower than those for trehalose (198 mM) and sucrose (164 mM), suggesting that
maltose is the optimum substrate. The maximum trehalose and trehalulose yields were 70% and >80%,
respectively. Active TreS-T.C is a trimer comprising three identical 60 kDa subunits. Homology modeling
analysis revealed that TreS-T.C had a GH13-typical (␤/␣)8 barrel catalytic domain. Two sites, one deter-
mining substrate specificity (L116) and the other affecting product formation (E330), were found near
the active center by homology modeling combined with site-directed mutagenesis. TreS-T.C may be used
effectively as a potential biocatalyst for the production of trehalose and trehalulose from maltose and
sucrose in a one-step reaction, respectively. This study also provides a feasible and effective method for
studying functional amino acid residues around TreS without performing crystal structure analysis and
high-throughput screening.
Keywords:
Thermomonospora curvata
Trehalose synthase
Trehalose
Trehalulose
Functional amino acid residue
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
application prospects in the food, cosmetics, and pharmaceutical
industries, thus the extensive interest of numerous researchers.
Five main enzymatic routes are involved in trehalose
biosynthesis: (1) trehalose-6-phosphate synthetase and trehalose-
6-phosphate phosphatase [13–15], (2) maltooligosyltrehalose
trehalohydrolase and maltooligosyltrehalose synthase [15–18],
(3) trehalose phosphorylase [14,15,19,20], (4) trehalose glycosyl-
transferring synthase (TreT) [20–22], and (5) trehalose synthase
(TreS) [23–25]. Among these routes, the conversion of maltose into
trehalose by TreS is a simple, fast, and low-cost method for the
industrial production of trehalose. Thus, an increasing number of
TreS or TreS genes have been purified or heterologously expressed
[23–29]. However, most studies on TreS have been confined to gene
cloning and enzymatic property determination; few have reported
on its catalytic mechanism or on functional amino acid residues.
The chemical synthesis of trehalulose is extremely difficult. Tre-
halulose is industrially produced exclusively from sucrose using
immobilized microorganisms. Through sucrose hydrolysis, several
organisms and enzymes, including trehalulose synthase, isomal-
tulose synthase, and sucrose isomerase, can convert sucrose into
trehalulose (a mixture of isomaltulose and trehalulose) to pro-
duce glucose and fructose in residual amounts. Depending on the
enzyme, the composition of enzyme products varies from mainly
isomaltulose at 66–91% [30–33] to predominantly trehalulose at
Trehalose (␣-d-glucopyranosyl-1,1-␣-d-glucopyranoside) is a
non-reducing disaccharide widely found in various organisms such
as bacteria, archaea, yeast, fungi, insects, non-mammalian animals,
and plants [1–5]. Trehalose is known as reserve energy that is a car-
bon source similar to glycogen and starch [6] as well as an active
preservative of proteins, biomass, pharmaceutical preparations,
and even organs for transplantation [7,8]. Therefore, trehalose is
widely used in the food, cosmetics, and pharmaceutical industries
[
3,9], thus attracting increasing interest among researchers. On the
other hand, trehalulose (1-O-␣-d-glucosylpyranosyl-␤-d-fructose)
is a structural isomer of sucrose (␣-d-glucosylpyranosyl-1,2-␤-d-
fructofuranoside) and a reducing disaccharide naturally present in
honey in small quantities [10,11]. Trehalulose is non-cariogenic,
exhibits a slower rate of monosaccharide release into the blood,
and has high solubility [12]. Relative to trehalose, fewer studies
have been conducted on trehalulose. However, both have broad
∗ Corresponding author at: College of Life Science & Technology, Guangxi Univer-
sity, 100 Daxue Road, Nanning, Guangxi 530004, China. Tel.: +86 771 3270336;
fax: +86 771 2503908.
E-mail addresses: jyliang86@163.com (J. Liang), weiyutuo@gxu.edu.cn (Y. Wei).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.01.014

J. Liang et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
27
∼
90% [34]. However, isomaltulose and trehalulose have similar
potassium phosphate buffer (pH 7.0), resuspended in the same
buffer, and then disrupted by sonication on ice for 10 min after 10 h
induction at 37 C. Cell debris was removed through centrifugation
at 12,000 × g for 20 min. The recombinant protein was purified
using nickel–nitrilotriacetic acid agarose resin (Qiagen, Hilden,
CA, Germany) according to manufacturer’s instructions. Enzyme
protein content was determined through the Bradford method,
with bovine serum albumin as standard. The purified protein
was analyzed through sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and native PAGE.
physical and chemical properties, such that the presence of isoma-
ltulose significantly increases the difficulty in the production and
purification of trehalulose. In particular, sucrose isomerase from
whiteflies converts sucrose into mainly trehalulose without iso-
maltulose [35]. However, this paper does not report trehalulose
yield.
◦
In this study, a new TreS gene was cloned and expressed from
Thermomonospora curvata. The expressed recombinant enzyme has
a novel action on sucrose that differs from that of other reported
enzymes, such as trehalulose synthase, isomaltulose synthase, and
sucrose isomerase. Two functional amino acid residues, which
determine substrate specificity and affect product formation, were
identified.
2.4. Enzyme assay
Standard reaction was conducted by adding 0.5 mg purified
enzyme to a 500 ␮l reaction mixture containing 100 mM phosphate
buffer (pH 6.5) and 1% substrate followed by incubation at 30 C for
2
. Materials and methods
◦
3
0 min. A volume of distilled water equal to that of the purified
2
.1. Bacterial strains, plasmids, and reagents
enzyme was added to the reaction mixture as control. The identity
of substrate products obtained from TreS-T.C catalysis was con-
firmed through high-performance liquid chromatography (HPLC),
T. curvata DSM 43183 was obtained from DSMZ (Germany).
The plasmid pSE380 (Invitrogen, WI, USA) was used as expres-
sion vector for TreS. E. coli XL10-Gold was used for routine cloning
and gene expression. T. curvata was grown under aerobic condi-
tions in a medium containing 30 g/l starch, 5 g/l peptone, 5 g/l yeast
extract, 0.5 g/l CaCl , 0.5 g/l MgCl ·7H O, 1 g/l K HPO , and 0.5 g/l
◦
performed at 30 C under a pressure of 96 bar using an Agilent 1100
series column (Alltima Amino 100A 5u, 250 mm × 4.6 mm) and
an Alltech 2000ES evaporative light scattering detector. Acetoni-
−
1
trile/water (79:21) was used as solvent at a flow rate of 1 ml min
.
2
2
2
2
4
With the control as the original substrate content, enzyme activ-
ity was assayed by measuring the amount of consumed substrate
through HPLC. One unit of enzyme activity was defined as the
amount of enzyme consuming 1 ␮mol of substrate per minute.
Kinetic analysis was performed at optimum temperature and
pH. The experiment was conducted for 30 min in a 100 mM phos-
phate buffer with substrate at various concentrations. Km and Vmax
values were obtained through a Lineweaver–Burk plot.
MnCl ·4H O at pH 7.0. The medium was incubated in a rotary
2
2
◦
shaker at 55 C and 200 rpm for 48 h. The E. coli and recombi-
◦
nant E. coli strains were routinely grown at 37 C and 200 rpm in
Luria–Bertani medium alone or with 100 ␮g/ml ampicillin. Restric-
tion enzymes, ligase, LA Taq DNA polymerase, and PrimeSTAR HS
DNA polymerase, were all obtained from Takara (Shiga, NJ, Japan).
Protein mixture molecular mass markers were purchased from Bio-
sciences (Amersham, CA, UK). Trehalulose was obtained from Wako
(
Osaka, MO, Japan). All other saccharides were purchased from
Sigma (St. Louis, MO, USA). All other chemicals were of analytical
grade.
2.5. Construction and analysis of protein models for TreS-T.C
Models were built through an online Automatic Modeling Mode
server at http://swissmodel.expasy.org. Obtained models were
analyzed through Swiss-Pdb Viewer and PyMOL.
2.2. TreS gene cloning and expression vector construction
Genomic DNA was extracted according to a previously described
method [36]. Based on the published coding DNA sequence of TreS
from T. curvata (GenBank accession no. YP 003301157), the primers
amplifying the TreS-T.C gene were designed with corresponding
restriction recognition sites (underlined) and His (6)-tag (bold
and italic bases). His-tagged primers were designed to facilitate
2
.6. Site-directed mutagenesis of TreS-T.C
Mutants were constructed through a fast cloning method [30]
with mutation primers containing mutation bases and 15–17 com-
plementary bases (forward and reverse primers) at the 5 -ends.
Linear mutation recombinant vectors were amplified by PCR with
PrimeSTAR HS DNA polymerase with TreS-T.C recombinant plas-
mids as parent DNA templates. Briefly, after gel confirmation of PCR
products, remaining unpurified PCR reactions were digested with
DpnI for 1 h at 37 C to remove the templates. The digested products
were then directly transformed into competent E. coli XL10-Gold
cells to obtain mutation recombinant plasmids.
ꢀ
one-step purification of resultant TreS-T.C. The sense primer was
ꢀ
ꢀ
5
3
-CTGAATTCATGCACCATCATCATCATCATCAGATGACCGGGGACC-
ꢀ
,
and the antisense primer was 5 -ACAAAGCTTTCACTT-
ꢀ
CGCCGTCTGCC-3 (EcoRI and HindIII restriction sites were under-
lined). The amplification conditions with LA Taq DNA polymerase
◦
◦
were as follows: 4 min at 94 C, followed by 30 cycles of 45 s at
◦
◦
◦
9
1
4 C, 45 s at 60 C, and 3 min at 72 C and a final extension of
0 min at 72 C. Polymerase chain reaction (PCR) products were
◦
analyzed using 0.8% agarose gel electrophoresis. PCR fragment
digested with corresponding restriction enzyme was inserted into
pSE380, which was linearized by the same enzymes. The fidelity of
inserted DNA sequence was confirmed by sequencing. Constructed
vectors were used to transform competent E. coli XL10-Gold cells.
3. Results
3.1. Purification and molecular mass analysis of TreS
TreS-T.C expressed in recombinant E. coli was isolated and puri-
fied. The result of SDS-PAGE analysis showed a single protein band
approximately 60 kDa (Fig. 1A, lane 3). On the other hand, active
enzymes had a molecular mass of approximately 180 kDa (Fig. 1B,
lane 4) according to native PAGE analysis, which suggested that
the native enzyme was a trimer of three identical 60 kDa subunits.
2
.3. Enzyme isolation, purification, and PAGE analysis
When the cell density (A600) of the incubated recombinant
cells
thiogalactopyranoside was added to a final concentration of
mM to induce gene expression. The cells were harvested by
centrifugation at 8000 × g for 10 min, washed twice with 100 mM
reached
approximately
0.6,
isopropyl-beta-d-
◦
1
The purified enzyme was stored at 4 C for more than one year in
imidazole elution buffer with no apparent loss in activity.

2
8
J. Liang et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
(
A) 110
100
90
80
70
60
5
6
7
8
pH
(
B) 110
Fig. 1. Purification and molecular mass analysis of TreS-T.C. (A) SDS-PAGE analysis
using 10% denaturing acrylamide separating gel and 4% stacking gel without SDS;
1
00
(
B) NATIVE-PAGE analysis using 5.0–12.5% native gradient acrylamide separating gel
and 4.0% stacking gel without SDS. Lanes M1 and M2 are protein molecular weight
markers. Lane 1 is the crude extract of the recombinant strain E. coli XL10-Gold
with pSE380. Lane 2 is the crude extract of the recombinant strain E. coli XL10-
Gold with pSE380-TreS-T.C. Lane 3 is the recombinant enzyme TreS-T.C purified
using nickel ion affinity chromatography. Lane 4 is the active purified recombinant
enzyme TreS-T.C.
9
0
80
70
60
50
3.2. Effects of pH, temperature, and metal ions on TreS-Sgris
activity
40
30
The optimum pH for TreS-T.C was 6.5. The enzyme maintained
high activity from pH 6.0 to 8.0 (Fig. 2A). The optimum tempera-
2
0
◦
ture was 35 C. Consequently, the enzyme maintained high activity
◦
◦
when reaction temperature ranged from 25 C to 50 C. However,
relative activity quickly decreased when temperature was above
5
10
◦ ◦
0 C or below 25 C (Fig. 2B). The effects of metal ions (KCl, FeCl ,
3
0
0
10
20
30
40
50
60
MnCl , CaCl , ZnCl , CuCl , BaCl , and FeCl ) were determined by
2
2
2
2
2
2
examining enzyme activity in the presence of 5 mM of these sub-
stances under optimum reaction conditions. Enzyme activity was
Temperature (ºC)
inhibited partly by Cu2+, Zn , and Fe (about 85% relative activ-
2+
2+
Fig. 2. Effects of pH and temperature on activity. (A) Reactions using 1% maltose as
◦
substrate (w/v) were assayed at 30 C with different pH values (5.5–8.0) for 30 min.
ity), whereas other metal ions had no significant effect on enzyme
activity (graphic ignored).
◦ ◦
(
B) Reactions were set at different temperatures, ranging from 5 C to 55 C to detect
the effects of temperature on enzyme activity with pH 6.5. Other reaction conditions
were the same as in (A). All enzyme activity was measured by monitoring the amount
of consumed substrate by HPLC. A 100% relative activity was denoted as the highest
conversion rate of maltose.
3.3. Substrate specificity of recombinant enzyme
3.4. Kinetic analysis of TreS-T.C
Different 1% sugars, including trehalose, lactose, sucrose,
cellobiose, isomaltose, isomaltulose, trehalulose, maltotriose,
maltotetrose, sorbitol, and dextrin, were used to detect the
substrate specificity of TreS-T.C under optimal pH and tem-
perature. As in the enzyme assay, a series of controls was
designed. Each reaction sample was analyzed by HPLC after
The results of the kinetics analysis on TreS-T.C are shown (see
Table 1). The Km value for maltose is approximately two degrees
Table 1
Kinetic analysis of TreS-T.C.
1
h or 5 h of incubation. TreS-T.C specifically catalyzed the
reversible conversion of maltose and trehalose and acted on
sucrose-catalyzed trehalulose formation without other disac-
charides, releasing small amounts of glucose and fructose as
by-products (Fig. 3). Other sugars were not catalyzed (graphic
ignored).
Substrate Km (mmol/l) Vmax a (U/mg) Product
Byproduct(s)
Maltose
Trehalose 198 ± 4
Sucrose
164 ± 5
96 ± 5
1549 ± 20
708 ± 30
911 ± 20
Trehalose
Maltose
Trehalulose Glucose and fructose
Glucose
Glucose
Note: Data represent the mean values and standard deviations of three independent
Finally, with sucrose as substrate, the optimum pH value, reac-
tion temperature, and influence of metal ions were consistent.
experiments.
a
Amount of substrates.

J. Liang et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
29
Fig. 3. HPLC results for maltose and sucrose production. Reaction samples were analyzed by HPLC after 1 h or 5 h incubation under other standard reaction conditions.
Sample concentration was diluted to approximately 0.1%, and 20 ␮l was loaded onto the column. (A) Standards for fructose (8.008), glucose (8.881), sucrose (12.596), maltose
(
14.624), trehalulose (15.534), and trehalose (16.197) shown as peaks; (B) peaks of reacted sample (maltose incubated 1 h) and of products [glucose (8.826), maltose (14.624),
and trehalose (116.166)]; (C) peaks of reacted sample (sucrose incubated 5 h) and of products [fructose (7.951), glucose (8.811), sucrose (12.596), and trehalulose (15.511)].
lower than those of trehalose and sucrose, indicating that TreS-T.C
preferred maltose as substrate instead of trehalose and sucrose.
Furthermore, under optimal reaction conditions, TreS-T.C has a rel-
atively higher catalytic rate with maltose as substrate compared
with trehalose and sucrose.
substrate into the catalytic center and as an export site of products.
Activity and substrate specificity were also directly related to the
central catalytic area of an enzyme [32–34]. Thus, each amino acid
was chosen to detect its function within 3.5 A˚ of active sites. These
amino acid residues included H120, D217, E259, H328, D329, E330,
L116, L32, L68, R408, N327, Y80, V117, M118, N119, T121, L216,
A218, V219, Y392, A260, N261, P220, P186, V411, R412, Y85, Q185,
E30, R215, and A258 (Fig. 5C).
3.5. Ranges of trehalose and trehalulose yields
TreS-T.C yield was different under different incubation times.
Maximum trehalose yield was 70% with glucose content of 8%
on maltose under optimum reaction conditions (Fig. 4). However,
glucose yield increased when the reaction was continued after
maximum trehalose yield was reached. Results were the same with
different substrate concentrations (from 2% to 80%), but not under
the same reaction times (data not shown). With sucrose as sub-
strate, the conversion rate into trehalulose was also independent of
substrate concentration. Maximum trehalulose yield from sucrose
exceeded 80% but required longer reaction time (Fig. 4B).
3
.7. Site-directed mutagenesis and saturation mutagenesis
Based on the opposite characteristics of amino acid, we designed
mutation primers to obtain various mutants, including H120A,
D217A, E259A, H328A, D329A, E330A, L116F, L32S, L68S, R408D,
N327S, Y80A, V117S, M118S, N119A, T121A, L216S, A218S, V219S,
Y392A, A260S, N261A, P220S, P186S, V411S, R412D, Y85A, Q185A,
E30H, R215D, and A258S.
◦
Enzyme assays were performed at pH 6.5 and 35 C with malt-
ose and sucrose as substrates. The results of enzymatic properties
showed that H120A, D217A, E259A, D329A, H328A, R408D, Y80A,
A218S, Y392A, P220S, P186S, R412D, N119A, N327S, V219S, Q185A,
E30H, R215D, and L68S had little to no enzyme activity on maltose
and sucrose compared with the wild type. Mutant L116Y showed
2.1-fold higher activity than the wild type on sucrose, but only
0.43-fold on maltose. On the other hand, mutant E330A only had
hydrolytic activity and no intermolecular transglycosylation activ-
ity on maltose and sucrose. Only 32% of mutations (V117S, M118S,
V411S, L32S, T121A, L216S, Y85A, A260S, N261A, and A258S) had
no significant effect on enzyme activity and substrate specificity.
Most amino acids near the active site significantly affected enzyme
activity or substrate specificity.
3
.6. Homology model building and structure analysis
Using trehalulose synthase catalyzing sucrose into trehalulose
as template (PDB ID: 2pwg), a TreS-T.C model was built through
SWISS-MODEL. The sequence identity of TreS-T.C and trehalulose
synthase was 29%, but both belonged to the GH13 family and had
the same substrate and product. The model and template had a
typical (␤/␣)8 barrel catalytic domain (Fig. 5A). Both trehalulose
synthase and TreS-T.C had five conserved key amino acids con-
stituting a catalytic pocket: H104, D200, E254, H326, and D327
[
31] and H120, D217, E259, H328, and D329, respectively. Three-
dimensional structures showed that all conserved amino acids
were in the center of the barrel catalytic domain (Fig. 5B). The cat-
alytic pocket had a deep groove on one side for binding substrates.
We speculated that the deep groove served as an entry point of
Given that site L116 significantly affects substrate specificity
and that E330 is vital to product formation, saturation mutagenesis
was performed. All mutants were purified to assay the enzymatic

3
0
J. Liang et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
4
. Discussion
(
A)
Glucose
Maltose
Trehalose
A trehalose synthase (TreS-T.C) from T. curvata was cloned and
80
expressed in E. coli XL10-Gold. TreS-T.C has some distinctive char-
acteristics: it exists in the form of a trimer of three identical 60 kDa
subunits, has a new reaction on sucrose, and is stored at 4 C for
70
◦
more than one year with no apparent loss of activity. In previous
papers, an active TreS purified from the cytosol of Mycobacterium
smegmatis is a hexamer of six identical 68 kDa subunits [23] and
from Thermobifida fusca is a tetramer of four identical 61 kDa sub-
units (data measured in the laboratory but not published). TreS has
a complex structure in active state. TreS from M. smegmatis [23]
and Thermus caldophilus GK24 [37] was also previously reported to
have unstable activity. However, TreS-T.C could be stored with very
6
0
50
40
◦
30
stable activity at 4 C for more than one year. Thus, TreS-T.C may
ideally be studied for its crystal structure.
Moreover, TreS-T.C had a new reaction: it could convert sub-
strate sucrose into only trehalulose without other disaccharides,
including isomaltulose, in contrast to other enzymes synthe-
sizing trehalulose, including trehalulose synthase, isomaltulose
synthase, and sucrose isomerase. All TreS reported previously could
specifically catalyze the reversible interconversion of maltose and
trehalose, but only a few acted on sucrose. A TreS obtained from
Thermus aquaticus also converted substrate sucrose into trehalu-
lose; however, the resulting by-products were glucose, fructose,
and palatinose, with only very low trehalulose-forming activity
[38]. As noted above, trehalulose synthase, isomaltulose synthase,
and sucrose isomerase also can convert sucrose into trehalulose
to produce glucose and fructose in residual amounts, but not on
maltose. However, the product was a mixture of trehalulose and
isomaltulose. Although TreS-T.C and those enzymes have some
similar characteristics, their amino acid sequence identities are
very low. The identities of TreS-T.C toward trehalose synthase
2
0
10
0
0
50
100
150
200
250
300
350
Reaction time (min)
(
B)
Sucrose
Fructose
Trehalulose
Glucose
80
70
(T. aquaticus), trehalulose synthase (Pseudomonas mesoacidophila
MX-45), isomaltulose synthase (Klebsiella sp. LX3), and sucrose
isomerase (Erwinia rhapontici) are 32%, 29%, 29%, and 28%, respec-
tively. These enzymes have some similar characteristics, but
protein molecules are extremely different. Their origin and evo-
lution need further exploration by researchers.
6
0
50
40
In this paper, maximum product yield from maltose and sucrose
was 70% and exceed 80%, respectively, under optimum conditions.
However, some TreS from other microorganisms have lower prod-
uct yield under optimum conditions, such as those from Picrophilus
torridus (61%) [26], Meiothermus rubber (47.2%) [39], Arthrobacter
aurescens (59.5%) [29], Thermus thermophilus (60%) [40], and Pseu-
domonas putida (∼45%) [27]. The formation of other minor products
also reduces the yield of major products about trehalulose syn-
thase, isomaltulose synthase, and sucrose isomerase. Indeed, the
product yield of TreS is associated with reaction temperature, pH,
and enzyme stability. According to the laws of thermodynamics
and the free energies of the hydrolysis of trehalose and maltose,
the calculation of equilibrium position between maltose and tre-
halose is allowed to be 82% in favor of trehalose [41]. Thus, all TreS,
given sufficient time, should give 82% trehalose under optimum
conditions.
3
0
20
10
0
0
5
10
15
20
25
30
Reaction time (h)
Fig. 4. Effects of reaction time on maximum product yield with maltose or sucrose
as substrate. Percentages of substrate and products in total sugars were monitored
by HPLC under different incubation times. (A) Maltose as substrate; (B) sucrose as
substrate. All data were average values from at least triplicates.
The crystal structure of TreS has not been obtained. Native and
carbohydrate complex structures of trehalulose synthase have been
studied to provide insight into sucrose isomerization [42]. Consid-
ering amino acid sequence identity with TreS-T.C, trehalulose
synthase is higher than isomaltulose synthase, sucrose isomerase,
and trehalose glycosyltransferring synthase (TreT) [43]. Similar to
all GH13 enzymes, they both use a two-step double-displacement
mechanism involving the formation and breakdown of a covalent
glucosyl-enzyme intermediate [44–46]. Thus, we chose trehalu-
lose synthase as the template of TreS-T.C to build a 3D model
using SWISS-MODEL. Five conserved amino acids (H120, D217,
properties. All 18 E330 mutants had only hydrolytic activity against
trehalose and sucrose. Compared with the wild type, almost all 18
L116 mutants had different catalytic activities and changes with
maltose or sucrose (Table 2). Although mutants L116G, L116K,
L116A, L116Y, and L116H had higher activity against sucrose, they
had lower activity against maltose. By contrast, the activity of
L116V, L116I, L116Q, L116T, L116M, and L116E decreased with
sucrose as substrate, but had minimal effect on maltose. Thus, these
two sites are important in determining substrate specificity and
formatting products.

J. Liang et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
31
Fig. 5. Comparative modeling of TreS-T.C based on known trehalulose synthase template. (A) Model of TreS-T.C (cyan) constructed using online SWISS-MODEL was super-
imposed with 2pwg (red). Five key amino acids (H104, D200, E254, H326, and D327) in the active center of 2pwg are indicated by blue marked sticks inside the (␤/␣)8 barrel
catalytic domain. (B) Side view of the surface model for TreS-T.C and its five conserved amino acids in the active center. The key amino acids are labeled with sticks and name
of residue. (C) All amino acids residues within 3.5 A˚ of the active sites for TreS-T.C. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of the article.)
E259, H328, and D329) that possibly construct the catalytic pocket
structure were decided and done in directed mutagenesis. The
mutants H120A, D217A, E259A, D329A, and H328A had little or
no enzyme activity against maltose and sucrose compared with
the wild type. Thus, the five conserved amino acids significantly
affect the catalytic activity of TreS-T.C. Furthermore, several con-
served amino acid residues and their functions have already been
identified about some GH13 enzymes. Five amino acid residues,
namely, H133 (not mentioned in paper), D230, E272, H341, and
D342, are highly conserved. D230 is a catalytic nucleophilic residue,
E272 is a general acid/base catalyst, and H341 and D342 are con-
served carboxylic acids [47]. The same conserved amino acids
were found on oligo-1,6-glucosidase from Bacillus cereus (Asp199,
Glu255, Asp329, His103, and His328) [48], amylosucrase from Neis-
seria polysacchareais (Asp286, Glu328, Asp393, His187, and His392)
[49], trehalulose synthase from P. mesoacidophila MX-45 (Asp200,
Table 2
Summary of saturation mutagenesis results.
Variants
Relative activity (%)
Sucrose
Variants
Relative activity (%)
Sucrose
Maltose
Maltose
L116
100.00
100.00
L116P
L116K
L116F
L116S
L116M
L116R
L116E
L116H
L116A
L116D
77.20 ± 0.34
180.00 ± 1.55
130.00 ± 1.04
110.32 ± 1.00
77.80 ± 0.09
0.00
73.56 ± 0.38
251.85 ± 1.99
256.05 ± 2.86
50.65 ± 0.04
81.11 ± 0.07
86.72 ± 0.05
83.98 ± 0.06
39.71 ± 0.04
118.20 ± 0.96
22.71 ± 0.03
101.74 ± 0.34
89.43 ± 0.17
21.48 ± 0.02
42.61 ± 0.02
L116G
L116V
L116W
L116I
L116Q
L116T
L116N
L116C
L116Y
143.54 ± 2.60
0.91 ± 0.06
58.27 ± 0.67
1.78 ± 0.02
14.53 ± 0.12
18.22 ± 0.18
115.51 ± 1.15
156.64 ± 1.50
210.24 ± 2.26
16.77 ± 0.16
63.01 ± 0.45
52.37 ± 0.07
75.60 ± 0.11
75.60 ± 0.20
75.12 ± 0.13
91.22 ± 0.30
92.44 ± 0.22
43.15 ± 0.09
Note: Data represent the mean values and standard deviations of three independent experiments.

3
2
J. Liang et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 26–32
Glu254, Asp327, His104, and His326) [42]. These amino acids are
vital for the catalytic center of enzymes. When mutation range was
expanded near the active center, site (L116) determined substrate
specificity, and another site (E330) affected product formation. The
spatial distance of L116 toward H120 and D217 is very close. A
hydrogen bond also exists between L116 and D217 (Fig. 5C). Sup-
posedly, H120 is a substrate binding site, and D217 is a nucleophilic
reagent. Therefore, the site of L116 would directly affect activ-
ity and substrate specificity, which was also evidenced by the
experimental results. On the other hand, E330 mutants affect tre-
halose formation. These possibly interfere with the second step of
the two-step double-displacement mechanism, glucose molecular
rearrangement within the enzyme molecule. These findings may
provide a theoretical basis for studying catalytic mechanism and
the transformation of industrial enzymes on TreS.
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