Enzymatic synthesis of propyl-α-glycosides and their application as emulsifying and antibacterial agents

Article abstract of DOI:10.1007/s12257-016-0013-z

Alkyl glycosides have been effectively used in many industries because of their biodegradable, emulsification and antibacterial properties. In this study, the alkyl glycoside of propyl glycosides (PG_n) was synthesized using β-cyclodextrin (β-CD) and 1-propanol through the transglycosylation reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from the Bacillus circulans A11. The optimal condition for the synthesis of propyl glycosides consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of CGTase in a water/propanol content containing 10% (v/v) 1-propanol at pH 6.0, 50°C for 96 h. Upon analysis of the product at the optimal condition by TLC, at least three products which move faster than glucose were observed. These transferred products were formed with molecular weights of 222.1, 384.1 and 546.4 daltons as determined by mass spectrometry analysis; these values were in accordance with propyl glucoside (PG₁), propyl maltoside (PG₂) and propyl maltotrioside (PG₃), respectively. PG₁ and PG₂ were produced and prepared on a large scale and subsequently purified by preparative TLC. The combined ¹H- and ¹³C-NMR analysis confirmed that the structures of PG₁ and PG₂ were propyl-α-D-glucopyranoside and propyl-α-D-maltopyranoside, respectively. Both PG₁ and PG₂ showed emulsification activity and stability in their formation in water and n-hexadecane. Furthermore, the antibacterial activity of both products was determined and it was found that PG₂ had a higher antibacterial activity against Staphylococcus aureus and Escherichia coli than that of PG₁.

Full text of DOI:10.1007/s12257-016-0013-z

Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
DOI 10.1007/s12257-016-0013-z
RESEARCH PAPER
Enzymatic Synthesis of Propyl-α-glycosides and Their Application as
Emulsifying and Antibacterial Agents
Rittichai Charoensapyanan, Kazuo Ito, Prakarn Rudeekulthamrong, and Jarunee Kaulpiboon
Received: 7 January 2016 / Revised: 4 April 2016 / Accepted: 27 April 2016
© The Korean Society for Biotechnology and Bioengineering and Springer 2016
Abstract Alkyl glycosides have been effectively used in structures of PG1 and PG2 were propyl-α-D-glucopyranoside
many industries because of their biodegradable, emulsification and propyl-α-D-maltopyranoside, respectively. Both PG1
and antibacterial properties. In this study, the alkyl and PG2 showed emulsification activity and stability in
glycoside of propyl glycosides (PGn) was synthesized their formation in water and n-hexadecane. Furthermore,
using β-cyclodextrin (β-CD) and 1-propanol through the the antibacterial activity of both products was determined
transglycosylation reaction of recombinant cyclodextrin and it was found that PG2 had a higher antibacterial activity
glycosyltransferase (CGTase) from the Bacillus circulans against Staphylococcus aureus and Escherichia coli than
A11. The optimal condition for the synthesis of propyl that of PG1.
glycosides consisted of an incubation of 1.5% (w/v) β-CD
and 500 U/mL of CGTase in a water/propanol content Keywords: antibacterial activity, cyclodextrin glycosyl-
containing 10% (v/v) 1-propanol at pH 6.0, 50°C for 96 h. transferase, intermolecular transglycosylation, non-ionic
Upon analysis of the product at the optimal condition by surfactant, propyl glycosides
TLC, at least three products which move faster than
glucose were observed. These transferred products were
formed with molecular weights of 222.1, 384.1 and 546.4
daltons as determined by mass spectrometry analysis; these
1. Introduction
values were in accordance with propyl glucoside (PG1), Alkyl glycosides are non-ionic surfactants which are
propyl maltoside (PG2) and propyl maltotrioside (PG3), composed of the hydrophilic sugar head group connected
respectively. PG1 and PG2 were produced and prepared on to the alkyl hydrophobic tail via a glycosidic bond. They
a large scale and subsequently purified by preparative TLC. become dominant components in products of the food,
1 13
The combined H- and C-NMR analysis confirmed that the detergent, cosmetic and pharmaceutical industries as a result
of their biodegradability, emulsification and antibacterial
properties [1]. Moreover, alkyl glycosides can be synthesized
Rittichai Charoensapyanan
by both chemical and enzymatic methods. However, the
Biochemistry and Molecular Biology Ph.D. Program, Faculty of Medicine,
enzymatic syntheses of alkyl glycosides, as shown from
Thammasat University, Pathumthani 12120, Thailand
many previous studies, are more advantageous than
Kazuo Ito
chemical syntheses. The enzymatic method proceeds under
Department of Biology, Graduate School of Science, Osaka City University,
Osaka 558-8585, Japan
non-toxic and mild conditions by a simple one-step reaction
process [2]. As reported, the alkyl glycosides could be
Prakarn Rudeekulthamrong
produced by several enzymes which differed in the kind
and length of the sugars and hydrocarbons involved as well
as the configuration of the linkages which depended on the
specific action of each enzyme [3-6]. The enzymatic synthesis
of alkyl glycosides can be achieved through several types
of enzymes as extracted from many organisms, such as β-
Department of Biochemistry, Phramongkutklao College of Medicine,
Phramongkutklao Hospital, Bangkok 10400, Thailand
*
Jarunee Kaulpiboon
Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine,
Thammasat University, Pathumthani 12120, Thailand
Tel: +662-926-9743; Fax: +662-926-9710
E-mail: jkaulpiboon@yahoo.com

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Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
mannosidase from Aspergillus niger [7], β-galactosidase from be used as effective protein-solubilizing surfactants and
Aspergillus oryzae [8], dextransucrase from Leuconostoc solubilizing agents for cell membranes [18-20]. Therefore,
mesenteroides [9] and, as indicated in most reports on α- or the aim of this study was to use the pBC recombinant
β-glucosidase, from various other sources [3-5]. However, CGTase from Bacillus circulans A11 to synthesize propyl
the group designated cylclodextrin glycosyltransferase glycosides using β-CD as the glycosyl donor and 1-
(CGTase, E.C. 2.4.1.19) has been studied less through alkyl propanol as an acceptor. The influence of various parameters
glycoside synthesis. CGTase has only been used to elongate on the production of propyl glycosides was investigated.
the carbohydrate group of commercially available dodecyl- The structure and biological properties of the propyl
β-D-maltoside via its coupling reaction using α-CD as a glycosides were also studied.
glycosyl donor [6].
CGTases belong to the α-amylase family, which consists
of multifunctional enzymes. CGTases catalyze mainly intra-
and inter-transglycosylation reactions (cyclization, coupling
2. Materials and Methods
and disproportionation), but can also exhibit, to a lesser 2.1. Materials
extent, α-amylase-like activity, hydrolyzing starches into Glucose (G1); maltose (G2); maltotriose (G3); maltotetraose
short linear saccharides [6,10]. On their first reactions, (G4); maltopentose (G5); maltohexose (G6); maltoheptaose
CGTases convert starch substrates to non-reducing cyclic (G7); methyl-α-D-glucopyranoside (MG1); sucrose; α-, β- and
maltooligosaccharides called cyclodextrins (CDs) through γ-CDs; soluble potato starch; corn starch; phenolphthalein,
the cyclization reaction. CDs are applied as stabilizers or n-hexadecane and bovine serum albumin were purchased
solubilizers in the food, pharmaceutical and cosmetic from Sigma-Aldrich (St. Louis, MO, USA). Mannitol salt
industries for their ability to form inclusion complexes with agar (MSA), yeast extract and tryptone were obtained from
hydrophobic molecules [11]. Besides the cyclization reaction, Difco (Bacton Dickinson and company, Sparks, MD, USA).
CGTases also catalyze a disproportionation reaction by Aquacide II, methanol, ethanol, silica gel 60 F254 glass plate
transferring linear α-1,4-linked oligosaccharides to another (20 cm in height) were purchased from Merck (Darmstadt,
oligosaccharide molecule; they also catalyze a coupling Germany). 1-Propanol was obtained from CARLO ERBA
reaction by opening CD rings and transferring them to Reagents (Val de Reuil, France). The commercial glucose
acceptors [6,10,11]. Naturally, the CGTase acceptors are oxidase kit was obtained from Human mti-diagnostics
saccharides or glycosides, which are very abundant in GmbH (Idstein, Germany). Other chemicals used were of
nature [12-14]. This transglycosylation reaction, transferring an analytical grade.
from donor to acceptor, proceeds via the major catalytic
machinery of enzymes for the synthesis of new glycosidic 2.2. Bacterial cultivation and enzyme production
bonds [15]. Thus, from this mechanism, CGTase also Escherichia coli BL21 (DE3) cells harboring the pBC
catalyzes transglycosylation to various compounds other recombinant plasmid, a pET19b-based plasmid containing
than saccharides, such as piceid [16] and ascorbic acid the CGTase gene with a signal peptide sequence from
[17]. Mathew et al. [16] have reported the efficient synthesis Bacillus circulans A11 (GenBank accession no. AF 302787),
of piceid glycosides through disproportionation catalyzed were prepared as previously described [21]. The recombinant
by CGTase from Bacillus macerans using maltodextrin as cells were grown in Luria Bertani (LB) medium containing
a glycosyl donor and piceid as a glycosyl acceptor. The 100 µg/mL ampicillin at 37°C for 24 h. The expression of
obtained piceid glycosides showed an increase in recombinant enzymes was induced with 0.2 mM IPTG
absorbability in the digestive tract. Aga et al. [17] reported when the OD600 of the culture reached 0.6. After 24 h, the
on the use of CGTase from Bacillus stearothermophilus in cells were removed and the culture broth containing crude
the production of ascorbyl glycosides. The result was a CGTase was collected by centrifugation at 12,000 × g at
greater stability under oxidative conditions through the 4°C for 2 h for further purification.
coupling reaction using α-CD and ascorbic acid as a
glycosyl donor and acceptor, respectively. In addition, 2.3. Purification of CGTase
CGTase can also use water as acceptor, resulting in a Recombinant CGTase was purified from the culture broth
hydrolysis reaction.
by starch adsorption [22] and DEAE-Toyopearl 650M
In this study, the catalytic mechanisms of CGTase were column chromatography. Corn starch was dried in an oven
exploited for the synthesis of a series of propyl glycosides. at 60°C for 3 h, cooled to room temperature and then
The CGTase enzyme can produce propyl glycosides that gradually added to a culture broth of crude CGTase at 4°C
have more than one glucose unit attached. There are to produce a final concentration of 5% (w/v). After 3 h of
numerous reports demonstrating that alkyl glycosides can continuous stirring, the starch cake was collected by

Enzymatic Synthesis of Propyl-α-glycosides and their Application as Emulsifying and Antibacterial Agents
391
centrifugation at 8,000 × g for 30 min at 4°C and washed 0.2 M phosphate buffer, pH 6.0. The reaction mixture was
twice with TB1 buffer (10 mM Tris-HCl, pH 8.5 containing incubated at 50°C for 5 min and was terminated by boiling.
10 mM CaCl2). The adsorbed CGTase was eluted from the Subsequently, the mixture was incubated with 10 µL of
starch cake by stirring for 30 min with 0.2 M maltose in glucoamylase (stock solution, 30 units) in 0.2 M phosphate
TB1 buffer (3 × 50 mL per liter of starting broth). The eluted buffer, pH 6.0, at 40°C for 30 min, and the reaction was
CGTase was collected by centrifugation at 10,000 × g for stopped by boiling for 5 min. An aliquot (10 µL) from the
30 min at 4°C. The CGTase solution was poured into dialysis reaction mixture was then added to 1 mL of glucose
tubing and concentrated by coating the tubing with a water- oxidase reagent and incubated at 37°C for 5 min before
absorbing agent (carboxymethylcellulose, Aquacide II), the absorbance was measured at 500 nm. The glucose
followed by dialyzation against TB1 buffer at 4°C three concentration was calculated from equation (1):
times before being subjected to DEAE-Toyopearl 650M
Glucose concentration (µmol/mL) = 5.55 × (ASample/AStd.)
column chromatography. The column (1.5 × 10 cm) was
(1)
equilibrated with TB2 buffer (10 mM Tris-HCl, pH 8.0).
CGTase was then applied to the column, and unbound
The absorbance of the standard solution (AStd.) was
proteins were eluted with TB2 buffer. After the column measured by adding 10 µL of a standard glucose solution
was washed thoroughly with TB2 buffer, bound proteins (5.55 µmol/mL) to 1 mL of glucose oxidase reagent. One
were eluted from the column using a linear salt gradient of unit of glucose oxidase activity was defined as the amount
0 ~ 0.2 M sodium chloride in TB2 buffer. Fractions of 2.0 mL of enzyme that produces 1 µmol of glucose per min under
were collected continuously. The protein and CGTase- the assay conditions used.
dextrinizing activity profiles of the eluted fractions were
monitored by measuring the absorbance at 280 and 600 nm, 2.4.2.2. Phenolphthalein assay
respectively. Fractions containing CGTase-dextrinizing The phenolphthalein assay measures the disappearance of
activity were pooled for further characterization. The purity β-CD in the reaction mixture [26]. A 250 µL portion of the
of the enzyme from each step in the purification was β-CD standard or sample solution was incubated with 750 µL
®
determined using 7.5% native-PAGE and a Coomassie
blue staining method [23].
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
∆A550 to µmoles of β-CD was performed using the β-CD
phenolphthalein calibration curve. The disappearance of
β-CD in the reaction mixture was calculated from the
2.4. Assay of CGTase activity
2.4.1. Dextrinizing activity
The dextrinizing activity of CGTase was determined by difference between β-CD concentration at 0 and 24 h
measuring the decrease in absorbance of a starch-iodine incubations with CGTase and acceptors. One unit of activity
complex at 600 nm [24]. The enzyme sample (50 µL) was was defined as the amount of enzyme capable of coupling
incubated with 0.15 mL of 0.2% (w/v) soluble potato 1 µmole of β-CD to alcohol per min.
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 2.5. Protein determination
of 0.2 M HCl and 0.25 mL of iodine reagent (0.02% (w/v) The protein concentration was determined by Bradford’s
I2) in 0.2% (w/v) KI). The mixture was adjusted to a final method [27], using bovine serum albumin (BSA) as a
volume of 5 mL with distilled water, and the absorbance at standard.
600 nm was measured. For the control, HCl was added
before the enzyme sample was taken. One unit of enzyme 2.6. Synthesis of alkyl glycosides
was defined as the amount of enzyme that produced a 10%
reduction in the intensity of the blue color of the starch- 2.6.1. Donor specificity
iodine complex per min under the described conditions.
2.4.2. Coupling activity
Selection of the appropriate donor was performed by
incubating 0.64% (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 (α-CD, β-CD and γ-CD) in a
reaction volume of 250 µL at 50°C for 24 h. The occurred
2.4.2.1. Glucose oxidase assay
A glucose oxidase kit was used to measure the enzymatic glucose from the CGTase transglycosylation reaction was
activity by exchanging β-CD for glucose [25]. The reaction determined using the glucose oxidase assay and the transfer
mixture (100 µL) contained 40 µL of 1% (w/v) β-CD, 40 µL products were analyzed by thin-layer chromatography
of 1% (w/v) cellobiose, 5 µL of the enzyme and 15 µL of (TLC) with the mobile phase of System I as described in

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Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
Section 2.7.
Erlenmeyer flask. The reaction mixture contained the β-CD
donor, 1-propanol acceptor and enzyme, all of which were
incubated under the optimum condition according to
2.6.2. Determination of transglycosylation efficiency
The efficiency of the CGTase transglycosylation reaction Section 2.8. After the incubation, the reaction mixture was
was determined from the transglycosylation product yield, stopped by boiling and then concentrated using a centrifugal
which was determined from the phenolphthalein assay and evaporator at 45°C prior to purification of the propyl
by TLC analysis. The transglycosylation reaction with glycosides by preparative thin-layer chromatography (PLC).
various alcohol types and contents were performed using
β-CD as a glycosyl donor. The reaction mixture consisted 2.10. Purification of propyl glycosides
of 0.64% (w/v) β-CD and various alcohol types and The concentrated reaction mixture was placed on a PLC
concentrations (methanol, ethanol or 1-propanol: 5 ~ 50% plate and developed at ambient temperature using solvent
(v/v)) and CGTase (75 U/mL by dextrinizing activity) in System II. One lane of the reaction mixture on the plate
20 mM phosphate buffer at pH 6.0 in a total volume of was then cut out and used to detect the propyl glycoside
250 µL. The reaction mixture was incubated at 50°C for products using a sulfuric acid/methanol (1:9 by vol)
24 h. The reaction was then terminated and assayed using developing solution. The preparative plate was dried and
the phenolphthalein method to determine the stability of heated at 110°C for 20 min. Each product from the PLC
CGTase in alcohols. In addition, aliquots from each reaction plate that had approximately the same relative mobility (Rf)
mixture were withdrawn and analyzed by TLC to determine values was scraped separately. The propyl glycoside
the transglycosylation yield using the mobile phase of products were extracted from the adsorbent (silica) by
System II as described in Section 2.7.
dissolving with methanol for 30 min at room temperature
and then centrifuging at 8,000 × g at 25°C for 1 h. The
methanol was further removed using a rotary evaporator at
2.7. TLC analysis
The transfer products were analyzed using Silica gel 60 45°C until the samples were thoroughly dry.
F254 (Merck, Germany) base TLC using a 5:5:3 (v/v/v)
ratio of n-butanol/ethyl alcohol/water as a mobile phase 2.11. Structural analysis of propyl glycosides
solvent (System I) to determine the best donor for the
synthesis of transglycosylation products. For the analysis 2.11.1. Digestion with amylolytic enzyme
of alkyl glycosides products, mobile phase System II (ethyl The structures of CGTase-catalyzed propyl glycosides were
acetate/acetic acid/water with a 3:1:1 (v/v/v) ratio) was preliminarily characterized by incubating with amylolytic
used. Spots on the TLC plate were visualized by dipping enzymes. Each dried propyl glycoside product was dissolved
into sulfuric acid/methanol (1:9 (v/v) ratio), followed by in 0.2 M acetate buffer, pH 5.5, followed by incubating
drying and heating at 110°C for 20 min. The intensity of with α-glucoamylase and α-glucosidase (20 U/mL) at 37°C
the synthesized product spots was quantified using a for 24 h. After incubation, the reaction was stopped by
®
scanning densitometer and a Quantity One 1-D analysis boiling and a 10 µL aliquot from each reaction was then
program. Glucose and methyl-α-D-glucopyranoside (MG1) analyzed by TLC (System II).
were used as standards.
2.11.2. Mass spectrometry (MS) analysis
2.8. Optimization of propyl glycoside production
The transfer products of interest were dissolved in 50% (v/v)
The optimal conditions for the production of propyl methanol, and their masses were determined using a
glycosides were decided based on the highest percentage microTOF spectrometer (Bruker, Germany). The products
yield of propyl glycoside products as determined from the were introduced into a mass spectrometer for processing in
TLC results. The effects of varying the 1-propanol acceptor the ESI-TOF-MS system, which ionizes by electrospray
(5 ~ 40% (v/v)), β-CD donor concentration (0.3 ~ 1.8% ionization (ESI) in the sodium positive ion mode using a
(w/v)), enzymatic units (50 ~ 500 U/mL), incubation time capillary voltage of 5,000 volts. A 4.0 L/min flow of nitrogen
(24 ~ 168 h), temperature (30 ~ 70°C) and pH (4.0 ~ 9.0) gas at a temperature of 150°C was used to nebulize the
were investigated using a sequential approach.
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)
2.9. Large-scale production
To obtain a higher yield of propyl glycoside products spectra of the products was detected as pseudo-molecular
+
for structural analysis and to determine their biological ion peaks [M+Na] from which the molecular weights
properties, large-scale production of propyl glycosides was were calculated using a Bruker Daltonics DataAnalysis 3.4
conducted in a total volume of 250 mL in a 1,000 mL software program.

Enzymatic Synthesis of Propyl-α-glycosides and their Application as Emulsifying and Antibacterial Agents
393
2.11.3. Nuclear magnetic resonance (NMR) analysis
on the surface of S. aureus-inoculated mannitol salt agar
Structural identification of the propyl glycosides that were and E. coli-inoculated LB agar. Standard antibiotics, i.e.,
1
13
synthesized by CGTase was performed by H- and C-NMR 10 µg/disc of ampicillin, 10 µg/disc of penicillin, 30 µg/disc
using a Bruker AVANCE III HD 600 NMR Spectrometer of tetracycline and 1 µg/disc of oxacillin, were also tested
in the Department of Biology, Graduated School of Science, as well as sterile water as a negative control. The plates were
Osaka City University, Japan. The spectrometer was operated incubated at 37°C for 24 h. The clear zone of inhibition
at 600 MHz at ambient temperature. Chemical shifts were was calculated by measuring the diameter of the inhibition
measured using sodium-4,4-dimethyl-4-sila-pentane sulfonate zone. The readings were taken in three different fixed
(DSS) as the internal standard. The purified products directions, and an average value of inhibition zone was
(2 mg) were freeze-dried and dissolved in 1 mL of CD3OD tabulated [31,32].
1 13
containing 0.1% DSS prior to H- and C-NMR analysis.
2.12.2.2. Minimal inhibitory concentration (MIC)
2.12. Characterization of the propyl glycoside products The minimal inhibitory concentration (MIC) value was
determined in liquid LB medium using the microdilution
method in a 96-well microtiter plate by incubating the
2.12.1. Emulsification activity and stability
The emulsification activity and stability of the propyl bacteria in LB broth with variable amounts of the sample
glycoside products were determined according to the method being tested. Briefly, the 6-mg starter content of propyl
of Cirigliano and Carman [28]. Purified propyl glycosides glycosides in 50 µL of sterile water was diluted by a two-
were dissolved in 4 mL of 0.5 mM Tris-HCl buffer fold serial dilution in multiple steps in the microtiter plate,
containing 0.05 M MgSO4 at pH 8.0 to a final concentration while the 0.5 McFarland culture was also diluted with LB
of 0.5 mg/mL. This solution was added to 100 µL of broth in a 1:200 (v/v) ratio. The 50 µL of culture was
n-hexadecane in a test tube and mixed continuously on a added to the 96-well microtiter plate. Following 18 h of
vortex mixer for 2 min to form an oil-in-water emulsion. incubation, 10 µL of resazurin solution was added to each
The emulsification activity was measured after the mixture well and the plate was incubated for 2 h. The minimum
was left to stand for 10 min. The activity was determined inhibitory concentration (MIC) value was defined as the
from the turbidity of an oil-in-water emulsion that was lowest concentration of propyl glycoside that prevented a
measured using a spectrophotometer at 540 nm. In addition, color change of resazurin from blue to pink. Resazurin, a
TM
other commercial non-ionic surfactants, such as Triton
blue dye, becomes pink when it is reduced to resorufin by
X-100 and methyl-α-D-monoglucoside (MG1), were also an oxidoreductase within a medium of viable bacteria. The
studied. The highest turbidity resulted when the surfactant MIC value was determined in triplicate experiments and
was set to 100% emulsification activity. For emulsification was expressed as mg/mL [31,32].
stability, the turbidity of an oil-in-water emulsion was
measured at 540 nm for 60 min. The log of the absorbance 2.12.2.3. Minimal bactericidal concentration (MBC)
at 540 nm was plotted vs time, and the dissociation constant The MBC was determined using a series of steps that were
(Kd) value was calculated from the slope of the line. The undertaken after the MIC test had been completed. The
most stable emulsion formed with the lowest Kd value and MBC test was performed by sub-culturing each well in
was set to 100% emulsification stability.
which no visible growth occurred (blue color) from a
previous MIC test to an agar medium. After 24 h of
incubation at 37°C, bacterial growth on the agar plate was
2.12.2. Antibacterial activity
Cultures of the following microorganisms were used in the evaluated. The lowest concentration at which the propyl
study: Gram-positive Staphylococcus aureus ATCC 25923 glycosides killed whole bacteria was determined to be the
and Gram-negative Escherichia coli ATCC 25922. The MBC value.
method of disc diffusion [29], using both the minimal
inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) [30], was applied.
3. Results and Discussion
2.12.2.1. Disc diffusion technique
3.1. Donor specificity
Filter paper discs (6 mm in diameter) were impregnated The donor specificity of the CGTase was evaluated using
with a solution containing 5 mg of each propyl glycoside α-CD, β-CD and γ-CD as the glycosyl donors in the
in sterile water. The inoculum density for a susceptibility transglycosylation reaction, with cellobiose as an acceptor.
test was adjusted to be equivalent to a 0.5 McFarland The appropriate glycosyl donor for the CGTase was
8
standard (1 × 10 CFU/mL). An air-dried disc was placed determined by the glucose oxidase method and TLC

394
Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
analysis (data not shown). The results indicated that CGTase
could transfer α-1,4-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 and resulted in the largest transglycosylation yield at
588.71 µg/mL, as 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
CGTase, other researchers have reported the use of starch
as a glycosyl donor for Bacillus macerans CGTase in the
synthesis of β-arbutin-α-glucoside and β-arbutin-α-maltoside
through its transglycosylation reaction [33]. Aga et al. [17]
found that Bacillus stearothermophilus CGTase could catalyze
the transglycosylation reaction using α-CD as a donor for
the synthesis of 2-O-α-D-glucopyranosyl L-ascorbic acid.
Thus, the donor 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.
Fig. 1. Relative coupling activity of CGTase in alcohol reaction
mixtures. Residual activity (%) was relative to the coupling
activity of CGTase incubated with 0.64% (w/w) β-CD and 5 ~
50% (v/v) alcohols in 20 mM phosphate buffer at pH 6.0, 50°C
for 24 h. All values were averaged from two experiments.
3.2. Effect of alcohol length and concentration on the
CGTase transglycosylation reaction
To investigate the capability of CGTase in the trans-
glycosylation of alkyl alcohols for the synthesis of alkyl
glycosides, the influence of the alcohol length and
concentration were studied in a buffer and in a mixture of
buffer/alcohol by varying the alcohol length and concentration
at the optimal temperature (50°C) and pH (6.0) of this
enzyme for 24 h. The alcohols used were 5 ~ 50% (v/v)
methanol, ethanol and 1-propanol. Following incubation, the
reaction was stopped by boiling and then subjected to the
phenolphthalein assay and TLC analysis. In this study, the
coupling activity of CGTase in buffer was 0.342 µmol/min/mg,
which was set at 100% relative activity. As shown in Fig. 1,
when the alkyl chain length and concentration of alcohol
increased, the activity of the enzyme decreased dramatically.
Many reports have suggested that long alkyl chain lengths
and high concentrations of alcohol have a direct effect on
the loss of the enzyme activity [3-5,7,9,34]. This could be
Fig. 2. Alkyl glycoside products from the transglycosylation
reaction of β-CD to various alcohol acceptors. Each of alkyl
glycoside spots was quantified relative to a standard MG spot in
1
the same TLC plate. All values were averages from duplicate
TLC plates.
a consequence of the negative influence of the reduction of was calculated from the intensity of the product spots
water activity, enzyme transition-state destabilization and relative to standard MG1 spots on the same TLC plate and
enzyme conformational modifications, which resulted in a was expressed in units of µg/mL. As shown in Fig. 2, the
decrease of enzyme activity, including the product yield in relationship between the alcohol concentration and yield of
the alcoholic solutions. Thus, the alcohol chain length and product was found to be a bell-shaped curve. This pattern
concentration had an important effect on the trans- explained the observation that when the concentration of
glycosylation activity of CGTase.
the alcohol as an acceptor substrate in the reaction mixture
The effect of the alcohol chain length and concentration was increased, the substrate was converted into more products
on alkyl glycoside synthesis was also determined by due to the higher turnover rate of the enzyme. However,
subjecting the sample from the above reaction to TLC higher alcohol concentrations decreased the product yield,
analysis (System II). The concentration of alkyl glycosides which may have resulted from the inactivation of the

Enzymatic Synthesis of Propyl-α-glycosides and their Application as Emulsifying and Antibacterial Agents
395
demonstrate that the pBC recombinant CGTase could
transfer glycosyl residues from β-CD to alkyl alcohols,
yielding alkyl glycosides. Furthermore, it is possible that
other longer carbohydrate chain glycosides were also
produced in reaction mixtures with each alcohol acceptor,
but they may have been obscured by the oligosaccharides.
Likewise, hydrolysis products were formed with similar Rf
values (Fig. 3, Lane 7, 9 and 11). 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
alkyl glycosides because of the high affinity of water for
the enzyme. In summary, the alkyl glycoside products were
produced by catalysis by the recombinant CGTase. This
process yielded at least 1-3 products (Fig. 3), which is
more than those produced by β-glucosidase. β-glucosidase
produced only one alkyl glycoside product from each
alcohol substrate [4,5]. To date, the production of propyl
glycosides with long chain oligosaccharides by CGTase has
never been studied. Thus, 1-propanol was chosen in the
present study as a suitable acceptor for further analysis.
Fig. 3. TLC analysis of the transglycosylation products from β-CD
to various alcohols by CGTase. TLC condition was System II, ethyl
acetate/acetic acid/water, 3:1:1 (v/v). Lane 1, maltooligosaccharide
standards; lane 2, β-CD standard; lane 3, standard MG ; lane 4-5,
1
3.3. Optimal condition for the synthesis of propyl
glycosides
product from enzyme reaction without alcohol acceptor at 0 and
24 h; lanes 6-7, product from enzyme reaction between β-CD and
30% methanol at 0 and 24 h; lanes 8-9, product from enzyme
reaction between β-CD and 20% ethanol at 0 and 24 h; lanes 10-11,
product from enzyme reaction between β-CD and 10% 1-propanol
at 0 and 24 h; AGs = alkyl glucosides or alkyl glycosides; MSs =
maltooligosaccharides.
To produce the highest yield of propyl glycosides, the
parameters involved in the reaction were optimized in a
sequentially independent manner by changing various
conditions in the transglycosylation reaction. These
parameters included the substrate concentration, enzyme
concentration, incubation time, temperature and pH. The
CGTase. Therefore, these results indicate that the maximum products that were obtained from each reaction were
yield was obtained using 30% (v/v) methanol, 20% (v/v) analyzed by TLC (System II) and quantified by comparison
ethanol or 10% (v/v) 1-propanol.
with the spot intensity of the standard MG1. As a result,
To ensure that the product observed as a spot on the TLC the optimum conditions that were obtained for the synthesis
was an alkyl glycoside product that was produced by of propyl glycosides were: incubation with 10% (v/v)
CGTase (Fig. 3), a reaction mixture without an alcohol 1-propanol and 1.5% (w/v) β-CD with 500 U/mL CGTase
acceptor (Fig. 3, Lane 5) and a reaction mixture containing in 20 mM phosphate buffer, pH 6.0, at 50°C for 96 h
both substrates incubated with CGTase for 0 h were used as (Fig. 4). Under these conditions, the propyl glycoside
control experiments. After incubating CGTase with the products comprised up to 37% (w/w) of the total products,
β-CD donor and alkyl alcohol acceptors for 24 h, the which was an increase of greater than 3.5-fold compared to
expected alkyl glycoside (AG) spots appeared at a higher the yield before optimization. This increase in product
position than the maltooligosaccharides (MSs) because of yield was an increase in all propyl glycosides, including
the non-polar property of the alkyl chains. In addition to both propyl glucoside and propyl maltooligoside products.
the expected alkyl glycosides, each alcohol acceptor also The synthesis of propyl glucosides by the action of
yielded other hydrolysis products, such as glucose, maltose other enzymes has, in general, been reported. For example,
and other maltooligosaccharides. No alkyl glycoside products β-glucosidase from Thai rosewood [4] and almond meal
were observed in the control experiments. The alkyl [5] could catalyze the synthesis of propyl β-glucosides with
glycoside products obtained using 30% (v/v) methanol yields of 59 and 93% (w/w), respectively. The reaction of
consisted of at least one product, while with 20% (v/v) Leuconostoc mesenteroides dextransucrase with sucrose
ethanol, at least two products were obtained. When 10% and propanol resulted in a 38% (w/w) yield of propyl
(v/v) 1-propanol was used as the acceptor, at least three α-glucoside [9]. However, all propyl glycoside productions
alkyl glycosides products were detected. Thus, these results have been previously reported as consisting of one unit of

396
Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
Fig. 4. Effects of (A) 1-propanol concentration, (B) β-CD concentration, (C) enzyme concentration, (D) incubation time, (E) temperature,
and (F) pH: acetate buffer (triangle), phosphate buffer (circle), and Tris-HCl buffer (square) on the production of propyl glycosides by
CGTase. The amount of propyl glycosides was calculated from the intensity of product spots as compared to 25 µg of standard MG spot.
1
glucose, which is linked by an α- or β-glycosidic bond. product was applied again onto the TLC plate to determine
Hence, the propyl glycoside yield that was obtained in this its purity. The TLC plate results showed that each propyl
study is quite good compared to the group involved in the glycoside product was isolated cleanly, based on their
bacterial enzyme-catalyzed synthesis.
polarities (Fig. 5, Lanes 3 and 4). In a previous study, the
synthesis of propyl glucoside from glucose as a glycosyl
3.4. Large-scale production and purification of propyl donor and 1-propanol as an acceptor using almond
glycosides β-glucosidase has been reported [5]. The propyl glucoside
The reaction mixture for the synthesis of propyl glycosides product was determined by high performance liquid
was scaled-up to a volume of 250 mL, and the reaction chromatography (HPLC) using an Aminex HPX-87H column
proceeded under the same optimum conditions. Propyl (300 × 7.8 mm, 9 mm, Bio-Rad). The product was eluted
glycoside products from the large-scale production were with 5 mmol/L H2SO4 at a flow rate of 0.6 mL/min at 65°C
then purified using PLC. The two propyl glycoside products and was detected using a refractive index detector, in
with different Rf values were scraped separately from the which propyl monoglucoside was the main product. In our
PLC plate and dissolved with methanol. The purified study, however, both monosaccharides and disaccharides

Enzymatic Synthesis of Propyl-α-glycosides and their Application as Emulsifying and Antibacterial Agents
397
Fig. 6. ESI-TOF mass spectrum of propyl glycosides.
hydrolyzed the linkage of the transfer product [35].
3.5.2. Mass spectrometry (MS) and nuclear magnetic
resonance (NMR) analysis
To determine the molecular weights of the products, the
obtained transglycosylation products were analyzed using
an ESI-TOF mass spectrometer in the positive mode. The
Fig. 5. TLC analysis of the purified propyl glycoside and
treatment with enzymes: Lane 1, standard glucose; lane 2, propyl
+
pseudomolecular ion peak [M+Na] of synthesized propyl
glycosides before purification; lane 3, isolated PG ; lane 4,
1
isolated PG ; lane 5, PG and PG mixtures; lane 6, PG and PG
2 1 2 1 2
glycosides appeared at m/z 245 (222 plus 23 of sodium
molecule), m/z 407 (384 plus 23 of sodium molecule) and
m/z 569 (546 plus 23 of sodium molecule) (Fig. 6). These
results corresponded to the molecular weights of propyl
glucoside (PG1, C9H18O6), propyl maltoside (PG2, C15H28O11)
and propyl maltotrioside (PG3, C21H38O16), respectively.
The structures of the alkyl glycosides were determined
mixtures with α-glucoamylase treatment; and lane 7, PG and
1
PG mixtures with α-glucosidase treatment.
2
of propyl glycoside were observed as major products.
3.5. Mass and structural analysis
13 1
by C- and H-NMR, as shown in Tables 1 and 2. For the
13
PG1 product, the C-NMR spectrum displayed nine carbon
3.5.1. Digestion with amylolytic enzymes
The structure of the synthesized propyl glycoside was signals. Six of these (δ 62.33 - δ 99.97) were assigned to
preliminarily investigated by enzyme treatment of the glucose. Another three signals (δ 11.05 - δ 70.70), which
reaction product with α-glucoamylase (E.C. 3.2.1.3) and increased when combined with glucose while comparing
α-glucosidase (E.C. 3.2.1.20), followed by TLC analysis of carbon signals of 1-propanol [36], were assigned to the
the final deglucosylated products. As shown in Fig. 5, prior propyl group. The C1' signal of the glucose unit changed
to α-glucoamylase digestion, two major glycoside products significantly from 92.77 to 99.97 ppm (+7.2 ppm) because
(PG1 and PG2) were observed; however, after digestion with of the glycosylation of 1-propanol with glucose [37]. The
1
α-glucoamylase, PG2 was eliminated, while the intensities types of glycosidic linkages were also investigated by H-
of PG1 and the glucose spots increased. This result implied NMR and were based on the coupling constant values of
that PG1 is a propyl monoglycoside, while PG2 is a propyl the anomeric protons, as shown in Table 2. The PG1
diglycoside consisting of disaccharide units with α-1,4- product gave a double signal at 4.79 ppm with a coupling
glycosidic linkages. Furthermore, the treatment of propyl constant (J) of 3.5 Hz, which suggested that the type of
glycosides with α-glucosidase produced only one spot of glycosidic linkage was an α-configuration. When PG1 was
glucose by TLC, while PG1 and PG2 were both eliminated. analyzed in terms of its structure and in combination with
The meaning of these observations is that the first glycosyl its molecular weight, we formed the summary conclusion
residue linked to propyl alcohol is of an α-configuration. that the PG1 product was propyl-α-D-glucopyranoside
Our results are consistent with the fact that CGTase is (Fig. 7A).
13
Likewise, structural analysis of PG1 and C-NMR
specific for α-1,4-linkage transfer. Thus, the amylolytic
enzymes α-glucoamylase and α-glucosidase could have spectrum analysis of PG2 were also performed and revealed

398
Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
13
Table 1. C-NMR data for PG and PG
1
2
Compound
a
1-Propanol Glucose
b
PG
PG
2
1
C-Alkyl
1
59.50
23.50
9.98
70.70
23.76
11.05
70.85
23.13
10.67
2
3
C-Glucose
1'
2'
92.77
72.15
73.43
70.32
72.10
61.27
99.97
73.38
74.86
71.65
73.57
62.33
99.75
73.32
74.44
78.32
72.85
62.20
97.58
72.47
74.55
71.55
73.51
62.25
3'
4'
5'
6'
1''
2''
3''
4''
5''
6''
Fig. 7. Structures of (A) propyl-α-D-glucopyranoside and (B)
propyl-α-D-maltopyranoside.
(Fig. 7B). To our knowledge, these findings are the first to
Values of chemical shift (δ) are stated in ppm.
a,b
The chemical shift values are taken from the previous reports
1
13
report the use of H and C-NMR techniques to resolve
the structure of propyl-α-D-glucopyranoside and propyl-α-
D-maltopyranoside. Generally, most of the propyl glycosides
in previous studies were generated by the action of β-
glucosidase from several sources, such as Thai rosewood
[4] and almond meal [5]. Other enzymes, such as β-
mannosidase from A. niger [7], were also used to produce
the propyl mannoside. However, the propyl glycosides that
were obtained using these enzymes yielded products with
β-linkages. Very few studies regarding the synthesis of
propyl glycosides with an α-linkage have been reported.
For example, L. mesenteroides α-dextransucrase was used
[36,37].
1
Table 2. H-NMR chemical shift values and coupling constants for
the glucosidic protons
Compound
Glucosidic protons
4.79 (3.5)
4.81 (3.5)
PG
1
PG
4.84 (3.5)
2
Values of chemical shift (δ) are stated in ppm. Coupling constants (Hz)
are shown in parentheses.
fifteen carbon signals (Table 1). Three of the signals were to produce propyl α-glycoside from sucrose and propanol
assigned to the propyl group (δ 10.67 - δ 70.85), while the [9]. However, the product obtained using this enzyme
remaining twelve signals (δ 62.20 - δ 99.75) were derived contained only one attached unit of monosaccharide. An
from two units of glucose. Compared to the sole glucose in-depth study of this report focused on the syntheses of
signals, the C1', C4' and C1'' signals in the glucose of PG2 methyl-α-D-glucopyranoside and ethyl-α-D-glucopyranoside
showed a downfield chemical shift of 6.98, 8.0 and 4.81 ppm, as surfactants. Thus, we were presented with a unique
respectively. These downfield shifts suggested that PG2 finding in propyl α-glycosides with oligosaccharides that
had two glycosidic linkages, one of which was between the were attached in large amounts. Specifically, we learned
propyl group and glucose and the other between glucose that CGTase from recombinant pBC cells is a suitable
1
and glucose. In addition, the H-NMR spectrum of PG2 enzyme that acts differently from other enzymes in the
revealed that a glucosyl or maltosyl residue was transferred to transglycosylation of oligosaccharides to 1-propanol for
the C1 of 1-propanol to provide an α-anomeric configuration, the synthesis of propyl glycosides.
based on the double signal at 4.81 ppm (J = 3.5 Hz).
Similarly, the last glucosyl residue of PG2 was transferred 3.6. Characterization of the propyl glycoside products
to the C4' position in the glucose unit of PG1 with an
α-configuration that had been confirmed by a double signal 3.6.1. Emulsification activity and stability
1
at 4.84 ppm (J = 3.5 Hz) of the anomeric proton by H- The emulsification activity of purified propyl glycosides
NMR analysis (Table 2). During the course of integrated was determined from their ability to form an oil-in-water
NMR and MS analyses, this product was determined to be emulsion with n-hexadecane and was also used to judge
propyl-α-D-maltopyranoside with an α-1,4 glycosidic linkage their surfactant properties. The highest turbidity was

Enzymatic Synthesis of Propyl-α-glycosides and their Application as Emulsifying and Antibacterial Agents
399
Table 3. Emulsification activity and stability of the commercial
propyl-α-D-diglucoside, propyl-α-D-monoglucoside and
methyl-α-D-monoglucoside. These data indicate that propyl-
α-D-glycosides can be used as an emulsion-stabilizing
agent. It is highly probable, as was reported previously, that
the emulsification activity of alkyl glycosides increases
according to the length of the hydrophobic alkyl chains and
the number of glucose groups. Furthermore, the alkyl chain
length has a much stronger influence on emulsification
activity compared to the number of glucose groups in the
alkyl glycoside [38].
products and transglycosylated products
Emulsification
a
activity (%)
Emulsification
b
stability (%)
Compound
TM
Triton X-100
100
1.4
30 1.6
36 1.2
100
MG
6
83.7 1.1
87.2 0.9
91.1 1.1
1
PG
1
PG
2
a
Determined by the turbidity of emulsification.
b
Calculated from the logarithmic plot of absorbance versus time.
All values are presented as the mean SD from three separate experi-
ments.
3.6.2. Antibacterial activity
The antibacterial activity of the synthesized propyl glycosides
TM
observed in the reaction mixture with Triton X-100 and was determined by the disc diffusion method. The results
was set as 100% emulsification activity. The turbidity of are expressed as the diameter of the inhibition zone (Table 4).
other glycosides was measured and compared to that with The study revealed that both propyl glucoside and propyl
TM
Triton X-100. As shown in Table 3, the results show that maltoside (5 mg/disc) exhibited moderate antibacterial activity
the emulsification activity of PG1 and PG2 was 30 and against S. aureus, with inhibition zones of 10 and 12 mm,
TM
36%, respectively, compared to that of Triton X-100. In and inhibited growth of E. coli, with inhibition zones of 12
addition, these synthesized glycoside products have better and 14 mm, respectively. As seen in Table 5, the lowest
emulsifying properties than commercial methyl glycoside MIC and MBC values for PG2 were found to be 1.88 and
(MG1) and exhibited a higher emulsification activity than 3.75 mg/mL against S. aureus, respectively, and 1.88 and
MG1. These results suggest that the emulsification activity 1.88 mg/mL against E. coli, respectively. Moreover, as a
of alkyl-α-D-glycosides could be affected by the alkyl and result, it showed that PG2 had greater antibacterial activity
carbohydrate chain lengths. The longer chains of both alkyl than PG1 and was the most effective agent for inhibiting
and carbohydrate groups exhibited better activity with n- E. coli growth. This pattern of activity of the propyl
hexadecane. These observations can be summarized by glycosides is likely a result of their properties as non-ionic
noting that the emulsion-forming properties are related to surfactants that can disrupt both the lipopolysaccharides
the physiochemical structure of an emulsifier. In addition to and phospholipids of the gram-negative bacterial cell wall,
the emulsification activity, the emulsification stability was resulting, in turn, in the inhibition of bacterial growth.
also determined using the dissociation constant (Kd) value. Similarly, Matin et al. [39] and Matsumura et al. [1] found
The results obtained demonstrated that the emulsification that other alkyl glycosides, such as butyl glycoside and
TM
stability of Triton X-100 was the best, followed by dodecyl mannoside, exhibited a broad spectrum of anti-
Table 4. Antibacterial activity of propyl glycoside products and standard antibiotic drugs
a
Antibacterial activity (Zone of Inhibition in mm of diameter )
b
b
b
b
Microorganism
AMP
TET
PEN
OXA
PG
PG
2
1
(10 µg)
30 0.2
22 0.1
(30 µg)
26 0.1
24 0.1
(10 µg)
40 0.2
0
(1 µg)
24 0.1
0
(5 mg)
10 0.1
12 0.2
(5 mg)
12 0.1
14 0.1
S. aureus ATCC 25923
E. coli ATCC 25922
a
Values of inhibitory zone in mm are the mean SD of three parallel measurements.
b
The concentration of antibiotics (AMP, Ampicillin; TET, Tetracycline; PEN, Penicillin; OXA, Oxacillin) was used according to the standard con-
centration shown in CLSI document [29].
Table 5. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of propyl glycosides against
microorganisms
a
MIC and MBC values
Microorganism
Ampicillin (µg/mL)
PG (mg/mL)
1
PG (mg/mL)
2
b
0.08 0 (0.08 0 )
S. aureus ATCC 25923
E. coli ATCC 25922
3.75 0 (3.75 0)
3.75 0 (3.75 0)
1.88 0 (3.75 0)
1.88 0 (1.88 0)
0.98 0 (0.98 0)
a
All data are shown as mean SD derived from triplicate experiments.
b
MBC values are shown in parentheses.

400
Biotechnology and Bioprocess Engineering 21: 389-401 (2016)
Glycosidase-catalysed synthesis of alkyl glycosides. J. Mol.
Catal. B-Enz. 6: 511-532.
microbial activity. They reported that the antimicrobial
activity resulted from the dominant role of alkyl glycosides
in penetrating microbial cell membranes [19,20,40]. In
addition, PG2 consisted of two glucose residues that enhanced
both the water solubility and surface activity [41,42]. Thus,
PG2 is more effective than PG1 as an antibacterial agent. In
contrast, an antibiotic, ampicillin, had a propensity to inhibit
peptidoglycan synthesis in the cell wall of gram-positive
bacteria (Table 5).
3. Ito, J., T. Ebe, S. Shibasaki, H. Fukuda, and A. Kondo (2007)
Production of alkyl glucoside from cellooligosaccharides using
yeast strains displaying Aspergillus aculeatus β-glucosidase 1. J.
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4. Lirdprapamongkol, K. and J. Svasti (2000) Alkyl glucoside
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4. Conclusion
The enzyme from the pBC recombinant cell containing the
CGTase gene from Bacillus circulans A11 can be used to
synthesize propyl α-D-glycosides, which has one-to-several
glucose residues from the glycosyl donor β-CD and 1-
propanol as acceptor through a transglycosylation reaction.
Under optimal conditions, the yield of propyl glycosides
obtained was 37% (w/w) of the total products. The acceptor
products for 1-propanol were confirmed as propyl-α-D-
glucopyranoside and propyl-α-D-maltopyranoside by ESI-
TOF-MS and NMR analyses. Moreover, the products
obtained from this study also exhibited an emulsification
potential and antibacterial activity against E. coli and
S. aureus.
7. Itoh, H. and Y. Kamiyama (1995) Synthesis of alkyl β-manno-
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haag, B. W. Dijkstra, and L. Dijkhuizen (2000) The three
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Acknowledgements
R. Charoensapyanan was supported by the Ph.D. Research-
Assistant Scholarship from Thammasat University. The
authors gratefully acknowledge the financial support provided
by the Thammasat University Research Fund under the TU
Research Scholar, Contract No. TP- 2/24/2557. In addition,
this work was performed under the Core-to-Core Program,
which was financially supported for the CCP exchange
program Year 2014-2015 by Japan Society for the Promotion
of Science (JSPS), National Research Council of Thailand
(NRCT), Vietnam Ministry of Science and Technology
(MOST), the National University of Laos, Beuth University
of Applied Sciences, Germany and Brawijaya University,
Indonesia. Partial support from the Phramongkutklao
College of Medicine Research Fund is also acknowledged.
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(1994) Transglycosylation to hesperidin by cyclodextrin glucan-
otransferase from an alkalophilic Bacillus Species in alkaline pH
and properties of hesperidin glycosides. Biosci. Biotechnol. Bio-
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intermolecular transglucosylation of cyclodextrin glycosyltrans-
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in cyclodextrin glycosyltransferase by use of activated substrates
and a mutant enzyme. Biochem. 36: 9927-9934.
16. Mathew, S., M. Hedström, and P. Adlercreutz (2012) Enzymatic
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