Construction of a recombinant thermostable β-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion of starch to trehalose

Article abstract of DOI:10.1021/jf062355t

A fusion gene that encoded a polypeptide of 1495 amino acids was constructed from the β-amylase (BA) gene of Clostridium thermosulfurogenes and trehalose synthase (TS) gene of Thermus thermophilus. The fused gene was overexpressed in Escherichia coli, and a recombinant bifunctional fusion protein with BA at the N-terminal (BATS) or C-terminal (TSBA) of TS having both β-amylase and trehalose synthase activities with an apparent molecular mass of 164 kDa was obtained. BATS or TSBA catalyzes the sequential reaction in which maltose is formed from starch and then is converted into trehalose. The K_m values of the BATS and TSBA fusion enzymes for the reaction from starch to trehalose were smaller than those of an equimolar mixture of BA and TS (BA/TS). On the other hand, the k_cat value of BATS approximated that of the BA/TS mixture, but that of TSBA exceeded it. TSBA showed much higher sequential catalytic efficiency than the separately expressed BA/TS mixture. The catalytic efficiency of TSBA or BATS was 3.4 or 2.4 times higher, respectively, than that of a mixture of individual enzymes, showing the kinetic advantage of the fusion enzyme. The thermal stability readings of the recombinant fusion enzymes BATS and TSBA were better than that of the mixture of individual recombinant enzymes. These results apparently demonstrate that fusion enzymes catalyzing sequential reactions have kinetic advantages over a mixture of both enzymes.

Full text of DOI:10.1021/jf062355t

1256 J. Agric. Food Chem. 2007, 55, 1256−1263
Construction of a Recombinant Thermostable
-Amylase-Trehalose Synthase Bifunctional Enzyme for
Facilitating the Conversion of Starch to Trehalose
â
JIA-HUNG WANG,†,‡ MENG-YIN TSAI, GUAN-CHIUN LEE,§ AND JEI-FU SHAW*,†,‡,|
‡
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 202, Institute
of Plant and Microbial Biology, Academia Sinica, Taipei, 115, Department of Biotechnology, Ming
Chuan University, Taoyuan, 333, and Department of Food Science and Biotechnology, National
Chung Hsing University, Taichung, 402, Taiwan
A fusion gene that encoded a polypeptide of 1495 amino acids was constructed from the â-amylase
(
BA) gene of Clostridium thermosulfurogenes and trehalose synthase (TS) gene of Thermus
thermophilus. The fused gene was overexpressed in Escherichia coli, and a recombinant bifunctional
fusion protein with BA at the N-terminal (BATS) or C-terminal (TSBA) of TS having both â-amylase
and trehalose synthase activities with an apparent molecular mass of 164 kDa was obtained. BATS
or TSBA catalyzes the sequential reaction in which maltose is formed from starch and then is converted
m
into trehalose. The K values of the BATS and TSBA fusion enzymes for the reaction from starch to
trehalose were smaller than those of an equimolar mixture of BA and TS (BA/TS). On the other
hand, the kcat value of BATS approximated that of the BA/TS mixture, but that of TSBA exceeded it.
TSBA showed much higher sequential catalytic efficiency than the separately expressed BA/TS
mixture. The catalytic efficiency of TSBA or BATS was 3.4 or 2.4 times higher, respectively, than
that of a mixture of individual enzymes, showing the kinetic advantage of the fusion enzyme. The
thermal stability readings of the recombinant fusion enzymes BATS and TSBA were better than that
of the mixture of individual recombinant enzymes. These results apparently demonstrate that fusion
enzymes catalyzing sequential reactions have kinetic advantages over a mixture of both enzymes.
KEYWORDS: Trehalose; Thermus thermophilus trehalose synthase; bifunctional enzyme; starch
INTRODUCTION
Recently, trehalose productions through fermentation of yeast
(
12) and Corynebacterium (25), through extraction from trans-
Trehalose (R-D-glucopyranosyl-1,1-R-D-glucopyranoside) is
a nonreducing disaccharide widely distributed in various organ-
isms, including bacteria, algae, fungi, yeasts, insects, and in
some plant and mammalian tissues (1-8). This compound may
serve as an energy reservoir, a protectant from a variety of
physical and chemical stresses, and a protein stabilizer in living
organisms (1, 9-14). It is an essential component for maintain-
ing cell viability. The absence of reducing ends in trehalose
makes it highly resistant to heat, pH, and Maillard’s reaction
formed plants (28), and through enzymatic processes from starch
26, 27) and maltose (15, 19) have been reported. Up to now,
(
several trehalose-synthesizing enzyme systems have been
reported in microorganisms (25, 29-32). Trehalose can be
metabolized in several ways, which invole different enzyme
systems including trehalose-6-phosphate synthetase and treha-
lose-6-phosphate phosphatase (27, 33, 34), trehalose synthase
(TS) (31, 35), maltose phosphorylase and trehalose phospho-
rylase (31), glycosyltransferase (36), and maltooligosyltrehalose
trehalohydrolase and maltooligosyltrehalose synthase (25). TS
(15-18). In addition, trehalose can protect proteins against
denaturation because of desiccation and freezing (19, 20), and
its water-holding capability makes its application possible in
additives, stabilizers, and sweeteners that are quite useful in
the food, cosmetic, and pharmaceutical industries (21-24). If
trehalose can be produced economically, it should have even
more potential commercial applications.
(E.C. 5.4.99.16), which converts maltose into trehalose in the
absence of coenzyme, has been identified in Thermus strains
and other microorganisms (31, 35).
Establishment of recombinant multifunctional fusion enzyme
has great potential in the protein engineering of enzymes (37,
3
8). Fusion of structural genes encoding enzymes that catalyze
sequential reactions has several advantages, such as simple
expression of a single recombinant unit containing multiple
genes and one-step purification of recombinant proteins. When
two enzymes are present in a complex, their physical proximity
catalyzing sequential reactions might increase the reaction rate
*
To whom correspondence should be addressed. Tel: +886-4-22840201.
Fax: +886-4-22853813. E-mail: boplshaw@gate.sinica.edu.tw.
†
National Taiwan Ocean University.
‡
Academia Sinica.
§
Ming Chuan University.
|
National Chung Hsing University.
1
0.1021/jf062355t CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/27/2007

Construction of Recombinant
â
-Amylase-Trehalose Synthase
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1257
by facilitating transfer of the reaction intermediate to the
catalytic sites of the next enzymes.
(5′-CCGGAATTCTTAGGATCTAGATCTGCCGAATTAATGAG-
CATAGCACCAAATTTCAAAG-3′) and PBA4 (5′-CCGCTCGAGT-
TAATTTTGCCATGTAATGGTGAC-3′), digested with EcoRI and
XhoI, and then ligated into EcoRI- and SalI-digested pET-TSd. This
plasmid, named pET-TSBA, was transformed into E. coli Tuner(DE3)-
pLysS.
In this study, we have successfully constructed a DNA
fragment that contains two genes encoding TS from Thermus
thermophilus and â-amylase (BA) (E.C.3.2.1.2) from Clostrid-
ium thermosulphurogenes (39-41), and this gene was expressed
in Escherichia coli. The recombinant bifunctional fusion
enzymes, TSBA and BATS, catalyze the sequential reactions
for the production of trehalose from starch, an abundant and
cheap raw material, more efficiently than a combination of
individual native enzymes.
Expression of Recombinant Enzymes. The E. coli strains Tuner-
(DE3)pLysS (Novagen) were used for expression studies. To express
the recombinant enzymes, transformed Tuner(DE3)pLysS was inocu-
lated into 6 L of Luria-Bertani (LB) medium supplemented with 100
µg/mL ampicillin and 34 µg/mL chloramphenicol in combination and
then grown at 37 °C. When the A590 reached 0.8, IPTG was added to
a final concentration of 1.0 mM. Incubation was continued for another
1
2 h at 20 °C, and the cells were harvested. The cell pellet was
MATERIALS AND METHODS
resuspended in 100 mL of lysis buffer (50 mM sodium phosphate buffer,
pH 7.0, 1 mM PMSF, and 2 mM EDTA), and cells were disrupted
with a French pressure cell press (Thermo Spectronic) for two cycles.
The soluble fractions of the lysed cells were collected using 30 min of
centrifugation at 15000g and 4 °C. The soluble protein fractions were
concentrated by ultrafiltration using a 30000 molecular weight cutoff
membrane (Millipore). The crude extracts of the recombinant enzymes
were harvested for purification.
Materials. All saccharides including maltose, trehalose, and starch
were purchased from Sigma. Protein assay reagents and dyes were from
Bio-Rad Lab. Columns for protein separation were obtained from
Pharmacia Biotech. Other chemicals and reagents were of analytical
grade.
Bacterial Strains and Cultivation. T. thermophilus and C. ther-
mosulfurogenes were obtained from the American Type Culture
Collection (ATCC). T. thermophilus ATCC 33923 was grown in 1260
Thermus B-P (ATCC medium), pH 7.0, at 70 °C. C. thermosulfurogenes
ATCC 33743 was grown in 1398 modified low phosphate-buffered
basal medium (ATCC medium), pH 7.3, at 60 °C under anaerobic
conditions.
Construction of Recombinant Expression Plasmids. Genes encod-
ing BA and TS were cloned from C. thermosulfurogenes and T.
thermophilus genomic DNA, respectively. The ORF of BA is 1563 bp
long and encodes 520 amino acids. The ORF of TS is 2898 bp long
and encodes 965 amino acid residues. The estimated molecular masses
of the two polypeptides are 57 and 106 kDa, respectively.
The BA gene was obtained from C. thermosulfurogenes genomic
DNA by the Pfu polymerase chain reaction (PCR) with the primers
Purification of Recombinant Enzymes. The crude extracts of the
recombinant enzymes were purified by the following steps. To purify
the recombinant TSase, solid (NH ) SO was added to the cell-free
4
2
4
extract to 70% saturation. The resulting precipitate was collected and
dissolved in 50 mM sodium phosphate buffer (pH 7.0) and then dialyzed
against the same buffer. The dialyzed solution was put on a DEAE-
Sepharose column (160mm × 10 mm, Pharmacia Biotech) equilibrated
with 50 mM sodium phosphate buffer (pH 7.0), and the adsorbed
enzyme was eluted with a linear gradient of 0-1 M NaCl in the same
buffer. The active fractions were pooled. Solid (NH ) SO was added
4
2
4
into the enzyme solution to reach a final concentration of 1 M. The
mixture was put on a Butyl-Sepharose column (160 mm × 10 mm)
equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing
P
BA1 (5′-TGAGCATAGCACCAAATTTCAAAG-3′) and PBA2 (5′-
1 M (NH
of 1 to 0 M (NH
4
)
2
SO4. The adsorbed enzyme was eluted with a linear gradient
SO in the same buffer. The active fractions were
CCGGAATTCTTAATTTTGCCATGTAATGGTGAC-3′), which con-
tain the translation initiation and termination codons (bold) of the C.
thermosulfurogene BA gene, respectively. NdeI and EcoRI sites
4
)
2
4
pooled and then dialyzed against 50 mM sodium phosphate buffer (pH
7.0) containing 0.1 M NaCl. The dialyzed solution was put on a Mono
Q-Sepharose column (160 mm × 10 mm) equilibrated with 50 mM
sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl. The adsorbed
enzyme was eluted with a linear gradient of 0.1-0.5 M NaCl in the
same buffer. The active fractions were pooled. The enzyme solution
was concentrated with ultrafiltration using a 50000 molecular weight
cutoff membrane (Amicon Ultra, Millipore) and then pooled as a
purified enzyme preparation.
(underlined) were introduced into PBA1 and PBA2, respectively. The
amplified DNA was ligated into NdeI- and EcoRI-digested pET-20b-
(+) (Novagen) to generate pET-BA.
The TS gene was obtained from T. thermophilus genomic DNA by
the Pfu PCR with the primers PTS1 and PTS2, which contain the
translation initiation and termination codons (bold) of the T. thermo-
philus TS gene, respectively. NdeI and SalI restriction enzyme sites
(
underlined) were introduced into PTS1 (5′-GGAATTCCATATGGAC-
The recombinant BA, BATS, and TSBA were purified by the method
of raw starch adsorption and desorption (42, 43). Solid (NH ) SO was
CCCCTCTGGTACAAGG-3′) and PTS2 (5′-GCCGACGTCGACCTAG-
GCTTTTCCGGCCTTGG-3′), respectively. The amplified DNA was
ligated into NdeI- and SalI-digested pET-20b(+) to produce pET-TS.
To make an expression construct for the BATS fusion enzyme, the
BA gene was amplified in the same manner with the primers PBA1 and
4
2
4
added to the cell-free extract to 70% saturation. The resulting precipitate
was collected and dissolved in 50 mM sodium phosphate buffer (pH
7.0) and then dialyzed against the same buffer. The dialyzed solution
was added to the raw starch, which was washed with 50 mM acetate
buffer (pH 6.0) at 4 °C, and the mixture was kept for 20 min with
occasional shaking. After centrifugation (5 min, 10000g), the residue
was washed twice with the same buffer. It was then suspended in the
elution buffer containing boiled soluble starch in 50 mM acetate buffer
P
P
BA5 (5′-CCGGAATTCATTTTGCCATGTAATGGTG-3′). The primer
BA5 does not contain the termination codon of the C. thermosulfurogene
BA gene. NdeI and EcoRI restriction enzyme sites (underlined) were
introduced into PBA1 and PBA5, respectively. The amplified DNA was
ligated into NdeI- and EcoRI-digested pET-20b(+) to produce pET-
BAd. The TS fragment was amplified with the primers PTS4 (5′-
CCGGAATTCTTAGGATCTAGATCTGCCGAATTAATGGACCC-
CCTCTGGTACAAGGACG-3′) and PTS2 and digested with EcoRI and
SalI , then ligated into EcoRI- and SalI-digested pET-BAd. This
plasmid, named pET-BATS, was transformed into E. coli Tuner(DE3)-
pLysS.
(pH 6.0) with 5 mM CaCl , shaken occasionally for 30 min, and
2
centrifuged. The supernatant solution containing recombinant enzyme
was pooled and dialyzed against 50 mM sodium phosphate buffer (pH
7.0). The dialyzed solution was put on a DEAE-Sepharose column (160
mm × 16 mm) equilibrated with 50 mM sodium phosphate buffer (pH
7.0), and the adsorbed enzyme was eluted with a linear gradient of
0-1 M NaCl in the same buffer. The active fractions were pooled.
To make an expression construct for the TSBA fusion enzyme, the
TS gene was amplified in the same manner with the primers PTS1 and
4 2 4
Solid (NH ) SO was added into the enzyme solution to a final
concentration of 1 M. The mixture was put on a Butyl-Sepharose
P
TS3 (5′-CCGGAATTCGGCTTTTCCGGCCTTGGCCTGC-3′). The
column (160 mm × 10 mm) equilibrated with 50 mM sodium phosphate
primer PTS3 does not contain the termination codon of the T. aquaticus
TS gene. NdeI and EcoRI restriction enzyme sites (underlined) were
introduced into PTS1 and PTS3, respectively. The amplified DNA was
ligated into NdeI- and EcoRI-digested pET-20b(+) to produce pET-
TSd. The BA fragment was amplified with the primers PBA3
4 2
buffer (pH 7.0) containing 1 M (NH ) SO4. The adsorbed enzyme was
4 2 4
eluted with a linear gradient of 1 to 0 M (NH ) SO in the same buffer.
The active fractions were pooled and then dialyzed against 50 mM
sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl. The dialyzed
solution was concentrated with ultrafiltration using a 30000 or 50000

1258 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Wang et al.
Figure 1. Expression cassettes of the enzymes BA, TS, BATS, and TSBA.
The recombinant DNAs for the BA, TS, BATS, and TSBA were inserted
into pET20b(+) and then transformed into Tuner(DE3)pLysS to express
targets. The linker between BA and TS encodes sequence EFLGSRSAEL.
The manners are described in the Materials and Methods.
molecular weight cutoff membrane (Amicon Ultra, Millipore) and then
pooled as a purified enzyme preparation.
Protein Assay. The molecular masses of the purified recombinant
proteins were determined in denaturing conditions by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electro-
phoresis was performed with a 12% separating gel. Proteins were
visualized by staining with Coomassie Brilliant Blue R-250 according
to standard procedures. Proteins were measured by the method of
Bradford using a protein assay kit from Bio-Rad Lab with bovine serum
albumin as the standard.
Figure 2. SDS-PAGE of the purified proteins BA, TS, BATS, and TSBA.
The recombinant enzymes were purified by a succession of steps as
described in the Materials and Methods. The purified proteins were
analyzed by SDS-12% PAGE and stained with Coomassie brilliant blue.
Lane M, protein size markers; lanes 1−4, 3 µg of purified BA, TS, BATS,
Enzyme Characterization. The activity of TS was assayed by
measuring trehalose produced from maltose. A reaction mixture
consisting of 50 mM sodium phosphate buffer (pH 6.0), 50 mM maltose,
and the enzyme in a final volume of 100 µL was incubated at 60 °C
for 2 h. The activity of BA was assayed by measuring maltose produced
from soluble starch. A reaction mixture consisting of 50 mM sodium
phosphate buffer (pH 6.0), 2% soluble starch, and the enzyme in a
final volume of 100 µL was incubated at 60 °C for 2 h. The activities
of fusion enzymes were assayed by measuring trehalose produced from
soluble starch, in the same reaction buffer and condition as used for
the BA assay. Then, this reaction mixture was heated at 100 °C for 10
min to stop the reaction. The trehalose or maltose produced was
measured by a high-performance liquid chromatography (HPLC)
system, as described below. One unit (U) of enzyme activity was
defined as the amount of enzyme that catalyzes the formation of 1
µmol of trehalose per min.
Purified enzymes were used for kinetic study with different substrates
at pH 6.0 and 60 °C. The enzyme activity was assayed with maltose
or soluble starch as substrate, respectively, and determined by the
production of trehalose as already mentioned. Then, the values of kinetic
parameters and standard deviations were calculated from each triplicate
sample with three independent experiments by nonlinear regression plots
of the Michaelis-Menten equation.
and TSBA, respectively. The numbers on the left indicate the sizes of
the markers (in kDa).
The estimated molecular masses of the recombinant BA, TS,
BATS, and TSBA proteins were about 57, 106, 164, and 164
kDa, respectively. The sizes of the proteins observed on the
gel were considered with those calculated from the deduced
amino acid sequence (40, 41).
Recombinant Enzyme-Catalyzed Trehalose Synthesis.
Enzymatic activities of the purified recombinant BA, TS, BATS,
and TSBA proteins were tested using maltose or starch as
substrates. As shown in Figure 3f,g, both BATS and TSBA
produced trehalose from starch. This demonstrated that BATS
and TSBA were functional and able to catalyze two sequential
reactions involving conversion from starch to maltose and then
to trehalose. This result is consistent with the activity of the
individual recombinant enzyme-catalyzed reactions. For ex-
ample, the recombinant BA catalyzed the hydrolysis of starch
to maltose, as shown in Figure 3c, and the recombinant TS
catalyzed the synthesis of trehalose from maltose (Figure 3d).
An equimolar mixture of these two enzymes produced trehalose
from starch by catalyzing the sequential reactions (Figure 3e).
The reactions catalyzed by the two enzymes are depicted in
Figure 4. The rates of trehalose synthesis from starch by the
BATS and TSBA fusion enzymes were at least 22 and 36%
faster than that by an equimolar mixture of the individual
enzymes (BA/TS), respectively (Figure 5).
Analysis of Carbohydrate. Quantitative analysis of sugar was
carried out by a HPLC system equipped with refractive index detector
(RID) and computerized processing unit (Chrom Manager multi-system,
Version 5.2, Taiwan). The columns used include an APS2 analytical
column (ThermoHypersil, 250 mm × 4.6 mm) and a guard column
(
ThermoHypersil, 10 mm × 4.6 mm). The solvent containing aceto-
nitrile/water/formic acid (80:20:1) was used as a mobile phase with a
flow rate of 1.0 mL/min.
Biochemical Properties of the Bifunctional Fusion En-
zymes TSBA and BATS. Biochemical parameters of the
recombinant TSBA and BATS were compared. The effect of
temperature on the enzyme activity of trehalose formation was
determined using starch as substrate at various temperatures
ranging from 10 to 90 °C. As shown in Figure 6, the optimal
temperature for all of the BATS, TSBA fusion enzyme, and
the BA/TS mixture was 70 °C (Figure 6a). Their temperature
dependence showed little difference even if the absolute
activities of trehalose synthesis of BATS, TSBA, and BA/TS
were different (data not shown). To determine differences in
their stability against thermal denaturation, BATS, TSBA, and
the equimolar mixture BA/TS were all incubated at various
temperatures ranged from 10 to 90 °C for 30 min and cooled
immediately (Figure 6b). At temperatures of 70 °C, both BATS
RESULTS
Expression and Purification of the Bifunctional Fusion
Enzymes BATS and TSBA. For the expression of the
bifunctional fusion enzyme, genes encoding BA and TS were
fused with a linker and expressed in E. coli. In the BATS fusion,
the C terminus of BA was fused with the N terminus of TS in
frame while the TSBA fusion enzyme was inverted, as shown
in Figure 1. The linker was designed to expedite the recom-
binant DNA construction without deleting any amino acids of
either protein moiety. This resulted in the insertion of 10 amino
acids, EFLGSRSAEL, during construction.
Recombinant enzymes produced in E. coli were purified to
homogeneity as determined by SDS-PAGE analysis (Figure 2).

Construction of Recombinant
â
-Amylase-Trehalose Synthase
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1259
Figure 3. Analysis of the products of the enzymatic reaction by HPLC.
The purified recombinant enzymes were incubated for 2 h at 60 C in 50
L of assay buffer containing 50 mM maltose or 2% soluble starch as
substrates. (a) A 12.5 mM concentration of each standard glucose,
maltose, and trehalose; (b) 2% of standard starch; (c) reaction product
of BA with 2% starch; (d) reaction product of TS with 50 mM maltose;
Figure 4. Overview of trehalose synthesis from starch by the fusion
enzyme BATS or TSBA. BA catalyzes the hydrolysis of starch to maltose,
which is further converted by TS to produce trehalose. The fusion protein,
BATS or TSBA, consisting of two individual enzymes, catalyzes the
sequential reactions.
°
µ
and (e−g) reaction products of BA/TS mixture, BATS, and TSBA with
2% starch, respectively.
and TSBA retained about 95% of their original activities, which
were obtained after incubation for 2 h at 60 °C, but the residual
activity of BA/TS was significantly reduced. The thermosta-
bilities of the recombinant fusion proteins were better than the
individual recombinant enzymes.
For the pH test, enzyme activities at various pH values were
assayed. As shown in Figure 7, the optimal pH was 6.0 for the
BATS, TSBA fusion enzymes, and the BA/TS mixture (Figure
7a). To examine pH stability, enzymes were incubated at various
pH values, and the residual activities were measured at pH 7.0.
All enzymes were stable from pH 5.0 to 7.0, as shown in Figure
7
b. The effects of metal ions and chemical reagents on the
activity are shown in Table 1. The enzyme activity of BATS
Figure 5. Time course for trehalose production from starch by the
2+
2+
2+
2+
or TSBA was inhibited by Cu , Hg , Pb , Zn , and Tris. It
recombinant enzymes. Ten picomoles of TSBA (
b
°
), BATS (
C in a final 70
L) were withdrawn
[
), or the
2+
3+
2+
was not significantly inhibited by Ca , Fe , Mg , DTT, or
EDTA.
BA/TS equimolar mixture ( ) was incubated at 60
4
µ
L of
assay buffer containing 2% soluble starch. Aliquots (10
at the indicated times and quantified by HPLC.
µ
Kinetic Properties of the Bifunctional Fusion Enzymes
BATS and TSBA. To depict the enzymatic properties of the
bifunctional fusion enzymes BATS and TSBA, the kinetic
parameters were determined as shown in Table 2. These values
were obtained using the Michaelis-Menten equation with Hyper
Software (Synergy Software). The kinetic parameters for the
overall synthesis of trehalose from starch were determined.
These results were compared with those of a parallel assay with
an equimolar mixture of BA/TS. The Km values of BATS and
TSBA for starch were 59 and 61%, respectively, lower than
those of BA/TS. The kcat value of TSBA for starch was 31%
higher than that of BA/TS, but the kcat value of BATS for starch

1260 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Wang et al.
Figure 6. Effects of temperature on the activity and stability of TSBA,
BATS, and the BA/TS equimolar mixture with starch as the substrate. (a)
The enzyme activities at various temperatures (10
b) To examine the thermal stability, the enzymes were incubated at
various temperatures (10 90 C) for 30 min and were immediately cooled.
The residual activities were measured at 60
BA/TS equimolar mixture are symboled in
−90 °C) were assayed.
(
Figure 7. Effects of pH on the activity and stability of TSBA, BATS, and
the BA/TS equimolar mixture with starch as substrate. Fifty millimolar
−
°
°
C. TSBA, BATS, and the
, and , respectively.
concentrations of acetate buffer (pH 4.0
.5), and NaHCO Na CO buffer (pH 8.5
enzyme activities at various pH values (pH 4.0
To examine pH stability, the enzyme was incubated at various pH values
pH 4.0 10.0) for 30 min at 60 C. The residual activities were measured
at pH 7.0. TSBA, BATS, and the BA/TS equimolar mixture are symboled
in , and , respectively.
−
6.5), phosphate buffer (pH 6.0
10.0) were used. (a) The
10.0) were assayed. (b)
−
b
,
[
4
8
3
−
2
3
−
−
was similar to that of BA/TS. The catalytic efficiencies (kcat/
Km) of the bifunctional fusion enzymes (BATS and TSBA) were
(
−
°
2.39 and 3.38 times greater than that of BA/TS, respectively.
The kinetic parameters for the TS activity were also measured
b,
[
4
at various concentrations of maltose. The catalytic efficiencies
of TS and BATS were similar because they had the similar Km
and kcat values. This suggested that the fusion pattern of BATS
did not affect the kinetic properties of the TS activity. Interest-
ingly, although the Km value of TS was similar to that of TSBA,
the kcat value of TSBA was 31% higher than that of TS. The
catalytic efficiency of TSBA for TS activity was 1.42 times
greater than that of TS, implying that the fusion pattern of TSBA
influenced the kinetic properties of the TS activity.
Table 1. Effects of Metal Ions and Chemical Reagents on the Activity
of the Fusion Enzymes BATS and TSBA with Starch as Substrate
a
relative activity (%)
BATS TSBA
effector
none
concentration (mM)
100
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.01
100
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.01
AlCl
3
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
10.0
88
96
82
16
92
22
100
86
68
54
0.08
0.21
0.01
0.06
0.03
0.11
0.38
0.68
0.12
0.26
0.61
0.08
0.18
0.09
90
98
82
14
96
28
102
81
70
49
44
99
98
28
0.12
0.18
0.22
0.02
0.16
0.09
1.78
0.68
0.56
0.02
0.14
0.28
0.46
0.21
CaCl
CoCl
2
2
CuSO
FeCl
HgCl
4
DISCUSSION
3
2
In the present work, we have successfully created a bifunc-
tional fusion enzyme for efficient production of trehalose from
starch at high temperature. First, we constructed the fusion
enzymes, BATS and TSBA, by in frame fusion of structural
genes for BA and TS (Figure 1) and overexpressed them in E.
coli. Then, the capabilities of purified fusion proteins to produce
trehalose at high temperatures were analyzed. Both fusion
proteins for trehalose synthesis from starch were more efficient
than an equimolar mixture of the two individual enzymes
MgCl
MnCl
2
2
NiCl
PbCl
2
2
ZnSO
DTT
4
41
100
96
EDTA
Tris
26
a
The enzyme activity was measured in the presence of metal ions and chemical
(
Figure 5).
reagents under the standard assay conditions. The relative activity was expressed
as a percentage of the enzyme activity in the absence of effectors. Data are the
mean values and standard deviations of three independent experiments.
Kinetic parameters of the fusion enzymes BATS and TSBA
were compared with those of a parallel assay with an equimolar
mixture of BA/TS (Table 2). The catalytic efficiency, kcat/Km,
of BATS and TSBA for both sequential reactions was 2.39 and
proximity effect. The proximity of enzymes allows the transfer
of a reaction intermediate directly to the active site of the next
enzyme in sequential reactions, and consequently, the overall
reaction rate of the whole enzymatic process could be increased
by preventing serious diffusion of intermediate (37, 38). It is
3
.38 times the BA/TS mixture, respectively. The BATS catalytic
efficiency was higher than that of BA/TS due to the reduced
Km value and an unchanged kcat value. The improved catalytic
activities of the BATS fusion enzymes might be due to a

Construction of Recombinant
â
-Amylase-Trehalose Synthase
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1261
Table 2. Kinetic Parameters of Recombinant Enzymes TS, TSBA, BATS, and BA/TS with Different Substratesa
substrate
starchb
maltose
cat (S-1)
kcat/K
(%- S-1)
1
K
m
(mM)
k
cat (S-1)
k
cat/K
m
(mM- S-
1
1
×
10-3)
enzyme
K
m
(%)
k
m
TS
37.63
35.36
38.46
±
±
±
3.89
2.96
0.86
10.74
14.40
11.07
±
±
±
1.02
2.88
1.06
285.40
407.20
287.70
±
±
±
18.98
38.12
16.89
TSBA
BATS
BA/TS
3.66
3.89
9.49
±
±
±
0.12
0.28
0.89
33.30
25.02
25.49
±
±
±
2.66
1.26
2.04
9.10
6.44
2.69
±
±
±
1.08
0.68
0.06
a
Data are the mean values and standard deviations of three independent experiments. b Parameters for overall sequential reaction from starch to trehalose.
Figure 8. Overview of trehalose synthesis by TSBA, BATS, and the BA/TS equimolar mixture. (a) The formation of trehalose from maltose, which is
related to intramolecular transglycosylation. TS converts maltose into trehalose in a reversible reaction, together with a small proportion of irreversibly
formed glucose. (b) TSBA, BATS, and the BA/TS all had a weak hydrolytic activity, which increases at higher temperatures. In the trehalose synthesis
reaction of TSBA, the glucose product was evidently reduced, and trehalose increased. These reactions were incubated for 24 h at 60
containing 2% soluble starch as substrate, and the reaction products were analyzed by HPLC.
°C in assay buffer
interesting to note that the catalytic efficiency of TSBA with
starch as substrate was 1.41 times that of BATS due to the
increased kcat value. The catalytic efficiency of TSBA with
maltose as substrate for TS activity was also 1.43 times greater
than that of TS due to the increased kcat value (Table 2).
Therefore, in addition to the proximity effect, the higher catalytic
efficiency of TSBA over BATS might be due to the conforma-
tion change of TS in the TSBA construct, resulting in further
improvement of kcat and catalytic efficiency. Further analyses
showed that the increase of TSBA catalytic efficiency over
BATS is due to the reduction of glucose formation (Figure 8b).
In another aspect, the yield of trehalose from maltose by TS
decreased with increasing reaction temperature. The reason
appears to be that TS has a weak maltose hydrolytic activity
reactions that cleave the R-(1-4)-linkage in maltose and form
the (1-1)-linkage in the nonreducing disaccharide trehalose. It
also readily converts maltose into trehalose in a reversible
reaction, together with a small proportion of irreversibly formed
glucose. In this study, glucose of the product was evidently
reduced, and trehalose increased in the trehalose synthesis
reaction of TSBA, in which the C terminus of TS was fused
with the BA in frame, as shown in Figure 8b. We suggest that
the TSBA configuration, which might have a more rigid
structure that was formed by BA fusing the C terminus of TS,
might decrease the minor glucose formation side reaction and
enhance efficiency of the major trehalose formation reaction.
Thermal stability experiments suggested no great differences
in the structures of each enzyme moiety in the fusion enzyme
and the individual enzymes even though BATS and TSBA were
slightly more stable than the BA/TS mixture. The molecular
(Figure 8a) (41), which increases at higher temperatures (44).
The thermostable TS from T. thermophilus catalyzes two-step

1262 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Wang et al.
(
5) Arisan-Atac, I.; Wolschek, M. F.; Kubicek, C. P. Trehalose-6-
phosphate synthase A affects citrate accumulation by Aspergillus
niger under conditions of high glycolytic flux. FEMS Microbiol.
Lett. 1996, 140, 77-83.
Table 3. Comparison of the Molecular and Enzymatic Properties of
Recombinant Enzymes BA/TS, BATS, and TSBA
property
BA/TS
57/106
70
6.0
BATS
164
70
6.0
TSBA
164
70
6.0
molecular mass (kDa)
optimum temperature (
(6) Wingler, A. The function of trehalose biosynthesis in plants.
°
C)a
Phytochemistry 2002, 60, 437-440.
b
optimum pH (pH)
thermal stability
(
7) Foster, A. J.; Jenkinson, J. M.; Talbot, N. J. Trehalose synthesis
and metabolism are required at different stages of plant infection
by Magnaporthe grisea. EMBO 2003, 22, 225-235.
a
up to 70
°
C
up to 70
°
C
up to 70 °C
c
c
c
(
80
pH 5.0
64:25:11
±
1.7%)
(92
pH 5.0
±
4.1%)
7.0
(95
pH 5.0
72:23:5
±
2.2%)
pH stability b
−7.0
−
−7.0
(
8) Marechal, L. R.; Belocopitow, E. Metabolism of trehalose in
Euglena gracilis. I. Partial purification and some properties of
trehalose phosphorylase. J. Biol. Chem. 1972, 247, 3223-3228.
sugar composition
66:23.5:10.5
d
of product (%)
a
b
(9) Richards, A. B.; Krakowka, S.; Dexter, L. B.; Schmid, H.;
Wolterbeek, A. P.; Waalkens-Berendsen, D. H.; Shigoyuki, A.;
Kurimoto, M. Trehalose: A review of properties, history of use
and human tolerance, and results of multiple safety studies. Food
Chem. Toxicol. 2002, 40, 871-898.
The enzyme activity was measured at pH 7.0 for 2 h. The enzyme activity
c
was measured at 60
activities at 70
trehalose, maltose, and glucose products and were obtained at 60
for 24 h of reaction.
°
C for 2 h. The percentages indicated the relative residual
d
°
C to that measured at 60
°
C. The data indicated the ratio of
°C and pH 6.0
(
(
(
(
10) Wiemken, A. Trehalose in yeast, stress protectant rather than
reserve carbohydrate. Antonie Van Leeuwenhoek 1990, 58, 209-
and enzymatic properties of the recombinant enzymes BA/TS,
BATS, and TSBA were quite similar, as shown in Table 3.
These results indicated that fusion of the two enzymes did not
cause significant structural perturbation or changes in physico-
chemical properties. We therefore inferred that two thermally
stable proteins joined by a suitable linker might form a
harmonious structure that provides more steady conformation
in reacting with substrates at high temperatures.
2
17.
11) Uritani, M.; Takai, M.; Yoshinaga, K. Protective effect of
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In conclusion, construction of a recombinant bifunctional
fusion enzyme catalyzing sequential reactions can increase the
efficiency of the enzymes and simplify the expression and
protein purification in one process, as demonstrated with BATS
and TSBA in this study. It also provides a convenient and
efficient way to produce trehalose for industrial applications.
The fusion BA and TS proteins significantly improves trehalose
productivity, increasing the reaction rate, as a result of one single
polypeptide chain, which has bifunctionality. Additionally, this
thermostable and thermophilic fusion enzyme has highly at-
tractive advantages when operating at high temperatures, thus
solving some severe problems, such as microbial contamination
and lower medium viscosity in the carbohydrate industry. In
combination with our previous invention, which produces high-
maltose syrup and a high-protein product simultaneously from
low-price raw materials such as rice (45, 46), this fusion enzyme
can be further used to efficiently produce high-value trehalose
and nutritious high-protein food from staples such as rice at a
low cost. Furthermore, the bifunctional genes can be transformed
directly into rice or other plants. In our previously reported
transgenic plant harboring a bifunctional thermostable amylop-
ullulanase gene (47, 48), the seeds are anticipated to produce
trehalose and protein-enriched flour for industrial uses and
human consumption. These works are currently underway.
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Received for review August 16, 2006. Revised manuscript received
December 4, 2006. Accepted December 8, 2006.
JF062355T