Production of l-rhamnulose, a rare sugar, from l-rhamnose using commercial immobilized glucose isomerase

Article abstract of DOI:10.1080/10242422.2017.1388374

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60 °C, 300 g?L^?1 l-rhamnose as a substrate, and 0.6 h^?1 dilution rate. The half-life of the immobilized enzyme at 60 °C was 809 h. Under the optimal conditions, the immobilized enzyme produced an average of 135 g?L^?1 l-rhamnulose from 300 g?L^?1 l-rhamnose after 16 days at pH 8.0, 60 °C, and 0.6 h^?1 dilution rate, with a productivity of 81 g/L/h and a conversion yield of 45% in a packed-bed reactor.

Full text of DOI:10.1080/10242422.2017.1388374

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Production of l-rhamnulose, a rare sugar, from l-
rhamnose using commercial immobilized glucose
isomerase
Yeong-Su Kim, Do-Yeon Kim & Chang-Su Park
To cite this article: Yeong-Su Kim, Do-Yeon Kim & Chang-Su Park (2017): Production of l-
rhamnulose, a rare sugar, from l-rhamnose using commercial immobilized glucose isomerase,
Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2017.1388374
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1388374
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Date: 15 October 2017, At: 16:51

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
https://doi.org/10.1080/10242422.2017.1388374
RESEARCH ARTICLE
Production of L-rhamnulose, a rare sugar, from L-rhamnose using commercial
immobilized glucose isomerase
aÃ
b
and Chang-Su ParkcÃ
Yeong-Su Kim
a
, Do-Yeon Kim
b
Division of Plant Resource Industry, Baekdudaegan National Arboretum, Bonghwa, South Korea; Department of Convergence
Industrialization, International Ginseng and Herb Research Institute, Geumsan, South Korea; Department of Food Science and
c
Technology, Catholic University of Daegu, Hayang, South Korea
ABSTRACT
ARTICLE HISTORY
A commercial immobilized D-glucose isomerase from Streptomyces murines (Sweetzyme) was
used to produce L-rhamnulose from L-rhamnose in a packed-bed reactor. The optimal conditions
Received 1 September 2017
Revised 22 September 2017
Accepted 28 September 2017
ꢀ
À1
for L-rhamnulose production from L-rhamnose were determined as pH 8.0, 60 C, 300 g L
À1
L-rhamnose as a substrate, and 0.6 h dilution rate. The half-life of the immobilized enzyme at
ꢀ
6
1
0 C was 809 h. Under the optimal conditions, the immobilized enzyme produced an average of
KEYWORDS
À1
À1
ꢀ
À1
35g L L-rhamnulose from 300 g L L-rhamnose after 16 days at pH 8.0, 60 C, and 0.6 h
l-rhamnulose; rare sugar;
glucose isomerase;
isomerization
dilution rate, with a productivity of 81 g/L/h and a conversion yield of 45% in a packed-bed
reactor.
Introduction
Menavuvu et al. 2006), Bacillus pallidus (Poonperm
et al. 2007), Bacillus halodurans (Prabhu et al. 2011),
Thermotoga mritima (Park et al. 2010), Mesorhizobium
loti (Takata et al. 2011), and Dictyoglomus turgidum
Rare sugars are defined by the International Society of
Rare Sugars (ISRS) as monosaccharides that rarely exist
in nature (Izumori 2002) and have recently attracted
much attention in the foods, pharmaceutical, and nutri-
tional industries due to their many uses such as low-cal-
orie sweeteners, bulking agents, antioxidants,
glycosidase inhibitors, nucleoside analogues, and immu-
nosuppressants (Doong et al. 1991; Levin et al. 1995;
Muniruzzaman et al. 1996; Hossain et al. 2000; Levin
(Kim et al. 2013). These L-rhamnose isomerases have
been applied in the production of rare sugars such as
L-lyxose, L-mannose, L-talose, D-glulose, D-allose, and
L-rhamnulose.
However, using a commercial immobilized enzyme
in the production of rare sugars provides enhanced
stability and better process control. Therefore, here we
used D-glucose isomerase (GI) from Streptomyces mur-
2
002; Matsuo et al. 2002). Over 50 kinds of rare sugars
exist in nature, including tetrose, pentose, hexose, and
D- and L-forms of sugars (Izumori 2002). D-Tagatose and
D-allulose have been formally approved by the US Food
and Drug Administration (FDA) as Generally Recognized
As Safe (GRAS) and are allowed for use in food indus-
tries (Levin 2002; Kim 2004; Mu et al. 2012).
One of the rare sugars, L-rhamnulose (6-deoxy-L-sor-
bose), is a precursor of furaneol, which has been used
in the flavour industry and it plays important roles in
sugar metabolism (Hecquet et al. 1996; Menavun et al.
TM
ines (xylose isomerase, Sweetzyme ), a commercially
available GI obtained from Novozyme, Denmark, and
characterized and optimized it for L-rhamnulose pro-
duction. Under the optimized conditions, continuous
L-rhamnulose production from L-rhamnose was per-
formed in a packed-bed reactor.
Materials and methods
Materials
2
006). L-Rhamnulose has been produced using L-rham-
nose isomerase, which has been isolated and charac-
terized from various microorganisms including
Escherichia coli (Badia et al. 1991), Pseudomonas stutzeri
Immobilized
GI
from
Streptomyces
murines
(Sweetzyme) was purchased from Novozyme
(Kalundborg, Denmark) under the trade name
Sweetzyme IT extra. Authentic standard sugars and all
(
Bhuiyan et al. 1999; Leang, Takada, Fukai, et al. 2004;
CONTACT Chang-Su Park
These authors contributed equally to this work.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
parkcs@cu.ac.kr
Department of Food Science and Technology, Catholic University of Daegu, Hayang, South Korea
Ã

2
Y.-S. KIM ET AL.
other regents were purchased from Sigma (St. Louis,
MO, USA).
(a) 120
100
Enzyme assay
80
The reaction was performed in 50 mM EPPS buffer (pH
À1
8
.0) containing 10 mM L-rhamnose and 1.5 UÁmL
60
40
ꢀ
enzyme at 60 C for 10 min. One unit (U) of enzyme
activity was defined as the amount of enzyme
required to produce 1 mmol L-rhamnulose from L-rham-
ꢀ
20
nose per minute at 60 C and pH 8.0.
0
6.0
Analytical methods
6.5
7.0
7.5
8.0
8.5
9.0
9.5
pH
The monosaccharide concentrations were determined
using a Bio-LC system (Dionex ICS-3000, Sunnyvale,
CA, USA) with an electrochemical detector and a
(b) 120
100
ꢀ
CarboPac PAI column. The column was eluted at 30 C
with 0.1 M NaOH (0–5 min), followed by a linear gradi-
8
0
ent (5–35 min) of sodium acetate (0–0.2 M) at
À1
1
mLÁmin
.
60
Results and discussion
40
Effects of pH and temperature on L-rhamnulose
production
20
The isomerization activity for L-rhamnulose production
using immobilized GI was examined in 50 mM PIPES
buffer (pH 6.5–7.5) and EPPS buffer (pH 7.5–9.0) con-
0
40
50
60
70
80
90
100
ꢀ
taining 10 mM L-rhamnose at 60 C for 10 min
Figure 1. Effect of pH (a) and temperature (b) the immobi-
lized glucose isomerase (GI) activity. (a) The reactions were
performed in 50 mM PIPES buffer (pH 6.5–7.5) and EPPS buffer
(
8
Figure 1(a)). Maximum activity was observed at pH
.0. The activity was >80% of the maximum at pH 7.0
ꢀ
(
pH 7.5–9.0) containing 10 mM L-rhamnose at 60 C for 10 min.
and 9.0. The optimum pH values for L-rhamnulose pro-
duction from L-rhamnose using L-rhamnose isomerase
from Bacillus pallidus Y25 (Poonperm et al. 2007),
Pseudomonas stutzeri (Leang, Takada, Fukai, et al. 2004,
Takada, Ishimura, et al. 2004), and Dictyoglomus tur-
gidum (Kim et al. 2013) were 7.0, 9.0, and 8.0,
respectively.
(
8
b) The reactions were performed in 50 mM EPPS buffer (pH
.0) containing 10 mM L-rhamnose at 45–90 C for 10 min.
ꢀ
Data represent the means of three experiments and error bars
represent standard deviation.
low for L-rhamnulose production. The optimum tem-
peratures for L-rhamnulose production from L-rham-
nose using L-rhamnose isomerase from B. pallidus Y25
(
2
Next, the isomerization activity for L-rhamnulose
production using immobilized GI was examined in a
Poonperm et al. 2007), P. stutzeri (Menavuvu et al.
ꢀ
006), and D. turgidum (Kim et al. 2013) are 65 C,
ꢀ
ꢀ
temperature range from 40 C to 95 C at pH 8.0.
ꢀ
ꢀ
6
0 C, and 70 C, respectively.
ꢀ
Maximum activity was observed at 90 C (Figure 1(b)).
These results suggested that the optimum pH and
The thermostability of the immobilized GI during
L-rhamnulose production from L-rhamnose was deter-
mined by measuring enzyme activity as a function of
time and temperature. Thermal inactivation of the
enzyme followed first-order kinetics. The half-lives of
temperature for L-rhamnulose production is pH 8.0
ꢀ
and 60 C, respectively, and the conversion yield of
L-rhamnose to L-rhamnulose was approximately 45%.
The conversion yield of L-rhamnulose from L-rhamnose
using L-rhamnose isomerase from B. pallidus Y25
(Poonperm et al. 2007), P. stutzeri (Shompoosang et al.
2014), and D. turgidum (Kim et al. 2013) were similar
at 40–45%. The optimum pH and temperature for
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
the enzyme at 50 C, 60 C, 70 C, 80 C, and 90 C
were 918, 809, 265, 24, and 13 h, respectively
(
Figure 2). The immobilized GI showed maximum activ-
ꢀ
ꢀ
ity at 90 C, but the thermostability at 90 C was too

BIOCATALYSIS AND BIOTRANSFORMATION
3
1
00
180
160
50
8
6
0
0
4
3
2
1
0
0
0
0
0
1
1
40
20
4
2
1
0
0
0
100
8
6
0
0
40
2
0
0
0
100
200
300
400
500
600
L-Rhamnose (g/L)
0
20
40
60
80
100
120
Figure 3. Effect of substrate concentration on L-rhamnulose
production (closed circle) from L-rhamnose, and conversion
yield of L-rhamnulose from L-rhamnose (open circle) in a
packed-bed reactor. The reactions were performed in 50 mM
Time (h)
ꢀ
ꢀ
Figure 2. Thermal inactivation of immobilized GI at 50 C
open circle), 60 C (open triangle), 70 C (open square), 80 C
closed diamond), and 90 C (closed inverted triangle). The
enzymes were incubated at temperatures ranging from 50 C
to 90 C for varying periods of time. A sample was withdrawn
at each time interval and assayed for the remaining enzyme
activity in 50 mM EPPS buffer (pH 8.0) containing 10 mM L-
ꢀ
ꢀ
(
(
ꢀ
À1
EPPS buffer (pH 8.0) containing 50–500 g L L-rhamnose at
ꢀ
ꢀ
À1
6
0 C with a dilution rate of 0.6 h . Data represent the means
ꢀ
of three experiments and error bars represent standard
deviation.
ꢀ
rhamnose at 60 C for 10 min. The experimental data for ther-
À1
was constant for 300 g L L-rhamnose, but decreased
mal deactivation of the enzyme were fitted to a first-order
curve and the half-lives of the enzyme were calculated using
Sigma plot 10.0 software (Systat software, San Jose, CA, USA).
Data represent the means of three experiments and error bars
represent standard deviation.
with increasing L-rhamnose concentration above
À1
300 g L . The decrease of the conversion yield with
increasing the concentration of L-rhamnose may be
due to substrate inhibition at high concentrations.
Next, we monitored L-rhamnulose production at dif-
D-fructose production from D-glucose using GI were pH
.0–8.5 and 60–70 C, respectively.
À1
ferent dilution rates ranging from 0.075 to 2.25 h
ꢀ
8
À1
using 300 g L
L-rhamnose in a packed-bed reactor
Figure 4). Until a dilution rate of 0.6 h , the product-
À1
(
Optimization of L-rhamnulose production using a
packed-bed reactor
ivity increased with constant conversion yield.
However, beyond 0.6 h , the conversion yield began
À1
to decrease. At higher dilution rate reduced L-rham-
nose retention time would lead to decrease productiv-
Next, the substrate concentration of L-rhamnose
required to produce L-rhamnulose using GI was investi-
gated. Immobilized GIs were packed into a bioreactor
ity. Thus, the optimal dilution rate for continuous
À1
L-rhamnulose production was determined as 0.6 h
.
(
XK 16; Amersham Pharmacia Biotech, Uppsala,
Consistent with this result, Kim et al. (2013) previously
reported the optimum conditions for L-rhamnulose
production from L-rhamnose using immobilized
Sweden) with a working volume of 40 mL. A solution
of 50 mM EPPS buffer (pH 8.0) containing 50–500 g L
À1
L-rhamnose in the feeding reservoir was fed continu-
ously into the reactor, and the effluent was allowed to
flow out of the reactor to the outside reservoir using a
peristaltic pump (Watson-Marlow 101 U/R, Cornwall,
À1
L-rhamnose isomerase from D. turgidum as 300 g L
L-rhamnose as a substrate with a dilution rate
À1
of 0.6 h
.
ꢀ
UK). The temperature was maintained at 60 C using a
water circulator (VTRC-620; Jeio Tech, Daejon, Korea).
The optimum concentration of L-rhamnose for L-rham-
nulose production using immobilized GI was deter-
Continuous L-rhamnulose production in a packed-
bed reactor
À1
The optimal conditions for L-rhamnulose production
mined as 300 g L (Figure 3). As the concentration of
À1
ꢀ
L-rhamnose varied from 50 to 500 g L , maximum
from L-rhamnose were determined as pH 8.0, 60 C,
À1
L-rhamnulose production was observed using
and a dilution rate of 0.6 h . We then used these con-
À1
4
00 g L L-rhamnose after 2 h. L-Rhamnulose produc-
ditions to evaluate the long-term operation of the
immobilized GI for continuous L-rhamnulose produc-
À1
tion reached a plateau at about 400 g L L-rhamnose.
The conversion yield of L-rhamnulose from L-rhamnose
tion in
a
packed-bed reactor for 30 days.

4
Y.-S. KIM ET AL.
1
1
1
1
60
140
L-rhamnulose has been continuously produced using
immobilized L-rhamnose isomerase from D. turgidum
in a packed-bed bioreactor, and an average of
40
20
00
1
1
8
6
4
2
0
20
00
0
À1
À1
130 g L L-rhamnulose was produced from 300 gL
L-rhamnose within 10 days, with a productivity of
78 g/L/h and a conversion yield of 43% (Kim et al.
8
6
4
2
0
0
0
0
0
0
2013). Thus, the two enzymes, immobilized GI and
L-rhamnose isomerase from D. turgidum, for L-rhamnu-
lose production show similar results with respect to
L-rhamnulose production, productivity, and conversion,
but the stability of the immobilized GI was three times
higher than that of L-rhamnose isomerase from D. tur-
gidum in a packed-bed reactor. The use of commercial
immobilized GI also has the advantage of producing
L-rhamnulose in an economical way without the
need for microbial culture, enzyme purification, and
immobilization.
0
0
0
.0
0.5
1.0
1.5
2.0
2.5
Dilution rate (h−1)
Figure 4. Effect of the dilution rate on L-rhamnulose produc-
tion (closed circle) and productivity (open circle) from L-rham-
nose in a packed-bed reactor. The working volume of the
reactor was 40 mL. A solution of 50 mM EPPS buffer (pH 8.0)
À1
containing 300 g L L-rhamnose in the feeding reservoir was
fed continuously into the reactor, and the effluent was allowed
to flow out of the reactor to the outside reservoir using a peri-
ꢀ
staltic pump. The temperature was maintained at 60 C using
Conclusions
a water circulator. Data represent the means of three experi-
ments and error bars represent standard deviation.
We describe optimal operating conditions and long
term stability for continuous L-rhamnulose production
from L-rhamnose in a packed-bed reactor using com-
mercial immobilized GI from Streptomyces murines
1
1
1
1
60
40
20
00
(Sweetzyme). The packed-bed reactor system produced
À1
À1
an average of 135 g L L-rhamnulose from 300 g L
L-rhamnose within an operation time of 16 days at pH
ꢀ
À1
8
.0, 60 C, and 0.6 h dilution rate, with a productivity
8
6
4
2
0
0
0
0
0
of 81 g/L/h and a conversion yield of 43%. This system
provides enhanced L-rhamnulose productivity from
L-rhamnose and offers long-term, continuous L-rham-
nulose production. This is a new approach for the pro-
duction of L-rhamnulose by commercial enzyme and
to the best of our knowledge, this is the first report
describing the enzymatic production of L-rhamnulose
using immobilized GI. Although immobilized GI
0
5
10
15
20
25
30
35
Time (days)
(
Sweetzyme) has been mainly used for high fructose
Figure 5. Continuous L-rhamnulose production from L-rham-
syrup production in the past, our findings indicate that
it shows potential for use in the production of various
rare sugars as well.
nose using immobilized GI in a packed-bed reactor. A solution
À1
of 50 mM EPPS buffer (pH 8.0) containing 300 g L L-rham-
nose in the feeding reservoir was fed continuously into the
reactor, and the effluent was allowed to flow out of the
À1
reactor, with a dilution rate of 0.6 h . The reaction was per-
ꢀ
formed at 60 C for 30 days. Data represent the means of
Acknowledgements
three experiments and error bars represent standard deviation.
This work was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(
NRF) funded by Ministry of Education under Grant [number
The immobilized enzyme continuously produced an
À1
À1
2015R1D1A1A01059570].
average of 135 g L
L-rhamnulose from 300 g L
L-rhamnose within 16 days, with a productivity of 81 g/
L/h and a conversion yield of 45% (Figure 5).
L-Rhamnulose concentration was reduced to approxi-
Disclosure statement
mately 55% at day 30. In
a
previous study,
No potential conflict of interest was reported by the authors.

BIOCATALYSIS AND BIOTRANSFORMATION
5
Funding
Leang K, Takada G, Ishimura A, Okita M, Izumori K. 2004b.
Cloning, nucleotide sequence, and overexpression of the
L-rhamnose isomerase gene from Pseudomonas stutzeri
in Escherichia coli. Appl Environ Microbiol 70:
This work was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by Ministry of Education under Grant [number
3298–3304.
2
015R1D1A1A01059570].
Levin GV, Zehner LR, Saunders JP, Beadle JR. 1995. Sugar
substitutes: their energy values, bulk characteristics, and
potential health benefits. Am J Clin Nutr 62:1161S–1168S.
Levin GV. 2002. Tagatose, the new GRAS sweetener and
health product. J Med Food 5:23–36.
Matsuo T, Suzuki H, Hashiguchi M, Izumori K. 2002. D-psicose
is a rare sugar that provides no energy to growing rats.
J Nutr Sci Vitaminol (Tokyo) 48:77–80.
ORCID
Yeong-Su Kim
Do-Yeon Kim
Chang-Su Park
http://orcid.org/0000-0002-9831-2883
http://orcid.org/0000-0001-8007-7347
http://orcid.org/0000-0002-0770-5068
Menavuvu BT, Poonperm W, Leang K, Noguchi N, Okada H,
Morimoto K, Granstrom TB, Takada G, Izumori K. 2006.
Efficient biosynthesis of D-allose from D-psicose by cross-
linked recombinant L-rhamnose isomerase: separation of
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