Biosynthesis of methyl glucoside and its antibacterial activity against Staphylococcus aureus and Escherichia coli

Article abstract of DOI:10.1016/j.bcdf.2019.100197

In this study, the methyl glucosides (MG_n) was synthesized using β-cyclodextrin (β-CD) and methanol through the coupling reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11. The optimal condition for the synthesis of MG_n consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of CGTase in a water/methanol solution containing 30% (v/v) methanol at pH 6.0, 50 °C for 120 h. Upon analysis by TLC, at least three MG products were observed. The molecular weight of the main transferred product was 217.08 Da; this value was in accordance with methyl monoglucoside (MG₁). MG₁ was produced and prepared on a large scale and subsequently purified by preparative TLC. The combined ¹H- and ¹³C-NMR analysis confirmed that the structure of MG₁ was methyl-α-D-glucopyranoside. In addition, MG₁ showed emulsification activity and stability in its formation in water and n-hexadecane. The antibacterial activity of MG₁ was also determined by agar disc diffusion method. The results found that the MG₁ (1, 5 and 10 mg/disc) showed antibacterial activity against E. coli ATCC 25922 only, with inhibition zones of 28, 38 and 40 mm, respectively. The MIC values (mg/mL) of MG₁ against S. aureus ATCC 25923 and E. coli ATCC 25922 were found to be 20.00 and 0.63, while MBC values (mg/mL) were 40.00 and 0.63, respectively.

Full text of DOI:10.1016/j.bcdf.2019.100197

Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
Contents lists available at ScienceDirect
Bioactive Carbohydrates and Dietary Fibre
journal homepage: http://www.elsevier.com/locate/bcdf
Biosynthesis of methyl glucoside and its antibacterial activity against
Staphylococcus aureus and Escherichia coli
a
b,*
Jarunee Kaulpiboon , Prakarn Rudeekulthamrong
a
Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand
Department of Biochemistry, Phramongkutklao College of Medicine, Phramongkutklao Hospital, Bangkok, 10400, Thailand
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, the methyl glucosides (MG ) was synthesized using β-cyclodextrin (β-CD) and methanol through the
n
Antibacterial activity
Cyclodextrin glycosyltransferase
Enzymatic synthesis
coupling reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11. The
optimal condition for the synthesis of MG
n
consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of
�
CGTase in a water/methanol solution containing 30% (v/v) methanol at pH 6.0, 50 C for 120 h. Upon analysis
by TLC, at least three MG products were observed. The molecular weight of the main transferred product was
Intermolecular transglycosylation
Methyl glucoside
2
17.08 Da; this value was in accordance with methyl monoglucoside (MG
a large scale and subsequently purified by preparative TLC. The combined 1H- and 13C-NMR analysis confirmed
that the structure of MG was methyl- -D-glucopyranoside. In addition, MG showed emulsification activity and
stability in its formation in water and n-hexadecane. The antibacterial activity of MG was also determined by
agar disc diffusion method. The results found that the MG (1, 5 and 10 mg/disc) showed antibacterial activity
against E. coli ATCC 25922 only, with inhibition zones of 28, 38 and 40 mm, respectively. The MIC values (mg/
mL) of MG against S. aureus ATCC 25923 and E. coli ATCC 25922 were found to be 20.00 and 0.63, while MBC
values (mg/mL) were 40.00 and 0.63, respectively.
1 1
). MG was produced and prepared on
1
α
1
1
1
1
1
. Introduction
synthesis of alkyl glycosides can be achieved through several types of
enzymes as extracted from many organisms, such as β-mannosidase from
Aspergillus niger (Itoh & Kamiyama, 1995), β-galactosidase from Asper-
gillus oryzae (Ooi, Hashimoto, Mitsuo, & Satoh, 1985), dextransucrase
from Leuconostoc mesenteroides (Kim et al., 2009) and, as indicated in
Methyl glucoside is an alkyl glycoside that is derived from sugar and
methanol. It is a non-ionic surfactant which is composed of the hydro-
philic sugar linked to the hydrophobic methanol via an -glycosidic
α
bond. The alkyl glycosides become dominant components in products of
the food, detergent, cosmetic and pharmaceutical industries as a result
of their biodegradability, emulsification and antibacterial properties
most reports on
α- or β-glucosidase, from various other sources (Ito et al.,
2007; Lirdprapamongkol & Svasti, 2000; Wang et al., 2012). For
cyclodextrin glycosyltransferase (CGTase, E.C. 2.4.1.19), it has been
studied less through alkyl glycoside synthesis. CGTase has been used
only to elongate the carbohydrate group of commercially available
(
Matsumura, Imai, Yoshikawa, Kawada, & Uchibor, 1990). Alkyl gly-
cosides can be synthesized by both chemical and enzymatic methods.
However, the enzymatic syntheses of alkyl glycosides, as shown from
many previous studies, are more advantageous than chemical syntheses.
The enzymatic method proceeds under non-toxic and mild conditions by
a simple one-step reaction process (Van Rantwijk, Oosterom, & Sheldon,
dodecyl-β-D-maltoside via its coupling reaction using
α
-CD as a glycosyl
donor (Svensson et al., 2009). In addition, Chotipanang, Bhunthumna-
vin, and Prousoontorn (2011) reported the synthesis of methyl gluco-
sides from β-CD and methanol using Paenibacillus sp. RB01 CGTase.
However, they have not been studied for their structure and biological
activity.
1
999). According to the reports, the alkyl glycosides can be produced by
several enzymes which differ in the kind and length of the sugars and
hydrocarbons involved as well as the configuration of the linkages which
depend on the specific action of each enzyme (Ito, Ebe, Shibasaki,
Fukuda, & Kondo, 2007; Lirdprapamongkol & Svasti, 2000; Svensson,
Ulvenlund, & Adlercreutz, 2009; Wang et al., 2012). The enzymatic
CGTases are multifunctional enzymes which catalyze mainly intra-
and inter-transglycosylation reactions (cyclization, coupling and
disproportionation). Moreover, they can also exhibit, to a lesser extent,
α-amylase-like activity, hydrolyzing starches into short linear
*
Corresponding author. Department of Biochemistry, Phramongkutklao College of medicine, Bangkok, 10400, Thailand.
E-mail addresses: prakarn_rudee@yahoo.co.th, prakarn_r@hotmail.com (P. Rudeekulthamrong).
https://doi.org/10.1016/j.bcdf.2019.100197
Received 10 July 2019; Received in revised form 30 August 2019; Accepted 4 September 2019
Available online 5 September 2019
2
212-6198/© 2019 Elsevier Ltd. All rights reserved.

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
saccharides (Svensson et al., 2009; Van der Veen, Van Alebeek, Uitde-
haag, Dijkstra, & Dijkhuizen, 2000). On their first reactions, CGTases
convert starch substrates to a mixture of non-reducing cyclic maltooli-
Germany). Methanol was obtained from CARLO ERBA Reagents (Val de
Reuil, France). The commercial glucose oxidase kit was obtained from
Human mti-diagnostics GmbH (Idstein, Germany). Other chemicals used
were of an analytical grade.
gosaccharides called CDs (
α-, β-, γ-CD) through the cyclization reaction
(
Loftsson, Jarho, M �a sson, & J €a rvinen, 2005). The proportions of CDs
produced are primarily determined by the type of enzyme employed,
concentration of starch, and degree polymerization of starch. CGTases
are often classified according to the main CD which they produce. Be-
sides the cyclization reaction, CGTases also catalyze a disproportion-
2.2. Bacterial cultivation and enzyme production
Escherichia coli BL21 (DE3) cells harboring the pBC recombinant
plasmid, a pET19b-based plasmid containing the CGTase gene with a
signal peptide sequence from Bacillus circulans A11 (GenBank accession
no. AF302787) (Kaulpiboon et al., 2010; Rimphanitchayakit, Tonozuka,
& Sakano, 2005), were prepared using a new pair of primers. The for-
ation reaction by transferring linear
α-1,4-linked oligosaccharides to
another oligosaccharide molecule; they also catalyze a coupling reaction
by opening CD rings and transferring them to acceptors (Loftsson et al.,
2
005; Svensson et al., 2009; Van der Veen et al., 2000). Generally, the
ward
and
reverse
primers
designed
were
0
0
CGTase acceptors are saccharides or glycosides, which are very abun-
dant in nature (Kitahata et al., 1992; Kometani, Terada, Nishimura,
Takii, & Okada, 1994; Wongsangwattana, Kaulpiboon, Ito, & Pongsa-
wasdi, 2010). This transglycosylation reaction, transferring from donor
to acceptor, proceeds via the major catalytic machinery of enzymes for
the synthesis of new glycosidic bonds (Mosi, He, Uitdehaag, Dijkstra, &
Withers, 1997). Thus, from this mechanism, CGTase also catalyzes
transglycosylation to various compounds other than saccharides, such as
piceid (Mathew, Hedstr o€ m, & Adlercreutz, 2012) and ascorbic acid
5 -CATGCCATGGAAAGATTTATGAAACTAACAGCCGTA-3 with NcoI
0
0
site and 5 -CCGCTCGAGTTAAGGCTGCCAGTTCACATTCATC-3 with
XhoI site (underlined), respectively. The DNA sequence of pBC trans-
formant showed a CGTase open reading frame of 2139 base pairs, which
encoded a polypeptide of 713 amino acids. The signal peptide consti-
tuted the N-terminal 27 amino acids. The pBC recombinant cells were
grown in Luria Bertani (LB) medium containing 100
μg/mL ampicillin at
�
37 C for 24 h. The expression of recombinant enzymes was induced
with 0.2 mM IPTG when the OD600 of the culture reached 0.6. After 24 h,
the cells were removed and the culture broth containing crude CGTase
(
Aga, Yoneyama, Sakai, & Yamamoto, 1991). Mathew et al. (2012) have
�
reported the efficient synthesis of piceid glycosides through dispropor-
tionation catalyzed by CGTase from Bacillus macerans using maltodex-
trin as a glycosyl donor and piceid as a glycosyl acceptor. The obtained
piceid glycosides showed an increase in absorbability in the digestive
tract. Aga et al. (1991) reported on the use of CGTase from Bacillus
stearothermophilus in the production of ascorbyl glycosides. The result
was a greater stability under oxidative conditions through the coupling
was collected by centrifugation at 12,000ꢀg at 4 C for 2 h for further
purification.
2.3. Purification of CGTase
Recombinant CGTase was purified from the culture broth by starch
adsorption (Kato & Horikoshi, 1984) and DEAE-Toyopearl 650M col-
�
reaction using
α-CD and ascorbic acid as a glycosyl donor and acceptor,
umn chromatography. Corn starch was dried in an oven at 60 C for 3 h,
respectively. In addition, CGTase can also use water as acceptor,
resulting in a hydrolysis reaction.
cooled to room temperature and then gradually added to a culture broth
�
of crude CGTase at 4 C to produce a final concentration of 5% (w/v).
For CGTase, transglycosylation occurs when either CD or starch is
used as the glycosyl donor. In describing the CGTase mechanism, the
coupling reaction is often referred to when the glycosyl donor used is
CD. Since the advantage of coupling reaction of CGTase in the formation
of glucosides of better quality, has been well accepted. So, in this study,
the catalytic mechanism of CGTase in term of coupling reaction as a type
of transglycosylation was exploited for the synthesis of a series of methyl
glucosides. The CGTase can produce methyl glucoside(s) that have one
and more than one glucose unit attached. There are numerous reports
demonstrating that methyl glucosides can be used as effective emulsi-
fying agents and surfactants (Aoudia & Zana, 1998; Knol, Sjollema, &
Poolman, 1998; Lichtenberg, Ahyayauch, & Go n~ i, 2013). So far, there
have been no reports on the use of methyl glucosides as antibacterial
agents. Therefore, the aim of this study was to use the pBC recombinant
CGTase from Bacillus circulans A11 to synthesize methyl glucoside(s)
using β-CD as the glycosyl donor and methanol as an acceptor. The in-
fluence of various parameters on the production of methyl glucoside(s)
was investigated. The structure and biological properties of the methyl
glucoside(s) including antibacterial activities were also studied.
After 3 h of continuous stirring, the starch cake was collected by
�
centrifugation at 8000ꢀg for 30 min at 4 C and washed twice with TB
1
buffer (10 mM Tris-HCl, pH 8.5 containing 10 mM CaCl ). The adsorbed
2
CGTase was eluted from the starch cake by stirring for 30 min with 0.2 M
maltose in TB buffer (3 ꢀ 50 mL per liter of starting broth). The eluted
1
�
CGTase was collected by centrifugation at 10,000ꢀg for 30 min at 4 C.
The CGTase solution was poured into dialysis tubing and concentrated
by coating the tubing with a water-absorbing agent (carboxymethyl-
�
cellulose, Aquacide II), followed by dialysis against TB1 buffer at 4 C
three times before being subjected to DEAE-Toyopearl 650M column
chromatography. The column (1.5 ꢀ 10 cm) was equilibrated with TB
buffer (10 mM Tris-HCl, pH 8.0). CGTase was then applied to the col-
2
umn, and unbound proteins were eluted with TB buffer. After the col-
2
umn was washed thoroughly with TB2 buffer, bound proteins were
eluted from the column using a linear salt gradient of 0–0.2 M sodium
chloride in TB buffer. Fractions of 2.0 mL were collected continuously.
2
The protein and CGTase-dextrinizing activity profiles of the eluted
fractions were monitored by measuring the absorbance at 280 nm and
600 nm, respectively. Fractions containing CGTase-dextrinizing activity
were pooled for further characterization. The purity of the enzyme from
each step in the purification was determined using 7.5% native-PAGE
and a Coomassie® blue staining method (Weber & Osborn, 1975).
2
. Materials and methods
2
.1. Materials
2
.4. Assay of CGTase activity
Glucose (G
1
); maltose (G
2
); maltotriose (G
); maltoheptaose (G
); sucrose;
3
); maltotetraose (G
); methyl- -D-
-, β-
4
);
maltopentose (G
5
); maltohexose (G
6
7
α
2.4.1. Dextrinizing activity
glucopyranoside and methyl-β-D-glucopyranoside (MG
1
α
The dextrinizing activity of CGTase was determined by measuring
the decrease in absorbance of a starch-iodine complex at 600 nm (Fuwa,
and γ-CDs; soluble potato starch; corn starch; phenolphthalein, n-hex-
adecane and bovine serum albumin were purchased from Sigma-Aldrich
1954). The enzyme sample (50
μ
L) was incubated with 0.15 mL of 0.2%
�
(
St. Louis, MO, USA). Mannitol salt agar (MSA), yeast extract and
(w/v) soluble potato starch in 0.2 M phosphate buffer, pH 6.0, at 40 C
for 10 min. The reaction was stopped by the addition of 2 mL of 0.2 M
tryptone were obtained from Difco (Bacton Dickinson and company,
Sparks, MD, USA). Aquacide II, Methanol, ethanol, silica gel 60 F254
glass plates (20 cm in height) were purchased from Merck (Darmstadt,
HCl and 0.25 mL of iodine reagent (0.02% (w/v) I
2
) in 0.2% (w/v) KI).
The mixture was adjusted to a final volume of 5 mL with distilled water,
2

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
and the absorbance at 600 nm was measured. For the control, HCl was
added before the enzyme sample was taken. One unit of enzyme was
defined as the amount of enzyme that produced a 10% reduction in the
intensity of the blue color of the starch-iodine complex per min under
the described conditions.
(10–50% (v/v)) and CGTase (75 U/mL by dextrinizing activity) in
20 mM phosphate buffer at pH 6.0 in a total volume of 250
μL. The re-
�
action mixture was incubated at 50 C for 24 h. The reaction was then
terminated and assayed using the phenolphthalein method to determine
the stability of CGTase in alcohols. In addition, aliquots from each re-
action mixture were withdrawn and analyzed by TLC to determine the
transglycosylation yield using the mobile phase of System II as described
in Section 2.7.
2
.4.2. Coupling activity
2
.4.2.1. Glucose oxidase assay. A glucose oxidase kit was used to mea-
sure the enzymatic activity by exchanging β-CD for glucose (Miwa,
Okudo, Maeda, & Okuda, 1972). The reaction mixture (100 L) con-
tained 40 L of 1% (w/v) cellobiose, 5 L of the
enzyme and 15
2
.7. TLC analysis
μ
μ
L of 1% (w/v) β-CD, 40
μ
μ
The transfer products were analyzed using Silica gel 60 F254 (Merck,
μ
L of 0.2 M phosphate buffer, pH 6.0. The reaction
Germany) base TLC using a 5:5:3 (v/v/v) ratio of n-butanol/ethyl
alcohol/water as a mobile phase solvent (System I) to determine the best
donor for the synthesis of transglycosylation products. For the analysis
of methyl glucoside products, mobile phase System II (ethyl acetate/
acetic acid/water with a 3:1:1 (v/v/v) ratio) (Charoensapyanan,Ito,
Rudeekulthamrong, & Kaulpiboon, 2016) and System III (ethyl aceta-
te/acetic acid/water with a 3:1.5:0.5 (v/v/v) ratio) were used. Spots on
�
mixture was incubated at 50 C for 5 min and was terminated by boiling.
Subsequently, the mixture was incubated with 10
μ
L of glucoamylase
�
(
stock solution, 30 units) in 0.2 M phosphate buffer, pH 6.0, at 40 C for
3
0 min, and the reaction was stopped by boiling for 5 min. An aliquot
(
10
μ
L) from the reaction mixture was then added to 1 mL of glucose
�
oxidase reagent and incubated at 37 C for 5 min before the absorbance
was measured at 500 nm. The glucose concentration was calculated
from equation (1):
the TLC plate were visualized by dipping into sulfuric acid/methanol
�
(
1:9 (v/v) ratio), followed by drying and heating at 110 C for 20 min.
The intensity of the synthesized product spots was quantified using a
Glucose concentration (
μ
m ol/m L) ¼ 5.55 ꢀ (ASam ple/AStd.
)
(1)
scanning densitometer and a Quantity One® 1-D analysis program.
The absorbance of the standard solution (AStd.) was measured by
α
1
Glucose and methyl- -D-glucopyranoside (MG ) were used as standards.
adding 10 L of a standard glucose solution (5.55 mol/mL) to 1 mL of
μ
μ
glucose oxidase reagent. One unit of glucose oxidase activity was
2
.8. Optimization of methyl glucoside production
Before optimizing the conditions, the reaction was performed by
defined as the amount of enzyme that produces 1
min under the assay conditions used.
μmol of glucose per
incubation of 0.6% (w/v) β-CD and 30% (v/v) methanol with 50 U/mL
2
.4.2.2. Phenolphthalein assay. The phenolphthalein assay measures the
disappearance of β-CD in the reaction mixture (Goel & Nene, 1995). A
50
L portion of the β-CD standard or sample solution was incubated
with 750 L of a phenolphthalein solution for 15 min. The decrease in
absorbance at 550 nm caused by complex formation between the dye
and β-CD was measured. Conversion of ΔA550 to moles of β-CD was
�
of CGTase in 20 mM phosphate buffer (pH 6.0) at 50 C for 24 h. In order
to increase the final productivity, the optimal conditions for the pro-
duction of methyl glucosides were decided based on the highest per-
centage yield of methyl glucoside products as determined from the TLC
results. The effects of varying the methanol acceptor (10–50% (v/v)),
β-CD donor concentration (0.3–1.8% (w/v)), enzymatic units (50–500
2
μ
μ
μ
performed using the β-CD phenolphthalein calibration curve. The
disappearance of β-CD in the reaction mixture was calculated from the
difference between β-CD concentration at 0 and 24 h incubations with
CGTase and acceptors. One unit of activity was defined as the amount of
�
U/mL), incubation time (24–168 h), temperature (30–70 C) and pH
(
5.0–8.0) were investigated using a sequential approach.
enzyme capable of coupling 1
μ
mole of β-CD to alcohol per min.
2.9. Large-scale production
To obtain a higher yield of methyl glucoside products for structural
analysis and to determine their biological properties, large-scale pro-
duction of methyl glucosides was conducted in a total volume of 250 mL
in a 1000-mL Erlenmeyer flask. The reaction mixture contained the β-CD
donor, methanol acceptor and enzyme, all of which were incubated
under the optimum condition according to Section 2.8. After the incu-
bation, the reaction was stopped by boiling and then concentrated using
2
.5. Protein determination
The protein concentration was determined by Bradford’s method
Bradford, 1976), using bovine serum albumin (BSA) as a standard.
(
2
2
0
.6. Synthesis of methyl glucosides
�
a centrifugal evaporator at 45 C prior to purification of the methyl
.6.1. Donor specificity
glucoside by preparative thin-layer chromatography (PLC).
Selection of the appropriate donor was performed by incubating
.64% (w/v) cellobiose and 75 U/mL CGTase in 20 mM phosphate
buffer at pH 6.0 with 0.64% (w/v) of the different glycosyl donors
2.10. Purification of methyl glucosides
�
(
α
-CD, β-CD and γ-CD) in a reaction volume of 250
μL at 50 C for 24 h.
The glucose produced from the CGTase transglycosylation reaction was
determined using the glucose oxidase assay and the transfer products
were analyzed by thin-layer chromatography (TLC) with the mobile
phase of System I as described in Section 2.7.
The concentrated reaction mixture was placed on a PLC plate and
developed at ambient temperature using solvent System II. One lane of
the reaction mixture on the plate was then cut out and used to detect the
methyl glucoside products using a sulfuric acid/methanol (1:9 by vol.)
�
dipping solution. The preparative plate was dried and heated at 110 C
2
.6.2. Determination of transglycosylation efficiency
for 20 min. Each product from the PLC plate that had approximately the
The efficiency of the CGTase transglycosylation reaction was deter-
f
same relative mobility (R ) values was scraped separately. The methyl
mined from the transglycosylation product yield, which was determined
from the phenolphthalein assay and by TLC analysis. The trans-
glycosylation reaction with various alcohol types and contents was
performed using β-CD as a glycosyl donor. The reaction mixture con-
sisted of 0.64% (w/v) β-CD and various concentrations of methanol
glucoside products were extracted from the adsorbent (silica) by dis-
solving with methanol for 30 min at room temperature and then
�
centrifuging at 8000ꢀg at 25 C for 1 h. The methanol was further
�
removed using a rotary evaporator at 45 C until the samples were
thoroughly dry.
3

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
2
2
.11. Structural analysis of methyl glucosides
2.12.2.1. Disc diffusion technique. Filter paper discs (6 mm in diameter)
μ
1
were impregnated with 15 L of the MG (concentration 1, 5 and 10 mg/
.11.1. Mass spectrometry (MS) analysis
disc) in sterile water. The inoculum density for a susceptibility test was
8
The transfer products of interest were dissolved in 50% (v/v)
adjusted to be equivalent to a 0.5 McFarland standard (1.5 ꢀ 10 CFU/
methanol, and their masses were determined using a microTOF spec-
trometer (Bruker, Germany). The products were introduced into a mass
spectrometer for processing in the ESI-TOF-MS system, which ionizes by
electrospray ionization (ESI) in the sodium positive ion mode using a
capillary voltage of 5000 volts. A 4.0 L/min flow of nitrogen gas at a
mL). An air-dried disc was placed on the surface of S. aureus-inoculated
mannitol salt agar and E. coli-inoculated LB agar. Standard antibiotics of
positive control, i.e., 10
of tetracycline and 1
μ
g/disc of ampicillin and penicillin, 30 g/disc
μ
μ
g/disc of oxacillin, were also tested as well as
�
sterile water as a negative control. The plates were incubated at 37 C for
24 h. The clear zone of inhibition was calculated by measuring the
diameter of the inhibition zone. The readings were taken in three
different fixed directions, and an average value of inhibition zone was
tabulated (Awwad, Salem, & Abdeen, 2013; Bhalodia & Shukla, 2011).
�
temperature of 150 C was used to nebulize the analytic solution to
droplets using a nebulizer pressure of 1.0 bar. The ions were detected
using linear time-of-flight mass spectrometry (TOF-MS), and the mass to
charge ratio (m/z) spectra of the products was detected as pseudo-
þ
molecular ion peaks [MþNa] from which the molecular weights
were calculated using a Bruker Daltonics DataAnalysis 3.4 software
program.
2.12.2.2. Minimal inhibitory concentration (MIC). The minimal inhibi-
tory concentration (MIC) value was determined in liquid LB medium
using the microdilution method in a 96-well microtiter plate by incu-
bating the bacteria in LB broth with variable amounts of the sample
2
.11.2. Preliminary structure analysis
The enzyme treatment method was used to make a preliminary check
being tested. Briefly, the 40-mg starter content of MG1 in 150 L of
μ
of the structure of CGTase-catalyzed methyl glucoside products. Each of
the methyl glucoside powders from reaction mixture at 24-h CGTase
incubation (2 mg) was dissolved and incubated with 20 U/mL of
sterile water was diluted by a two-fold serial dilution in multiple steps in
the microtiter plate, while the 0.5 McFarland culture was also diluted
with LB broth in a 1:200 (v/v) ratio. The 150
μ
L of culture was added to
Rhizopus sp. glucoamylase (E.C. 3.2.1.3) and Aspergillus niger
α
-glucosi-
the 96-well microtiter plate. Following 18 h of incubation, 30
μL of
�
dase (E.C. 3.2.1.20) in 0.2 M acetate buffer, pH 5.5 at 37 C for 24 h. The
reaction was heated to inactivate enzyme activity and analyzed by TLC
as described in Section 2.7 (System III).
resazurin solution was added to each well and the plate was incubated
for 2 h. The minimum inhibitory concentration (MIC) value was defined
as the lowest concentration of MG1 that prevented a color change of
resazurin from blue to pink. Resazurin, a blue dye, becomes pink when it
is reduced to resorufin by an oxidoreductase within a medium of viable
bacteria. The MIC value was determined in triplicate experiments and
was expressed as mg/mL (Awwad et al., 2013; Bhalodia & Shukla,
2011).
2
.11.3. Nuclear magnetic resonance (NMR) analysis
Structural identification of the methyl glucoside that was synthesized
1
13
by CGTase was performed by H- and C-NMR using a Bruker AVANCE
III HD 600 NMR Spectrometer. The spectrometer was operated at
6
00 MHz at ambient temperature. Chemical shifts were measured using
sodium-4,4-dimethyl-4-sila-pentane sulfonate (DSS) as the internal
2
.12.2.3. Minimal bactericidal concentration (MBC). The MBC was
standard. The purified products (2 mg) were freeze-dried and dissolved
determined using a series of steps that were undertaken after the MIC
test had been completed. The MBC test was performed by sub-culturing
each well in which no visible growth occurred (blue color) from a pre-
OD containing 0.1% DSS prior to 1H- and C-NMR
13
in 1 mL of CD
analysis (Roslund, T a€ htinen, Niemitz, & Sj o€ holm, 2008).
3
�
vious MIC test to an agar medium. After 24 h of incubation at 37 C,
2
2
.12. Characterization of the methyl glucoside product
bacterial growth on the agar plate was evaluated. The lowest concen-
tration at which the MG
MBC value.
1
killed whole bacteria was determined to be the
.12.1. Emulsification activity and stability
The emulsification activity and stability of methyl- -D-glucoside
α
were performed according to the method of Cirigliano and Carman
1985), and compared to other alkyl glycosides. The final concentration
of 2.5 mM purified alkyl glucosides was prepared by dissolving in
0 mM Tris-HCl buffer containing 0.01 M MgSO , pH 8.0. The 4 mL
solution was mixed with 100 L of n-hexadecane to form an oil-in water
(
2.13. Data analysis
5
4
All data are expressed as the mean ꢁ SD from at least three separate
experiments and the differences were calculated using unpaired Stu-
dent’s t-test (GraphPad Prism version 4 software). The p-values < 0.05
were considered statistically significant.
μ
emulsion by vigorous mixing on a vortex for 2 min. The emulsification
activity was obtained after the reaction was left to stand for 10 min. The
turbidity was determined by a spectrophotometer at 540 nm. For
emulsification stability, the turbidity of the emulsion that had formed
was determined at 10, 20, 30, 40, 50 and 60 min. The absorbance at
3. Results and discussion
5
40 nm was calculated as a logarithmic function. The dissociation con-
3.1. Donor specificity
stant was calculated from the slope obtained from plotting the time
versus log (OD540). Commercial non-ionic surfactant, Triton™ X-100
The donor specificity of the CGTase was evaluated using
α-CD, β-CD
and the standard methyl-β-D-monoglucoside (M-β-G
1
) were used as the
and γ-CD as the glycosyl donors in the coupling reaction, with cellobiose
as the best acceptor of the B. circulans A11 CGTase according to
Wongsangwattana et al. (2010). The appropriate glycosyl donor for the
CGTase was determined by the glucose oxidase method and TLC analysis
control group.
2
.12.2. Antibacterial activity
Cultures of the following microorganisms were used in the study:
(data not shown). The results indicated that CGTase could transfer -1,
α
Gram-positive Staphylococcus aureus ATCC 25923 and Gram-negative
Escherichia coli ATCC 25922. The method of disc diffusion (CLSI,
4-linked glucosyl residues from all of the CDs donors to a cellobiose
acceptor, which was a β-1,4-glucosidic residue. The highest percentage
of the coupling activity was observed when β-CD was used as a donor
2
014), expressing both the minimal inhibitory concentration (MIC) and
minimal bactericidal concentration (MBC) (Sarker, Nahar, & Kumar-
and resulted in the largest transglycosylation yield at 588.71 g/mL, as
μ
asamy, 2007), was applied.
determined by TLC analysis. Judging from the enzyme activity and
product yields, β-CD is a suitable glycosyl donor for transglycosylation to
an alcohol acceptor. In addition to using β-CD as a glycosyl donor for
4

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
Fig. 1. (A.) Relative coupling activity of CGTase in
methanol and (B.) methyl glucoside products from
the transglycosylation reaction of β-CD to 10–50% (v/
v) methanol acceptors.
The relative coupling activity was performed as re-
ported in Section 2.6.2. and methyl glucoside prod-
1
ucts were quantified relative to a standard MG spot
in the same TLC plate. All values are averages from
triplicate TLC plates.
CGTase, other researchers have reported the use of starch as a glycosyl
donor for Bacillus macerans CGTase in the synthesis of β-arbu-
tin-
α-glucoside and β-arbutin-α-maltoside through its transglycosylation
reaction (Sugimoto, Nishimura, Nomura, Sugimoto, & Kuriki, 2003). For
B. circulans A11 CGTase, Aramsangtienchai, Chavasiri, Ito, and Pong-
sawasdi (2011) reported that starch, β-CD and maltoheptaose (G7) do-
nors were equally effective using glucose oxidase assay. However, β-CD
was the specific substrate of the β-CGTase and had a low degree on
hydrolysis reaction of this enzyme when compared to the starch sub-
strate. Moreover, the use of β-CD donor gave higher transglycosylation
yields than starch and formed fewer by-products. Another example, Aga
et al. (1991) found that Bacillus stearothermophilus CGTase could cata-
lyze the transglycosylation reaction using
α
-CD as a donor for the syn-
thesis of 2-O- -D-glucopyranosyl-L-ascorbic acid. Thus, the donor
α
specificity of CGTase depends on the unique property of each CGTase
that is used in the catalytic reaction to form a new covalent bond with
the acceptor of interest.
3
.2. Effect of the methanol concentration on the CGTase
transglycosylation reaction
To investigate the capability of CGTase in the transglycosylation of
methyl alcohol for the synthesis of methyl glucosides, the influence of
the methanol concentration was studied in a buffer and in a mixture of
buffer/methanol by varying the methanol concentration at the optimal
Fig. 2. TLC analysis of the transglycosylation products from β-CD to 30% (v/v)
methanol by CGTase.
TLC condition was System II, ethyl acetate/acetic acid/water, 3:1:1 (v/v). Lane
�
temperature (50 C) and pH (6.0) of this enzyme for 24 h. The methanol
1
, maltooligosaccharide
concentrations used here as single phase reactions were varied in the
range of 10–50% (v/v). The coupling activity of CGTase in the methanol
solution was determined in relative to that in 20 mM phosphate buffer,
pH 6.0 which was set as 100% (Fig. 1A). The results was, that when the
concentration of methanol increased, the activity of enzyme was
dramatically decreased. This could be a consequence of the negative
influence of the reduction of water activity, enzyme transition-state
destabilization and enzyme conformational modifications, which
resulted in a decrease of enzyme activity, including the product yield in
the alcoholic solutions. Thus, the alcohol concentration had an impor-
tant effect on the transglycosylation activity of CGTase. The enzyme
retained approximately 50% of its maximal coupling activity after
incubating in a co-solvent system of 20% (v/v) methanol. The effect of
methanol on the enzyme activity has generally been observed by many
investigators. For instance, the rate of starch hydrolysis by Aspergillus
standards; Lane 2, β-CD standard; Lane 3, standard MG
1
; Lane 4–5, product
from enzyme reaction without alcohol acceptor at 0 and 24 h; Lane 6–7,
product from enzyme reaction between β-CD and 30% methanol at 0 and 24 h;
MOSs ¼ maltooligosaccharides.
in the reaction mixture (Larsson, Svensson, & Adlercreutz, 2005). In
addition, the rate of sucrose consumption was also found to be lowered
with the rising concentration of methanol in the reaction catalyzed by
Rahnella aquatilis levansucrase (Kim, Kim, Lee, Song, & Rhee, 2000).
The effect of the methanol concentration on methyl glucoside syn-
thesis was also determined by subjecting the sample from the above
reaction to TLC analysis (System II). The concentration of methyl glu-
cosides was calculated from the intensity of the product spots relative to
standard MG spots on the same TLC plate and was expressed in units of
1
μg/mL. As shown in Fig. 1B, the relationship between the methanol
oryzae -amylase decreased with increasing concentration of methanol
α
5

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
Fig. 3. Effects of (A.) methanol concentration, (B.) β-CD concentration, (C.) enzyme concentration, (D.) incubation time, (E.) temperature and (F.) pH: acetate buffer
circle), phosphate buffer (square) and Tris-HCl buffer (triangle) on the production of methyl glucoside by CGTase.
The amount of methyl glucoside was calculated from the intensity of product spots as compared to 25 g of standard MG
(
μ
1
spot.
concentration and yield of product was found to be a bell-shaped curve.
This pattern explained the observation that when the concentration of
the methanol as an acceptor substrate in the reaction mixture was
property of the methyl group. In addition to the expected methyl
glucoside, the methanol acceptor also yielded other hydrolysis products,
such as glucose, maltose and other maltooligosaccharides. No methyl
glucoside product was observed in the control experiments. The methyl
glucoside product obtained using 30% (v/v) methanol consisted of at
least one product. Thus, these results demonstrate that the pBC recom-
binant CGTase could transfer glycosyl residues from β-CD to methyl
alcohol, yielding methyl glucoside. Furthermore, it is possible that other
longer carbohydrate chain glucosides were also produced in reaction
mixtures with methanol acceptor, but they may have been obscured by
the oligosaccharides. Likewise, hydrolysis products were formed with
i
increased, the initial velocity (V ) of the enzyme-catalyzed reaction
increased, leading to more products. However, over 30% (v/v) methanol
concentration decreased the product yield, which may have resulted
from the inactivation of the CGTase. Therefore, these results indicate
that the maximum yield was obtained using 30% (v/v) methanol. From
this point, the need for enzyme which has the high stability in organic
medium become a demand for this research. To the best of our knowl-
edge, the immobilization of enzyme is an important strategy to over-
come this problem. As previously reported (Adlercreutz, 2013;
Rodrigues, Ortiz, Berenguer-Murcia, Torres, & Fern a� ndez-Lafuente,
f
similar R values (Fig. 2, Lane 7). The hydrolysis products resulted from
the transfer of β-CD to water, which was due to the multifunctional
CGTase. In addition, hydrolysis products occurred in the reaction
mixture in preference to methyl glucosides because of the high affinity
of the enzyme for water. In summary, the methyl glucoside products
were produced by catalysis by the recombinant CGTase. This process
yielded at least 1 product (Fig. 2). To our knowledge, until now,
although CGTase has previously been used for methyl glucoside syn-
thesis, the focus of work was at the synthesis and optimization of the
alkyl glucoside production (Chotipanang et al., 2011). There have been
no reports on the structural and biological characterization of them.
Thus, methanol should be chosen here as acceptor to synthesize the
methyl glucosides for further biological study.
2
013), the immobilized enzyme could improve its enzyme stability due
to broad ranges of optimal temperatures and pH levels. In addition, the
immobilized enzyme could be also reused several times without enzyme
loss. Thus, this led to the increase of product yield.
To ensure that the product observed as a spot on the TLC was a
methyl glucoside product that was produced by CGTase (Fig. 2), a re-
action mixture without an alcohol acceptor (Fig. 2, Lane 4–5) and a
reaction mixture containing both substrates incubated with CGTase for
0
h (Fig. 2, Lane 6) were used as control experiments. After incubating
CGTase with the β-CD donor and methanol acceptor for 24 h, the ex-
pected methyl glucoside (MG ) spot (Product I, R
¼ 0.45) appeared at a
higher position than the maltooligosaccharides (MOSs) (G1-3, R
¼ 0.28,
.17, 0.1, respectively; β-CD, R ¼ 0.03) because of the non-polar
1
f
f
0
f
6

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
3
.4. Large-scale production and purification of methyl glucoside
Large scale production (250 mL) was performed after the reaction
conditions were optimized as described in methods Section 2.9. The
�
reaction mixture was concentrated by evaporation at 50 C. The
concentrated reaction was spotted onto preparative TLC. Preparative
TLC was employed to purify transglycosylation products by application
of an intense reaction mixture on the Merck preparative plate (1 and
2
mm). Mobile phase used here was the same as in TLC analysis. The
expected products at the indicated R value on preparative TLC were
f
scraped out separately and then, they were subjected to solvent
extraction with methanol. Prior to the mass spectrophotometry (MS)
and Nuclear magnetic resonance (NMR) analyses, the isolated product
was checked again by TLC. Only methyl monoglucoside (MG
enough yield for further characterization.
1
) had
3
3
.5. Structural analysis of methyl glucoside
.5.1. Mass spectrophotometry (MS) analysis
The molecular weight of synthesized product was elucidated by mass
spectrometer with the positive mode as described in Section 2.11.1. The
þ
pseudomolecular ion [MþNa] of synthesized methyl-
α
-monoglucoside
from the CGTase displayed at m/z 217.08 (194.08 plus 23 sodium
molecule). This product corresponded to the size of methyl mono-
1 7 14 6
glucoside (MG , C H O ).
3
.5.2. Degradation of reaction products with glucoamylase/
To confirm that the product was a glucosyl derivative, the reaction
mixture was treated with glucoamylase from Rhizopus sp. This enzyme
cleaves the glycosidic bond of -1,4-glucan. After the reaction mixture
α
-glucosidase
Fig. 4. TLC analysis of amylolytic enzyme treatment of CGTase reaction
products.
TLC condition was System III, ethyl acetate/acetic acid/water, 3:1.5:0.5 was
α
used to separate other methyl glucosides i.e. methyl maltoside (MG
methyl maltotrioside (MG ). Lane 1: Standard methyl glucoside (MG ) (20
Lane 2: Standard G1-4 (20 g), Lane 3: Reaction mixture at 0-min incubation,
2
) and
was incubated for 24 h, glucoamylase (20 U/mL) was added and then
3
1
μg),
�
incubated at 37 C for 24 h, and analyzed by TLC (Fig. 4, Lane 5). The
μ
results showed that after treatment with glucoamylase, the spots of
transfer product (Product II and III) and hydrolysis product (maltooli-
Lane 4: Reaction mixture at 24-h incubation, Lane 5: Reaction mixture at 24-h
incubation and treatment with glucoamylase for 24 h, Lane 6: Reaction mixture
gosaccharides) disappeared and only the intensities of MG
increased. So, it implied early that the bonds which were linked between
methanol and sugars i.e maltose or maltotriose of Product II (MG ) and
III (MG ), were -glycosidic linkages, corresponding to MG where n > 1
Nakamura, Haga, & Yamane, 1994). In addition to Rhizopus sp. glu-
1
and glucose
at 24-h incubation and treatment with
.3. Optimal condition for the synthesis of methyl glucosides
To produce the highest yield of methyl glucosides, the parameters
α-glucosidase for 24 h.
2
3
3
α
n
(
coamylase treatment, the glucoside products were also checked for the
configuration between methanol and first linked-glucosyl unit with
involved in the reaction were optimized in a sequentially independent
manner by changing various conditions in the transglycosylation reac-
tion. These parameters included the substrate concentration, enzyme
concentration, incubation time, temperature and pH. The products that
were obtained from each reaction were analyzed by TLC (System II) and
A. niger
α
-glucosidase. This enzyme hydrolyzes the
α
-linkage of the
non-reducing terminal of glucoside substrate (Yao, Mauldin, & Byers,
2
003). The 24 h-reaction mixture of CGTase was further treated with
�
A. niger
α-glucosidase (20 U/mL) for 24 h at 37 C before TLC analysis.
1
quantified by comparison with the spot intensity of the standard MG . As
As a result in Fig. 4 (Lane 6), only glucose was observed, so it means that
the linkage of methanol and first glucose was -form type.
a result, the optimum conditions that were obtained for the synthesis of
methyl glucosides were: incubation with 30% (v/v) methanol and 1.5%
α
(
w/v) β-CD with 500 U/mL CGTase in 20 mM phosphate buffer, pH 6.0,
�
3.5.3. Nuclear magnetic resonance (NMR) analysis
The 13C-NMR and 1H-NMR spectra of MG
(Product I, R
at 50 C for 120 h (Fig. 3). Under these conditions, the total yield of
methyl glucoside was increased to 13-fold (3.2 mg/mL or 39% (w/w)
yield) compared to the yield obtained before optimization (0.24 mg/mL
or 3% (w/w) yield). The synthesis of alkyl glucosides by the action of
other enzymes has been reported. For example, β-glucosidase from Thai
rosewood (Lirdprapamongkol & Svasti, 2000) and almond meal (Wang
et al., 2012) could catalyze the synthesis of methyl β-D-monoglucoside
with yields of 97 and 28% (w/w), respectively. However, the transfer
reaction catalyzed by both glucosidases could only transfer one mono-
saccharide unit. The reaction of Leuconostoc mesenteroides dex-
transucrase with sucrose and methanol resulted in a 38% (w/w) yield of
1
f
¼ 0.45)
were performed as described in Section 2.11.3. According to Fig. 5A, a
doublet signal at 4.65 ppm (J ¼ 3.67 Hz) was assigned to the anomeric
proton (Kapaev, Egorova, & Toukach, 2014), which suggests that only
one glucose unit was
-linked to the methyl group. The 1H-NMR spec-
α
trum displayed methyl protons at 3.41, suggesting the presence of
methanol. The 13C-NMR spectrum also showed seven carbon signals,
including carbon signals of both glucose and methanol (Fig. 5B). The
α
-configuration of the D-glucosyl residue in MG
anomeric carbon of glucosyl signal at δ ¼ 99.86 ppm. From these results
with ESI-TOF MS and enzymatic treatment, the structure of MG
1
was confirmed by the
1
methyl
sp. RB01 could catalyze the synthesis of methyl
product with a maximum yield of 37% (w/w) (Chotipanang et al.,
011). Hence, the methyl monoglucoside yield that was obtained in this
α
-D-monoglucoside (Kim et al., 2009). CGTase from Paenibacillus
(
Product I, R
MG ).
f
¼ 0.45) was identified as methyl-α-D-monoglucoside
α
-D-monoglucoside
(
1
2
study is quite good compared to the group involved in the bacterial
enzyme-catalyzed synthesis.
7

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
1
3
1
Fig. 5. The (A.) C-NMR and (B.) H-NMR of methyl- -D-monoglucoside (400 MHz).
α
3
3
.6. Characterization of the methyl glucoside products
of alkyl glycosides was found in PenG
M-β-G , respectively. These results suggested that the emulsification
activity of alkyl- -D-glycosides increased when increasing number of
hydrophobic alkyl chain length and hydrophilic moiety from glucose
residue. Furthermore, the alkyl- -D-glycosides with linear alkyl chain
showed better emulsification activity than that of branched chain alkyl-
-D-glycosides. In addition, the stability of emulsion formed by alkyl
1 1 1
, followed by IsoPenG , MG and
1
.6.1. Emulsification activity and stability
α
Emulsification activity of purified alkyl glycosides was determined
from their ability to form an oil-in-water emulsion with the n-hex-
adecane substrate. The commercial Triton™ X-100 and M-β-G , and the
synthetic pentyl- and isopentyl glucoside (PenG and IsoPenG ) were
α
1
1
1
α
also checked for emulsification activity. The highest turbidity was found
in Triton™ X-100 and set as 100% emulsification activity. The turbidity
of other alkyl glycosides was measured and compared to that with
Triton™ X-100. As shown in Table 1, the highest emulsification activity
glycosides was also studied as a function of time. The emulsion of n-
hexadecane was performed and the turbidity was measured every
10 min for 60 min at OD540. The logarithms of the absorbance were
d
plotted versus time and the dissociation constant (K ) value was
8

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
Table 1
Table 2
Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration
Emulsification activity and stability of commercial products and trans-
glycosylated products.
(MBC) of MG
1
and ampicillin against microorganisms.
a
b
a
Compound
Emulsification activity (%)
Emulsification stability (%)
Microorganism
MIC and MBC values
1
MG (mg/mL)
Triton™ X-100
100 ꢁ 0.0
100 ꢁ 0.0
12.5 ꢁ 0.4
79.1 ꢁ 1.0
84.7 ꢁ 0.9
92.7 ꢁ 1.2
89.1 ꢁ 1.1
Ampicillin ( g/mL)
μ
Methanol
4.41 ꢁ 0.7
24.41 ꢁ 0.8
26.40 ꢁ 0.5
36.32 ꢁ 0.9
31.14 ꢁ 1.0
b
S. aureus ATCC® 25923
E. coli ATCC® 25922
0.08 ꢁ 0 (0.08 ꢁ 0)
0.98 ꢁ 0 (0.98 ꢁ 0)
20.00 ꢁ 0 (40.00 ꢁ 0)
0.63 ꢁ 0 (0.63 ꢁ 0)
M-β-G
MG
PenG
1
1
c
a
1
All data are shown as mean ꢁ SD derived from triplicate experiments.
d
IsoPenG
1
b
MBC values are shown in parentheses.
a
Determined by the turbidity of emulsification.
b
Calculated from logarithmic plot of absorbance versus time.
number of hydrophobic alkyl chain lengths and number of glucose
groups. Moreover, the alkyl chain length has a much stronger influence
on emulsification activity and stability compared to the number of
glucose groups in the alkyl glycoside.
c-d
The products were synthesized as the previous report (Charoensapyanan
et al., 2017).All values are the mean of three separate experiments.
3
.6.2. Antibacterial activity
The antibacterial activity of MG
1
was determined using agar diffu-
(1, 5 and 10 mg/disc) was
sion method. The antibacterial activity of MG
1
tested against two bacterial strains, 1 g-positive (Staphylococcus aureus
ATCC 25923) and 1 g-negative (Escherichia coli ATCC 25922). These
strains have been selected for their usefulness in further study applica-
tion. Normally, they are found to be skin flora and food-borne patho-
gens, respectively (Awwad et al., 2013). Antibacterial potential of the
MG
Zone of inhibition of the MG
standards i.e. ampicillin (10 g/disc) and tetracycline (30
results revealed that MG (1, 5 and 10 mg/disc) exhibited no antibac-
1
was assessed in terms of zone of inhibition of bacterial growth.
was compared with that of different
g/disc). The
1
μ
μ
1
terial activity against S. aureus, with inhibition zones of 0 mm, but
inhibited strongly the growth of E. coli, with inhibition zones of 28, 38
and 40 mm, respectively (Fig. 6). As seen in Table 2, the lowest MIC and
MBC values for MG
S. aureus, respectively, and 0.63 and 0.63 mg/mL against E. coli,
respectively. As a result, it showed that MG was the most effective agent
for inhibiting E. coli growth. This pattern of activity of the MG is likely a
1
were found to be 20.0 and 40.0 mg/mL against
1
1
result of its property as non-ionic surfactants that can disrupt both the
lipopolysaccharides and phospholipids of the gram-negative bacterial
cell wall, resulting, in turn, in the inhibition of bacterial growth. In
contrast, an antibiotic drug, ampicillin (10
inhibit peptidoglycan synthesis in the cell wall of gram-positive bacteria
Fig. 6). Similarly, Matin, Bhuiyan, and Azad (2013) and Matsumura
μ
g/disc), had a propensity to
(
et al. (1990) found that other alkyl glycosides, such as butyl glycoside
and dodecyl mannoside, exhibited a broad spectrum of antimicrobial
activity. They also reported that the antimicrobial activity resulted from
the dominant role of alkyl glycosides in penetrating microbial cell
membranes (Knol et al., 1998; Lichtenberg et al., 2013; Wenk, Alt,
Seelig, & Seelig, 1997).
4
. Conclusion
The enzyme from the pBC recombinant cell containing the CGTase
gene from Bacillus circulans A11 can be used to synthesize methyl -D-
α
Fig. 6. The inhibition zone of MG1 against (A.) S. aureus ATCC 25923 and (B.)
E. coli ATCC 25922.
glucosides, which have one to several glucose residues from β-CD donor
and methanol acceptor through a transglycosylation reaction. Under
optimal conditions, the yield of methyl glucosides obtained was
increased compared to pre-optimization levels. The acceptor product for
d
calculated from the slope of the graph. The smaller K is the greater
stability. The emulsification stability of Triton™ X-100 was set as 100%
due to its being the most effective in stabilization of the oil-in-water
methanol was confirmed as methyl-
α
-D-glucopyranoside by ESI-TOF-MS
and NMR analyses. Moreover, the obtained MG
1
product from this study
emulsion. The addition of alkyl-
revealed that they were able to stabilize the emulsion over a period of
time tested (Table 1). PenG exhibited the best ability to stabilize an oil-
in-water emulsion. Although, MG showed lower emulsifying properties
than PenG , it was better than that of methanol. So, these data indicated
that MG could likely be used as an emulsion-stabilizing agent. This was
α-D-glycosides to the emulsion
exhibited emulsifying properties and good antibacterial activity against
gram-negative bacterium, E. coli. Finally, for its application to industry,
1
1
MG may be used as an alternative antibacterial agent to replace anti-
1
biotic for preventing attachment of pathogens.
Conflicts of interest
1
1
highly probable as Von Rybinski (1996), report that the emulsification
activity and stability of alkyl glycosides increased according to the
The authors declare that they have no conflict of interest.
9

J. Kaulpiboon and P. Rudeekulthamrong
Bioactive Carbohydrates and Dietary Fibre 20 (2019) 100197
Acknowledgements
Knol, J., Sjollema, K., & Poolman, B. (1998). Detergent-mediated reconstitution of
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Kometani, T., Terada, Y., Nishimura, T., Takii, H., & Okada, S. (1994).
Transglycosylation to hesperidin by cyclodextrin glucanotransferase from an
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The authors gratefully acknowledge the financial support provided
by the Faculty of Medicine, Thammasat University, Thailand under the
research fund Contract No. TP-2-16/2016. In addition, partial support
from the Phramongkutklao College of Medicine Research Fund,
Thailand is also acknowledged.
Larsson, J., Svensson, D., & Adlercreutz, P. (2005). -Amylase-catalysed synthesis of
α
alkyl glycosides. Journal of Molecular Catalysis B: Enzymatic, 37, 84–87.
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solubilization of lipid bilayers. Biophysical Journal, 105, 289–299.
Lirdprapamongkol, K., & Svasti, J. (2000). Alkyl glucoside synthesis using Thai rosewood
β-glucosidase. Biotechnology Letters, 22, 1889–1894.
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