Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste receptor

Article abstract of DOI:10.1016/j.phytochem.2016.03.004

Steviol glycosides (SG's) from Stevia rebaudiana (Bertoni) have been used as a natural low-calorie sweeteners. Its aftertaste bitterness restricts its use for human consumption and limits its application in food and pharmaceutical products. In present study, we have performed computational analysis in order to investigate the interaction of two major constituents of SG's against homology model of the hTAS2R4 receptor. Molecular simulation study was performed using stevioside and rebaudioside A revealed that, sugar moiety at the C-3′′ position in rebaudioside A causes restriction of its entry into the receptor site thereby unable to trigger the bitter reception signaling cascade. Encouraged by the current finding, we have also developed a greener route using β-1,3-glucanase from Irpex lacteus for the synthesis of de-bittered rebaudioside A from stevioside. The rebaudioside A obtained was of high quality with percent conversion of 62.5%. The results here reported could be used for the synthesis of rebaudioside A which have large application in food and pharmaceutical industry.

Full text of DOI:10.1016/j.phytochem.2016.03.004

Phytochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Synthesis of rebaudioside A from stevioside and their interaction model
with hTAS2R4 bitter taste receptor
⇑
Ramit Singla, Vikas Jaitak
Centre for Pharmaceutical Sciences and Natural Products, School of Basic and Applied Sciences, Central University of Punjab, Bathinda(Pb) 151 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Steviol glycosides (SG’s) from Stevia rebaudiana (Bertoni) have been used as a natural low-calorie sweet-
eners. Its aftertaste bitterness restricts its use for human consumption and limits its application in food
and pharmaceutical products. In present study, we have performed computational analysis in order to
investigate the interaction of two major constituents of SG’s against homology model of the hTAS2R4
receptor. Molecular simulation study was performed using stevioside and rebaudioside A revealed that,
sugar moiety at the C-3 position in rebaudioside A causes restriction of its entry into the receptor site
thereby unable to trigger the bitter reception signaling cascade. Encouraged by the current finding, we
have also developed a greener route using b-1,3-glucanase from Irpex lacteus for the synthesis of
de-bittered rebaudioside A from stevioside. The rebaudioside A obtained was of high quality with percent
conversion of 62.5%. The results here reported could be used for the synthesis of rebaudioside A which
have large application in food and pharmaceutical industry.
Received 16 October 2015
Received in revised form 15 January 2016
Accepted 7 March 2016
Available online xxxx
0
0
Keywords:
Stevia rebaudiana (Asteraceae)
Steviol glycosides
Homology model
Ramachandran plot
hTAS2R4
b-1,3-Glucanase
Transglycosylation
Ó 2016 Elsevier Ltd. All rights reserved.
1
. Introduction
are extracted from a native shrub of Brazil and Paraguay, Stevia
rebaudiana Bertoni (Stevia). The sweetener mixture mainly consists
Gustatory reception in human has five widely accepted Descrip-
tors: sweet, umami, bitter, salty, and sour. Taste receptor cells
TRCs) and signaling elements have been recognized in the oral
cavity as well as taste buds present on the tongue
Chandrashekar et al., 2006). Sweet taste receptor (STR) belong to
class C of G-protein coupled receptor (C-GPCR) family (Adler
et al., 2000). Sweet taste reception is commenced by the activation
of heterogenic receptor, made up by a combination of hTAS1R2 and
hTAS1R3 protein (Mayank and Jaitak, 2015). In contrast to the sin-
gle receptor-based detection of sweet taste, bitter taste receptors
belong to frizzled receptor family of G-protein coupled receptor
of stevioside (6–10%), rebaudioside A (2–4%) and other minor
constituents up to 0.1–1%. (Jaitak et al., 2008; Singh et al., 2011).
Stevioside is reported to be 250–300 times and rebaudioside-A is
300–400 times sweetener than sucrose (Kochikyan et al., 2006).
Although, concentration of stevioside in S. rebaudiana leaves is
higher than rebaudioside-A but aftertaste bitterness of stevioside
and its low solubility in water restricts its use for human consump-
tion and limits its application in food and pharmaceutical products.
Rebaudioside-A, one of the other major constituents next to ste-
vioside in S. rebaudiana leaves is preferred than stevioside, being
more sweetener, high solubility in water and devoid of aftertaste
bitterness (McChesney, 2006; Nabors, 2001; O’Donnell and
Kearsley, 2012). In Dec 2008, US Food and Drug Administration
(FDA) granted approval to stevia containing a minimum of 95%
rebaudioside-A (Generally Recognized As Safe, GRAS) as general
purpose sweetener for use in foods and beverages (Carakostas
et al., 2008). The French Food Safety Agency (AFSSA), Food
Standards Australia New Zealand (FSANZ) EFSA, European Food
Safety Authority (EFSA) evaluated rebaudioside-A and certified a
positive safety assessment (Boileau et al., 2012; GRAS notice
000354). In September 2009, France authorised the use of rebau-
dioside-A as a sweetener at the national level. In comparison to
stevioside, the percentage of rebaudioside-A in S. rebaudiana leaves
is low, and also non-availability of rebaudioside-A genotypes limits
(
(
(Fredriksson et al., 2003). They exhibit unique but partially over-
lapping molecular receptive ranges because the transduction of
bitter taste in humans is mediated by 25 receptors of hTAS2R gene
family that are cluster of chromosomes 5p15, 7q31 and 12p13
(Singh et al., 2011). hTAS2Rs are between 290 and 333 amino acids
long, have seven transmembrane helices (TM1-TM7), a short extra-
cellular amino terminal and an intracellular carboxyl-terminal
(Upadhyaya et al., 2015).
Steviol glycosides (SGs) are natural sweeteners and belongs to
the category of ent-kaurene diterpenes (Jaitak et al., 2008) and
⇑
Corresponding author.
E-mail address: vikasjaitak@gmail.com (V. Jaitak).
http://dx.doi.org/10.1016/j.phytochem.2016.03.004
031-9422/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Singla, R., Jaitak, V. Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste
receptor. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.004

2
R. Singla, V. Jaitak / Phytochemistry xxx (2016) xxx–xxx
Table 1
Dock scores, protein–ligand interactions of hTAS2R4 for SG.
S. No.
Ligand
Dockscore
kcal/mol)
Ligand interaction
(
1
2
Stevioside
Rebaudioside A
ꢀ13.385
ꢀ8.761
Val 70 , Tyr 147, Glu 172, Asn 169
Ile 170. Lys 262, Gln 249, Met 257,
Asp 258
Models of hTAS2R4 were generated by I-TASSER, that utilizes
threading, ab initio replica-exchange Monte Carlo modeling
simulations and structural refinement (Yang et al., 2015b). Eight
templates having PDB id 4GRV, 4BWB, 4N6H, 4IAR, 2Z73, 2DJH,
4
BUO and 4EA3 were found to be optimum to build the homology
model. Models were evaluated from C-score.
Subsequently final model was selected by the assessment of
Ramachandran plot (Fig. 1) created by RAMPAGE server to deter-
mine if the model is folded correctly about the solved crystal struc-
ture of proteins available in protein data bank (Lovell et al., 2003).
The overall G-score as evaluated by Procheck was -0.11 for
hTAS2R4. The quality factor calculated by ERRAT was 91.409.
These outcomes suggested that the model constructed is of high
quality. All GPCR family proteins shares common structural fea-
tures of seven hydrophobic transmembrane helices with an extra-
cellular amino (N) terminus and an intracellular carboxyl (C)
terminus. The constructed model consisted of an extracellular
topological domain, a transmembrane domain and cytoplasmic
topological domain (Fig. 2a).
Fig. 1. Ramachandran plot for hTAS2R4.
its commercialization. The choice therefore, falls on the synthesis
of rebaudioside-A from stevioside.
Out of 25 receptors, the hTAS2R4 receptor is involved in bitter
taste reception activation by stevioside and rebaudioside
A
(
Hellfritsch et al., 2012). But, the molecular/structural mechanism
behind the bitterness is still unknown. In the present study, we
have developed a homology model for bitter receptor and deter-
mined the structural requirements of a ligand for bitter taste
receptor activation. We have also proposed a strategy for the syn-
thesis of highly de-bittered rebaudioside A from stevioside by
enzymatic biotransformation.
2.2. Binding site analysis
The binding site of homolog was determined using sitemap
2
. Results and discussion
application of maestro 9.0. The primary amino acid residues that
are participating in the binding site were Phe 84, Val 85, Phe 88,
Met 89, Phe 168, Leu 177, Ser 180, Ser 184, Ser 243, Thr 246, Leu
2.1. Homology model construction and validation
2
47, Tyr 250, Leu 266 and Thr 270 (Fig 2b). Upon 3D analysis of
Due to the unavailability of the crystal structure of hTAS2R4
receptor, a homology model of hTAS2R4 receptor was prepared.
the binding site, it was revealed that the binding cavity was
smaller in size and volume.
N-terminal
Transmembrane
Domain
C-terminal
a)
b)
Fig. 2. 3D structure of constructed homology model of hTAS2R4 receptor, helix 1(Red), helix 2 (orange), helix 3 (yellow), helix 4 (light green), helix 5 (dark green), helix 6
blue) and helix 7 (purple) (a) Binding site in constructed model of h hTAS2R4, Hydrophobic map (yellow mesh), hydrogen-bond donor map (blue mesh), hydrogen-bond
(
acceptor map (red mesh) (b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Singla, R., Jaitak, V. Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste
receptor. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.004

R. Singla, V. Jaitak / Phytochemistry xxx (2016) xxx–xxx
3
Fig. 3. Ligand interaction diagram of stevioside (a) and rebaudioside A (b); stevioside (c) and rebaudioside A (d) in the binding site of hTAS2R4 receptor.
Molecular interactions of stevioside and rebaudioside A was
analysed with bitter taste receptor hTAS2R4. Stevioside was having
docking score of ꢀ13.587 kcal/mol (Table 1). The van der waals
forces contribute 30.47%, and H-bond contributes 44.2% of the dock
score. Stevioside display an H-bond interaction with backbone via
revealed that the SGs interact with the binding site of the bitter
receptor (Fig 3c and d). Upon analysing the interaction profile of
stevioside and rebaudioside A, it came to light that due to struc-
tural similarity, rebaudioside A and stevioside lies in the same
binding cavity sharing a couple of amino acids including Phe 69,
Phe 168. Phe 169, Ile 170, Glu 172, Met 259, Gly 260 and Lys
0
000
Val 70 (OH O@C, bond length 1.85 Å) and side chain via Tyr 147
0
000
0000
00
(
2
OH OH, bond length 2.34 Å), Glu 172 (OH OH, bond length
262. But due to the presence of sugar residue at the C-3 position,
0
000
.00 Å) and Asn 169 (O NH, bond length 2.43 Å) of the receptor
rebaudioside A was unable to enter deep inside the binding cavity
which is needed for the receptor activation (Pydi et al., 2012, 2014)
(Fig 3c and d). Whereas, in case of stevioside the absence of addi-
cavity (Fig. 3a). Rebaudioside A showed very feeble binding affinity
with the receptor cavity as reflected by docking score of
0
0
ꢀ
8.761 kcal/mol. Rebaudioside A display an H-bond interaction
tional sugar residue at the C-3 position, it was able to make a
strong contact with the receptor site and through extensive
hydrophobic interactions initiate a cascade of signaling involved
in the taste reception. As a significant proportion of stevioside in
steviol glycoside, thereby it can be speculated that the characteris-
tic after bitterness is associated with the higher content of stevio-
side. Rebaudioside A was unable to trigger the bitter taste signaling
due to the presence of additional glucose moiety.
0
000
with the backbone, via Ile 170 (OH OH, bond length 2.00 Å) and
0
000
Met 257 (OH OH, bond length 1.74 Å). It showed H-bond interac-
tion with side chain and participating amino acid residue were Gln
0
000
0000
2
49 (HO HN, bond length 2.06 Å), Asp 258 (OH OH, bond length
0
000
0000
2
.10 Å) and Lys 262 (C@O HN, bond length 2.27 Å and HO HN,
bond length 2.12 Å) (Fig 3b). Due to the spatial arrangement of
the sugar moieties, the rebaudioside-A was not able to interact
optimally with the receptor cavity.
Numerous reports have been published on the limited bitter-
ness profile of rebaudioside A, (Boileau et al., 2012; Carakostas
et al., 2008; Hellfritsch et al., 2012; Kochikyan et al., 2006) which
is the outcome of its restricted interaction with the bitter receptor.
Current molecular simulation study also suggests its limited inter-
action with the hTAS2R4 receptor. The docking experiment
In a recent in vitro study, it has been observed that stevioside
stimulate the hTAS2R4 bitter receptor and the result obtained were
found to corroborate with our molecular simulation study
(Hellfritsch et al., 2012). Therefore, preparation of rebaudioside A
lies in an important niche, where its semisynthetic intervention
not only involve importance in the food industry but also in
pharmaceutical industry.
Please cite this article in press as: Singla, R., Jaitak, V. Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste
receptor. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.004

4
R. Singla, V. Jaitak / Phytochemistry xxx (2016) xxx–xxx
OH
OH
HO
OH
HO
OH
HO
OH
O
OH
HO
OH
OH β-1,3 glucanase
3''''
β-1,3 glucanase
3''' 2'''
2''''
OH
O
4
'''
1'''
1''''
4''''
5''''
2''
O
HO
HO
HO
O
O
OH
OHO
OH
O
O
O
5'''
o
OH
O
3''
5''
O
HO
O
OH
55 C, pH=4.5,
hr
55 C, pH=4.5,
hr
o
6'''
O
1''
OH
HO
O
4''
OH 6'''' OH
OH
hydrolyse
3
OH
OH
3
n
13
12
O
17
O
transglycosylase
11
O
2
0
14
6''
OH
Curdlan
16
1
2
3
9
6
15
10
8
4
5
7
18
1
9
HO
HO
HO
O
O
O
O
O
HO
HO
1'
2'
O
3'
5'
4'
OH
Stevioside
6'
HO
OH
Rebaudioside A
Fig. 4. Transglycosylation of stevioside to rebaudioside A.
0
0
2
.3. Synthesis of rebaudioside-A
attached to the C-3 position were observed to be d
4.35, 73.59, 70.01 and 61.02. Melting point was found to be
245–247 °C.
C
78.46,
7
In recent times, the importance of developing greener route for
the synthesis of pharmaceutically cardinal moieties has been a
center of interest in the medicinal chemists due to its industrial
and environmental importance (Roschangar et al., 2015).
Enzymatic synthesis attracted a considerable attention and con-
tributed a significant role in the development of greener routes. By
the process of enzymatic transglycosylation, there is a formation of
the glycosidic bond during the transfer of a sugar residue from
one glycoside to another (Jaitak et al., 2009a,b; Nishihashi et al.,
3
. Conclusion
In the present study, we have investigated the binding mode of
two SG’s, stevioside and rebaudioside A against hTAS2R4 receptor
via the computational method. Stevioside and rebaudioside A have
structural similarity thereby bind at same receptor site hTAS2R4
receptor. Rebaudioside-A was not able to enter the receptor site
effectively thereby failing to initiate bitter taste response due to
the presence of a sugar residue at the C-3 position. By this reason,
the rebaudioside A has shown superior quality taste profile that
makes it a valuable additive in food and pharmaceutical industry.
Encouraged by our findings, we have developed a highly efficient
and greener route for the semi-synthesis of rebaudioside A from
stevioside using enzymatic reaction. The developed protocol could
be used for the synthesis of high-quality rebaudioside A from bit-
tered stevioside for meeting the rising demands of low calorific
natural sweeteners in food and pharmaceutical industry.
1
983; Wang et al., 2015; Yang et al., 2015a)
0
0
Amount of rebaudioside A formed
Percent Conversion ¼
Amount of stevioside decreased ꢁ 1:2
ꢁ
100
Molecular weight of rebaudioside A
Molecular weight of stevioside
where\1:2"is calculated as ¼
In the present study, we have synthesized rebaudioside A from
stevioside by enzymatic biotransformation using b-1,3-glucanase
from Irpex lacteus. The transglycosylation executed by the used
enzyme is involved of two in-situ steps. In the first step by the
property of b-1,3-glucanase it cleaves the glucose moiety from
4. Experimental
4.1. Homology model development and validation
0
0
the donor curdlan followed by selectively transferring at the C-3
position of stevioside with b-configuration in the second step
Fig. 4). The temperature, pH, time, the concentration of curdlan
Primary sequence of hTAS2R4 taste receptor (taste receptor
type 2, member 4) from Homo sapiens (Q9NYW5) was retrieved
from the Universal Protein Resource (http://www.uniprot.org/)
and used as targets for homology modeling. To find suitable tem-
plates for hTAS2R4, BlastP search was performed against Protein
Data Bank (PDB). From searching results, it was inspected that no
appropriate template was found that share more than 35% of
sequence identity, and they were not able to satisfy query coverage
of 100%. Erstwhile studies on GPCR have been reported that homol-
ogy models of improved quality and reliability can be achieved
using multiple templates (Larsson et al., 2008; Yarnitzky et al.,
2010). Homology modeling was performed using the I-TASSER
(Iterative Threading ASSEmbly Refinement) web server of
University of Michigan, USA maintained by Zhang Lab (Roy et al.,
2010). Upon submission of the query sequence, I-TASSER retrieves
templates of proteins having similar folds (or super-secondary
structures) from the PDB library by LOMETS (Local Meta-threading
Server) (Wu and Zhang, 2007). LOMETS identified eight templates
with PDB id 4GRV, 4BWB, 4N6H, 4IAR, 2Z73, 2DJH, 4BUO and 4EA3.
I-TASSER server excises the continuous fragments from the
selected/ identified PDB templates and then reassembles them into
(
and enzyme plays a significant role in the product yields.
Stevioside and curdlan were taken in a ratio of 1:2. The reaction
condition was optimized at 55 °C, in a citric acid buffer of pH 4.5,
reaction time was 3hrs. The reaction product was formed in max-
imum yield with enzyme activity of 3.425 units/g. The reaction
product was purified by column chromatography, and the percent
conversion rate was calculated as 62.5%. The characterization of
the product was performed by NMR, HRMS, melting point and data
was found to be consistent with previous literature (Singh et al.,
1
2
009). In H NMR, anomeric proton at d
H
5.28 (1H, d, J = 8 Hz) of
0
000
H-1 indicates the b configuration of glucose moiety attached to
C-3 . The other three anomeric values 5.64 (1H), 5.02 (1H), 4.95
1H) represent the three more glucose moieties in rebaudioside-
A. In C NMR, dc at 14.89 (C-20) and dc 28.04 (C-18) indicates
the presence of two methyl groups. dc value at 175.6 and dc 104
indicating the presence of carbonyl carbon (C-19) and for the exo-
cyclic C@C (C-17) group respectively. The shifting of the dc from
0
0
(
1
3
7
8.0 to dc 86.27 indicated the attachment of a glucose moiety at
0
0
C-3 (Jaitak et al., 2009a,b). Additional peaks for the sugar moiety
Please cite this article in press as: Singla, R., Jaitak, V. Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste
receptor. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.004

R. Singla, V. Jaitak / Phytochemistry xxx (2016) xxx–xxx
5
a complete models by utilizing replica-exchange Monte Carlo sim-
4.5. Enzymes and substrates
ulations with the threading unaligned regions (mainly loops) built
by ab initio modeling (Zhang, 2008).
Stevioside for the synthesis was isolated from S. rebaudiana and
characterized according to our earlier published protocol (Jaitak
et al., 2009a,b). b-1,3-Glucanase from I. lacteus and Curdlan from
Alcaligenes faecalis were obtained from Sigma Chemical Co. Sodium
citrate and citric acid of AR grade were obtained from SDFCL.
Low free-energy states of the models are identified by SPICKER
through clustering the decoys (Zhang and Skolnick, 2004b). Next
step proceeds with fragment assembly simulation that is guided
by TM-align for removing any steric clashes and to refine the global
topology of the cluster decoys (Zhang and Skolnick, 2004a). The
final five complete model of hTAS2R4 is obtained by REMO that
optimises the hydrogen-bonding network (Li and Zhang, 2009).
I-TASSER server also evaluates the quality of the models from
C-score, which is based on the impact of threading template align-
ments and the convergence parameters of the structure assembly
simulations. TM-score is also calculated which is a recently pro-
posed scale of measuring the structural similarity between two
structures. In TM-score, the small distance is weighted stronger
than the big distance that makes the score insensitive to the local
modeling error as with the case of RMSD calculations. A TM-score
4.6. Synthesis of rebaudioside A
Stevioside (200 mg) and curdlan (400 mg) was dissolved in
citrate buffer (0.1 M with pH 4.5) and b-1,3-glucanase 3.425 U/g
was added. The reaction mixture was shaking in an incubator for
3
hr at 55 °C. The reaction mixture was cooled to room temperature
and the enzyme was deactivated by boiling at 100 °C. Progress of
the reaction was monitored by TLC using solvent system ethyl
acetate/ethanol/H O (8:2:1.2, v/v/v) (Jaitak et al., 2009a,b). The
2
developed plate was dried and spots were visualized by spraying
with 5% sulfuric acid. Reaction mixture was passed through the
Dianion HP-20, followed by washing with methanol. Methanol
fraction was dried over rota vapor and recrystallized using cold
ethanol to yield 150 mg of pure Rebaudioside A. Product was con-
firmed by, NMR, HRMS and melting point determination.
>0.5 indicates a model of correct topology and a TM-score <0.17
means a random similarity. The refined models were checked
using procheck, ERRAT available at http://services.mbi.ucla.edu/
SAVES/ and RAMPAGE. The ones with best structural features and
geometry were selected for the molecular simulation study.
Rebaudioside A: MP 245–247 °C, R
f
value 0.45, ethyl acetate:
ethanol:water (8:2:1.2, v/v/v), H NMR (400 MHz, DMSO): 5.64
1H, d, J = 8 Hz), 5.28 (1H, d, J = 8 Hz), 5.02 (1H, t, J = 8 Hz) and
4
1
4
6
7
9
4
.2. Ligand modeling
1
(
The 2D structure of ligands were constructed using the maestro
.0 software and saved in sdf format (standard data format). The
D structures were converted to the 3D structure using the Ligprep
13
.95 (1H, d, J = 8 Hz).
C-NMR (100 MHz, DMSO): dc 14.89,
9
2
8.56, 19.79, 21.14, 28.04, 37.48, 38.70, 39.20, 40.03, 41.04,
1.64, 43.10, 43.20, 47.0, 53.23, 56.52, 60.56, 61.02, 61.34, 68.90,
9.48, 70.01, 70.32, 72.44, 73.59, 74.35, 75.98, 76.49, 76.88,
6.98, 77.42, 78.46, 78.79, 78.88, 79.12, 85.40, 86.27, 94.45,
module of maestro 9.0. This module adds hydrogens, eliminates
any discrepancies between bond length and angle.
6.45, 102.47, 102.98, 104.0, 152.78, 175.63. HRMS (ESI): m/z calc.
ꢀ
4
.3. Ligand-binding pocket determination
for C44
H
70
O
23 [MꢀH] : 965.4230; found 965.5040.
Ligand binding site was calculated using sitemap application of
Acknowledgements
maestro 9.0. This application performs calculations on the total
protein to locate binding sites whose size, functionality, and extent
of solvent exposure using the OPLS-2005 force field to determine
the binding pocket.
Authors are grateful to Department of Science and Technology,
New Delhi, India for providing financial assistance during the work.
Authors are also thankful to the Honourable Vice - Chancellor for
providing the necessary facilities for the Central University of
Punjab, Bathinda, India.
4
.4. Molecular docking
The minimized homolog protein prepared by protein prepara-
Appendix A. Supplementary data
tion wizard was utilized in the docking simulation. Molecular
docking calculations were carried out using glide package of mae-
stro 9.0. Affinity grid of 46 ꢁ 46 ꢁ 46 grid points and spacing 0.25
was generated using the Grid Generation module of maestro 9.0
was generated for the homolog protein around the binding site
detected using sitemap module of maestro 9.0. Glide docking
was performed using the extra percision mode analysis of Maestro
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2016.
0
3.004.
References
9
.0. The van der waals radii of ligand was taken as 0.5 Å and charge
cut off as 0.15. This program calculates the van der waals forces,
hydrogen bond interactions and electrostatic forces. Docking sim-
ulation was performed using the OPLS-2005 force field. Force field
refined ten ligand-receptor possess which were further minimized
to yield a single pose, selected on the basis of scoring function. The
scoring function combines weighted Coulomb/van der Waals
protein–ligand interaction energies, the terms favoring binding
affinity (hydrophobic enclosure, neutral–neutral hydrogen-bond
motifs, charged-charged hydrogen-bond motifs, pi-cation interac-
tions), and the various penalty terms (desolvation penalties and
ligand strain). Ligand interaction module was utilized for to calcu-
late the schematic representation of the residue–ligand interaction
between ligands and the active site of hTAS2R4 taste receptor
model.
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Please cite this article in press as: Singla, R., Jaitak, V. Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste
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