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.