Practical Preparation of Lacto-N-biose I
2103
crystalline LNB (99.6% purity) was obtained after
1
700
600
500
400
300
200
100
0
filtration and vacuum drying. The H-NMR spectum of
the compound was taken in D2O using an Avance 800
spectrometer (Bruker Biospin, Rheinstetten, Germany)
at 298 K. It was identical with that of authentic LNB.
Anomeric analysis using HPLC19,20) revealed that the
crystal was a ꢁ-anomer of LNB.
Fru
LNB
The above results indicate that LNB was produced by
a one-pot enzymatic reaction. All the unit processes used
in this method were scale-up ready. It should be noted
that the reaction time can easily be shortened only by
increasing the amounts of the four enzymes at the same
ratio. Although this method requires expensive UDP-
Glc, the amount of UDP-Glc used was only 1/600 of
GlcNAc, indicating that UDP-Glc acted as a catalyst
with a turn-over number of 500. Considering these facts,
this method can be used in industrial production of LNB
by constructing a bio-reactor system using immobilized
enzymes. Since LNB has been hypothesized to be a
bifidus factor in HMOs, LNB may be applicable as an
additive to formula milk to achieve bifidus flora in
bottle-fed infants as well as breast-fed infants.
Suc
GlcNAc
Glc
0
100 200 300 400 500 600 700
Time (h)
Fig. 2. The Time Course of LNB Production.
The concentrations of LNB, sucrose, fructose, GlcNAc, and
glucose are shown by closed square, closed circle, open circle, open
triangle, and open square respectively.
(pH 7.0), 10 mM MgCl2, 26 mg/l (1716 U/l) SP, 22
mg/l (3080 U/l) GalE, 17 mg/l (3570 U/l) GalT, and 43
mg/l (150 U/l) LNBP at 30 ꢀC. The concentrations of
LNB, GlcNAc, sucrose, fructose, and glucose were
monitored by high performance liquid chromatography
(Shimadzu, Kyoto, Japan) with a Corona Charged
Aerosol Detector (ESA Inc., Chelmsford, MA) using a
Shodex Asahipak NH2P50-4E column (4.6 mm ID ꢂ
250 mm, Showa Denko, Tokyo, Japan) with acetonitrile-
H2O (75:25 by volume) as the solvent at a flow rate of
1 ml/min. The time course of the reaction is shown in
Fig. 2. Production of LNB was observed with decreases
in sucrose and GlcNAc at an early stage of the reaction.
The LNB concentration was reached 500 mM after 600 h
of reaction. The yield was 83% based on the GlcNAc
used.
Acknowledgments
This work was supported in part by the Program for
Promotion of Basic Research Activities for Innovative
Biosciences (PROBRAIN) of Japan.
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After the reaction, 50 g of DEAE-cellulose (Wako
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the reaction mixture to adsorb the enzymes. The mixture
was stirred at room temperature for 90 min. After
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natant was concentrated to 4.8-liter using a rotary
evaporator. After crystallization at 4 ꢀC for 12 h, 1.5 kg
of crystalline LNB (96.9% purity) was recovered by
filtration, followed by vacuum drying. In addition, 0.3 kg
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the filtrate after concentration to 1.8-liter and crystal-
lization under the same conditions.
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The two LNB samples (1.5 kg and 0.3 kg) were mixed
and dissolved in 3.6-liter water at 60 ꢀC, followed by the
addition of 3.6-liter of ethanol at 4 ꢀC for crystallization
with filtration and vacuum drying. Finally, 1.4 kg of
9) Moro, G., Arslanoglu, S., Stahl, B., Jelinek, J., Wahn, U.,