Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Article abstract of DOI:10.1021/jf061318z

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85°C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85°C. The k_cat values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s^-1, respectively, whereas the k_cat values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s^-1, respectively. The K_M values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.

Full text of DOI:10.1021/jf061318z

J. Agric. Food Chem. 2006, 54, 7105−7112 7105
Expression, Purification, and Characterization of the
Maltooligosyltrehalose Trehalohydrolase from the Thermophilic
Archaeon Sulfolobus solfataricus ATCC 35092
TSUEI-YUN FANG,*,† WEN-CHI TSENG,§ MENG-SHIN GUO,†
†
TONG-YUAN SHIH,† AND XING-GUANG HUNG
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, and Department of
Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan
The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the R-1,4-glucosidic linkage
next to the R-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the
maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was
PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A
high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence
of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid
precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent
optimal pH of 5 and an optimal temperature at 85 °C. The enzyme was stable at pH values ranging
from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 °C. The kcat values
of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were
193, 1030, 1190, and 1230 s-1, respectively, whereas the kcat values for glucose formation during
hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s-1, respectively. The
K
M values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligo-
saccharides are similar at the same corresponding DPs. These results suggest that this MTHase
could be used to produce trehalose at high temperatures.
KEYWORDS: Maltooligosyltrehalose trehalohydrolase; trehalose; thermophilic enzyme; Sulfolobus; E.
coli; starch
INTRODUCTION
widely present in insects, fungi, and bacteria. Because trehalose
can protect proteins and lipid membranes from desiccation,
freezing, high temperature, and osmotic stress, the applications
of trehalose have been found in many different areas, such as
the use as a preservative or stabilizer for cells, organs, food,
cosmetics, and medicines (10). As the applications of trehalose
are increasing, a productive process with a high yield of
trehalose is gaining more attention.
Maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141,
MTHase, also known as glycosyltrehalose trehalohydrolase,
glycosyltrehalose-hydrolyzing enzyme, and trehalose-forming
enzyme) mainly cleaves the R-1,4-glucosidic linkage next to
the R-1,1-linked terminal disaccharide of maltooligosyltrehalose
to produce trehalose and the maltooligosaccharide with lower
molecular mass. Several bacteria are capable of producing
MTHase, such as Arthrobacter sp. Q36 (1), Arthrobacter
ramosus S34 (2), Corynebacterium glutamicum (3), Deinococ-
cus radiodurans (4), Rhizobium sp. M-11 (5), Sulfolobus
solfataricus MT4 (6), Sulfolobus solfataricus KM1 (7), Sul-
folobus acidocaldarius ATCC 33909 (8), and Sulfolobus shi-
batae DMS 5389 (9).
In addition to MTHase, maltooligosyltrehalose synthase (EC
5.4.99.15, MTSase, also known as glycosyltrehalose synthase,
glyosyltrehalose-producing enzyme, and trehalosyl dextrin-
forming enzyme) is also used in the process of trehalose
production from starch (1-3, 5-9). MTSase mainly catalyzes
an intramolecular transglycosylation reaction to produce a
nonreducing maltooligosyltrehalose by converting the R-1,4-
glucosidic linkage at the reducing end of maltooligosaccharide
to an R-1,1-glucosidic linkage. Trehalose is currently synthesized
from starch on an industrial scale by using MTSase and MTHase
obtained from mesophilic bacterium Arthrobacter sp. Q36 or
Rhizobium sp. M-11 (11).
Trehalose is a nonreducing disaccharide that contains two
glucose molecules linked by an R-1,1-glucosidic linkage and is
* Address correspondence to this author at the Department of Food
Science, National Taiwan Ocean University, 2 Pei-Ning Rd., Keelung 202,
Taiwan (telephone 886-2-2462-2192, ext. 5141; fax 886-2-2462-2586; e-mail
tyfang@mail.ntou.edu.tw).
Using the thermophilic enzymes to produce trehalose from
starch has several advantages. The reaction is generally carried
† National Taiwan Ocean University.
§ National Taiwan University fo Science and Technology.
10.1021/jf061318z CCC: $33.50
© 2006 American Chemical Society
Published on Web 08/29/2006

7106 J. Agric. Food Chem., Vol. 54, No. 19, 2006
Fang et al.
were confirmed by DNA sequencing. To express a wild-type MTHases
which contains no extra His-tag coding sequence derived from the
cloning stepssthe pET-15b-treZ was digested with NcoI and XhoI to
remove the His-tag coding sequence, made blunt by mung bean nuclease
and ligated, resulting in a recombinant clone designated pET-15b-∆H-
treZ. DNA sequencing was also performed to ascertain the removal of
the His-tag coding sequence. All DNA automatic sequencings were
carried out by Mission Biotech Corp. (Taipei, Taiwan).
out at a high temperature, which can accelerate the reaction rate,
lower the degree of retrogradation of starch, and decrease the
risks of microbial contaminations in the reaction mixtures (12,
13). In addition, starch is a relatively inexpensive substrate;
therefore, using starch could lower the production cost. In a
previous process, thermophilic MTSase and MTHase were
combined with a debranching enzyme to produce trehalose from
starch with a yield of around 82% (13, 14). This limit in yield
of trehalose was due to the side-hydrolysis reactions of
maltooligosaccharides catalyzed by MTSase and MTHase (13,
14). However, the substrate specificity of MTHase on this side-
hydrolysis reaction has received little attention.
Recently the complete genome of thermophilic archaeon S.
solfataricus ATCC 35092, also known as P2, has been
sequenced (15). The MTSase from this strain has been examined
in our previous study (16), but the MTHase, encoded by the
treZ gene, from this strain remained uncharacterized. In the
present study the treZ gene of MTHase was PCR-cloned from
the genomic DNA of S. solfataricus ATCC 35092, and the
cloned gene was overexpressed in Escherichia coli. In addition
to characterizing the general properties of this recombinant
MTHase, we also investigated the substrate specificity and
kinetic parameters of the enzyme on both hydrolysis reactions
of maltooligosyltrehaloses and maltooligosaccharides, respec-
tively.
Expression of MTHase by E. coli. The pET-15b-∆H-treZ vector
was transformed into E. coli BL21-CodonPlus (DE3)-RIL to express
MTHase. One single colony from a newly transformed culture plate
was inoculated into 10 mL of terrific broth (TB) medium supplemented
with 100 µg/mL ampicillin plus 34 µg/mL chloramphenicol and grown
at 37 °C until the OD600 reached around 0.6. Cells were collected by
centrifugation and resuspended in 4 mL of fresh medium. A volume
of 3 mL of the resuspended culture was then added to 600 mL of fresh
medium containing 100 µg/mL of ampicillin plus 34 µg/mL chloram-
phenicol and grown until the OD600 reached around 0.6. The culture
was then induced by an addition of IPTG to a final concentration of
0.5 mM. After a further culture at 20 °C for 16 h, the cells were then
harvested by centrifugation and stored at -70 °C before further
processing.
Preparation of Cellfree Extract. Frozen cells (6 g) expressing wild-
type and His-tagged MTHases were suspended in 18 mL of lysis buffers
A and B, respectively. Lysis buffer A contained 20 mM Tris-HCl (pH
8.0) and 1 mM benzamidine. Lysis buffer B contained 20 mM Tris-
HCl (pH 7.9), 5 mM imidazole, 0.5 M NaCl, and 1 mM benzamidine.
The suspended cells were disrupted using a French Press disruptor (Sim-
Aminco, Rochester, NY) at 20000 psi (1360 atm). The cellfree extract
was then prepared by removing the insoluble fractions from the
supernatant of the above mixture by centrifugation at 10000g for 2 h.
MATERIALS AND METHODS
Materials. The genomic DNA of S. solfataricus ATCC 35092 was
obtained from the American Type Culture Collection (Manassas, VA).
E. coli BL21-CodonPlus (DE3)-RIL was obtained from Stratagene (La
Jolla, CA). Plasmid pET-15b was from Novagen (Madison, WI). Vent
DNA polymerase and mung bean nuclease were purchased from New
England BioLabs (Beverly, MA). T4 DNA ligase and restriction
enzymes were supplied by Promega (Madison, WI). Glucose (G1),
maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5),
maltohexaose (G6), maltoheptaose (G7), glucoamylase, 3,5-dinitrosali-
cylic acid (DNS), bovine serum albumin (BSA), isopropyl-â-D-
thiogalactoside (IPTG), and phenylmethanesulfonyl fluoride (PMSF)
were from Sigma (St. Louis, MO). Ultrafree-15 and Microcon
centrifugal filter units were obtained from Millipore (Bedford, MA).
Sephacryl S-200 HR and protein low molecular mass standards were
from Amersham Pharmacia Biotech (Piscataway, NJ).
Purification of Wild-Type Enzyme. Heat treatment was first used
to precipitate most of the undesired proteins by incubating the cell-
free extract in an 80 °C water bath for 1 h followed by centrifugation
to remove the heat-labile proteins. A 10% (w/v) streptomycin sulfate
stock solution was then added to a final concentration of 1% (w/v) to
precipitate the nucleic acids. After centrifugation at 10000g for 1 h,
the supernatant was dialyzed against 20 mM Tris-HCl buffer (pH 8.5)
and was subsequently loaded onto a Q-Sepharose column (1.6 × 10
cm), which was pre-equilibrated with the same dialysis buffer. The
column was first washed thoroughly with the same buffer until the
absorbance of 260 nm reached baseline. Finally, a linear gradient of
0-0.4 M NaCl in the above buffer was used to elute the bound proteins.
The eluted fractions containing enzyme activity were collected and
dialyzed against a buffer containing 20 mM Tris-HCl (pH 8.0).
Amplification of the treZ Gene. The treZ gene was amplified by
using the Polymerase Chain Reaction (PCR). Two primers were
designed on the basis of the treZ sequence of S. solfataricus (GenBank
accession no. AE006815 Region: 7425 .. 9110). To clone the treZ
gene into the pET-15b vector, the XhoI and BamHI restriction sites
were included in the forward and reverse primers, respectively. The
primer sequences are as follows: 5XtreZ (forward primer), 5′-CCC
GGG TCG ACT CGA GAT GAC GTT TGG TTA TAA ATT AGA
TGA-3′; 3BtreZ (reverse primer), 5′-TTA GCA GCC GGA TCC CTA
AAG TTT ATA TAA AGC AAA TCC CT-3′; the XhoI and BamHI
restriction sites are in boldface. The reaction was carried out in 100
µL of reaction mixture containing the genomic DNA of S. solfataricus
ATCC 35092, two primers, dNTP, Vent DNA polymerase, and Vent
DNA polymerase buffer, and was performed by using a GeneAmp PCR
system 2400 (Perkin-Elmer, Wellesley, MA) according to the following
conditions in sequence: 95 °C for 5 min, an amplification, and a final
extension at 72 °C for 10 min. The amplification profile was 1 min at
95 °C, 1 min at 60 °C, and 2 min at 72 °C.
Construction of Expression Vector for MTHase. The 1.7 kbp
PCR-amplified fragment was purified and then digested with XhoI and
BamHI. The digested fragment was inserted into the pET-15b vector,
resulting in a recombinant vector designated pET-15b-treZ. The
amplified treZ gene was fused in frame with the His-tag coding
sequence on pET-15b; therefore, the expressed recombinant MTHase
possessed a His-tag on its N-terminal region. The sequences of the
entire treZ gene and the fused His-tag coding region on pET-15b-treZ
Purification of His-Tagged Enzyme. The cell-free extract was
diluted with an equal volume of binding buffer (5 mM imidazole, 0.5
M NaCl, 20 mM Tris-HCl, pH 7.9) prior to being loaded onto a
Novagen His‚Bind column (1.6 × 5 cm), which was previously
equilibrated with the same binding buffer. The column was first washed
with the binding buffer, then washed with a wash buffer (60 mM
imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), and finally eluted
with an elute buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl,
pH 7.9) according to the protocols provided in the Novagen His‚Bind
Kits. The eluted fractions containing enzyme activity were collected
and dialyzed against a buffer containing 20 mM Tris-HCl (pH 8.0).
HPLC Analysis of the Molecular Masses of Wild-Type and His-
Tagged Enzymes. The molecular masses of both wild-type and His-
tagged MTHases were measured using a BioSep-SEC-S4000 column
(300 × 7.8 mm), which is a silica-based gel filtration column. The
mobile phase contained 50 mM sodium phosphate buffer (pH 6.8), and
the flow rate was controlled at 0.5 mL/min. The analysis was carried
out under a Hitachi HPLC L-7000 (Tokyo, Japan) equipped with an
UV detector. The molecular masses of wild-type and His-tagged
MTHases were determined by comparison with the calibration curve
derived from the molecular mass standards.
Preparation of Maltooligosyltrehaloses. The preparation of mal-
tosyltrehalose (G2T), maltotriosyltrehalose (G3T), maltotetraosyltreha-
lose (G4T), and maltopentaosyltrehalose (G5T) was according to the
procedures described by Kato et al. (17). The MTSase reactions were

Maltooligosyltrehalose Trehalohydrolase from S. solfataricus
J. Agric. Food Chem., Vol. 54, No. 19, 2006 7107
carried out at 75 °C for 2-4 h in 0.05 M citrate-phosphate buffer
(pH 5) by using 100 mM G4-G7 as substrates. At the end of each
MTSase reaction, a 0.1 N NaOH treatment was used to decompose the
remaining reducing sugars, and then the reaction mixture was deionized
by Amberlite IR-120 A and IRA-900 (Fluka, Buchs, Switzerland). The
purified MTSase was prepared according to the method given in our
previous study (16).
Table 1. Comparison of the Deduced Amino Acid Sequences of
MTHase from S. solfataricus ATCC 35092 with Those of Other
MTHases from the Sulfolobus Genus
no. of
residues
identityb
(%)
source
accession no.a
S. solfataricus ATCC 35092
S. solfataricus MT4
S. solfataricus KM1
S. shibatae B12
S. acidocaldarius ATCC 33909
P95867
nonec
Q55088
Q9UWN9
Q53641
561
561
559
559
556
100
99.4
79.9
78.5
60.0
Enzyme Activity Assay. The MTHase activity was assayed at 60
°C for 10 min by using 2.8 mM G4T as substrate in 0.05 M citrate-
phosphate buffer (pH 5). One unit of MTHase activity was defined as
the amount of enzyme required to produce 1 µmol of trehalose in 1
min. MTHase cleaves G4T into equimolar amounts of trehalose and
G4, and because the latter oligosaccharide is the only reducing sugar
in the mixture, the concentration of G4, and hence the activity of the
enzyme, can be determined by the DNS assay (18). In determining the
effects of pH and temperature on the activity and stability of enzyme,
the following buffers with a final concentration of 50 mM were used
in different pH ranges: citrate-phosphate buffer (pH 3.5-7), Tris-
HCl buffer (pH 7.5-8.5), and NaHCO3-Na2CO3 buffer (pH 9.5-11).
For determination of the optimal pH, the enzyme (0.311 µg/mL) was
assayed at different pH values under the standard conditions. For
determination of pH stability, the enzyme (3.11 µg/mL) was incubated
at various pH values at 4 °C for 24 h, and the remaining activities
were assayed under the standard conditions. For determination of the
optimal temperature, the enzyme (0.311 µg/mL) was assayed at different
temperatures under the standard conditions. For determination of
thermostability, the enzyme (3.11 µg/mL) was incubated at various
temperatures at pH 5 for 2 h, and the remaining activities were assayed
under the standard conditions. For determination of the effects of metal
ions on the activity, the enzyme (0.311 µg/mL) was assayed at 60 °C
and pH 5 as in the standard conditions except that the buffers contained
EDTA or a metal ion at a final concentration of either 0.4 or 4 mM.
a The deduced amino acid sequences were obtained from the TrEMBL protein
sequence database. b The identity values were analyzed by SIM-Local similarity
program on the website http://tw.expasy.org/tools/sim-prot.htmL (22). c The deduced
amino acid sequence of MTHase from S. solfataricus MT4 was from de Pascale
et al. (23).
RESULTS AND DISCUSSION
Gene Analysis and Comparison of Amino Acid Sequences
of Several MTHases from the Sulfolobus Genus. MTHase
from S. solfataricus ATCC 35092 possesses 561 amino acids
and a calculated molecular massof 64371 Da, which was
deduced from the nucleotide sequence of the treZ gene
and analyzed by using the Compute pI/Mw tool at http://
tw.expasy.ch/tools/pi_tool.htmL (22), respectively. MTHase
from S. solfataricus ATCC 35092 shares a >75% identity with
other MTHases from the Sulfolobus genus except S. acidocal-
darius (Table 1). In addition, a 99.4% identity of amino
sequences existed between MTHase from S. solfataricus ATCC
35092 and that from S. solfataricus MT4 (23) with the following
residues indicating the only changes: [G4A; L7I; C461Y].
By the information from CAZy, an Internet resource on
glycosyl hydrolases at http://afmb.cnrs-mrs.fr/CAZY/, these
MTHases have been previously classified in family 13 of
glycosyl hydrolases on the basis of their amino acid sequence
similarities according to the classification developed by B.
Henrissat (24-26). Families 13, 70, and 77 of glycosyl
hydrolases constitute Clan GH-H, which contains functionally
and structurally related enzymes (27). Many enzymes from Clan
GH-H acting on starch, such as R-amylase, pullulanase, isoamy-
lase, and cyclodextrin glucanotransferase, are known as the
R-amylase family of enzymes (27). There are four highly
conserved regions of amino acid sequences in the R-amylase
family of enzymes (27). Except for one residue, [Ser257] of
MTHase from S. shibatae B12, and three residues, [Leu250;
Phe280; Phe371] of MTHase from S. acidocaldarius, the amino
acid sequences of the four highly conserved regions in these
MTHases are identical. The four conserved regions also contain
three essential amino acid residues, Asp252, Glu283, and
Asp377 (numberings according to the enzyme from S. solfa-
taricus KM1), which play a catalytic role in the active site of
the R-amylase family (27, 28). The three-dimensional structures
of MTHases from S. solfataricus KM1 and Deinococcus
radiodurans also suggested that residues Asp252, Glu283, and
Asp377 are catalytic residues (4, 29). Asp252 acts as the
catalytic nucleophile/base, and Glu283 is the catalytic proton
donor (4, 29). Asp377 is not directly involved in the catalysis
but has been proposed to stabilize the transition state and to
maintain Glu283 in the correct protonation state (4).
HPLC Analysis of the Hydrolysis Products of MTHase Using
Maltooligosaccharides as Substrates. The hydrolysis products of
MTHase using maltooligosaccharides as substrates were measured by
a Hitachi HPLC L-7000 equipped with a refractive index detector. The
enzyme reactions were carried out at 60 °C for 1.5-36 h in 0.05 M
citrate-phosphate buffer (pH 5) by using 14 mM G2-G7 as substrates.
The reactions were stopped by incubating the mixture in boiling water
for 10 min, and then the mixtures were filtered through the Microcon
centrifugal filter unit with a YM-3 membrane (MWCO 3000) to remove
the enzyme. The filtrates were analyzed by a Vercopak Nucleosil 5
µm NH2 column (4.6 × 250 mm) with a mobile phase consisting of
30% (v/v) H2O and 70% (v/v) acetonitrile.
Mass Spectrometric Analysis. The mass spectrometric analysis was
performed on a MicroMass (Manchester, U.K.) Quattro-BIOQ mass
spectrometer as previously described (19).
Protein Concentration Measurement. Protein concentration was
determined according to Bradford’s method (20) with BSA as standard.
Measurement of Glucose Formation from the Hydrolysis of
Maltooligosaccharides. The substrates, G4-G7 (14 mM), were incu-
bated with MTHase in 50 mM citrate-phosphate buffer at pH 5 and
60 °C for 10 min. The reactions were stopped by adding 0.4 volume
of 4 M Tris-HCl buffer at pH 8.0 followed by incubation of the mixtures
in boiling water for 10 min. The glucose that was released from the
hydrolysis of the substrates was measured according to the glucose
oxidase method (21).
Enzyme Kinetics. The initial rates of hydrolysis of G2T-G5T and
G4-G7 were determined at 60 °C in 50 mM citrate-phosphate buffer
at pH 5 by using 8-10 substrate concentrations ranging from 2 to 50
mM. Samples taken at five different time intervals were stopped by
adding DNS or 4 M Tris-HCl buffer (pH 8.0). In the hydrolysis of
G2T-G5T, the molar concentration of the released trehalose was equal
to that of the released G2-G5 measured by the DNS method (18) as
described in the method of enzyme activity assay. In the hydrolysis of
G4-G7, the released glucose was measured by the glucose oxidase
method (21). Values of kcat and KM were calculated by fitting the initial
rates as a function of substrate concentration to the Michaelis-Menten
equation using Enzfitter software (Elsevier-Biosoft).
Expression and Purification of MTHase. The protein
expression levels of both wild-type and His-tagged MTHases
were higher in the absence of IPTG than in the presence of 0.5
mM IPTG induction, and these expression levels of wild-type
were higher than those of His-tagged MTHases under the same
induction conditions. In the absence of IPTG induction, the

7108 J. Agric. Food Chem., Vol. 54, No. 19, 2006
Fang et al.
Table 2. Summary of Purification Steps of Wild-Type and His-Tagged MTHases Expressed in E. coli BL21-CodonPlus (DE3)-RIL
total
protein (mg)
total
activity (units)
activity
recovery (%)
specific
activity (units/mg)
purification
fold
enzyme
purification step
wild-type
crude extract
heat treatmenta
Q Sepharose
1180
145
25.0
79600
54100
20300
100
67.9
25.4
67.5
373
812
1
5.53
12.0
His-tagged
crude extract
924
41.0
19500
12700
100
65.3
21.1
310
1
14.7
His
‚
Bind
a The crude extract of wild-type enzyme was heated at 80
°C for 1 h and then centrifuged to remove the precipitates.
Figure 1. SDS-PAGE analysis of purifications of wild-type and His-tagged
MTHases in E. coli BL21-CodonPlus (DE3)-RIL carrying the pET-15b-
∆
H-treZ and pET-15b-treZ, respectively: (lane M) molecular mass
standards; (lanes 1 and 6) total cellular proteins of the E. coli cells carrying
the pET-15b- H-treZ and pET-15b-treZ, respectively; (lanes 2 and 7)
insoluble proteins for the E. coli cells carrying the pET-15b- H-treZ and
Figure 2. Determination of the molecular masses of wild-type and His-
tagged MTHases by HPLC analysis. The protein standards ( ) included
a, thyroglobin (669 kDa); b, apoferritin (443 kDa); c, ferritin (416 kDa); d,
catalase (219 kDa); e, -amylase (200 kDa); f, adolase (176 kDa); g,
alcohol dehydrogenase (150 kDa); h, albumin (67 kDa); i, ovalumin (43
kDa); j, chymotrypsinogen A (25 kDa); and k, ribonuclease A (13.7 kDa).
b
∆
∆
â
pET-15b-treZ, respectively, after cell lysis; (lanes 3 and 8) crude cell-free
extracts of wild-type and His-tagged MTHases; (lane 4) partially purified
fraction of wild-type MTHase after heat treatment; (lane 5) purified fraction
of wild-type MTHase after ion-exchange chromatography; (lane 9) partially
purified fraction of His-tagged MTHase after metal-affinity chromatography.
Wild-type (
standards. The KD values were calculated using the equation KD
(Ve V0)/(VT V0), where Ve is the elution volume and VT and V0 represent
0) and His-tagged (4) MTHases were plotted with the protein
)
−
−
the total liquid volume and the void volume of the column, respectively.
yields of the active wild-type and His-tagged MTHases ex-
pressed in E. coli BL21-CodonPlus (DE3)-RIL were 13300 and
3250 units/g of wet cells, respectively. The yield of 13300
units/g of wet cells was much higher than the previously reported
yields of 2822 and 726 units/g of wet cells for the E. coli JM109
and Rb-791 expressing S. solfataricus KM1 and MT4 enzymes,
respectively (11). However, in the presence of IPTG induction,
the yields of the active wild-type and His-tagged MTHases
expressed in E. coli were only 3230 and 2280 units/g of wet
cells, respectively. The expression level of target protein can
be affected by many factors, such as the gene toxicity,
proteolytic degradation of target protein, secondary structure
in the mRNA transcript, and excessive rare codon usage (30,
31). In general, IPTG is used to inactivate lac repressor and to
induce the overexpression of target gene. In the absence of
IPTG, the expression of the target gene is not induced and under
its basal expression level due to the gene leakage. The expression
of MTHase from Sulfolobus solfataricus MT4 studied by de
Pascale et al. also had shown that the highest level of protein
expression was obtained in the absence of IPTG induction (23).
Because the gene of MTHase contains many rare codons, such
as AGG and AGA, we suspect that the overexpression of
MTHase gene induced by IPTG may conversely inhibit the
machinery of protein synthesis.
matography, whereas His-tagged MTHase was purified from
the cell-free extract directly by metal chelating chromatography
(Table 2). The purified wild-type MTHase showed a single band
on SDS-PAGE, indicating that a high purity of protein was
obtained (Figure 1, lane 5). However, the His-tagged enzyme
was only partially purified after metal chelating chromatography
(Figure 1, lane 9). The apparent molecular masses of wild-
type and His-tagged MTHases estimated under denaturing
conditions by SDS-PAGE were around 64 and 66 kDa,
respectively (Figure 1), which are in good agreement with the
calculated molecular masses. Because there are 23 amino acid
residues inherited from the His-tag fragment, the molecular mass
of the His-tagged enzyme is about 2.5 kDa larger than that of
wild-type enzyme. The molecular masses of the purified wild-
type and the partially purified His-tagged MTHases under non-
denaturing conditions were then analyzed by HPLC using the
BioSep-SEC-S4000 gel filtration column. The purified wild-
type MTHase had a peak at 18.99 min, whereas His-tagged
MTHase appeared as a peak at 20.33 min (data not shown). By
comparison with the calibration curve derived from the molec-
ular mass standards, the molecular mass of wild-type MTHase
under non-denaturing conditions was around 128.8 kDa and that
of His-tagged MTHase around 68.7 kDa (Figure 2). Because
the theoretical molecular masses of one subunit for wild-type
and His-tagged MTHases are around 64.4 and 66.9 kDa, the
estimated 128.8 and 68.7 kDa corresponded to 2 and 1.03
subunits, respectively. These results suggest that the purified
wild-type MTHase is a homodimer, whereas the purified His-
tagged MTHase is a monomer. The three-dimensional structure
The expressed wild-type and His-tagged MTHases were
mostly soluble and constituted about 80 and 60%, respectively,
of the total expressed MTHases that appeared in the cell-free
extract (Figure 1, lanes 1-3 and 6-8). Wild-type MTHase was
purified from the cell-free extract of E. coli sequentially by heat
treatment, nucleic acid precipitation, and ion-exchange chro-

Maltooligosyltrehalose Trehalohydrolase from S. solfataricus
J. Agric. Food Chem., Vol. 54, No. 19, 2006 7109
Table 3. Substrate Specificity of Wild-Type MTHase from S. solfataricus ATCC 35092a
DP of
substrate
maltooligosyl-
trehalose
trehalose formation rate
maltooligo-
saccharide
glucose formation rate
ratio of
G/Tb (%)
-1
-
1
-1
-1
[mol
‚
(mol of MTHase
)‚
s
]
[mol
‚
(mol of MTHase
)
‚
s
]
4
5
6
7
G2T
G3T
G4T
G5T
165
996
1090
1250
±
±
±
±
10c
55
80
30
G4
G5
G6
G7
5.46
12.3
12.9
6.49
±
±
±
±
0.47
0.5
0.3
3.31
1.23
1.18
0.52
0.95
a G2T
enzyme (0.311
−
G5T and G4
−
G7 were used as substrates to determine the trehalose and glucose formation rates, respectively. The substrate (14 mM) was incubated with the
µ
g/mL for G2T G5T and 3.11 g/mL for G4-G7) at 60 SD from triplicate experiments.
−
µ
°
C, pH 5. b Ratio of glucose to trehalose formation. c Mean
±
of MTHase from S. solfataricus KM1 had also revealed that
the enzyme is a homodimer covalently linked by an intermo-
lecular disulfide bond at residue C298 (29), which corresponds
to C301 of MTHase from S. solfataricus ATCC 35092. Several
previous studies suggested that a His-tag at the N or C terminus
of protein affected the oligomerization state of protein (32-
34). Because the His-tagged MTHase contains 23 additional
amino acid residues in its N terminus, this extra fragment
probably does not contain any secondary element (32) and hangs
around residue C324, which corresponds to C301 of wild-type
MTHase; it may, therefore, interfere with the formation of the
intermolecular disulfide bond at residue C324.
Originally, the His-tag enzyme was designed for purification
directly from metal chelating chromatography to obtain apparent
homogeneity by taking advantage of the His-tag. However, the
His-tagged enzyme could be only partially purified by metal
chelating chromatography. Because the protein expression level
and active enzyme yield of His-tagged MTHase were less than
those of wild-type MTHase and because the His-tagged enzyme
was in a monomer form, which is different from the homodimer
form of wild-type enzyme, we did not conduct further purifica-
tion steps to obtain apparent homogeneity after metal chelating
chromatography, and further studies on only the wild-type
MTHase were carried out.
The molecular mass of the purified wild-type MTHase was
also analyzed by electrospray ionization mass spectrometry and,
after treatment with 50 mM DTT, it showed a molecular mass
of 64.24 kDa. These data are consistent with the theoretical
molecular mass of one subunit of the MTHase after the cleavage
of the N-terminal methionine. These observations suggested that
the MTHase from S. solfataricus ATCC 35092 might be also a
homodimer covalently linked by an intermolecular disulfide
bond similar to the MTHase from S. solfataricus KM1.
Effects of pH and Temperature on the Activity and
Stability of Wild-Type MTHase. The recombinant enzyme
showed an optimal activity at pH 5.0-5.5 and remained stable
in the pH range from 3.5 to 11 (Figure 3a). The enzyme had
an optimal activity at 85 °C and retained virtually all of its
activity at that temperature for 2 h (Figure 3b). The good
thermostability of this enzyme indicated that the purified
recombinant MTHase was well folded and should have the same
structure as that produced directly from S. solfataricus ATCC
35092.
Effects of Metal Ions on the Activity of Wild-Type
MTHase. The addition of EDTA, Na+, K+, Mg2+, Ca2+, and
Zn2+ had no activation or inhibition effect on MTHase activity.
Adding 0.4 and 4 mM Hg2+ strongly inhibited MTHase, whereas
Cu2+ inhibited it only at 4 mM. These results indicated that
these metal ions were not required for MTHase activity.
Although the catalytic domain of the R-amylase family of
enzymes generally contains one conserved calcium ion to
stabilize the interface of domains A and B, there is no metal
ion bound to the domain interface in the three-dimensional
structure of MTHase from S. solfataricus KM1 (29).
Figure 3. Effects of pH and temperature on the activity (
b) and stability
(
O
) of wild-type MTHase: (a) effects of pH; (b) effects of temperature.
Substrate Specificity. Maltooligosyltrehaloses and malto-
oligosaccharides of various DPs were used as substrates to study
the substrate specificity of MTHase because both types of
oligosaccharides could occupy the active site of MTHase for
enzymatic reactions. MTHase converts maltooligosyltrehaloses
to trehalose and a maltooligosaccharide with lower DP; for
example, G5T is converted to trehalose and G5. The side reaction
of this maltooligosaccharide (Gn) with MTHase would then
produce glucose and a maltooligosaccharide (Gn-1). As shown
(Table 3) the trehalose formation rate increased as the DP value
of the substrate increased, and G5T was the most preferred
substrate tested in this study for trehalose formation. However,
for glucose formation, G6 exhibited the highest formation rate.
The ratios of glucose formation to trehalose formation, which
represented the ratios of the side reaction to the major reaction
of MTHase, were 3.31, 1.23, 1.18, and 0.52% for DP 4-7
substrates, respectively. These results suggested that the hy-
drolysis reaction toward the low DP maltooligosaccharides might
be one of the major reasons that caused the decreased yield of
trehalose production from starch.
HPLC Analysis of the Hydrolysis Products of Wild-Type
MTHase Using Maltooligosaccharides as Substrates. To
further examine the product profiles of the side reaction of
MTHase, the enzyme reactions were carried out at 60 °C for
0-36 h by using G2-G7 as substrates. Figure 4 shows the
ln(residual concentration of substrate) versus time. The residual

7110 J. Agric. Food Chem., Vol. 54, No. 19, 2006
Fang et al.
Table 4. Comparison of Kinetic Parameters of MTHases from S. solfataricus ATCC 35092, S. solfataricus KM1, and S. acidocaldarius ATCC 33909
for Hydrolysis of G2T
−G5T
recombinant
S. solfataricus ATCC 35092a
natural
S. solfataricus KM1b
S. acidocaldarius ATCC 33909c
-
1
-
1
-1
-
1
-
1
-
1
-
1
-
1
-1
substrate
k
cat (s
)
K
M (mM)
k
cat/KM (s
‚
mM
)
k
cat (s
)
K
M (mM)
k
cat/KM (s
15.1
129
‚
mM
)
k
cat (s
)
K
M (mM)
k
cat/KM (s
‚
mM
)
G2T
G3T
G4T
G5T
193
1030
1190
1230
±
±
±
±
7d
30
30
40
11.1
7.22
5.66
5.89
±
±
±
±
0.1
17.4
143
210
198
±
±
±
±
0.5
6
8
339
747
866
756
22.4
5.8
5.6
4.7
NRe
NR
NR
NR
16.7
2.7
3.7
4.9
NR
NR
NR
NR
0.48
0.37
0.45
155
161
9
a This study. Experiments were carried out at 60
and pH 5.5. c Nakada et al. (31). Experiments were carried out at 60
°
C and pH 5. The enzyme concentration was 0.311
C and pH 6. d Standard error from the curve fitting. e Not recorded.
µ °C
g/mL. b Kato et al. (30). Experiments were carried out at 60
°
Figure 4. Residual concentrations of substrates after hydrolysis by wild-
type MTHase. After hydrolysis of 14 mM DP 2 7 maltooligosaccharides
by wild-type MTHase (0.311 g/mL) at 60 C, pH 5, the residual
concentrations of substrates were determined from the HPLC analysis:
) G2; ( ) G3; ( ) G4; ( ) G5; ( ) G6; ( ) G7.
−
µ
°
(
O
1
4
9
0
[
Table 5. Kinetic Parameters of Wild-Type MTHase from S. solfataricus
ATCC 35092 for Glucose Formation in Hydrolysis of G4
−G7 at 60 °C
and pH 5a
-1
-1
-1
substrate
kcat (s
)
KM (mM)
kcat/KM (s
‚
mM
)
G4
G5
G6
G7
5.49
17.7
18.2
6.01
±
±
±
±
0.20
0.5
0.7
10.9
5.86
5.63
2.63
±
±
±
±
0.9
0.50
3.02
3.24
2.29
±
±
±
±
0.20
0.13
0.22
0.13
0.40
0.51
0.20
0.13
a The enzyme concentration was 3.11
fitting.
µ
g/mL. b Standard error from the curve
concentrations of G2 and G3 stayed almost unchanged, suggest-
ing that G2 and G3 were poorly hydrolyzed. On the other hand,
G6 was hydrolyzed most quickly among the substrates because
the ln(residual concentration) of G6 had the steepest decline with
the reaction time. These data were consistent with the previous
observations of maltooligosaccharide hydrolysis that was indi-
cated by the glucose formation rate as shown in Table 3. Figure
5 shows the formations of hydrolysis products of MTHase using
G4-G7 as substrates. When G4 was hydrolyzed, the same
concentrations of G1 and G3 were produced, whereas no G2 was
detected, indicating that G4 was not hydrolyzed to maltose.
When G5 was hydrolyzed, the concentrations of G1 and G2 were
nearly the same as those of G4 and G3, respectively, and the
concentrations of G1 and G4 were higher than those of G2 and
G3 at each sampling time. These data suggest that G5 was
hydrolyzed more likely to glucose than to maltose. The G1
formation rate was the highest, whereas the G2 formation rate
was approaching the level of G1 formation rate as the DP of
substrate increased, indicating that the substrate of higher DP
tended to be hydrolyzed to maltose. In addition to the hydrolysis
Figure 5. Hydrolysis products of wild-type MTHase using DP
maltooligosaccharides as substrates: (a d) hydrolysis products of wild-
type MTHase (0.311 g/mL) using 14 mM G4, G5, G6, or G7, respectively,
as substrate at 60 C, pH 5. The concentrations of hydrolysis products
were determined from the HPLC analysis: ) G1; ( ) G2; ( ) G3; (
G4; ( ) G5; ( ) G6.
4−7
−
µ
°
(b
O
1
4)
9
0
activity toward maltooligosyltrehaloses, an earlier study indi-
cated that MTHase from S. solfataricus KM1 could also
hydrolyze maltooligosaccharides from the reducing end to
release either glucose or maltose (35). The above observations
suggest that MTHase from S. solfataricus ATCC 35092 could
also hydrolyze maltooligosaccharides to release glucose or
maltose, similar to the properties of MTHase from S. solfataricus
KM1.
Enzyme Kinetics. Hydrolysis of G2T-G5T by wild-type
MTHase from S. solfataricus ATCC 35092 was carried out at
60 °C and pH 5. A comparison of kinetic parameters (kcat and

Maltooligosyltrehalose Trehalohydrolase from S. solfataricus
J. Agric. Food Chem., Vol. 54, No. 19, 2006 7111
Table 6. Comparison of Some Enzymatic Properties of MTHases from Sulfolobus Genus (Strains S. solfataricus ATCC 35092, S. solfataricus MT4,
S. solfataricus KM1, S. acidocaldarius ATCC 33909, and S. shibatae DSM 5389)
recombinant
natural
ATCC 35092a
MT4b
KM1c
85
5.5
C, 6 h (100%)
ATCC 33909d
DSM 5389e
optimum temperature (
°
C)
85
5
85
5
75
85
70
4.5
85
−
−
°
75
85
4.5
75
85
4.5
NR
NR
NR
optimum pH
5.5−6
thermostabilityf
80
85
3.5
°
°
C, 2 h (100%)
C, 2 h ( 94%)
°
°
C, 2 h (100%)
80
85
°
°
C, 1 h (100%)
C, 1 h ( 93%)
°
C, 2 h (100%)
C, 2 h ( 78%)
9.5
∼
C, 2 h (
10.5
∼
80%)
∼
°
−
∼
pH stability
−
11
4
−
3.5
Cu2
no
−10
5.5
−
9.5
+
+
+
+
+
metal ion inhibition
Cu2 , Hg2
NRg
NR
NR
Cu2 , Hg2
+
Ca2 activation
no
no
side-hydrolysis reactionh
yes
yes
yes
a This study. b de Pascale et al. (23). c Kato et al. (30). d Nakada et al. (31). e Di Lernia et al. (9). f The residual activities are shown in parentheses. g Not recorded in
the published literature. h The hydrolysis reaction toward maltooligosaccharides.
KM) of MTHases from different strains of S. solfataricus is given
in Table 4. In general, the kcat values of these MTHases
increased, whereas the KM values decreased, as the DP of
substrates increasedsa phenomenon observed for most amy-
lolytic enzymes. Nevertheless, kinetic parameters of these three
MTHases are very different. The MTHase from S. solfataricus
ATCC 35092 has higher kcat values for hydrolysis of
G3T-G5T compared to those of the enzyme from S. solfataricus
KM1 and marginally higher KM values for the hydrolysis of
G3T-G5T by those enzymes from S. solfataricus KM1 and
S. acidocaldarius ATCC 33909. The catalytic efficiencies
(kcat/KM) of MTHase from S. solfataricus ATCC 35092 are
111-135% of those of the enzyme from S. solfataricus KM1.
These higher catalytic efficiencies suggest the MTHase from
S. solfataricus ATCC 35092 can be a more efficient enzyme
that could be used in trehalose production from starch.
genus. The results from this study also suggest that this MTHase
can be used to produce trehalose at high temperatures.
ABBREVIATIONS USED
MTSase, maltooligosyltrehalose synthase; MTHase, malto-
oligosyltrehalose trehalohydrolase; G1, glucose; G2, maltose; G3,
maltotriose; G4, maltotetraose; G5, maltopentaose; G6, malto-
hexaose; G7, maltoheptaose; DNS, 3,5-dinitrosalicylic acid;
BSA, bovine serum albumin; IPTG, isopropyl-â-D-thiogalac-
toside; DP, degree of polymerization; G2T, maltosyltrehalose;
G3T, maltotriosyltrehalose; G4T, maltotetraosyltrehalose; G5T,
maltopentaosyltrehalose.
ACKNOWLEDGMENT
We thank Dr. Ming F. Tam at the Institute of Molecular Biology
of Academia Sinica for carrying out the electrospray ionization
mass spectrometric analysis.
Kinetic parameters for glucose formation in hydrolysis of
G4-G7 by wild-type MTHase from S. solfataricus ATCC 35092
at 60 °C and pH 5 are given in Table 5. In the hydrolysis of
G4-G7, the KM values of wild-type MTHase decrease as the
DP of substrates increase, whereas the kcat values of MTHase
increase as the DP of substrates increase except for hydrolysis
of G7. The KM values for hydrolysis of maltooligosaccharides
are similar to those for hydrolysis of maltooligosyltrehaloses
at the same DP except for G7. However, the kcat and kcat/KM
values for hydrolysis of maltooligosaccharides are only 0.5-
2.8 and 1.2-2.9% of those of maltooligosyltrehaloses at the
same DP. These results suggest that both maltooligosyltrehaloses
and maltooligosaccharides can access the binding site of
MTHase, but the latter has comparably lower hydrolysis rates.
Previous studies reported similar observations of the hydrolysis
of maltooligosaccharides by the MTHases from S. solfataricus
KM1 and S. acidocaldarius ATCC33909 (35, 36); however,
the kinetic parameters were not determined in those studies.
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Received for review May 9, 2006. Revised manuscript received
July 21, 2006. Accepted July 28, 2006. This work was supported by
Center for Marine Bioscience and Biotechnology at National Taiwan
Ocean University and Grant NSC 93-2313-B-019-008 from the National
Science Council at Taiwan.
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