Bioconversion of sucralose-6-acetate to sucralose using immobilized microbial cells

Article abstract of DOI:10.1016/j.molcatb.2013.02.007

Bioconversion of sucralose-6-acetate to sucralose, an artificial sweetener has been carried out using Arthrobacter sp. (ABL) and Bacillus subtilis (RRL-1789) strains isolated at IIIM, Jammu, India. Biotransformation of sucralose-6-acetate to sucralose involves use of microbial whole cells, immobilized whole cells and immobilized whole cell bioreactor. Immobilized whole cells packed bed reactor has shown much superior biotransformation process in aqueous system using green technology, where purification of the final product is not required. The final sucralose bioproduct was directly concentrated under vacuum to get white crystalline powder. The immobilized whole cell bioreactor was used for more than three cycles continuously, thus provided much cheaper, less time consuming and easy down streaming process. Moreover, the method does not require any purification steps, which is otherwise requisite for presently available methods for sucralose production, resulting in even lower cost of overall process.

Full text of DOI:10.1016/j.molcatb.2013.02.007

Journal of Molecular Catalysis B: Enzymatic 91 (2013) 81–86
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Bioconversion of sucralose-6-acetate to sucralose using immobilized
ଝ
microbial cells
∗
Asha Chaubey, Chand Raina, Rajinder Parshad , Abdul Rouf, Pankaj Gupta, Subhash C. Taneja
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 October 2012
Received in revised form 6 February 2013
Accepted 25 February 2013
Available online 5 March 2013
Bioconversion of sucralose-6-acetate to sucralose, an artificial sweetener has been carried out using
Arthrobacter sp. (ABL) and Bacillus subtilis (RRL-1789) strains isolated at IIIM, Jammu, India. Biotrans-
formation of sucralose-6-acetate to sucralose involves use of microbial whole cells, immobilized whole
cells and immobilized whole cell bioreactor. Immobilized whole cells packed bed reactor has shown
much superior biotransformation process in aqueous system using green technology, where purifica-
tion of the final product is not required. The final sucralose bioproduct was directly concentrated under
vacuum to get white crystalline powder. The immobilized whole cell bioreactor was used for more than
three cycles continuously, thus provided much cheaper, less time consuming and easy down streaming
process. Moreover, the method does not require any purification steps, which is otherwise requisite for
presently available methods for sucralose production, resulting in even lower cost of overall process.
© 2013 Elsevier B.V. All rights reserved.
Keywords:
Sucralose
Sucralose-6-ester
Biotransformation
Packed bed reactor
Immobilization
1
. Introduction
Sucralose is generally synthesized by chemical methods involv-
ing five-step process by selective substitution of three chlorine
ꢀ
ꢀ
Sucralose,
trichlorogalactosucrose
or
4,1 ,6 -
atoms for three hydroxyl groups in the sucrose molecule. These
ꢀ
ꢀ
trichlorogalactosucrose is known as an artificial sweetener
having 600 times sweetening ability than sucrose. It is used in
place of sugar to eliminate or reduce calories in a wide variety
of products, including beverages, baked goods, desserts, dairy
products, canned fruits and syrups. Sucralose is chemically known
as 1,6-dichloro-1,6-dideoxy-beta-d-fructofuranosyl-4-chloro-4-
deoxy-alpha-d-galactopyranoside and has been derived from
methods for synthesis of sucralose (4,1 ,6 -trichlorogalactosucrose;
TGS) involve the chlorination through multistep protection and de-
protection strategy to obtain final sucralose. The reagents used in
the reaction are hazardous and involve the formation of different
byproducts, which need to be removed [1–7]. Involvement of sev-
eral purification steps, reduce the overall yield of the process for
production of sucralose.
ꢀ
ꢀ
sucrose by replacing the hydroxyl groups in the 4,1 , and 6
The enzymatic methods of sucralose synthesis involve removal
of 6-chloro-6-deoxygalactosyl moiety from the 6-position of chlo-
rinated sugar tetrachlororaffinose (TCR) [8]. Luo et al. [9] described
improved synthesis of sucralose from sucrose by chemical synthe-
sis, which again needs purification to obtain pure product. Ratnam
et al. [10] reported enzymatic process for sucralose production
wherein they could achieve maximum 95% deacetylation using
free or immobilized enzyme. Such process essentially requires sev-
eral downstream processing and product purification steps. Thus,
the process is quite expensive and time consuming. Alternative
methods involve deacetylation of sucrose-6-ester followed by chlo-
rination and purification to get pure sucralose [11]. To the best of
our knowledge, this is only one report of enzymatic deacetylation
of sucralose-6-acetate, however Arthrobacter sp. or Bacillus sublilis
strains were not used. We have therefore, used these two indige-
nous strains for a single step hydrolysis of sucralose-6-acetate to
obtain the sucralose (TGS) with 100% purity without any purifi-
cation. The method is green and does not involve any hazardous
reagents.
positions with chlorine.
First report on sucralose appeared in 1987 by Queens Eliza-
beth College in London and Tate & Lyle, a private company where,
chemical modification of sucrose by selective replacement of three
hydroxy groups with chlorine atoms was reported. Canada was the
first country to approve use of sucralose in foods in 1991 followed
by the United States in 1998. It is now being used in at least 28
countries as sweetening agent. Sucralose is being sold under the
brand name Splenda by McNeil Specialty Products Company, New
Brunswick, New Jersey. About 120 products using sucralose as a
sweetener are in the U.S. market.
ଝ
IIIM Communication number: IIIM/1515/2012.
Corresponding author. Tel.: +91 191 2569000 09x237.
E-mail addresses: achaubey@iiim.ac.in (A. Chaubey), rparshad@iiim.ac.in
∗
(
R. Parshad).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.02.007

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A. Chaubey et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 81–86
Table 1
Present report demonstrates the enzymatic hydrolysis of
Screening of various indigenous and commercial enzymes for bioconversion of
sucralose-6-acetate to sucralose.
sucralose-6-acetate to sucralose in a single step using acyl
hydrolase enzyme from IIIM Jammu, India microbial isolates;
Arthrobacter sp. (ABL) and Bacillus subtilis (RRL 1789). The bio-
conversion of sucralose-6-ester has been carried out in aqueous
system without pH adjustment or use of any buffering salts using
free enzyme/whole cells/immobilized enzyme/immobilized whole
cells. The final bioproduct does not require any purification and can
be directly concentrated to get crystalline powder.
Enzyme
Time (h)
Conversion (%)
Arthrobacter sp. lipase (ABL)
Bacillus subtilis (RRL-1789)
Trichosporon beigelii (Y-15)
Pig Liver Esterase (PLE)
Candida antarctica (CAL-B)
Pseudomonas fluorescence (PSF)
Pseudomonas sp. lipase (PSL)
Mucor miehei lipase (MM)
Mucor javanicus (MJ)
96
48
24
50
96
24
24
24
24
5
100
100
5
100
85
ND
5
ND
2
. Experimental
5
Amano AS
90% + 10% side product
Porcine pancreas lipase (PPL)
Candida rugosa lipase (CRL)
Candida cylindracea (CCL)
24
24
24
20
30
25
2
.1. Materials and general experimental conditions
The reagents and solvents used in the present study were mostly
LR grade. Substrate sucralose-6-acetate was a kind gift from Dr.
V. Arosker, Mumbai. Silica gel coated aluminum plates from M/s
Merck were used for TLC. Commercial enzymes were purchased
2
.5. Biotransformation of sucralose-6-ester to sucralose using
free isolated enzyme
from Sigma. 1H NMR spectra in CD OD were recorded on Bruker
3
5
00 MHz spectrometers with TMS as the internal standard. Chem-
Biotransformation of sucralose-6-ester to sucralose was
performed using several commercial lipases as shown in
Table 1 and indigenous lipase/esterase enzymes B. subtilis (RRL-
ical shifts were expressed in parts per million (ı ppm). MS were
recorded on LC MS Agilent 1100 series. IR was recorded on a FT-IR
Hitachi (270-30) spectrophotometer. Optical rotations were mea-
1
789)/Arthrobacter sp. (ABL), T. beigelii (Y-15) for comparative
◦
sured on Perkin-Elmer 241 polarimeter at 25 C using sodium D
performance (Table 1). Enzymatic reaction was performed by
suspending sucralose-6-ester and enzymes (purified lyophilized
powder) in 1:1 ratio in water/buffer under shaking. Biotransfor-
mation reaction was monitored by TLC at different time intervals
till complete biotransformation is achieved. On completion of
biotransformation, reaction was terminated by centrifugation to
remove insoluble impurities and the product was extracted with
chloroform, dried and purified by column chromatography in
5:95%, methanol:DCM solvent system. Bioproduct was finally ana-
lyzed by HPLC, optical rotation and NMR for its purity.
light.
2.2. Enzymes
Commercial enzymes such as Candida rugosa lipase (CRL),
Candida cylindracea lipase (CCL), Candida antarctica lipase (CAL),
Pseudomonas sp. lipase (PSL), Mucor miehei lipase (MM), Porcine
pancreas lipase (PPL), Mucor javanicus lipase (MJL) were procured
from Sigma. Indigenous lipase such as Arthrobacter sp. lipase (ABL),
B. subtilis lipase (RRL-1789) and Trichosporon beigelii lipase (Y-15)
were grown in specified media and conditions followed by enzyme
isolation by ultrasonication as described previously [12–14].
2.6. Biotransformation of sucralose-6-ester to sucralose using
microbial cells
Biotransformation of sucralose-6-ester to sucralose (Scheme 1)
was carried out using freshly harvested microbial cells from
indigenous strains B. subtilis/Arthrobacter sp. Reaction mixture was
prepared by suspending sucralose-6-ester in requisite amount of
water to obtain 10–50 g/L substrate concentration. Enzymatic reac-
tion was initiated by addition of enzyme/cell biomass/immobilized
enzyme in equal amount of substrate. Biotransformation was
monitored on TLC at different time intervals. On completion of bio-
transformation, the reaction was terminated by centrifugation and
concentrated under vacuum, purified by column chromatography
followed by crystallization. The product purity was tested by HPLC,
optical rotation and NMR.
2
.3. Production of biomass
Biomass from Arthrobacter sp. was grown in 1% peptone, 0.5%
beef extract and 0.5% NaCl, pH 7.0 as described previously [12]. B.
subtilis biomass was grown in 1% peptone, 0.1% yeast extract, 0.5%
NaCl, and 0.5% sucrose at pH 7.2 for 30–36 h [13].
2
2
.4. Immobilization of microbial cells
.4.1. Entrapment in sol–gel supports
Arthrobacter sp./B. subtilis microbial cells were immobilized in
sol–gel supports prepared from tetraethylorthosilicate precursor
as previously reported [15]. Entrapment was carried out by adding
ABL cells suspension/free enzyme to the homogenized sol during
polymerization process. Gelation was carried out further for 24 h.
The gel was filtered and washed several times with buffer to remove
any adhered whole cells/free enzyme. The entrapped ABL was then
further used for biotransformation studies.
2.7. Biotransformation of sucralose-6-acetate to sucralose in
immobilized whole cells/immobilized whole cell reactor
Reaction mixture was prepared by dissolving 1.0 g of sucralose-
6
-ester in water to get 10–50 g/L concentration. The reaction was
initiated by addition of immobilized whole cells (beads) equivalent
to requisite amount of immobilized/entrapped whole cells beads
so as to make S:E ratio 1:1. To carry out reaction with 1.0 g of sub-
strate, 30.0 g of entrapped whole cell beads (equivalent to 1.0 g of
whole cells) were used under shaking at 50 rpm. Biotransformation
was monitored on TLC at different time intervals till complete bio-
transformation was achieved. Aqueous product was separated by
filtration, beads were washed with water and concentrated under
vacuum to obtain white crystalline powder.
2
.4.2. Entrapment of whole cells in calcium alginate
Freshly harvested B. subtilis/Arthrobacter sp. cells were mixed
with sodium alginate (sodium alginate:cell biomass used was 1:1,
:2, 1:3) and passed through syringe into 2% calcium chloride
1
solution as previously described [16]. The entrapped cells thus
obtained were washed with water and further used for hydrolysis
of sucralose-6-acetate at different substrate concentrations.

A. Chaubey et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 81–86
83
Scheme 1. Bioconversion of sucralose-6-acetate to sucralose.
Immobilized whole cell bioreactor was fabricated by packing
50 g of immobilized whole cell calcium alginate beads in a reactor
3.71–3.75 (m, 2H), 3.96–3.85 (m, 2H), 3.87–3.92 (m, 2H), 3.99–4.07
(m, 1H), 4.08 (dd, J = 3.5 Hz and 10.2 Hz, 1H), 4.11–4.19 (m, 1H),
4.24 (dd, J = 3.83 Hz and 6.59 Hz, 1H), 4.31 (d, J = 8.39 Hz, 1H),
4.35–4.41 (m, 1H), 4.52–4.60 (m, 1H), 5.38 (d, J = 3.9 Hz, 1H); 13 C
NMR, ı: 20.9, 44.8, 46.2, 65.0, 66.1, 69.1, 69.5, 69.6, 77.3, 77.6, 83.6,
1
(
ꢀ
ꢀ
1.5 × 15 ). 5 g substrate was suspended in water to prepare 10 g/L
concentration and circulated through the reactor continuously at a
flow rate of 10 mL/min until 100% bioconversion was obtained. The
design of the reactor has been shown in Scheme 2.
93.9, 104.8, 172.4. ESI-MS (m/z): 438. Anal. Calc. for C14H21Cl O :
3
9
The aqueous product was separated from the reactor, the reac-
tor was washed well with water and bioproduct was concentrated
under vacuum providing a cost effective and easy downstream
processing. There is no need to separate soluble impurities or pro-
teins which is essential in case of soluble enzymes. The reactor was
used continuously three times for the biotransformation process.
C, 38.24; H, 4.81. Found C, 38.73; H, 5.24.
ꢀ
ꢀ
ꢀ
ꢀ
4,1 ,6 -trichloro-4,1 ,6 -trideoxygalactosucrose:
HPLC
Purity
>99%; [␣]D25 +71.5 (c 1, MeOH) 1H NMR, ı: 3.63–3.68 (m, 2H),
3.71 (s, 2H), 3.79 (dd, J = 11.1 Hz and 3.0 Hz, 1H), 3.82 (dd, J = 3.9 Hz
and 10.1 Hz, 1H), 3.85–3.93 (m, 2H), 4.02–4.09 (m, 2H), 4.25–4.30
(m, 1H), 4.34 (dt, J = 1.0 Hz and 6.7 Hz, 1H), 4.38–4.40 (m, 1H), 5.37
(
7
d, J = 3.91 Hz, 1H); 13C NMR, ı: 44.9, 46.3, 63.1, 65.1, 69.4, 69.8,
1.8, 77.3, 77.6, 83.8, 93.9, 104.8. ESI-MS (m/z): 396. Anal. Calc. for
C12H19Cl O : C, 36.25; H, 4.82. Found C, 36.01; H, 5.00.
2.8. Characterization of product
3
8
Biotransformation process was monitored initially by TLC and
HPLC until 100% conversion is achieved. The purified crystallized
product obtained was tested for its purity by TLC, HPLC, NMR and
optical rotation. Conversion and purity profile of the reaction prod-
uct was determined by Perkin-Elmer HPLC on C-8 column using RI
3. Results and discussion
3.1. Enzymatic biotransformation of sucralose-6-ester to
sucralose
(
refractive index) detector. Mobile phase for HPLC was ACN:Water,
◦
1
7:83; flow rate 1 mL/min and column temperature 40 C. Elemen-
tal analysis of purified product was carried out using Elementar
Vario EL III.
Various lipases/esterases from commercial and indigenous
source were screened and tested for biocatalytic efficiency for
the production of sucralose from sucralose-6-ester. As shown in
Table 1, most of the commercial enzymes presented significant
hydrolysis of the substrate. Amano AS hydrolyzed the ester very
fast in (5 h), but the product contained side products/impurities.
However, enzymes from indigenous source Arthrobacter sp. lipase,
B. subtilis and commercial PLE enzymes demonstrated complete
bioconversion of sucralose-6-ester at a low speed without any side
products. Since these biotransformation reactions were carried out
using soluble enzymes, the protein impurities were present in the
reaction mixture. The required purification steps resulted in an
expensive and time consuming process. The product obtained from
the experiments using whole cell biomass from indigenous strains
was fairly good but still some purification was required.
ꢀ
ꢀ
ꢀ
ꢀ
6
-O-acyl-4,1 ,6 -trichloro-4,1 ,6 -trideoxygalactosucrose:
HPLC
2
5
Purity >99%; [␣]D +74.0 (c 1, MeOH) 1H NMR, ı: 2.01 (s, 3H),
3.2. Biotransformation using microbial whole cells of B. subtilis
and Arthrobacter sp. at various substrate concentrations
In order to avoid the purification of product while using
enzymatic biotransformations, freshly prepared biomass from
lipase/esterase producing indigenous strains Arthrobacter sp. and B.
subtilis were used for biotransformation reactions. Fig. 1(a) shows
B. subtilis whole cells catalyzed biotransformation at different sub-
strate concentrations (10–300 g/L). Best results were obtained with
1
4
0–20 g/L concentration where 100% product was observed in
8 h whereas using 50 g/L concentration, complete conversion was
observed in 72 h. On further increasing the substrate concentration
(100 g/L) complete conversion was observed in 96 h.
Arthrobacter sp. Lipase could result in complete biotrans-
formation in 72 h, whereas increase in substrate concentration
slowed down the reaction rates due to which, complete
Scheme 2. Design of immobilized whole cell packed bed reactor for bioconversion
of sucralose-6-acetate to sucralose.

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A. Chaubey et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 81–86
a 120
a 120
100
100
80
60
40
20
0
1
2
5
0g/l
0g/l
0g/l
80
6
0
0
100g/l
4
Whole cells
2
3
00g/l
00g/l
Immobilized
whole cells
20
0
0
50
100
150
200
250
300
0
50
100
150
200
Time (h)
Time (h)
b
120
00
b
1
20
1
100
8
6
4
2
0
0
0
0
0
8
0
0
1
0g/l
20g/l
6
5
1
2
3
0g/l
Whole cells
00g/l
00g/l
00g/l
40
Immobilized w hole
cells
20
0
0
50
100
150
200
250
300
350
0
50
100
150
200
250
Time (h)
Time (h)
Fig. 1. (a) Biotransformation using microbial whole cells of Bacillus subtilis at vari-
ous substrate concentrations. (b) Biotransformation using microbial whole cells of
Arthrobacter sp. at various substrate concentrations.
Fig. 2. (a) Biotransformation using immobilized whole cells of Bacillus subtilis. (b)
Biotransformation using immobilized whole cells of Arthrobacter sp.
hydrolysis of sucralose-6-acetate to sucralose was obtained in
1
20–168 h (Fig. 1b).
a
120
00
3.3. Biotransformation using B. subtilis and Arthrobacter sp.
1
biomass with different substrate to enzyme ratio
8
0
1
0g/l
Bioconversion of sucralose-6-acetate to sucralose at 10–100 g/L
concentration was carried out at different substrate:enzyme ratio,
i.e. 1:0.25, 1:0.5, 1:0.75, 1:1, 1:2, 1:3 using Arthrobacter sp. and
B. subtilis cell biomass. The results showed that 1:1 ratio of
substrate to enzyme ratio was suitable for biotransformation of
sucralose-6-ester at 10–100 g/L substrate concentrations using
whole cells//immobilized whole cells of B. subtilis (Fig. 2a) and
Arthrobacter sp. (Fig. 2b). Therefore, S:E ratio of 1:1 was used for
all biotransformation studies.
60
20g/l
50g/l
4
0
20
0
0
50
100
150
200
250
Time (h)
b
120
1
00
3.4. Biotransformation of sucralose-6-ester to sucralose using
calcium alginate entrapped microbial cells
80
6
4
2
0
0
0
0
Biotransformation of sucralose-6-acetate (1.0 g) was carried out
under shaking conditions. It was observed that calcium alginate
entrapped B. subtilis cells resulted in complete bioconversion in
10g/l
20g/l
50g/l
7
2–96 h at 10–20 g/L substrate concentration (Fig. 3a). On further
increasing the substrate concentration up to 50 g/L, rate of reaction
was much slower resulting in 100% biotransformation in 7 days
0
50
100
150
200
250
(
168 h). Calcium alginate entrapped microbial cells from Arthrobac-
Time (h)
ter sp. (Fig. 3b) showed slower reaction rates with production of
pure product in 9 days (216 h). The reaction was terminated by
filtration and concentrated under vacuum for crystallization of
product followed by purity testing.
Fig. 3. (a) Biotransformation of sucralose-6-acetate to sucralose in immobilized
whole cells reactor of Bacillus subtilis. (b) Biotransformation of sucralose-6-acetate
to sucralose in immobilized whole cell reactor of Arthrobacter sp.

A. Chaubey et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 81–86
85
Fig. 4. (a) HPLC chromatograph of standard mixture sucralose and sucralose-6-acetate. (b) HPLC chromatograph reaction mixture during biotransformation. (c) HPLC
chromatograph of bioproduct from immobilized whole cell bioreactor.
3
.5. Biotransformation of sucralose-6-acetate to sucralose in
crystallization of product. This process for biotransformation was
found to be the best among all, as there was no requirement of
filtration or removal of biocatalyst. Thus downstream process is
much easier and less time consuming when immobilized whole
cell bioreactor was used.
immobilized whole cell bioreactor
Immobilized B. subtilis whole cells bioreactor was fabricated by
packing of calcium alginate entrapped whole cell beads in a reactor.
Biotransformation was carried out with 10–50 g/L concentration
at a flow rate of 10 mL/min (Scheme 2). Biotransformation was
monitored on TLC/HPLC at different time intervals. Fig. 4 shows
HPLC chromatograms of the standard substrate/product mixtures
3.6. Reuse of immobilized whole cell reactor for
biotransformation
(
Fig. 4a), during bioconversion process (Fig. 4b) and the bioprod-
The immobilized whole cell bioreactor of B. subtilis and
Arthrobacter sp. was reused for biotransformation of sucralose-6-
ester at 5.0 g batch scale. Fig. 5 shows the biotransformation results
for B. subtilis (Fig. 5a) and Arthrobacter sp. (Fig. 5b) immobilized
whole cell reactor. The continuous flow reactor was continuously
uct (Fig. 4c). On achieving complete conversion, the reaction was
terminated by taking out the aqueous product from the reactor.
The reactor was thoroughly washed with water to remove adhered
bioproduct. The product was concentrated under vacuum for

8
6
A. Chaubey et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 81–86
1
1
20
00
days. Reuse of immobilized Arthrobacter sp. reactor in batch process
could provide 100% conversion in 8, 10 and 12 days respectively in
consecutive cycles.
a
4
. Conclusions
8
6
4
2
0
0
0
0
0
Biotransformation process for the conversion of sucralose-
-acetate to sucralose has been successfully developed using
Cycle 1
Cycle 2
Cycle 3
6
immobilized whole cell reactor of Arthrobacter sp. and B. subtilis.
B. subtilis strain proved to be better as it took less time for com-
plete bioconversion of substrate as compared to Arthrobacter sp.
strain. The immobilized whole cell packed bed reactor has shown
easy and inexpensive batch biotransformation process for sucralose
production in aqueous system. The immobilized whole cell packed
bed reactor was used more than three cycles with equal quality
of product. Thus, a single step hydrolysis provides pure sucralose
0
100
200
300
400
Time (h)
(TGS) with 100% purity without any purification.
b
120
00
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used for at least three consecutive cycles for three batch biotrans-
formations. On reusing the same batch of immobilized B. subtilis,
(
2008) 552–562.
1
00% hydrolysis of substrate was obtained in 7–8 days whereas
on third time reuse complete bioconversion was observed in 12