Licorice extract and its principle component, Glycyrrhizinic acid (GL) have
extensively used in food as a sweetener and a flavoring ingredient and finds
wide application in both traditional and herbal medicine (Amin
et al., 2011). GL is composed of one molecule of 18-β Glycyrrhetinic
Acid (GA) as aglycone and two molecules of glucuronic acid (Hennell
et al., 2008). The pharmacological activity is due to the presence
of triterpene aglycone 18-β Glycyrrhetinic Acid (GA) and in lesser measures,
to its glycoside Glycyrrhizin (GL) (Gisbon, 1978; Samuelsson,
1993). Biotransformation of GL into GA can be implemented through the function
of β-glucuronidase which is produced by Escherichia coli MTCC 1652
and can hydrolyze glucoside bond. β-glucuronidase from E. coli MTCC
1652 have been produced and analyzed and the specificity and productivity of
conversion of GL to GA was achieved successfully and has a wide range of pH,
high substrate specificity and high efficiency for GL conversion.
Alginate is a naturally occurring binary linear heteropolymer that contains
1, 4-linked β-D-mannuronic acid and α-L-glucuronic acid residue. Because
of its good biocompatibility and processing capacity, it is one of the best
entrant matrixes for cell entrapment (Zhang et al.,
2010). Entrapment is one of the simplest methods available for enzyme or
cell immobilization under mild conditions and results in minimal denaturation
of the immobilized biocatalyst compared with other immobilization methods (Roy
et al., 2004).
To the best of our knowledge, no studies have yet reported on the use of immobilized whole cells for the bioconversion of GL to GA. In the present research, E. coli MTCC 1652 whole cells were immobilized with alginate. Very stable immobilized cells were achieved that allowed the continuous production of GA.
MATERIALS AND METHODS
Plant materials, microorganism and chemicals: The roots of G. glabra were obtained from local market of New Delhi, India. The crude drug was identified and a specimen voucher No./NISCAIR/RHMD/consult/2012-2013/2085-92 is issued by NISCAIR, CSIR, New Delhi, India. Bacterial strain E. coli MTCC 1652 were collected from MTCC, IMTECH, Chandigarh, India. The bacterial strain were grown and maintained in nutrient agar slants at 4°C. Glycyrrhetinic acid (GA) and glycyrrhizin 98% pure (GL) obtained from Sigma Aldrich, Bangalore, India. All the chemicals, reagents and microbiological medium obtained from Hi-media, Bombay, India.
Cell suspension production: E. coli MTCC 1652 cell suspension was prepared in nutrient broth. To the 50 mL of nutrient broth consisting of 5 g peptone, 5 g NaCl, 1.5 g beef extract, 1.5 g yeast extract and 1000 mL distilled water, pH was adjusted to 7.2, bacterial inoculums was added and incubate at 37°C, for 24 h in orbital shaker at 150 RPM. The cells were harvested from the culture broth by centrifugation at 5000 RPM for 10 min and the pellets were stored at 4°C for use.
Immobilization of E. coli MTCC 1652 whole cell in calcium alginate:
Sodium alginate powder in the range of 4-10% w/v was added to 100 mM Tri-HCl
buffer, pH 7. The sodium alginate powder was added slowly to the buffer solution
in order to prevent flocculation. Stirring was performed by magnetic stirrer
for 45 min to obtain a homogenous solution of sodium alginate. The cell suspension
was then slowly added (volume ratio of 1:1) to the sodium alginate solution.
The resulting mixture was added drop wise using a syringe to CaCl2
solution of 0.1-0.5 M. Each drop led to the formation of a calcium alginate
bead inside which the E. coli cells were entrapped. The beads were stabilized
by stirring for 1 h and were stored at 4°C (Barin et
Determination of entrapment efficiency in beads: To determine the total
number of immobilized cells within the beads, three beads were selected at random
from a sample. The selected beads were ruptured to make homogenous solution.
The homogenous solution was diluted 10 times. The cells within the diluted solutions
were allowed to grow in petri dishes containing nutrient agar and incubated
at 37°C for 24 h. The cell density was obtained by colony counts on petri
dishes (Greenberg et al., 1995).
Effect of pH, volume and time on bioconversion rate: Effect of pH, volume
and time of the synthetic medium (0.05% NH4Cl, 0.005% of (NH4)2•SO4,
0.4% dextrose, 0.01% NaCl, 0.01% MgCl2•6H2O, 0.6%
Na2HPO4 and 0.3% KH2PO4), pH of
the medium was adjusted to 7.2 (Spizzizen et al.,
1951) on bioconversion were determined by incubating the alginate entrapped
cells. Different concentrations (1, 2 and 3%) w/v of pure GL in different reaction
volume (2, 4 and 6 mL) of synthetic medium was used for bioconversion. The optimum
pH for bioconversion was determined by incubating the GL at different pH of
5.5, 6.5 and 7.5 at 37°C for 4, 8 and 12 h, respectively with 10 no of beads.
Enzyme assay: β-glucuronidase activity was measured in terms of
Hydrolytic Unit (HU) by incubating enzyme with 3 mM GL solution for 10 min at
35°C. The enzymatic reaction was blocked by adding 200 mM glycine buffer
solution pH 10.4. The amount of GA formed after hydrolysis was monitored by
High Performance Liquid Chromatography (HPLC). One HU is defined as μg
of GA released per 10 min from 1 μg μL-1 of GL solution.
Analysis of 18-β glycyrrhetinic acid: Samples consisting of unconverted
GL and GA were analyzed by high performance liquid chromatography (Analytical
technologies, Boroda, India). The chromatography was carried out by column Lichrospher
100 RP C18 (temperature 25°C), the mobile phase consist of methanol: water
(85:15v/v) at flow rate of 1 mL min-1 with run time 10 min. Detection
of GL and GA was carried out by UV detector at 254 nm (Wang
et al., 2010).
Optimization of the immobilization conditions: For better entrapment
of microbial cells, optimization of different parameters improves the rigidity
and permeability of the beads (Anisha and Prema, 2008).
The effects of alginate concentration and Ca2+ concentration were
investigated in the current study. The concentration of sodium alginate and
molarity of calcium chloride was optimized at different range. When sodium alginate
powder in range of 1-10% was added into 0.1 to 0.5 M CaCl2 solution,
the beads formed were stable only at 8% with 0.4 M CaCl2 solution.
The optimal conditions for maximal immobilization efficiency were determined
to be 0.4 M Ca2+ and 8% alginate concentration, the entrapment efficiency
was 900 CFU/beads and size of the beads were in the range of 3-5 mm.
Bioconversion of GL to GA: The effect of percentage of standard GL i.e.,
1, 2 and 3% under different volume and pH was observed in the present study.
The standard chromatogram of GA, GL and extracted GA from the broth is presented
in Fig. 1a-c.
At 1% GL concentration at pH 5.5, with 10 beads the conversion rate and GA%
was undetectable under all the reaction volume i.e., with 2, 4 and 6 mL after
4, 8 and 12 h incubation. However, the HU of enzyme was observed with 4 and
8 mL reaction volume with maximum 40.779 HU and there is decrease in GL concentration
in the reaction mixture. At pH 6.5 with 10 beads only bioconversion was observed
after 12 h incubation in 4 and 6 mL reaction volume with GA concentration of
48.503 and 42.774 μg per mL with 35.048 and 31.570 HU, respectively (Fig.
||(a) HPLC Chromatograms of pure GL and GA (b) Degradation of
3% GL into GA at pH 6.5 and 12 h and (c) Degradation of 3% plant extract
of G. glabra root powder into GA at pH 6.5 and 12 h
||Bioconversion of 1% of GL to GA at 12 h with pH of 6.5
At pH 7.5, the conversion rate and % GA was undetectable under all the reaction
volume i.e., with 2, 4 and 6 mL after 4, 8 and 12 h incubation with very less
At 2% GL concentration at pH 5.5, the concentration of GA was undetectable
under all the conditions with very less enzyme activity. However, there is sharp
decrease in the GL concentration to 13.455 μg mL-1 after 12
||Bioconversion of 2% of GL to GA at 12 h with pH of 6.5 and
||Bioconversion of 3% of GL to GA at 12 h with pH of 5.5 and
pH 6.5 with reaction volume of 2, 4 and 6 mL
At pH 6.5, 28.522 μg of GA per mL was observed after 12 h of incubation
with enzyme activity of 29.981 HU in 6 mL reaction volume. At pH 7.5, very less
amount of GA was observed in 4 mL reaction volume and GL was completely exhausted
from the reaction mixture. However at 6 mL reaction volume 33.875 μg of
GA was observed per mL of the reaction volume after 4 h of incubation with enzyme
activity of 20.192 HU, thereafter i.e., at 8 and at 12 h GA was undetected in
the reaction mixture (Fig. 3).
At 3% GL concentration at pH 5.5, considerable amount of GL is converted in to GA after 12 h incubation with 59. 387 μg, 61.426 μg and 60.818 μg per mL with enzyme activity of 40.003, 39.021 and 24.010 HU in 2, 4 and 8 mL reaction volume, respectively. At 6.5 pH, maximum amount of GL is converted to GA with a concentration of 72.694 μg mL-1 and enzyme activity of 46.039 HU in 4 mL reaction volume at 12 h incubation (Fig. 4).
Under optimized condition the immobilized cell produces 58.663 μg per mL of GA, in plant extract containing 95.118 μg of GL mL-1 of the extract with 61.67% conversion rate at 12 h.
Bioconversion of GL to GA is efficiently carried out by entrapped E. coli
cells in alginate beads. The enzyme responsible for bioconversion is β-glucuronidase.
The conversion rate is highly depend on the immobilization method, materials
used for cell entrapment, bead size, no of cell entrapped in each bead, reaction
volume, pH of the medium and substrate concentration (Zhang
et al., 2010).
In the present research, alginate method was selected for immobilization of
E. coli cells since alginate is a widely used method of choice for whole
cell immobilization (Barin et al., 2007). The
immobilization procedure is optimized for better entrapment of E. coli
cell and bead stability. The bioconversion reaction was optimized for substrate
concentration form low to high under diverse pH condition with diverse reaction
mixture volume with different time of incubation. At very less concentration
of GL the conversion is undetectable under acidic condition however the enzyme
was activity was observed. Since the β-glucuronidase activity was optimum
at pH 6 (Amin et al., 2011), as there is lower
enzyme activity at acidic condition less amount of GL is converted in to GA.
A very similar conversion rate was observed under slight alkaline pH i.e., at
7.5. However under low acidic conditions i.e. at pH of 6.5, maximum bioconversion
was observed with all the concentration of GL irrespective of reaction mixture.
This shows that bioconversion of GL to GA is optimum at lower acidic condition.
At pH 5.5 and at 7.5 the GL concentration is decreases as the time of incubation
increases, this may be due to conversion of GL to other unknown molecules.
Enzyme activity and continuous conversion of GL into GA in immobilized whole cell system depends on cell concentration, % of GL, pH, reaction volume and incubation time. Alginate immobilized E. coli cell showed higher enzyme activity with 46.039 HU and 72.649 μg per mL of GA production at 12 hr of incubation time. Under actual condition, 61.67% conversion was achieved with crude water extract of G. glabra root containing 95.118 μg of GL per mL.
We acknowledge Dr. H. B Singh, taxonomist and chief scientist, NISCAIR, CSIR, New Delhi, India for authenticating our herbal crude drugs.