42
B.-H. Lee et al. / Carbohydrate Polymers 132 (2015) 41–49
1996). Once it enters the enterocyte, the absorbed 2-DG is rapidly
efficient molecule for the glucose transporter systems (Gallagher,
trace the location of glucose entry into a range of target cells using
different types of imaging systems (Durham & Woolsey, 1985;
Nedergaard & Astrup, 1986; Yamato, Kataoka, Mizuma, Wada, &
Watanabe, 2009).
of WCS at 35 ◦C for 10 min (Kim et al., 2011). After the reaction, the
amount of released fructose was measured by the dinitrosalicylic
acid (DNS) method, using fructose as a standard (Sumner & Howell,
1935). One unit of AS was defined as the amount of enzyme that
catalyzes the production of 1 mol of fructose per min.
Enzymatic synthesis of 2-DG-MOs was carried out in 50 mM
Tris–HCl buffer (pH 7.0) containing 2-DG as an acceptor (3%, w/v)
and sucrose (1 M) as a donor molecule. The pre-warmed substrate
mixture was reacted at 35 ◦C at different times (0, 3, 6, 12, 24, and
24 h) with purified recombinant AS (5 U) to produce 2-DG-MOs. The
enzyme reaction was terminated in a boiling water bath for 10 min.
As an analytical tool to study the physiological effect of loca-
tional deposition of dietary glucose in the small intestine, 2-DG
holds promise; however 2-DG itself is absorbed in the proximal part
ride with the idea that its digestion rate could be controlled using
a technique of selective inhibition of the mucosal ␣-glucosidases
(AS; E.C. 2.4.1.4) with 2-DG as an acceptor molecule (Fig. 1). The AS
catalyzes a consecutive transglucosyl reaction from sucrose onto
the acceptor molecule, and produces ␣-1,4-linked ␣-glucans (Kim
et al., 2013; Kim, Kim, & Yoo, 2015; Potocki de Montalk et al., 2000).
The synthesized 2-DG-MOs were hypothesized to be hydrolyzed
similarly to glucose by the mucosal ␣-glucosidases as they com-
pare in size with homogenous maltooligosaccharides. 14C-labeled
2-DG-containing synthesized products will be applied to test the
correlation between the location of digestion and absorption, and
physiological response in vivo.
2.5. Analysis of linear chain length distribution by HPAEC
Chain length distributions of enzyme-synthesized 2-DG-MOs
were measured using a high-performance anion-exchange chro-
matograph (HPAEC) equipped with an electrochemical detector
(ED40; Dionex, Sunnyvale, CA, USA). Filtered samples (with
matographic separation of the linear oligosaccharides from the
sample was achieved by gradient elution from 100% eluent A to
100% eluent B with 600 mM sodium-acetate in 150 mM sodium-
hydroxide (Lee et al., 2007). Differences among the means of
number-average degree of polymerization (DPn) were evaluated
using ANOVA and statistical significance was considered at P < 0.05.
2.6. MALDI-TOF-MS analysis
Molecular mass spectrum of 2-DG-MOs was obtained by Voy-
ager DESTR matrix-assisted laser desorption/ionization-time of
a matrix. One microliter of sample (0.1–0.001 mg/mL) was mixed
with 1 mL of matrix (0.1–5 pmol/mL) and dropped on a sample
plate, which was followed by air-drying until homogeneous crys-
tals formed (Park et al., 2007).
2. Materials and methods
2.1. Materials
Sucrose and 2-DG were obtained from Sigma–Aldrich Chemi-
cal Co. (St. Louis, MO, USA). Waxy corn starch (WCS) was a gift
from Tate and Lyle, Inc. (Decatur, IL, USA). Chemical components
for Luria-Bertani (LB) medium were obtained from Difco Labora-
tories (Detroit, MI, USA). Other chemical reagents were purchased
from Sigma–Aldrich Chemical Co.
2.7. Hydrolysis properties by human pancreatic ˛-amylase and
mammalian mucosal ˛-glucosidases
human pancreatic ␣-amylase was investigated. The synthesized-
products were solubilized in 10 mM PBS buffer (pH 6.9, 10 mg/mL,
2013). The product was hydrolyzed using each of four recombinant
mammalian mucosal ␣-glucosidases. The mucosal ␣-glucosidases
(ctMGAM, ntMGAM, ctSI, and ntSI) were prepared as described
(Jones et al., 2011). Each mucosal ␣-glucosidase [1000 U, one unit
that released 1 g of glucose from 1% maltose per 10 min at 37 ◦C]
was reacted with 1% (w/v) of 2-DG-MOs in 10 mM PBS buffer (pH
6.8) at 37 ◦C for 6 h. The amount of glucose released was analyzed
by the glucose oxidase/peroxidase (GOPOD) method (Vasanthan,
2001).
polysaccharea
The E. coli BL21(DE3) transformant harboring recombinant AS
gene from Neisseria polysaccharea (NpAS), which was prepared as
previously described (Jung et al., 2009), was grown in LB broth with
20 g of kanamycin per 1 mL at 37 ◦C until absorbance reached 0.6
at 600 nm. The cells were collected by centrifugation (5000 × g for
20 min at 4 ◦C) after isopropyl--d-thiogalactoside (IPTG) induc-
ity column chromatography (Qiagen, Hilden, Germany) was used
to purify recombinant AS by increasing imidazole concentration
(0–250 mM). The purified protein concentration was determined
by the Bradford method (Bradford, 1976).
For enzyme kinetic study of the hydrolysis of individual mucosal
␣-glucosidases on 2-DG-MO, each enzyme (5 g/mL) was reacted