Enzyme and Microbial Technology 38 (2006) 855–859

Large scale production of d-allose from d-psicose using continuous

bioreactor and separation system
Kenji Morimoto a , Chang-Su Park b , Motofumi Ozaki b , Kei Takeshita c ,
Tsuyoshi Shimonishi b , Tom Birger Granström a , Goro Takata a ,
Masaaki Tokuda a , Ken Izumori a,∗
a Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
b Kagawa Industry Support Foundation, Hayashi-cho, Takamatsu, Kagawa 761-0301, Japan
c Fushimi Pharmaceutical Co., Ltd., Nakatsu-cho Marugame, Kagawa 763-8605, Japan

Received 11 April 2005; received in revised form 9 August 2005; accepted 10 August 2005

Abstract
l-Rhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172 can convert d-psicose to d-allose. Partially purified recombinant l-RhI from
Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce d-allose. Total 20,000 units of immobilized enzyme
converted d-psicose to d-allose without remarkable decrease in the enzyme activity over 17 days. When 50% d-psicose (w/w) was applied to a
column with a flow rate of 0.8 ml/min at 42 ◦ C, approximately 30% d-psicose was isomerized to d-allose for 17 days. However, by reducing the flow
rate to 0.4 ml/min after 17 days, d-allose was transformed at the same rate for 13 days. The total of 27 l reaction mixture was separated by Simulated-
Moving-Bed Chromatograph system. Approximately 2.2 l/d of 50% (w/w) reaction mixture was separated continuously. After separation, d-allose
and d-psicose fractions were 3 l of approximately 10% (w/w) with 95% purity and 10 l of approximately 8% (w/w) with 95% purity per day,
respectively. The separated d-allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature.
Crystals of d-allose were separated from the syrup by filtration and 1.65 kg crystals of 100% purity were obtained. The d-allose crystal yield from
the d-psicose substrate was approximately 10%.
© 2005 Elsevier Inc. All rights reserved.

Keywords: Rare sugars; d-Allose; l-Rhamnose isomerase; Simulated-Moving-Bed Chromatograph

1. Introduction We established the bioproduction strategy route named as

Rare sugars including monosaccharides are rare in nature
and knowledge of their biological and physiological functions
have consequently been known little so far. We are working
on production of various kinds of rare sugars using microbial
enzymes and whole cells. It has been proven that some rare
hexose sugars have exhibited physiologically active functions
[1–3]. Rare sugars have received increasing attention in recent
years for a variety of usages, such as low-calorie carbohydrate
sweeteners, inhibitor of microbial growth and bulking agents
[1,2]. For instance, d-allose, one of rare aldo-hexose, has been
reported to have a possibility to be applied in pharmaceutical
industry, such as inhibitor of ischemia/reperfusion injury [3].
∗ Corresponding author. Tel.: +81 87 891 3290; fax: +81 87 891 3289.
E-mail address: izumori@ag.kagawa-u.ac.jp (K. Izumori).
“Izumoring” [4]. To produce d-allose in large quantities, one of
the best route is a two step reaction as follows: first d-psicose
is produced from inexpensive monosaccharide d-fructose, and
in the second step, d-psicose is isomerized to d-allose. We
reported that mass production of d-psicose from d-fructose was
achieved by using d-tagatose 3-epimerase (d-TE) from Pseu-
domonas cichorii ST-24 [5], d-psicose was separated from sugar
mixture by Simulated-Moving-Bed Chromatograph (JPN Pat.
No. P2001-354690A, 2001). l-Rhamnose isomerase (l-RhI),
l-rhamnose ketol-isomerase [EC 5.3.1.14], which isomerizes
l-rhamnose to l-rhamnulose, has been found in Escherichia
coli [6,7], Salmonella and Pseudomonas [8–10]. l-Rhamnose
isomerase, which was produced constitutively, from Pseu-
domonas stutzeri LL172was reported to catalyze isomerizations
not only between l-rhamnose–l-rhamnulose but also between
d-allose–d-psicose (Fig. 1; [10,11]). We had reported that small
scale of d-allose production was successful using this enzyme

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.08.014
856 K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
Fig. 1. Schematic diagram of d-allose and d-psicose production from d-fructose catalyzed by l-rhamnose isomerase.
[10]. Moreover, l-rhi gene from this strain was successfully
cloned into E. coli and over-expressed [12].
In this paper, we describe continuous d-allose production
from d-psicose by use of immobilized enzyme of recombi-
nant l-RhI under the optimized condition. The immobilized
enzyme has advantages such as reusability, less by-product
formation, and long operation time. Furthermore, we estab-
lished the separation condition for reaction mixture contain-
ing d-allose by using Simulated-Moving-Bed Chromatograph
system.
2. Materials and methods
2.1. Chemicals
All chemical agents were purchased from Wako Pure Chemicals (Tokyo,
Japan). Chitopearl BCW-2510 resin was purchased from Fuji Spinning Co.,
Ltd. (Tokyo, Japan). It has primary, secondary and quaternary amine groups
and binds to proteins with electrovalent mode. d-Psicose was prepared by our
laboratory as described previously [5].
2.2. Microorganism culture condition
The l-rhi gene of Pseudomonas stutzeri LL172, which was expressed contin-
uously, was inserted in pQE 60, named as pOI-01. This plasmid was transformed
in E. coli JM 109 [12]. The recombinant E. coli JM109 was cultured in a medium
composed of 3.5% polypepton, 2.0% yeast extract, 0.5% NaCl and 100 ␮g/ml
ampicillin in the four 15 l jar-fermentor at 28 ◦ C for 18 h, and recombinant
enzyme was induced by addition 0.5 mM IPTG and 1 mM MnCl2 in the culture
medium. After 5 h period, the cells were collected by continuous centrifugation
(8000 × g, 4 ◦ C).
2.3. Partial purification of the enzyme
When l-RhI was completely purified, it became to be unstable according to
formation of aggregation. Therefore, we used stable partially purified enzyme
which was prepared as follows. The collected cells were suspended in 10 mM
Tris–HCl buffer (pH 9.0), and disrupted by ultrasonicator. The cell free extract
was heat-treated at 50 ◦ C for 10 min. The supernatant was collected by cen-
trifugation at 8000 × g for 30 min. The supernatant was slowly added with 1M
MnCl2 to a final concentration 10 mM. And polyethylene glycol #6000 was
slowly added to a final concentration 5% (w/v) and the mixture was stirred for
1 h at 4 ◦ C. This mixture was centrifuged at 12,000 × g for 30 min and precip-
itation was discarded. More polyethylene glycol #6000 was added to a final
concentration 20% (w/v). After stirring for 1 h, the precipitation was collected
by centrifugation at 12,000 × g for 30 min and suspended in a small quantity of
10 mM Tris–HCl buffer (pH 9.0). This solution was used as partially purified
enzyme.
2.4. Enzyme and protein assay
It is difficult to detect an isomerase activity from ketose to aldose, because
the amount of reducing ketose is not detected easily. Isomerase activity is gen-
erally detected from aldose to ketose, and ketose conversion from aldose is
easy to know enzyme activity using cysteine–carbazole method [13]. Appro-
priately, diluted partially purified enzyme, 50 ␮l, was incubated with 50 ␮l of
50 mM d-allose as a substrate at 30 ◦ C for 10 min. Accumulation of product,
d-psicose, was measured by cysteine-carbazole method. One unit of l-RhI is
defined as the amount of enzyme that converts 1 ␮mol of d-allose to d-psicose
in 1 min. The reaction mixture was assayed by the HITACHI High-performance-
liquid-chromatography (HPLC), which is constructed by L-7490 refractive index
detector, D-2500 chromato-integrator and Hitachi HPLC column GL-C611.
Chromatography was carried out at 60 ◦ C using 10−4 M NaOH solution at a
flow rate of 1.0 ml/min. Protein concentration was measured according to the
method of Bradford [14] with bovine serum albumin as a standard.
2.5. Optimization of l-RhI immobilization condition
The partially purified enzyme (100 units) was immobilized on 1, 2, 3 and
5 g of BCW-2510 Chitopearl beads at 4 ◦ C for 1 day. Immobilization efficiency
was calculated as follows: residual activity in the supernatant was analyzed
with d-allose as a substrate and then d-psicose amount was measured by
cysteine–carbazole method as described above.
2.6. d-Allose mass production
The immobilized enzyme, which was prepared under the optimized condi-
tion, was charged in a column (4 cm × 40 cm) tempered at 42 ◦ C. Substrate 50%
 K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859 857

(w/w) d-psicose dissolved in 10 mM Tris–HCl buffer (pH 9.0) was applied by

flow rate of 0.8 ml/min. When the enzyme activity was decreased, the flow rate
was reduced to 0.4 ml/min.

2.7. Separation of d-psicose and d-allose

Before separation, reaction mixture was desalted by passing through Amber-

lite IRA-411 (CO3 2− form) and DIAION SK1B (H+ form) ion-exchanged resin.
After desalting, the reaction mixture was separated to obtain mainly d-allose
and d-psicose by TREZONE system (Organo Corp.) which can operate the
Simulated-Moving-Bed Chromatograph. It consisted of 2 pumps, 26 valves and
4 columns. Each column was filled with 750 ml of ion exchanged resin (Ca2+
form). This system can separate a reaction mixture which is composed of two
main components.

2.8. Crystallization of d-allose
After separation of reaction mixture, d-allose fraction was evaporated to the
concentration of 50% (w/w). The syrup was kept at room temperature to allow
d-allose crystals to grow without adding seed crystals.
Fig. 2. Effect of the Chitopearl BCW-2510 beads volume on d-allose production
from d-psicose. d-Psicose in 50 mM Tris–HCl (pH 9.0) was used as a substrate at
42 ◦ C. After incubation, converted d-allose was measured by HPLC as described
in Section 2. Symbols: open circles, 1 g beads; open squares, 2 g beads; close
squares, 3 g beads; open triangles, 5 g beads.

3. Results inactivated in the column and enzyme was gradually detached it

3.1. Immobilization of l-RhI
When 100 U of the enzyme was immobilized, not less than
90% of l-RhI was bound to 2, 3, or 5 g of BCW-2510 Chito-
pearl beads. By contrast, only approximately 50% of enzyme
was bound to 1 g beads (Table 1). In this study, we found that
the maximum equilibrium point (33% d-allose) was achieved
after 5 h in all conditions (Fig. 2). The conversion efficiency
was, however, the lowest among all in the case of using of 1 g
beads. The best condition for immobilization of l-RhI on Chi-
topearl beads was achieved when 2 or 3 g of beads were used for
100 U of enzyme. Based on this result and cost-effectiveness,
we determined that we utilized 2 g beads for 100 U of
l-RhI.
from resin, it became impossible to keep an equilibrium point.
We made preliminary tests whether bioreactor could be used
continuously after enzyme activity was decreased. As a result,
the flow rate of 0.4 ml/min was able to maintain the optimal con-
version rate. When the flow rate was dropped to 0.4 ml/min after
17 days, d-allose was produced with the some ratio for 13 days
more in this study (Fig. 4). Eventually, bioreactor was able to
maintain 1 month for continuous operation and to produce reac-
tion mixture containing 5.02 kg of d-allose from 27 l (16.6 kg)
of 50% (w/w) d-psicose syrup for 1 month (Table 2). When the
same amount of l-RhI, which was used for bioreactor construc-
tion in this study, was let to react as soluble enzyme, the d-allose
yield was approximately 30%.

3.2. Continuous conversion from d-psicose to d-allose

The partially purified l-rhamnose isomerase which was pre-

pared from 40 l culture has 20,000 U of enzyme activity. Based
on the result of the optimized immobilization condition, we pre-
pared a bioreactor using 400 g beads. As shown in Figs. 2 and 3,
reaction equilibration was 30 (d-allose): 70 (d-psicose) toward
50% (w/w) d-psicose as a substrate, although a small amount of
some by-products detected. The immobilized l-rhamnose iso-
merase was able to convert d-psicose to d-allose in the flow rate
of 0.8 ml/min without significant decrease in the enzyme activity
over the first 17 days. Since immobilized enzyme was gradually

Table 1
Immobilization efficiency of l-Rhl on Chitopearl BCW-2510
Chitopearl resin Residual activity in Efficiency (%)
volume (g) supernatant (U)

1 42.7 51.3
2 5.98 94.0
3 3.19 96.8
5 3.78 96.2 Fig. 3. HPLC analysis of d-allose from d-psicose by immobilized l-rhamnose
isomerase.
858 K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859

3.3. Separation of d-psicose and d-allose

As a result of the optimized separation condition, contin-

uously the 50% (w/w) mixture of a maximum approximately
2.2 l in 1 day could be processed. The separated d-allose frac-
tion volume was 3 l/d containing 10% (w/w) d-allose with 95%
purity and a minor amount of by-products (Fig. 5). d-Psicose
fraction volume was 10 l/d containing 8% (w/w) d-psicose with
95% purity (Fig. 5).

3.4. Crystallization of d-allose

Fig. 4. Bioconversion of d-allose from d-psicose by immobilized l-rhamnose
isomerase. Bold arrow and broken arrow indicate a period of flow rate of 0.8
and 0.4 ml/min, respectively.
The separated d-allose solution was concentrated up to about
50% (w/w) and crystallized gradually at room temperature. The
crystals of d-allose were separated from the solution by filtration.
Finally, we could obtain approximately 1.65 kg d-allose crystal
with 100% purity (Fig. 5).

Table 2
The yield of d-allose mass production

Reacted mixture (kg)a Separated d-allose (kg)a Separated d-psicose (kg)a Loss of sugars (kg)a Crystallized d-allose (kg)b

Total sugarc 16.6 3.50 9.61 3.49

d-Allosec 5.02 3.33 0.64 1.05 1.65
d-Psicose 11.36 0.00 8.96 2.40
By-productsc 0.22 0.17 0.00 0.05
a Reaction mixture indicates total weight after enzyme reaction. “Separated d-allose” and “separated d-psicose” indicate total weights after separation, respectively.

“Loss of sugars” indicates total weight of Loss by desalting and separation processes.
b “Crystallized d-allose” indicates obtained weight of d-allose crystal from separated d-allose fraction.
c “Total sugar”, “d-allose”, “d-psicose” and “by-product” indicate sugar weights contained in “reacted mixture”, “separated d-allose”, “separated d-psicose” and

“loss of sugars”, respectively.

Fig. 5. HPLC analysis of reaction mixture before and after separation and d-allose crystal: (A) indicates a deionized solution mixture; (B and C) indicate fractions
separated d-allose and d-psicose by TREZONE system, respectively; (D) indicates finally obtained d-allose crystal.
 K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
4. Discussions
We previously reported d-psicose mass production from d-
fructose by d-tagatose 3-epimerase from Pseudomonas cichorii
ST-24 [5], and separation of d-psicose and d-fructose sugar
mixture by Simulated-Moving-Bed Chromatograph (Organo
Corp, JPN Pat. No. P2001-354690A 2001). In this paper, we
have reported d-allose mass production from d-psicose by l-
rhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172.
P. stutzeri LL172 expressed constitutively l-rhi gene. How-
ever, expression level was too low to construct an efficient
bioreactor. In order to increase the l-RhI expression level and
construct the bioreactor, we carried out the over-expression of
l-rhi gene in E. coli JM109 [12]. This recombinant could pro-
duce l-RhI of 20-fold toward the volumetric yield in comparison
with expression level of wild type P. stutzeri LL172 [12]. Par-
tially purified enzyme prepared from recombinant was able to
produce mainly d-allose from d-psicose, although it produced
some d-fructose and other by-product (Fig. 3).
In the previous report [10], d-allose was produced by batch
reaction using l-RhI immobilized on Chitopearl beads. The
batch reaction has two problems as follows: (1) we have to sep-
arate reaction mixture from immobilized enzyme after reaction;
(2) l-RhI was damaged by drop of pH due to high concentra-
tion d-psicose. Consequently, it was not possible to use it for a
long period. Therefore, we constructed a continuous bioreactor
where substrate was injected by a pump as described in Section
2. As a result, declining rate of immobilized enzyme activity was
decreased even under high substrate condition and immobilized
enzyme was able to produce d-allose continuously. The solu-
tion after the reaction was very clean, thereby eliminating the
separation step to recover the immobilized enzyme for further
reactions. The reason why the substrate concentration was 50%
(w/w) of d-psicose was that it is the best condition to reduce the
formation of by-products as much as possible. Although we tried
to use 20% (w/w) d-psicose, much more by-products were gen-
erated as compared with 50% (w/w) d-psicose (data not shown).
The bioreactor constructed in this study was able to convert d-
psicose to d-allose to the equilibrium point at the flow rate of
0.8 ml/min at 42 ◦ C, however, conversion ability was remark-
ably decreased after 17 days (Fig. 4), suggesting that the half
life of this bioreactor was approximately a half month in these
conditions. After 17 days, the bioreactor could convert d-psicose
into d-allose until equilibrium point for more 13 days, when the
flow rate was decreased down to 0.4 ml/min. Although, it was
possible to use for 10 more days, we did not try to use more 30
days to consider productivity.
We tried to separate the reaction mixture to obtain pure d-
allose crystal. The TREZONE system could separate 2.2 l of
this reaction mixture per day. By this system, we can separate
reaction mixture continuously for 10 days and obtained 3.6 l of
purity 95% d-allose solution, although it contained d-psicose
859
and some by-product. However, crystallization of d-allose was
performed in advance of d-psicose in the syrup mixture because
d-allose has a property to crystallize easier than d-psicose. Con-
sequently, d-allose is crystallized specifically in the mixture. The
crystallized d-allose was able to be harvested easily by filtration
using 5-␮m filter paper, and the purity was 100% as determined
by HPLC analysis (Fig. 5). The yield of d-allose in this study
was summarized in Table 2. Finally the crystal of d-allose was
obtained 10% efficiency of reacted d-psicose weight. In this
study, we established the mass production of d-allose by con-
tinuous bioreactor and Simulated-Moving-Bed Chromatograph.
From now on, we will carry out improvements of bioreactor per-
formance and Simulated-Moving-Bed Chromatograph in order
to increase reaction efficiency and d-allose yield from the mix-
ture of d-allose and d-psicose.
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