Cloning, expression, and characterization of a d-psicose 3-epimerase from clostridium cellulolyticum H10

Article abstract of DOI:10.1021/jf201356q

The noncharacterized protein ACL75304 encoded by the gene Ccel-0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co²⁺. The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co²⁺, respectively, suggesting that this enzyme was extremely thermostable in the presence of Co²⁺ but readily inactivated without metal ion. The Michaelis-Menten constant (K_m), turnover number (k_cat), and catalytic efficiency (k_cat/K_m) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min^-1, and 186.4 mM min^-1, respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.

Full text of DOI:10.1021/jf201356q

ARTICLE
pubs.acs.org/JAFC
Cloning, Expression, and Characterization of a D-Psicose 3-Epimerase
from Clostridium cellulolyticum H10
,†
†
†
†
‡
†
Wanmeng Mu,* Feifei Chu, Qingchao Xing, Shuhuai Yu, Leon Zhou, and Bo Jiang
†
State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, Jiangsu, People’s Republic of China
‡
Roquette America, Keokuk, Iowa 52632, United States
ABSTRACT: The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC
3
5319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli.
The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as
2+
D-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co . The optimum
pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and
2
+
1
0 min when incubated with and without Co , respectively, suggesting that this enzyme was extremely thermostable in the presence of
2+
Co but readily inactivated without metal ion. The MichaelisꢀMenten constant (K ), turnover number (k ), and catalytic efficiency
m
cat
ꢀ
1
ꢀ1
(
k /K ) values of the enzyme for substrate D-psicose were estimated to be 17.4 mM, 3243.4 min , and 186.4 mM min ,
cat m
respectively. The enzyme carried out the epimerization of D-fructose to D-psicose with a conversion yield of 32% under optimal
conditions, suggesting that the enzyme is a potential D-psicose producer.
KEYWORDS: D-psicose 3-epimerase, Clostridium cellulolyticum, D-psicose, purification, characterization
18
’
INTRODUCTION
and deposited the enzyme as GenBank accession no. ACO59490.
Although these three DTEase family enzymes can all produce D-
psicose from D-fructose, a low homology in amino acid sequence
was found among them.
Rare sugars were defined by the International Society of Rare
Sugars (ISIR) as monosaccharides and their derivatives that exist
in nature but are present in only limited quantities (First
International Symposium of ISRS, Takamatsu, Japan, 2002). D-
Psicose (D-ribo-2-hexulose or D-allulose), an epimer of D-fructose
18
In this study, the noncharacterized protein previously pro-
posed as xylose isomerase domain protein TIM barrel, which is
encoded by a hypothetical open reading frame Ccel_0941 in the
genome of the Clostridium cellulolyticum H10 (ATCC 35319),
was expressed in Escherichia coli and then characterized as
DPEase. The enzymatic properties, substrate specificity, and
kinetic parameters were also investigated and compared to other
DTEase family enzymes.
1
at the C3 position, is considered to be a rare sugar. This rare
2
sugar is absorbed poorly in the digestive tract, has zero energy
1
,3,4
for growth, and is a useful low-calorie sweetener.
It can be also
5
used as an inhibitor of hepatic lipogenic enzyme and intestinal
R-glycosidase for reducing body fat accumulation. It exists in
6
extremely small quantities in commercial carbohydrate or agri-
cultural products and is difficult to chemically synthesize. Inter-
conversion between D-fructose and D-psicose by epimerization
using D-tagatose 3-epimerase (DTEase) family enzymes has been
focused on as a commercially attractive enzymatic reaction for
’ MATERIALS AND METHODS
Chemicals and Reagents. Taq DNA polymerase, deoxynucleo-
side triphosphate (dNTP), chemicals for PCR, T4 DNA ligase, and
plasmid miniprep kit were obtained from Takara (Dalian, China). The
resin for protein purification, the Chelating Sepharose Fast Flow, was
obtained from GE (Uppsala, Sweden). The pET-22b(+) expression
vector was obtained from Novagen (Darmstadt, Germany). Electro-
phoresis reagents were purchased from Bio-Rad. Isopropyl β-D-1-
thiogalactopyranoside (IPTG) and all chemicals used for enzyme assays
and characterization were at least of analytical grade obtained from
Sigma (St. Louis, MO) and Sinopharm Chemical Reagent (Shanghai,
China). Oligonucleotides were synthesized by Sangon Biological En-
gineering Technology and Services (Shanghai, China).
7
ꢀ10
the production of D-psicose.
The genes of DTEase family enzymes are widely presumed in
various microorganisms and show relative sequence similarity,
indicating that the enzymes possibly have a common catalytic
mechanism; however, there are only several studies that char-
acterize the enzymes. DTEase was first purified and characterized
11,12
from Pseudomonas cichorii,
showing that it can efficiently
catalyze the epimerization of ketohexoses, and P. cichorii DTEase
7,8
has been successfully used in D-psicose mass bioproduction.
Kim et al. reported another characterization study of the putative
DTEase from Agrobacterium tumefaciens that can specifically
Bacterial Strains and Culture Conditions. C. cellulolyticum
H10 (ATCC 35319) was obtained from American Type Culture
13
catalyze the interconversion of D-fructose and D-psicose. Due
to its high substrate specificity for D-psicose, it was named D-
psicose 3-epimerase (DPEase). Soon after, the three-dimen-
19
Collection (ATCC, USA) and cultivated as described previously.
14
sional structures of A. tumefaciens DPEase and P. cichorii
Received: April 5, 2011
15,16
DTEase
were solved one after another, and the active sites were
Revised:
June 6, 2011
well identified. Recently, we have characterized a third DTEase
family enzyme, the DTEase from Rhodobacter sphaeroides SK011,
Accepted: June 13, 2011
Published: June 13, 2011
17
r 2011 American Chemical Society
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ARTICLE
The E. coli strains DH5R and BL21(DE3) were purchased from Sangon
Biological Engineering Technology and Services (Shanghai, China) and
used as host strains for transformation of plasmid and expression. They
were routinely cultured in LuriaꢀBertani (LB) medium supplemented
with ampicillin (50 μg/mL) at 37 °C with rotary shaking at 200 rpm.
Gene Cloning and Protein Expression. The genomic DNA of
C. cellulolyticum H10 was extracted by using the DNAzol reagent
according to the manufacturer’s protocol. The whole genome of this
strain was sequenced and released in GenBank (NCBI accession no.
NC_011898), which revealed that there is a putative gene, Ccel_0941,
encoded the putative protein, ACL75304. The protein was proposed as
the product xylose isomerase domain protein TIM barrel. According to
0
the nucleotide sequence of the putative gene, forward (5 -CGGG-
0
TGCATATGAAACATGGTATATACTACG-3 ) and reverse primer
0
0
(5 -GCTGGTCTCGAGGGAGTGTTTATGACATTCT-3 ) were de-
signed to introduce the NdeI and XhoI restriction sites (underlined).
Table 1. Amino Acid Sequence Homology of DTEase Family
Enzymes
sequence
A
length
(Aa)
sequence
B
length
(Aa)
amino acid
homology (%)
a
b
c
CC-DPEase
CC-DPEase
CC-DPEase
CC-DPEase
AT-DPEase
AT-DPEase
AT-DPEase
PC-DTEase
PC-DTEase
RS-DTEase
293
293
293
293
289
289
289
290
290
295
AT-DPEase
289
290
295
270
290
295
270
295
270
270
60
41
28
23
38
30
17
31
20
PC-DTEase
d
RS-DTEase
e
TM0416p
PC-DTEase
RS-DTEase
TM0416p
RS-DTEase
TM0416p
TM0416p
18
a
b
C. cellulolyticum DPEase, GenBank accession no. ACL75304. A.
c
tumefaciens DPEase, GenBank accession no. AAL45544. P. cichorii
DTEase, GenBank accession no. BAA24429. R. sphaeroides DTEase,
GenBank accession no. ACO59490. T. maritima TM0416p.
d
Figure 2. SDS-PAGE analysis of the purified C. cellulolyticum DPEase
(lane 2) and protein markers stained with Coomassie blue.
e
Figure 1. Multiple sequence alignment of DTEase family enzymes and their homologues. The amino acid sequence for DPEase from C. cellulolyticum
H10 (CC-DPEase; GenBank accession no. ACL75304) was aligned with those of A. tumefaciens DPEase (AT-DPEase; AAL45544), P. cichorii DTEase
(PC-DTEase; BAA24429), R. sphaeroides DTEase (RS-DTEase; ACO59490), and T. maritima TM0416p. The alignment was performed using the
ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Amino acid residues that are identical in all of the displayed sequences are
marked by asterisks (*); strongly conserved or weakly conserved residues are indicated by colons (:) or dots (.), respectively. The residues involved in the
metal coordinating site (9), the residues responsible for the interaction between the enzyme and O1, O2, and O3 of D-fructose (b), and the residues
providing a hydrophobic environment around the substrate around the 4-, 5-, and 6-position of D-fructose (2) are symbolized according to the crystal
14
15
structures of A. tumefaciens DPEase and P. cichorii DTEase. .
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Journal of Agricultural and Food Chemistry
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PCR amplification was performed by Taq Plus DNA polymerase for
The recombinant E. coli for protein expression was cultivated with
shaking (200 rpm) in 400 mL of LB medium containing 50 μg/mL
ampicillin at 37 °C. When the OD600 reached 0.6, IPTG was added at
1 mM, and DPEase was induced and overexpressed at 30 °C for 5 h and
then harvested by centrifugation at 4 °C for 10 min at 10000g. The
enzyme was expressed as 6ꢁhistidine-tagged fusion protein, which was
available for affinity chromatography.
3
1
5 cycles consisting of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for
min, followed by a final extension step of 10 min at 72 °C. The 0.9 kb
PCR-amplified fragment was purified and then digested with NdeIandXhoI.
The digested fragment was inserted into the pET-22b(+) vector with NdeI
and XhoI sites; therefore, an in-frame fusion 6ꢁhistidine-tag sequence at the
C-terminus was provided in the reconstructed plasmid, named pET-Cc-dpe,
and the pET-Cc-dpe was transformed into E. coli BL21(DE3).
Protein Purification. To purify the recombinant DPEase, the
centrifuged cell pellets were resuspended in lysis buffer (50 mM Tris-
HCl, 100 mM NaCl, pH 7.5) and disrupted by sonication at 4 °C for
6
min (pulsations of 3 s, amplify 90) using a Vibra-Cell 72405 sonicator,
and cell debris was removed by centrifugation (20000g, 20 min, 4 °C).
The cell-free extract was applied onto a Chelating Sepharose Fast Flow
2+
resin column (1.0 cm ꢁ 10.0 cm), previously chelating Ni , and
equilibrated with a binding buffer (50 mM Tris-HCl, 500 mM NaCl,
pH 7.5). Unbound proteins were eluted from the column with a washing
buffer (50 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 7.5).
Then, the DPEase was eluted from the column with an elution buffer
(50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5). The
active fractions were pooled and dialyzed overnight against 50 mM Tris-
HCl buffer (pH 7.5) containing 10 mM ethylenediaminetetraacetic acid
(
5
EDTA) for 48 h at 4 °C. Subsequently, the enzyme was dialyzed against
0 mM EDTA-free Tris-HCl buffer (pH 7.5).
Enzyme Assay. The activity was measured by the determination of
the amount of produced D-psicose from D-fructose. The reaction mixture of
1
0
5
mL contained D-fructose (50 g/L), Tris-HCl buffer (50 mM, pH 8.0),
.1 mM Co , and 0.5 μM enzyme. The reaction mixture were incubated at
5 °C for 2 min, and the reaction was stopped after 10 min by boiling. The
2+
generated D-psicose was determined by the HPLC method. One unit of
enzyme activity was defined as the amount of enzyme catalyzing the
formation of 1 μmol of D-psicose/min at pH 8.0 and 55 °C.
Effect of Temperature and pH. The optimum temperature of
enzyme activity was measured by assaying the enzyme samples over the
range of 35ꢀ70 °C for 2 min. Two buffer systems, sodium phosphate
(
50 mM, pH 6.0ꢀ7.0) and Tris-HCl (50 mM, pH 7.5ꢀ9.0), were used
for measuring the optimum pH of enzyme activity. The thermal stability
of the enzyme was studied by incubating the enzyme in Tris-HCl buffer
(50 mM, pH 8.0) at various temperatures. At given time intervals,
samples were withdrawn and the residual activity was measured under
standard assay conditions. To determine the pH stability, the enzyme
was incubated at pH 6.0ꢀ9.0 at 4 °C for up to 2 h, and the remaining
enzyme activity was measured at time intervals under standard assay
conditions.
Figure 3. Effect of pH (A) and temperature (B) on C. cellulolyticum
DPEase. Values are the mean of three replications ( standard deviation.
Table 2. Comparison of Biochemical Properties of DTEase Family Enzymes
strain for enzyme source
1
3
12
18
C. cellulolyticum
A. tumefaciens
P. cichorii
R. sphaeroides
optimal temperature (°C)
optimal pH
55
50
60
40
8.0
Co
8.0
7.5
9.0
Mn
2
+
2+
2+
metal ion required
Mn
no
substrate with highest specificity
D-psicose
24 min (55 °C, without metal ion)
D-psicose
D-tagatose
D-fructose
a
half-life
63.5 min (50 °C)
8.90 min (55 °C)
3.99 min (60 °C)
NR
approximately 60 min (50 °C)
1
9
6
0 min (60 °C, without metal ion)
2
+
.5 h (55 °C, with 0.1 mM Co
.8 h (60 °C, with 0.1 mM Co
)
)
2
+
equilibrium ratio between D-psicose
and D-fructose
32:68 (55 °C)
32:68 (30 °C)
33:67 (40 °C)
20:80 (30 °C)
23:77 (40 °C)
a
NR, not reported.
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Journal of Agricultural and Food Chemistry
ARTICLE
enzyme solution purified by affinity chromatography was dialyzed
against 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA
for 48 h at 4 °C. Subsequently, the enzyme was dialyzed against 50 mM
EDTA-free Tris-HCl buffer (pH 7.5). Enzyme activity was then assessed
2+
as described above in the presence of the following ions at 1 mM: Co ,
2
+
2+
2+
2+
2+
2+
Mn , Mg , Fe , Ni , Zn , and Cu , respectively.
Determination of Kinetic Parameters. Kinetic parameters of
C. cellulolyticum DPEase were determined in 50 mM Tris-HCl buffer
2
+
(
pH 8.0) containing 0.1 mM Co and 5ꢀ200 mM substrate for reaction
at 55 °C. The enzyme reactions were stopped after 10 min by boiling,
and the amount of D-psicose was determined by the HPLC assay. Kinetic
parameters, such as the MichaelisꢀMenten constant (K
m
) and turnover
number (kcat) values for substrates, were obtained using the Line-
weaverꢀBurk equation and quantification of enzyme concentration.
Analytical Methods. The concentrations of D-fructose and D-psicose
were analyzed by HPLC equipped with a refractive index detector and a
2+
Ca -carbohydrate column (Waters Sugar-Pak 1, Waters Corp., Milford,
MA), which was eluted with water at 85 °C and 0.4 mL/min. Protein
concentration was determined according to the method of Bradford using
bovine serum albumin as a standard. SDS-PAGE was carried out according
to the method of Laemmli. Gels (12% w/v polyacrylamide) were stained
with Coomassie Brilliant Blue and destained with an aqueous mixture of
10% (v/v) methanol/10% (v/v) acetic acid.
’
RESULTS AND DISCUSSION
Amino Acid Sequence Alignment of DTEase Family En-
zymes. DTEase family enzymes catalyze C3 epimerization of
various ketohexoses, including rare sugars that exhibit physiolo-
gical functions and promote beneficial health for humans. They
have been confirmed as the key enzymes for the bioproduction of
valuable rare sugars and, in particular, commercially used for the
production of D-psicose from D-fructose. Although many genes of
the DTEase family enzymes have been predicted from various sources
(
GeneBank accession no. ACO59490, BAA24429, F72381,
AL939126, BAB50266, AAL45542, NP_865388, BX294153,
NC_005126, and NP_435986), to date, however, only three
enzymes have been conclusively characterized as DTEase family
13
12
enzymes, including P. cichorii DTEase, A. tumefaciens DPEase,
18
and R. sphaeroides DTEase. A DTEase-related protein (TM0416p)
from the hyperthermophilic bacterium Thermotoga maritima was
purified and its crystal structure resolved; however, it was
suggested that the substrate specificity of TM0416p was likely
to differ substantially from those of DTEase family enzymes, and
it was also not confirmed whether the protein could convert D-
20
fructose to D-psicose. The amino acid sequence alignment
among P. cichorii DTEase, A. tumefaciens DPEase, R. sphaeroides
DTEase, and T. maritima TM0416p was developed, and the
results revealed that the protein sequence homology of DTEase
family enzymes previously characterized was not significant, with
the highest value of 38% similarity between P. cichorii DTEase
and A. tumefaciens DPEase, as shown in Table 1 and Figure 1.
The C. cellulolyticum H10 genomic sequence was recently com-
pleted and deposited as GenBank accession no. NC_011898.
The protein with ProteinID ACL75304 encoded by the hypothe-
tical open reading frame Ccel_0941 was supposed as the product
xylose isomerase domain protein TIM barrel in the C. celluloly-
ticum H10 genome. However, the ACL75304 was found to be
highly homologous in amino acid sequence with A. tumefaciens
DPEase (60% identity), as shown in Table 1 and Figure 1. Also,
ACL75304 exhibited 41, 28, and 23% amino acid sequence
similarities with P. cichorii DTEase, R. sphaeroides DTEase, and
T. maritima TM0416p, respectively.
Figure 4. Effect of pH and temperature on the stability of C. cellulolyticum
DPEase. (A) Effect of pH. The pH stability was investigated by preincubat-
ing the enzyme buffers of different pH values at 4 °C for 2 h and measuring
the remaining activity at 55 °C and pH 8.0. (B) Effect of temperature on
enzyme stability without metal ion. The temperature stability was investi-
gated by exposing the enzyme at 45 °C (9), 50 °C (b), 55 °C (2), and
6
0 °C (1) for different time intervals at pH 8.0. (C) Effect of temperature
2+
on enzyme stability with 0.1 mM Co . The temperature stability was
investigated by exposing the enzyme at 55 °C (9) and 60 °C (b) for
2+
different time intervals at pH 8.0 in the presence of 0.1 mM Co .
Effect of Various Metallic Ions. Before the effects of various
metallic ions on C. cellulolyticum DPEase activity were studied, the
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Journal of Agricultural and Food Chemistry
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Table 3. Comparison of the Epimerization Activities of DTEase Family Enzymes
relative activity (%)
a
13
12
18
ketose
D-psicose
C. cellulolyticum DPEase
A. tumefaciens DPEase
P. cichorii DTEase
R. sphaeroides DTEase
100 ( 0.4
4.9 ( 0.4
48.2 ( 2.3
0.9 ( 0.2
0
100
33.7
100
167
33.3
33.3
0
100
109.9
181.3
34.1
0
D-tagatose
D-fructose
50.7
0.7
0
D-sorbose
D-fructose-6-phosphate
D-ribulose-5-phosphate
0
0
0
0
a
2+
Experiments were carried out at 55 °C and pH 8.0 in the presence of 0.1 mM Co . Values are the mean of three replications ( standard deviation.
Table 4. Comparison of the Kinetic Parameters of DTEase Family Enzymes
a
13
12
C. cellulolyticum DPEase
A. tumefaciens DPEase
P. cichorii DTEase
k
cat
ꢀ
K
m
k
cat/ K
ꢀ1
m
k
cat
ꢀ1
K
m
k
cat/ K
ꢀ1
m
k
cat
ꢀ1
K
m
k
cat/ K
ꢀ1
m
1
ꢀ1
ꢀ1
ꢀ1
substrate
(min
)
(mM)
(mM min
)
(min
)
(mM)
(mM min
)
(min
)
(mM)
(mM min
)
b
D-psicose
D-tagatose
D-fructose
3243.5 ( 56.5
184.8 ( 9.6
17.4 ( 0.2
244 ( 5
186.4 ( 1.9
0.75 ( 0.01
62.7 ( 1.5
2381
12
762
24
205
NR
NR
55
NR
NR
NR
270
0.35
NR
NR
3354.5 ( 47.2
53.5 ( 1.8
2068
85
2+
NR
a
This study. Experiments were carried out at 55 °C and pH 8.0 in the presence of 0.1 mM Co ; values are the means of three replications ( standard
b
deviation. NR, not reported.
1
4
The crystal structures of A. tumefaciens DPEase and P. cichorii
pET-22b(+), revealed the presence of large amounts of protein
around 31 kDa, which was in agreement with the predicted
molecular mass for the DPEase protein. It was suggested that
DPEase could be overexpressed by IPTG induction. The E. coli
cells were harvested and disrupted by sonification. Cell debris
was removed by centrifugation (20000g, 20 min, 4 °C). The
supernatant was the crude extract of the enzyme, which was then
further purified by Ni -Chelating Sepharose Fast Flow resin.
Recombinant DPEase with a C-terminal 6ꢁhistidine-tag was
purified to electrophoretic homogeneity with a single band on
SDS-PAGE by metal chelating affinity chromatography (Figure 2).
The enzyme exhibited 32 and 130 kDa as apparent molecular
mass through SDS-PAGE and gel filtration, respectively. It was
indicated that the enzyme is a tetramer with four identical
subunits.
15
2+
DTEase have both been determined. The Mn ion is coordinated
by Glu150, Asp183, His209, and Glu244 in A. tumefaciens DPEase
or with Glu152, Asp185, His211, and Glu246 in P. cichorii
DTEase, and these residues are found to be strictly conserved in
ACL75304 as Glu150, Asp183, His209, and Glu244, respectively. In
addition, the amino acid residues responsible for the interaction
between the enzyme and O1, O2, and O3 of D-fructose (Glu156,
14
15
2+
14
His186, and Arg215 in A. tumefaciens DPEase or Glu158, His188,
15
and Arg217 in P. cichorii DTEase ) are also conserved in
ACL75304 as Glu156, His186, and Arg215, respectively. As for
the amino acid residues providing a hydrophobic environment
around the substrate, ACL75304 is completely conserved with A.
tumefaciens DPEase, in which Tyr6, Trp14, Gly65, Ala107,
Trp112, and Phe246 create a hydrophobic pocket around the 4-,
14
5
-, and 6-positions of D-fructose, but less well conserved with P.
Effects of Metal Ions on C. cellulolyticum DPEase. The
relative activity of C. cellulolyticum DPEase was investigated in
the presence of several divalent metal ions, which were added at
the final concentration of 1 mM. In previous findings, the enzyme
activities of A. tumefaciens DPEase and R. sphaeroides DTEase
were enhanced in the presence of metal ions. Interestingly, the P.
cichorii DTEase provided by Phe7, Trp15, Cys66, Leu108, Trp113,
15
and Phe248. Due to the high similarities with A. tumefaciens
DPEase and P. cichorii DTEase in amino acid sequence, especially
the key amino acid residues of the active site, metal coordinating site,
and substrate combinant binding site, ACL75304 is suggested as a
possible member of the DTEase family of enzymes.
1
2
cichorii DTEase does not require any cofactor for its activity.
In this study, we characterized this protein by experimental
approaches and confirmed it as DTEase family enzyme with high
substrate specificity for D-psicose. Therefore, we named
ACL75304 as C. cellulolyticum DPEase.
Expression and Purification of C. cellulolyticum DPEase.
To investigate the biochemical properties of C. cellulolyticum
DPEase, we expressed the enzyme with a C-terminal 6ꢁhistidine-
tag in E. coli BL21(DE3) and purified the protein to homo-
geneity. Bacteria transformed with the expression vector and
induced with IPTG abundantly expressed the 6ꢁhistidine-
tagged protein. SDS-PAGE analyses on the extracts of E. coli
BL21(DE3) harboring pET-Cc-dpe induced by IPTG, compared
to that of the control E. coli BL21(DE3) cells harboring plasmid
Opposite from P. cichorii DTEase, C. cellulolyticum DPEase
showed null activity in the absence of any ions, which indicated
that it is an enzyme with a strict divalent metal ion requirement.
C. cellulolyticum DPEase showed a maximal activity in the
2
2+
2+
presence of Co ; however, when Co was replaced with Mn
+ 2+ 2+ 2+
, Fe , Ni , or Mg , the enzyme activity was reduced to 92, 67,
2+
2+
65, and 38% of that in presence of Co , respectively. For Zn
2+
and Cu , the enzyme activity was barely detected. Therefore, C.
cellulolyticum DPEase is obviously different from other DTEase
family enzymes in metal ion dependence. Every reported
DTEase family enzyme could display activity without metal
ion, but C. cellulolyticum DPEase is strictly metal-dependent. It
is interesting that for aldose isomerase or ketose epimerase some
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Journal of Agricultural and Food Chemistry
ARTICLE
2
1
enzymes must require metal ions as cofactors to display activity,
some do not require metal ions,
12,22,23
and others could display
activity without metal ion, but their activity could be remarkably
13,18
enhanced by metal ions.
Effects of Temperature and pH on C. cellulolyticum DPEase.
Figure 3 shows pH and temperature profiles for C. cellulolyticum
DPEase activity. The optimum pH and temperature for enzyme
activity are 8.0 and 55 °C. Comparison of the biochemical properties
of C. cellulolyticum DPEase and other reported DTEase family
enzymes is shown in Table 2. All four of these enzymes’ optimal pH
values were found to be weakly alkaline, ranging from 7.5 to 9.0, and
optimal temperatures ranged from 40 to 60 °C.
Furthermore, pH and thermal stabilities of the enzyme were
investigated. More than 90% activity of the purified C. celluloly-
ticum DPEase was retained at pH 6.5ꢀ9.0 after incubation at
4
°C for 2 h (Figure 4A), indicating that the C. cellulolyticum
DPEase was relatively stable under neutral and weakly alkaline
conditions. In the absence of metal ion, the C. cellulolyticum
DPEase was relatively stable below 45 °C but decreased above
5
0 °C with increasing incubation time (Figure 4B). The results
13
were very similar to those of A. tumefaciens DPEase.
2+
In addition, we found that Co could remarkably enhance the
thermostability of C. cellulolyticum DPEase (Figure 4C), and it
was the first study about the effect of metal ion on the thermo-
stability of DTEase family enzymes. When incubated with
2
+
0
.1 mM Co , the half-lives for the enzyme could extend to 9.5
and 6.8 h at 55 and 60 °C, respectively, which indicated that the
2+
enzyme is extremely thermostable in the presence of Co .
However, without metal ion, the half-lives for the enzyme were
only 24 and 10 min at 55 and 60 °C, respectively, so C.
2+
cellulolyticum DPEase requires Co for both enzyme activity
and thermostability.
Substrate Specificity and Enzyme Kinetics. Epimerization
activities of C. cellulolyticum DPEase with various substrates were
carried out (Table 3). It was concluded that the substrate spe-
cificity of C. cellulolyticum DPEase was prior to D-psicose and
decreased for other substrates in the following order: D-fructose,
D-tagatose, and D-sorbose. As reported for other DTEase family
1
2,13,18
enzymes,
C. cellulolyticum DPEase also could not catalyze
D-fructose-6-phosphate or D-ribulose-5-phosphate. The substrate
specificity of C. cellulolyticum DPEase was similar to that of A.
13
tumefaciens DPEase, but different from that of P. cichorii
DTEase and R. sphaeroides DTEase. The optimal substrates of
1
2
18
P. cichorii DTEase and R. sphaeroides DTEase were D-tagatose
and D-fructose, respectively. Interestingly, C. cellulolyticum
DPEase activity on D-psicose was 10-fold higher than for D-tagatose,
which showed a higher multiple than that of A. tumefaciens
13
DPEase at 2.7-fold. However, the epimerization activity on D-
tagatose was 1.67- and 1.1-fold higher than that on D-psicose for
1
2
18
P. cichorii DTEase and R. sphaeroides DTEase, respectively.
The kinetic parameters of C. cellulolyticum DPEase were
determined (Table 4). The K , k , and catalytic efficiency
Figure 5. HPLC analysis profiles of epimerization products and equi-
librium ratio between D-fructose and D-psicose: (A, B) standards of
D-fructose and D-psicose, respectively; (C) reaction products after the
reaction equilibrium.
m
cat
(
k /K ) values of C. cellulolyticum DPEase for substrate D-
cat m
1
ꢀ
psicose were estimated to be 17.4 mM, 3243.4 min , and
ꢀ
1
1
86.4 mM min , respectively. The k /K of C. cellulolyticum
cat m
DPEase for D-psicose was 2.97- and 248-fold higher than those
for D-fructose and D-tagatose, respectively, indicating that the
enzyme was not DTEase, but DPEase. These results showed a
DPEase. However, the catalytic efficiency of other DTEase family
enzymes was not reported.
Bioconversion of D-Psicose by C. cellulolyticum DPEase.
The equilibrium ratio between D-psicose and D-fructose of C.
cellulolyticum DPEase was determined to be 32:68 when D-psicose
and D-fructose were at a total concentration of 50 g/L, with three
13
relatively similar tendency with A. tumefaciens DPEase, in which
the k /K for D-psicose was 2.41- and 586-fold higher than for
cat
m
D-fructose and D-tagatose, respectively. It was concluded that
we had characterized the second DPEase after A. tumefaciens
7
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Journal of Agricultural and Food Chemistry
ARTICLE
(
(
No. BE2010678 and BE2010626), and the PIRTJiangnan Project
No. 2008CXTD01).
’
ABBREVIATIONS USED
DTEase, D-tagatose 3-epimerase; DPEase, D-psicose 3-epimer-
ase; LB, LuriaꢀBertani; IPTG, isopropyl β-D-1-thiogalacto-
pyranoside; HPLC, high-performance liquid chromatography;
SDS-PAGE, sodium dodecyl sulfateꢀpolyacrylamide gel electro-
phoresis; EDTA, ethylenediaminetetraacetic acid.
’
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(
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’
AUTHOR INFORMATION
(
14) Kim, K.; Kim, H. J.; Oh, D. K.; Cha, S. S.; Rhee, S. Crystal
Corresponding Author
structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and
its complex with true substrate D-fructose: a pivotal role of metal in
catalysis, an active site for the non-phosphorylated substrate, and its
conformational changes. J. Mol. Biol. 2006, 361, 920–931.
*
Phone: (86) 510-85919161. Fax: (86) 510-85919161. E-mail:
wmmu@jiangnan.edu.cn.
Funding Sources
This work was supported by the NSFC Project (No. 20906040),
the Fundamental Research Funds for the Central Universities
(
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