Characterization of a novel D-arabinose isomerase from Thermanaeromonas toyohensis and its application for the production of D-ribulose and L-fuculose

Article abstract of DOI:10.1016/j.enzmictec.2019.109427

D-Ribulose and L-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of D-arabinose and L-fucose byDD-arabinose and L-fucose by D-arabinose isomerase from bacterial sources for the production of D-ribulose and L-fuculose have not yet become industrial due to the shortage of biocatalysts, resulting in poor yield and high cost of production. In this study, a thermostable D-ribulose- and L-fuculose producing D-arabinose isomerase from the bacterium Thermanaeromonas toyohensis was characterized. The recombinant D-arabinose isomerase from T. toyohensis (Thto-DaIase) was purified with a single band at 66 kDa using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight of 310 kDa, and the specific activities for both D-arabinose and L-fucose were observed to be 98.08 and 85.52 U mg^?1, respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn²⁺ was added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both D-arabinose and L-fucose at 75 °C after 10 h. The Michaelis-Menten constant (K_m), the turnover number (k_cat) and catalytic efficiency (k_cat/K_m) for D-arabinose/L-fucose were 111/81.24 mM, 18,466/10,688 min^?1, and 166/132 mM^?1 min^?1, respectively. When the reaction reached to equilibrium, the conversion rates of D-ribulose from D-arabinose and L-fuculose from L-fucose were almost 27% (21 g L^?1) and 24.88% (19.92 g L^?1) from 80 g L^?1 of D-arabinose and L-fucose, respectively.

Full text of DOI:10.1016/j.enzmictec.2019.109427

Enzyme and Microbial Technology 131 (2019) 109427
Contents lists available at ScienceDirect
Enzyme and Microbial Technology
journal homepage: www.elsevier.com/locate/enzmictec
Characterization of a novel D-arabinose isomerase from Thermanaeromonas
toyohensis and its application for the production of D-ribulose and L-fuculose
Muhammad Waheed Iqbala,b, Tahreem Riaz , Hinawi A.M. Hassanina,b, Dawei Nia,b
,
a,b
Imran Mahmood Khan , Abdur Rehman , Shahid Mahmood , Muhammad Adnana,b,
Wanmeng Mu
a
a,b
a,b
a,b
a,b,⁎
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu, 214122, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
D-arabinose isomerase
Thermostable
Thermanaeromonas toyohensis
D-arabinose
L-fucose
D-Ribulose and L-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the
agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are
generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of D-
arabinose and L-fucose byDD-arabinose and L-fucose by D-arabinose isomerase from bacterial sources for the
production of D-ribulose and L-fuculose have not yet become industrial due to the shortage of biocatalysts,
resulting in poor yield and high cost of production. In this study, a thermostable D-ribulose- and L-fuculose
producing D-arabinose isomerase from the bacterium Thermanaeromonas toyohensis was characterized. The re-
combinant D-arabinose isomerase from T. toyohensis (Thto-DaIase) was purified with a single band at 66 kDa
using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight
of 310 kDa, and the specific activities for both D-arabinose and L-fucose were observed to be 98.08 and
Characterization
−
1
2+
8
5.52 U mg , respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn
added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both D-
arabinose and L-fucose at 75 °C after 10 h. The Michaelis-Menten constant (K ), the turnover number (kcat) and
catalytic efficiency (kcat/K ) for D-arabinose/L-fucose were 111/81.24 mM, 18,466/10,688 min , and 166/
min , respectively. When the reaction reached to equilibrium, the conversion rates of D-ribulose
was
m
−
1
m
−1
−1
132 mM
−
1
−1
from D-arabinose and L-fuculose from L-fucose were almost 27% (21 g L ) and 24.88% (19.92 g L ) from
−
1
80 g L
of D-arabinose and L-fucose, respectively.
1. Introduction
anticancer drugs. Hence, our focus was to extract rare sugars such as D-
ribulose and L-fuculose by using a simple method from economical and
easily available sources [7].
Recently, rare sugars have fascinated the attention of researchers in
terms of their uses and numerous purposes. The enzyme D-arabinose
isomerase (D-AIase; EC 5.3.1.3), known as D-arabinose aldo-ketose iso-
merase, is very useful for producing D-ribulose from D-arabinose and L-
fuculose from L-fucose (Fig. 1). D-Ribulose and L-fuculose are applicable
in a variety of applications in different fields, but the production of
these rare sugars requires a chemical method that is time-consuming
and laborious. D-Ribulose is a rare aldopentose that plays a crucial role
in antineoplastic, antiviral and antitumoral activities [6], while L-fu-
culose is one of the most important rare sugars and has been applied for
a variety of purposes in pharmaceutical industrial molecules and ma-
terials. This rare sugar has been used to combat different kinds of dis-
eases such as hepatitis B, HIV, cardioprotective, antiviral and
According to the BRENDA enzyme database, D-arabinose isomerase
(D-AIase) is usually produced by Klebsiella pneumoniae (K. pneumoniae)
and Escherichia coli (E. coli) and recent studies also showed that D-ara-
binose could be metabolized by Salmonella typhimurium (S. typhi-
murium). The pH range for K. pneumoniae is 7.0–10, and manganese
(Mn2 ) metal is applied to enhance the enzyme activity [8]. Therma-
naeromonas toyohensis (Thto) is a strictly anaerobic, gram-positive,
thermophilic and thiosulfate-reducing bacterium that was screened
from a geothermal aquifer in the Toyoha Mines in Japan at a depth of
550 m, which was used for this research to produce D-AIase [9]. D-Xy-
lose isomerase and L-arabinose isomerase have been studied in different
kinds of microorganisms, such as numerous thermophiles, mesophiles
+
⁎
Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
E-mail address: wmmu@jiangnan.edu.cn (W. Mu).
https://doi.org/10.1016/j.enzmictec.2019.109427
Received 30 May 2019; Received in revised form 9 August 2019; Accepted 10 September 2019
Available online 11 September 2019
0141-0229/ © 2019 Elsevier Inc. All rights reserved.

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
Fig. 1. Structural illustration for enzymatic production of D-ribulose and L-fuculose from D-arabinose and L-fucose, respectively, with the help of recombinant Thto-
DaIase.
and hyperthermophiles [10,11]. Only three kinds of bacterial strains for
D-AIase have been examined until now, such as Aerobacter aerogenes (A.
aerogenes also known as K. aerogenes), K. pneumoniae and Bacillus pal-
lidus (B. pallidus) [6]. Although there have only been a few bacterial
strains used to characterize D-arabinose isomerase [12], T. toyohensis
was reported for the first time as a strictly thermostable bacterial strain
encoding a D-arabinose isomerase named Thto-DaIase.
The thermostable enzyme has a number of benefits due to its ability
to withstand high temperatures, resistant to chemical and biochemical
denaturation, increased reaction velocities, increased substrate solubi-
lity at high temperature, reduced risk of contamination and reduced
number of undesirable byproducts during reactions [13]. The Thto-
DaIase used in the catalytic reaction to isomerize aldose to ketose also
promotes the formation of D-allulose from D-altrose (rare sugars), which
is naturally present in very small amounts, via opening the ring of the
substrate. Rare sugars have many advantages in the pharmaceutical,
food, and agriculture industries and have various physiological func-
tions. D-AIase, used against fucosidosis (human lysosomal disease), is
affected by glycolipid and glycoprotein degradation. There are 36 kinds
of hexoses and pentoses; among the 36 (24 aldoses and 12 ketoses),
only seven are present in nature, while all others are called rare sugars
2. Materials and methods
2.1. Materials and chemicals
The resin column used to purify overexpressed T. toyohensis D-ara-
binose isomerase from recombinant E. coli, a fast flow chelating se-
pharose column, was obtained from GE Healthcare (Uppsala, Sweden).
The isopropyl-β-D-1-thiogalactopyranoside (IPTG), D-arabinose, L-fu-
cose and all other chemicals and reagents were purchased from Sigma-
Aldrich (St Louis, MO, USA) and Bio-Rad (Hercules, CA, USA). The
plasmid was synthesized and constructed by Sangon Biological
Engineering Technology and Services (Shanghai, China). The E. coli
strain of DH5α (Invitrogen, Carlsbad, CA, USA) was used in this study
for cloning and propagating the plasmid.
2.2. Gene cloning and expression
The putative enzyme was obtained from the NCBI-BLAST tool
(
public database), and the protein sequence was acquired from an
earlier deposited protein in GenBank (WP_084665866.1). ESPript soft-
ware was used to generate the protein sequence alignment. According
to NCBI, the complete genome of T. toyohensis was discovered, se-
quenced, and then published in GenBank (NCBI) with the reference
sequence or accession No. WP_084665866.1. The sequence has dif-
ferent site interfaces: trimer interfaces for polypeptide binding, sub-
strate binding sites for chemical binding and Mn2+ binding sites for ion
binding. The DNA of the target gene and encoded protein of T. toyo-
hensis (protein ID No. WP_084665866.1) was constructed by a com-
mercial synthesis company (Generay Biotech Co., Ltd, Shanghai, China)
and cloned into vector pET-22b (+) (Novagen, Darmstadt, Germany)
with NdeI and XhoI sites to construct an in-frame fusion with a 6 × His-
tag sequence at the C-terminus [17,18]. The resulting plasmid was
Thto-DaIase in pET-22b (+), and D-AIase was transformed and
[
14]. Currently, new strategies have been completely developed for the
bioproduction of rare sugars; so, D-AIase is considered to have much
potential for industrial application. Earlier reports showed that D-psi-
cose/D-allulose, D-ribulose, L-fuculose, L-mannose, D-talitol, L-ribose are
rare sugars produced by enzymatic methods [15,16].
In this study, a highly thermostable D-arabinose isomerase from the
novel thermophilic bacterium T. toyohensis (Thto-DaIase) was cloned
and overexpressed in E. coli. The biochemical properties (molecular
mass, pH, optimum temperature, metal ions and thermostability) were
examined. Kinetic parameter analysis, molecular modeling and se-
quence similarity were identified by amino acid sequence alignment.
2

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
overexpressed in E. coli BL21. The cells harboring Thto-DaIase were
cultivated in Luria–Brentani (LB) medium with ampicillin at a final
2.5. Effects of pH and temperature on the activity of recombinant Thto-
DaIase
−
1
concentration of 100 μg mL
and incubated at 37 °C until the optical
−1
density (OD600) reached 0.6-0.8. IPTG (1 mM L ) was added for in-
duction, and after 6–10 h, D-AIase expressing cells were grown at 28 °C
with shaking at 200 rpm.
The enzyme activity was determined by using a variety of buffer
solutions with various pH ranges (5–10.5), and the optimal pH values
were detected. Three different buffers with different pH were used:
sodium phosphate buffer (50 mM, pH 5.0–7.0), Tris−HCl (50 mM, pH
7.0–9.0) and glycine-NaOH (50 mM, pH 9.0–10.5). The pH stability was
2
.3. Protein purification and molecular mass determination of recombinant
tested by incubating the enzyme in various pH ranges (5.0–10.5) at 4 °C
and 75 °C for 24 and 1 h, respectively. The optimal temperature for the
enzyme activity with glycine-NaOH buffer at pH 9.0 was evaluated at
35–95 °C. The thermostability of Thto-DaIase was studied by incubating
the enzyme in 50 mM glycine-NaOH buffer at pH 9.0 and various
temperatures 45, 55, 65, 75, 85 and 95 °C within specific intervals of
times. The effects of thermostability on Thto-DaIase were determined
by log k (k: rate constant of thermal denaturation) and 1/T with an
Arrhenius plot using the k = 0.693/t1/2 equation.
Thto-DaIase
The purification steps of D-arabinose isomerase from T. toyohensis
Thto-DaIase) were executed at 4 °C. The recombinant protein-expres-
sing cells were collected from the broth culture by centrifugation at
0,000 × g for 15 min at 4 °C, and then these cells were resuspended in
(
1
lysis buffer (50 mM Tris−HCl buffer, 100 mM NaCl) at pH 7.0. After
mixing, the cell disruption was performed at 4 °C by using an ultra-
sonication Vibra-Cell 72,405 Sonicator (Bio-Block Scientific, Illkirch,
France). The denatured and unlysed cells were discarded by cen-
trifugation (10,000 × g, 20 min, 4 °C), and the supernatant was used as
a crude extract of Thto-DaIase. The crude enzyme was applied to a resin
column (chelating sepharose fast flow 1 cm × 10 cm) charged with
2.6. Effects of metal ions on recombinant Thto-DaIase activity
As previously described, reactions were achieved in 50 mM glycine
buffer (pH 9.0) comprising substrates (D-arabinose/L-fucose) and re-
combinant enzyme (Thto-DaIase) for 10 min at 75 °C. To inspect the
influence of metal ions on the activity of Thto-DaIase, the recombinant
enzyme was tested after treatment with EDTA and individual metal
2
+
Ni
NaCl, pH 7.0),
Novagen). The bound nondenatured protein was detached using a
followed by binding buffer (50 mM Tris−HCl buffer, 500 mM
a
process called nickel-affinity chromatography
(
2
+
2+
2+
2+
2+
2+
2+
2+
washing buffer (50 mM Tris−HCl buffer, 50 mM imidazole, 500 mM
NaCl, pH 7.0). The enzyme was eluted with elution buffer (50 mM
Tris−HCl buffer, 500 mM imidazole, 500 mM NaCl, pH 7.0), and the
solution containing purified enzyme was dialyzed overnight at 4 °C
against dialysis buffers A (50 mM Tris−HCl buffer, 10 mM EDTA, pH
ions, including Ca , Ni , Mn , Mg , Ba , Co , Fe , Zn and
Cu
2+
at concentrations of 1 mM. The measured activities in the pre-
sence of metals were compared with the enzyme activity without the
2
+
addition of a metal ion, which was used as a control. The effect of Mn
concentration for both D-arabinose and L-fucose was determined by
7
.0) and B (50 mM Tris buffer, pH 7.0).
The instinctive molecular mass for Thto-DaIase was determined by
incubating Thto-DaIase with different concentrations (1–5 mM) of
2+
Mn
at pH 9.0 at 75 °C for 10 min.
analyzing the purified enzyme with high-performance liquid chroma-
tography (HPLC, TSK G3000SW) column (Tosoh. Co., Ltd, Tokyo,
Japan). By using differential refractometer (Optilab T-rEX, CA, USA)
with a light-scattering detector (Dawn Heleos II, CA, USA) and a UV
detector (Waters 2489, USA), the purified enzyme was eluted in 50 mM
Tris−HCl buffer (pH 7.0) containing 300 mM NaCl with a flow rate of
2.7. Homology modeling and amino acid sequence comparisons of
recombinant Thto-DaIase
The homology structural and substrate docking of recombinant
Thto-DaIase was designed by using the SWISS-MODEL server (https://
swissmodel.expasy.org/), and the structure of Thto-DaIase was de-
termined experimentally by using a template of A. pallidus (PDB ID:
3A9T). The secondary structure of PDB ID: 3A9T was designed by
ESPript server (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) [4].
To verify and validate the structure, the verification server (http://
services.mbi.ucla.edu/SAVES/) and structure analysis were used, while
Discovery-studio were used to visualize and access the 3-dimensional
structure.
−1
1
mL min . The subunit molecular mass of purified Thto-DaIase was
calculated by SDS-PAGE (5% stacking gels and 12% resolving gels)
under denaturing conditions using the prestained ladder as a reference
protein (MBI Ferments, Glen Burnie, MD, USA). For the staining of
protein bands, Coomassie Brilliant Blue 250 was used, and the de-
staining solution was used for visualization.
2
.4. Determination of protein concentration and enzyme assay
To further analyze the amino acid sequence of Thto-DaIase, the
sequence alignment tool ClustalW2 (http://www.ebi.ac.uk/Tools/
clustalw2/index.html) was used to compare D-AIases from previously
reported bacteria. A phylogenetic relationship of recombinant Thto-
DaIase and previously identified bacteria with known accession num-
bers was created with MEGA-X using the neighbor-joining method. The
scale of the tree indicates the branch length of the same unit, which is
used to determine evolutionary distances based on phylogenetic trees
formed by the Poisson correction method, and the branch length (op-
timum sum) of the tree was 0.85878910. The phylogenetic tree analysis
involved 6 amino acid sequences [1–3].
The protein concentrations of Thto-DaIase were analysed by the
Bradford method using bovine serum albumin (BSA) as a standard
protein [19]. The activity of Thto-DaIase was determined by calculating
the amount of D-ribulose from D-arabinose and L-fuculose from L-fucose.
Experiments were performed on a high-performance liquid chromato-
graphy (HPLC) system (1200 Series; Agilent Technologies, Santa Clara,
CA, USA) linked with an infrared (IR) detector (Shodex, RI-101), and
the column was a SUGAR-PAK (6.5 × 300 mm) at 85 °C; the mobile
−1
phase was double distilled water at a 0.4 mL min
flow rate. D-Ara-
binose and L-fucose (substrates) were used to examine the activity of
Thto-DaIase, and the substrates were transformed into D-ribulose and L-
fuculose, respectively. The reaction was continued for 10 min at 75 °C
in 50 mM glycine buffer, pH 9.0, with D-arabinose and L-fucose and
purified Thto-DaIase. “The one unit of enzyme activity (U) was defined
as the amount of enzyme required to increase 1 μ mol of D-ribulose/L-
The number of amino acids, isoelectric point, molecular weight,
aliphatic index, instability index, estimated half-life and grand average
of hydropathicity (GRAVY) were assessed by ExPASy-Compute pI/M
tools at https://web.expasy.org/cgi-bin/protparam/protparam [20].
w
2.8. Specific activities, substrate specificities and kinetic parameters of
recombinant Thto-DaIase
−
1
fuculose min
at 75 °C and pH 9.0″.
Different substrates were used to analyze the specific activities and
3

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
specificities of Thto-DaIase; D-arabinose, L-fucose, D-altrose, L-xylose, L-
galactose, D-galactose and L-arabinose were used to a final concentra-
tion of 50 mM.
Different concentrations of D-arabinose, L-fucose, and L-xylose
(
10–300 mM) were applied to calculate the kinetics of recombinant
Thto-DaIase. The reactions were executed in 50 mM glycine-NaOH (pH
.0) at 75 °C for 10 min. The kinetic parameters, K (mM) and kcat
min ), were calculated using the Michaelis-Menten equation by a
nonlinear-regression method using GraphPad Prism software analysis.
9
(
m
−
1
2.9. Enzymatic production of D-ribulose andL-fuculose
The products D-ribulose and L-fuculose from D-arabinose and L-fu-
cose, respectively, by recombinant Thto-DaIase, were examined by
using HPLC with a sugar column (Waters SUGAR-PAK 1, Waters Corp.,
Milford, MA, USA). The mobile phase used to detect the sugars was
deionized water with an 85 °C column temperature and a flow rate of
−1
0.4 mL min
with the help of an attached refractive index detector.
The reaction mixture was prepared for the biological synthesis of D-
ribulose and L-fuculose in 50 mM glycine-NaOH, pH 9.0 at 75 °C with
2
+
1
8
mM Mn , and three different concentrations were used: 30, 50,
0 g L
−1
D-arabinose and 36, 50 and 80 g L−1 L-fucose.
3. Results and discussion
3.1. Homology and amino acid sequence comparison of putative Thto-
DaIase among other D-AIases
The hypothetical protein from T. toyohensis has 600 amino acids and
a
6
predicted isoelectric point and molecular weight of 5.5 and
5,800.97 Da, respectively. The derived sequenced amino acids of the
hypothetical protein from T. toyohensis (WP_084665866.1) showed
4.62, 71.67 and 69.32% identities with D-AIases from A. pallidus
7
(
(
WP_063389244.1), Dictyoglomus turgidum (D. turgidum) DSM6724
YP_002352328.1) and E. coli W (ADT76410.1), respectively (Table 1).
Thto-DaIase showed an aliphatic index of 84.08, which is higher than
2.82, 78.16 and 76.55 of D. turgidum, E. coli W and A. pallidus, re-
spectively. The instability index computed by ExPASy-Compute pI/M
8
w
tools was 33.30, which is less than 40, classifying the protein as stable,
and was lower than other comparable bacterial strains, such as D. tur-
gidum and E. coli W, which had instability indexes of 33.97 and 37.46,
respectively, and higher than that of A. pallidus at 29.18 (Table 1). The
estimated half-lives presented in Table 1 were 30 h, 20 h, and 10 h,
which corresponded to in vivo for yeast, in vitro for mammalian re-
ticulocytes, and in vivo for E. coli, respectively. The half-life values were
similar to D. turgidum, E. coli W and A. pallidus strains. The GRAVY was
−
0.183, which was closer to positive than those of D. turgidum and E.
coli W, which were -0.253 and -0.256, respectively (Table 1) [20].
The sequence alignment of Thto-DaIase with theD-AIases protein
sequences from previously characterized D-AIases-producing thermo-
stable bacteria is given in Fig. 2. The secondary structure was designed
from the previously reported 3-dimensional structure of A. pallidus
(
PDB ID: 3A9T) by the ESPript server [4]. The multiple sequence
alignment from T. toyohensis (GenBank: WP_084665866.1) compared
with A. pallidus (GenBank: WP_063389244.1), D. turgidum DSM6724
(
GenBank: YP_002352328.1), and E. coli W (GenBank: ADT76410.1),
showed that all the sequences have more than 65% similarity. The red
background showed the residues that were strictly conserved, the red
type in blue boxes revealed highly conserved residues, and elements for
the secondary structure were reported by comparing Thto-DaIase with
the previously discovered structure of D-AIase from A. pallidus with the
PDB ID: 3A9T, shown as star symbols on the residues (Fig. 2).
The phylogenetic relationship among different D-AIase-producing
bacteria, which was previously characterized, showed that T. toyohensis
producing D-arabinose isomerase (Thto-DaIase) has a closer relationship
with Caldicellulosiruptor saccharolyticus (C. saccharolyticus) and D.
4

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
Fig. 2. Amino acid alignment of recombinant Thto-
DaIase. The GenBank accession No. of the D-arabi-
nose isomerases were Aepa-DaIase: A. pallidus
(
(
WP_063389244.1); Thto-DaIase: T. toyohensis
WP_084665866.1); Ditu-DaIase: D. turgidum DSM
6724 (YP_002352328.1); Esco-DaIase: E. coli
W
(ADT76410.1). The red background represents
strictly conserved residues and the red type in blue
boxes represent highly conserved residues. The star
symbols above residues indicate secondary structure
elements by the template A. pallidus with PDB ID:
3A9T [4]. ESPript was used to perform the alignment
[5]. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web
version of this article.)
5

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
Fig. 3. (A) The phylogenetic relationship of recombinant
Thto-DaIase with previously identified bacteria with known
accession numbers. The tree was generated with MEGA-X by
the neighbour-joining method. The branching point numbers
show bootstrap values with genetic distance values on the
substitution position [1–3]. (B) SDS-PAGE examination of
purified Thto-DaIase overexpressed in E. coli BL21; lane 1 is
protein marker, lane 2 indicates crude extract, lane 3 is a heat-
treated enzyme and lane 4 is the purified His-Trap column
product. (C) The native molecular mass of recombinant Thto-
DaIase determined by HPLC using the reference standards
apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehy-
drogenase (150 kDa), albumin (66 kDa), and carbonic anhy-
drase (29 kDa). The red circle at 310 kDa shows the native
molecular mass of recombinant Thto-DaIase. (For interpreta-
tion of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
−
1
turgidum (approximately 98%), therefore T. toyohensis was used to
characterize L-fucose isomerase (L-FIase) and D-AIase. The sequence si-
milarity between C. saccharolyticus and D. turgidum was more than 70%,
which provided evidence that their ancestors had a close relationship
among themselves. The phylogenetic tree revealed that T. toyohensis
also has similar phylogeny to B. pallidus, E. coli W, K. pneumoniae and A.
pallidus (Fig. 3A).
specific activities of 93 and 76 U mg
for D. turgidum and C. sacchar-
olyticus, respectively [13,21]. The protein obtained on each purification
step was used to evaluate its subunit molecular mass by SDS-PAGE,
which displayed a single band near 66 kDa (Fig. 3B). This value was
larger than the theoretical calculated molecular weight (65,800.97 Da)
of recombinant Thto-DaIase with 600 amino acid residues and a His-tag
on its C-terminus that was obtained by using ExPASy-Compute pI/M
w
tools. The molecular mass of recombinant Thto-DaIase was measured
by HPLC with a single peak at approximately 7.19 min in the elusion
profile. Fig. 3C shows that the molecular mass of Thto-DaIase measured
under nondenaturing circumstances was approximately 310 kDa by
comparing Thto-DaIase with standard curves of different reference
proteins, such as carbonic anhydrase (29 kDa), albumin (66 kDa), al-
cohol dehydrogenase (150 kDa), β-amylase (200 kDa) and apoferritin
(443 kDa), and native enzyme corresponding to 310 kDa. While the
theoretical molecular mass of one unit of recombinant Thto-DaIase is
approximately 66 kDa, the native molecular mass of the enzyme was
calculated to be 310 kDa, corresponding to four subunits. These results
specify that recombinant Thto-DaIase is a homotetramer that is similar
to the previously characterized D-arabinose from B. pallidus [22].
3
.2. Overexpression and purification of the recombinant Thto-DaIase
D-Arabinose isomerase from T. toyohensis, a putative gene with the
identical sequence as that from GenBank accession No.
WP_084665866.1 was emulated and expressed in E. coli. The enzyme
showed activity for both substrates D-arabinose andL-fucose. The un-
characterized Thto-DaIase was purified by solubilizing the protein with
heat treatment and affinity chromatography, obtaining a final pur-
ification of 32-fold, 98.08 U mg specific activity and 44% yield for D-
arabinose, and 35-fold, 85.52 U mg
for L-fucose (Table 2). The yield of active recombinant Thto-DaIase in E.
coli from cell culture was 2960 U L
−
1
−
1
specific activity and 48% yield
−
1
−1
for D-arabinose and 2360 U L
for L-fucose (Table 4), which had not been previously described. The
specific activity of Thto-DaIase was higher from the previously reported
Table 2
Purification of Thto-DaIase.
Substrates
D-Arabinose
L-fucose
Steps
Total Protein (mg)
Total Activity (U)
Specific Activity (U mg−1)
Yield
%)
Purification Folds
(
Crude Extract
Heat-Treatment
His-Trap
Crude Extract
Heat-Treatment
His-Trap
290
21.7
4.0
290
21.7
4.00
887
563
392
707
490
342
3.06
100
63
44
100
69
48
1
8
32
1
9
25.98
98.08
2.44
22.60
85.52
35
6

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
Fig. 4. Effect of pH, temperature and thermostability on
the activity of recombinant Thto-DaIase. (A) Effect of pH;
three different types of buffers at 50 mM [phosphate (pH
5
9
.0–7.0), Tris−HCl (pH 7.0–9.0) and glycine (pH
.0–10.5)] were used to determine the optimum pH of
recombinant Thto-DaIase for D-arabinose and L-fucose.
B) pH stability of recombinant Thto-DaIase for D-arabi-
(
nose and L-fucose; enzyme was incubated at different pH
values (5.0–10.5) at 4 °C for 24 h to evaluate the residual
activities. (C) Residual activities of recombinant Thto-
DaIase for D-arabinose and L-fucose incubated at different
pH values (5.0–10.5) at 75 °C for 1 h. (D) Effect of tem-
perature on the activity of recombinant Thto-DaIase for
D-arabinose and L-fucose; 50 mM glycine-NaOH with pH
9.0 was used. (E) Effect of the thermostability of re-
combinant Thto-DaIase for D-arabinose and L-fucose;
different time intervals 0–20 h and temperatures 45, 55,
65, 75, 85 and 95 °C were used to incubate the enzyme.
Data represent the mean of three replicates.
3
.3. Influences of pH, temperature, and thermostability on the activity and
DaIase exhibited 90% activity at 65 °C and 70% activity at 55 °C, which
demonstrated that Thto-DaIase is highly thermostable. The effect of
temperature on Thto-DaIase for the L-fucose substrate was measured to
be 50% activity at 55 °C and 80% at 65 °C (Fig. 4D). The results were
closely related to the findings of Ju and Oh (2010), as they found that
the optimum temperature for C. saccharolyticus D-AIase, which iso-
merizes D-arabinose and L-fucose, was 75 °C.
stability of recombinant Thto-DaIase
The influence of pH, temperature, and thermostability were mea-
sured and are demonstrated in Fig. 4. The activity of recombinant Thto-
DaIase was measured by using three different kinds of buffers: sodium
phosphate (pH 5.0–7.0), Tris−HCl (pH 7.0–9.0) and glycine-NaOH (pH
9
.0–10.5). The variation in pH values in the reaction mixture had sig-
Thto-DaIase was further tested at different temperatures (45, 55, 65,
75, 85 and 95 °C), and the activity was observed from 0, 5, 10, 15 and
20 h (Fig. 4E). The results showed that Thto-DaIase was more than 60%
active and stable with D-arabinose and L-fucose at 45, 55, 65, 75 and
85 °C for 5 h, while at 95 °C, it showed sudden inactivation and de-
creased the activity. After 10 h, the enzyme showed more than 60%
activity at 45, 55 and 65 °C, which is a property of thermostable en-
zymes. Moreover, the activity reduced when the temperature was in-
creased to 75, 85, and 95 °C. By increasing time and temperature, the
activity of Thto-DaIase with D-arabinose and L-fucose decreased; after
20 h, it retained approximately 40% activity at 65 °C (Fig. 4E). The half-
life (t1/2) of Thto-DaIase for D-arabinose, based on first-order kinetics,
was measured as 133, 32, 13, 5, 4 and 2 h at 45, 55, 65, 75, 85 and
95 °C, respectively. The present results revealed that recombinant Thto-
DaIase was more stable than the enzyme from D. turgidum, which ex-
hibited half-lives of 20, 12, 7, 5 and 2 h at 65, 70, 75, 80 and 85 °C,
respectively [13]. In the case of the L-fucose substrate, Thto-DaIase
exhibited 111, 27, 11, 5, 4 and 3 h half-lives at 45, 55, 65, 75, 85 and
95 °C, respectively. Ju and Oh (2010) reported that the half-life values
of L-fucose isomerase (L-FIase) from C. saccharolyticus at 60, 65, 75 and
80 °C were 62, 13, 6, 2 and 1 h, respectively, which were lower than
those of the present findings; therefore, the present findings are good
for industrial applications.
nificant effects on the dissociation state of the substrate and enzyme
complex that changed the enzyme activity. Thto-DaIase exhibited op-
timum activity at pH 9.0 in the glycine-NaOH buffer for D-arabinose and
L-fucose (Fig. 4A), which was similar to D-AIase from K. pneumoniae and
D-AIase from B. pallidus [6,12]. The enzyme exhibited minimum activity
at pH 5.0 in sodium phosphate for both D-arabinose and L-fucose, while
at pH 7.0, both substrates retained more than 50% activity, which was
increased by increasing the pH values. In Tris−HCl buffer (pH 7.0), the
enzyme retained about 70% activity for D-arabinose and L-fucose sub-
strates, which decreased by increasing the pH up to pH 9.0 (Fig. 4A). As
shown in Fig. 4A, Thto-DaIase displayed optimum catalytic activity
under alkaline conditions in a glycine-NaOH buffer (pH 9.0) for both
substrates D-arabinose and L-fucose, which was decreased by increasing
the pH from 9.0-10.5. The enzyme stability at different pH values was
assessed by incubating purified enzyme at different pH values
(
5.0–10.5) at 4 °C for 24 h (Fig. 4B). The enzyme fully retained its ac-
tivity from pH 5.0–10.5 in sodium phosphate buffer, Tris−HCl and
glycine-NaOH buffers, which revealed no significant effect on activity,
while at pH 9.0, the residual activity was increased for both substrates.
The enzyme was also incubated at 75 °C in different pH values for 1 h to
measure the thermal effects of pH on Thto-DaIase stability. The enzyme
fully retained its activity at all pH values and showed the highest ac-
tivities of at pH 9.0 for D-arabinose and L-fucose (Fig. 4C). The effects of
temperature on Thto-DaIase activity were assessed from 35 to 95 °C for
D-arabinose and L-fucose (Fig. 4D). The optimum activity was observed
at 75 °C for both substrates, although in the case of D-arabinose, Thto-
3.4. Effects of metal ions on recombinant Thto-DaIase activity
It was necessary to remove metal ions from the purified enzyme
7

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
Fig. 5. Effects of metal ions, the concentration of metal
ions, and production of D-ribulose/L-fuculose from re-
combinant Thto-DaIase (A) Influence of metal ions on the
activity of recombinant Thto-DaIase for D-arabinose and L-
fucose; reactions were performed in 50 mM of glycine
buffer at 75 °C for 10 min. The control showed a purified
2
+
enzyme without metal. (B) Effect of Mn
concentration
on the activity of recombinant Thto-DaIase for D-arabi-
nose and L-fucose. (C) D-ribulose production from D-ara-
binose by recombinant Thto-DaIase at different con-
−
1
centrations (30, 50 and 80 g L ) of D-arabinose. (D) L-
Fuculose production from L-fucose by recombinant Thto-
−
1
DaIase at different concentrations (36, 50 and 80 g L ) of
L-fucose.
with EDTA to attain the accurate effects of metals on enzyme activity
and subsequently dialyze against Tris−HCl buffer (pH 7.5) to remove
on Mn2+ to enhance the activity [12,22].
Among all other metal ions, Mn2+ significantly heightened the ac-
2
+
2+
2+
the effect of EDTA. Few metal ions Ca and Mg are already involved
in enzyme purification after affinity chromatography, which forces
tivity of Thto-DaIase; subsequently, different concentrations of Mn
(from 1 to 5 mM) were applied with D-arabinose and L-fucose (Fig. 5B).
Thto-DaIase showed the highest activity at 1 mM Mn , which was
2
+
2+
Mn
to bind to the binding site of the enzyme molecule. D-Arabinose
isomerase is a metalloenzyme in which divalent metal ions act as a
cofactor for the isomerization of rare sugars [21]. The recombinant
Thto-DaIase was incubated with various divalent metal ions (EDTA,
considered the optimal concentration for both substrates
(Fig. 5B). Mn2+ is a cofactor for L-fucose and D-arabinose isomerization
by L-FIase from C. saccharolyticus [21]. All reported D-AIases and L-
2
+
2+
2+
2+
2+
2+
2+
2+,
2+
2+
Ni , Fe , Zn , Mn , Co , Ca , Ba , Mg
and Cu ) at
FIases from C. saccharolyticus, B. pallidus and E. coli displayed Mn
concentrations of 1 mM (Fig. 5A). The results demonstrated that man-
ganese (Mn ) increased the activity of Thto-DaIase by 133% and 76%
for D-arabinose and L-fucose, respectively, with respect to the control
metal ion was used to activate and characterize the enzyme [6,13,21].
2
+
2
+
2+
2+
2+
2+
2+
2+
3.5. Substrate specificity of recombinant Thto-DaIase and enzyme kinetics
(
100%), while Ni , Fe , Zn , Ba , Ca , Mg , and Cu
de-
2
+
creased the activity of Thto-DaIase (Fig. 5A). Copper (Cu ) and EDTA
strongly declined the activity of the enzyme for both substrates. How-
enhanced the activity of Thto-DaIase significantly, which
corroborated that this enzyme is more dependent on Mn
Various reports are available on rare sugar production, such as D-
arabinose from D-ribulose, L-galactose from D-tagatose and L-galactose
from L-tagatose, that elaborate the catalytic reactions of several sub-
strates [23–26]. The hydroxyl groups present in aldose substrates or-
iented in the left-handed configuration at the C2 position and in the
right-handed configuration at the C3 and C4 positions, such as D-ara-
binose, L-fucose, D-altrose, and L-galactose. But the C5 has different
configuration for all of these substrates which is inactive during the
catalytic reaction. The open-chain structures of the aldose substrates
responsible for the aldose-ketose isomerization reactions catalyzed by
the D-arabinose isomerase. The specificity of recombinant Thto-DaIase
for different monosaccharides (substrate) of D- and L- forms (pentoses
2
+
ever, Mn
2
+
than the
also increased the
2
+
other metals tested to increase its activity. Co
enzyme activity by approximately 43% and 50% compared with the
control for D-arabinose and L-fucose respectively, which was highly
2
+
2+
dependent after Mn
(Fig. 5A). A previous study showed that Mn
and Co2 metal ions were highly effective for L-FIase from D. turgidum,
+
which promotes the isomerizing reactions of L-fucose and D-arabinose
and is highly thermostable [13]. D-Arabinose isomerase from K. pneu-
moniae and B. pallidus showed that this enzyme was highly dependent
Table 3
Substrate specificity and Kinetic parameters of recombinant Thto-DaIase.
m
Kcat/K )
(mM−1 min-1
Substrate
Product
Specific Activity (U mg−1)
K
m
(mM)
K
cat (min−1
)
D-Arabinose
L-fucose
D-Altrose
L-Galactose
L-Xylose
D-Galactose
L-Arabinose
D-ribulose
L-Fuculose
D-Allulose
L-Tagatose
L-Xylulose
D-Tagatose
L-Ribulose
98.0 ± 0.97
85.5 ± 1.81
15.0 ± 1.50
01 ± 0.32
01 ± 0.02
0.63 ± 0.26
0.00 ± 0.00
111 ± 1.7
81.2 ± 2.1
69 ± 2.4
ND
ND
ND
18,466 ± 27
10,688 ± 30
7328 ± 11
ND
ND
ND
ND
166 ± 2.8
132 ± 3.5
106 ± 2.0
ND
ND
ND
ND
ND
ND: not displayed.
8

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
and hexoses), such as D-galactose, D-altrose, L-fucose, L-galactose, D-
arabinose, L-xylose, and L-arabinose, was investigated (Table 3).
The recombinant Thto-DaIase exhibited the highest activity for D-
arabinose and L-fucose and moderate activity against D-altrose, whereas
the lowest activity was towards L-galactose, L-xylose, and D-galactose,
while no activity was observed against L-arabinose. The specific activ-
ities of recombinant Thto-DaIase for D-arabinose, L-fucose and D-altrose
were 1.6-fold, 1.12-fold and 1-fold higher, respectively from C. sac-
charolyticus. The results revealed that recombinant Thto-DaIase is more
efficient for the isomerization reaction of these substrates than D-AIase/
L-FIase from C. saccharolyticus [21]. The specific activity of recombinant
Thto-DaIase for D-arabinose and D-galactose was higher (3- and 1.25-
fold) and lower (1.08- and 1.53-fold) for L-fucose and D-altrose com-
pared to L-FIase/D-AIase from D. turgidum, respectively. Highly ther-
mostable recombinant Thto-DaIase showed higher activity for D-arabi-
nose, L-fucose and D-altrose, which were 2.64-, 2.25- and 15-fold,
respectively, and lower activity for D-galactose, which was 1.9-fold that
of D-AIase from K. pneumoniae [12].
The results shown in Table 3 prompted the researchers to further
study the isomerization and transformation processes to produce rare
sugars that are useable in the food and pharmaceutical industries. Thto-
DaIase enzymatically has the ability to isomerize two kinds of sub-
strates D-arabinose and L-fucose to produce D-ribulose and L-fuculose,
the most expensive sugars, respectively. This enzyme also introduced
activity towards D-altrose to D-allulose, which corroborates that Thto-
DaIase has different substrate binding sites than D-AIase from K. pneu-
moniae. Although similar to other kinds of isomerases used for the
transformation of rare sugars, Thto-DaIase has the ability to be used in
the formation of many rare sugars, such as D-ribulose from D-arabinose,
L-fuculose from L-fucose, D-allulose from D-altrose, D-tagatose from D-
galactose, L-xylulose from L-xylose and L-tagatose from L-galactose by an
isomerization process (Table 3). We revealed that Thto-DaIase is the
most promising enzyme for producing D-altrose from D-allulose and
needs further study.
The kinetic parameters of recombinant Thto-DaIase are given in
Table 3 for D-arabinose, L-fucose, and D-altrose. The Michaelis-Menten
constant (K ) and Kcat/K were higher for D-arabinose than for L-fucose
m m
and D-altrose, which indicates that the enzyme is a D-arabinose iso-
merase. Although, Thto-DaIase has a higher affinity for D-altrose than D-
arabinose and L-fucose but the isomerization rate of L-fucose and D-al-
trose was slower than that of D-arabinose. Table 4 demonstrates the
comparison of the biochemical and kinetic parameters of recombinant
Thto-DaIase with L-FIase/D-AIase from other microorganisms
[
12,13,21,22,27].
3.6. Bioproduction of D-ribulose and L-fuculose by recombinant Thto-DaIase
D-ribulose and L-fuculose were produced by recombinant Thto-
DaIase in 0.5 mL of the reaction mixture at pH 9.0 and 75 °C. By ap-
−1
plying 30, 50 and 80 g L of D-arabinose substrate, after 3, 5 and 10 h,
the enzymatic reaction reached approximate equilibrium by exhibiting
conversion ratios of 27.66, 27.04 and 26.25%, respectively (Fig. 5C).
After the reaction reached equilibrium, recombinant Thto-DaIase pro-
−
1
−1
duced 8.29, 13.52 and 21 g L of D-ribulose from 30, 50 and 80 g L
of D-arabinose, respectively, which was relatively high, while by ap-
−
1
plying 36, 50 and 80 g L
DaIase produced 9.31, 12.56 and 19.92 g L of L-fuculose with 25.87,
4.88 and 24.9% conversion ratios, respectively (Fig. 5D). The results
of L-fucose substrate, recombinant Thto-
−1
2
were compared with other bacteria producing the same isomerizing
enzyme, such as L-FIase from C. saccharolyticus used to isomerize L-fu-
cose and D-arabinose [21], which demonstrated a conversion ratio of
24% for both D-arabinose and L-fucose substrates at pH 7.0 and 70 °C
after 3 h. The D-AIase from K. pneumonia showed a 10% conversion ratio
for both substrates at pH 9.0 and 40 °C [12]. Reaction conditions such
as pH and temperature affect the conversion ratio and productivity.
Therefore, comparing the conversion yields of recombinant Thto-DaIase
9

M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
Fig. 6. 3-Dimensional structure of the active site of re-
combinant Thto-DaIase complexed with substrates D-
arabinose (a) and L-fucose (b) and the enzyme-substrate
complexes (c). The molecular model indicates the hy-
pothetical active-site residues of D-arabinose and L-fucose
that were closer to substrates and were used to enhance
the activity of recombinant Thto-DaIase. The dashed
lines show hydrogen bonds, while the green sticks show
the substrates. (For interpretation of the references to
colour in this figure legend, the reader is referred to the
web version of this article.)
with other D-ribulose- and L-fuculose-producing enzymes showed that
the present enzyme was acceptable, and no byproducts were detected.
Therefore, it tends to be a very important property for industrial ap-
plication because it can lower the cost of the downstream industry by
simplifying the purification process.
arabinose was applied, the conversion ratio of D-ribulose from D-ara-
−1
binose was approximately 27.66% and produced 21 g L of D-ribulose,
while for L-fuculose from L-fucose, a 24.88% conversion ratio was ob-
−1
served, with 19.92 g L
of production yield and no byproducts were
observed. The results showed that recombinant Thto-DaIase has great
potential for D-ribulose and L-fuculose production in industries with a
single enzyme by a simple purification method, which is good to lower
the cost for the downstream industry.
3.7. Docking and molecular modeling for active site residues of recombinant
Thto-DaIase
5. Ethical statement
The docked molecular model of Thto-DaIase for both substrates D-
arabinose and L-fucose was created by a previously known template
structure of A. pallidus (PDB ID: 3A9T) (Fig. 6) [4]. The recombinant
Thto-DaIase has 74.62% similarity with the template organism A. pal-
lidus; therefore, it was completely docked. Sometimes, environmental
changes affected the amino acid arrangements and cause problems
during mutation and docking [28,29].
The molecular model was used to identify specific residues with a
ligand-docking tool using a linear form of D-arabinose and L-fucose. The
molecular model of Thto-DaIase exhibiting the active site residues for D-
arabinose are Phe306, Gln307, Gly308, Gln309, Asn318, Gly319,
Asp320, Glu323, Thr343, Glu344, Asn399, Ser400, Gly401 and Ser402
This study does not involve any human testing.
Declaration of Competing Interest
The authors declare that they do not have any conflict of interest.
Acknowledgments
This work was funded by the National Natural Science Foundation
of China (No. 31801583), the Natural Science Foundation of Jiangsu
Province (No. BK20181343 and BK20180607), and the National First-
Class Discipline Program of Food Science and Technology (No.
UFSTR20180203)
(
Fig. 6A), and for L-fucose, Met192, Gln309, Glu344, Asp368, Ser400,
Asn533 and His534 (Fig. 6B), which showed a close relationship with
the substrates. These residues may impart a decisive role to enhance the
activity of the enzyme and could be designated as promising candidates
of enzyme activity enhancer. The active site residues presented parallel
geometry with the associated protein (Fig. 6C).
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