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Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin
Accepted Manuscript Original article Purification and Characterization of Alliinase produced by Cupriavidus necator and its Application for Generation of Cytotoxic Agent: Allicin Sagar Chhabria, Krutika Desai PII: DOI: Reference:
S1319-562X(16)00005-X http://dx.doi.org/10.1016/j.sjbs.2016.01.003 SJBS 620
To appear in:
Saudi Journal of Biological Sciences
Received Date: Revised Date: Accepted Date:
12 September 2015 1 December 2015 2 January 2016
Please cite this article as: S. Chhabria, K. Desai, Purification and Characterization of Alliinase produced by Cupriavidus necator and its Application for Generation of Cytotoxic Agent: Allicin, Saudi Journal of Biological Sciences (2016), doi: http://dx.doi.org/10.1016/j.sjbs.2016.01.003
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1
Purification and Characterization of Alliinase produced by Cupriavidus necator and its Application for
2
Generation of Cytotoxic Agent: Allicin
3
Sagar Chhabria1, Krutika Desai2*
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6
(W), Mumbai 400056, India.
7
2
8
College of Commerce & Economics, Vile Parle (W), Mumbai 400056, India
Dept. of Biological Sciences, Sunandan Divatia School of Science, SVKM’s NMIMS University, Vile Parle
Dept. Of Microbiology, SVKM’s Mithibai College of Arts, Chauhan Institute of Science &Amrutben Jivanlal
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*Address all correspondence to:
11 12 13 14 15 16 17 18 19
Krutika B. Desai, Ph.D. Dept. of Microbiology, SVKM’s Mithibai College of Arts, Chauhan Institute of Science & Amrutben Jivanlal College of Commerce & Economics, Vile Parle (W), Mumbai 400056, India Tel: +91-22-42339000 Fax: +91-22-26130441 E-mail: [email protected]
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1
Abstract
2
Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the
3
enzyme alliinase (EC 4.4.1.4). A bacterium Cupriavidus necator with the ability of alliinase production was
4
isolated from a soil sample and was identified by morphological, biochemical and 16 S rRNA sequence.
5
Alliinase production was optimized and further it was purified to apparent homogeneity with 103 fold
6
purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100
7
chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular
8
weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum
9
temperature was 35 oC. The enzyme exhibited maximum reaction rate (Vmax) at 74.65 U/mg and Michaelis-
10
Menten constant (Km) was determined to be 0.83 mM when alliin was used as a substrate. The cytotoxic activity
11
of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT
12
assay and Acridine orange-ethidium bromide staining. This approach of in-situ allicin generation suggests a
13
novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent
14
allicin.
15 16
Keywords: Alliinase; Characterisation, Cupriavidus necator; Purification, MIA Pa Ca-2 cells
17 18
Abbreviations: Diethylaminoethyl (DEAE), β-mercaptoethanol (β-ME), Pyridoxal 5’ Phosphate (PLP)
2
1
1.
Introduction
2
Allicin (Diallyl thiosulphate) is the best-known active compound of freshly crushed garlic extract and is known
3
to possess a wide range of pharmacological activities which include antimicrobial, anti-inflammatory,
4
antithrombotic, antifungal, antiviral, antioxidant, anticancer, and antiatherosclerotic (Ilić et al. 2011; Oommen et
5
al. 2004). This allyl-sulphur compound is synthesized as a result of C-S lyase activity of alliinase (Alliin Lyase;
6
EC 4.4.1.4) on alliin (S-allyl-L-cysteine sulphoxide), a naturally-occurring diastereomer molecule (Jones et al.
7
2004) (Fig. 1). Alliinase catalyses the release of volatile sulphur compounds which is responsible for the
8
characteristic flavour and odour of Allium species.
9
Alliinase has been purified from garlic, onion, and other plants of the genus Allium (Schwimmer and Mazelis
10
1963; Mazelis and Crews 1968; Tobkin and Mazelis 1979; Nock and Mazelis 1987; Landshuter et al. 1994;
11
Lohmuller et al. 1994; Rabinkov et al. 1994; Manabe et al. 1998). The enzymatic production of allicin occurs in
12
garlic, because of the injury of the plant tissue that enables interaction of the enzyme in vacuoles with alliin
13
gathered in the cytosol (Yamazaki et al. 2002); hence allicin production has been discussed as a defense
14
mechanism of the plant against microbial infection or insect attack (Slusarenko et al. 2008). Allicin generated
15
from alliinase which was purified from garlic, has been studied for fungicidal activity against Magnaporthe
16
grisea (Fry et al. 2005).
17
In the past, some microorganisms have also been studied for their alliinase producing ability, viz. Durbin and
18
Uchytil (1971) reported the presence of enzyme in Penicillium corymbiferum and Yutani et al. (2011) reported
19
the presence of an intracellular alliinase in Ensifer adhaerens; further allicin generated from this enzyme
20
exhibited potent fungicidal activity against Saccharomyces cerevisiae.
21
In the last few years, alliinase had found its application in agriculture, pharmaceutical and food industry.
22
Alliinase has been immobilised on N-succinyl chitosan polymer (Zhou and Wang 2009), calcium alginate beads
23
(Anifantaki et al. 2010), Monoclonal antibody (Chhabria et al. 2015), porous aluminium oxide (Milka et al.
24
2000) and alginate microparticles (Ko et al. 2012), further used to catalyse the conversion of alliin to allicin, an
25
active ingredient of pharmaceutical compositions and food additive. The immobilized alliinase in alginate beads
26
can be further used for the development of a biosensor for monitoring cysteine sulphoxides which could prove
27
valuable in several applications in the food industry.
28
Chemical instability and low miscibility of allicin in water obstruct its practical use; hence a binary system
29
consisting of purified alliinase and alliin, in which allicin was generated in-situ, had been demonstrated to be a
3
1
promising potential anticancer approach for inhibition of the gastric carcinoma cell line in vitro and in vivo by
2
Miron et al (2003). Arditti et al (2005) reported the application of purified alliinase, for in-situ generation of
3
allicin which further inhibited the growth of B-chronic lymphocytic leukemia tumour in vivo.
4
Currently, much attention is paid to the development of microbial enzyme technology, as these enzymes are
5
relatively more stable than the enzymes derived from plants. In addition, the microorganisms represent an
6
attractive source of enzymes because they are economically feasible, can be cultured in large quantities in a
7
short time by fermentation and enzyme yield is predictable and controllable.
8
In the present study, we report for the first time, purification and characterization of an intracellular alliinase
9
enzyme from Cupriavidus necator. The isolated enzyme was further used for on-site generation of cytotoxic
10
agent-allicin; which was assessed on pancreatic cancer (MIA PaCa-2) cell line using MTT assay and Acridine
11
orange-ethidium bromide staining.
4
1
2.
Material and methods
2
2.1 Chemicals and Reagents
3
Alliin & Allicin were purchased from LKT Laboratories, Inc (St. Paul, MN). DEAE Cellulose, Sephadex G-100
4
and Pyridoxal 5’ Phosphate (PLP) were purchased from Sigma-Aldrich. Molecular weight markers for
5
Sephadex G-100 chromatography were obtained from Bio-Rad laboratories, Inc, California. USA.
6 7
2.2 Isolation of Alliinase-Producing Microorganism
8
A 1:10 diluted suspension of soil sample collected from the garlic field was plated onto a synthetic medium (2
9
% Na2 HPO4, 0.3 % KH2PO4, 0.1 % D-glucose, 0.05 % NaCl, 0.002 % CaCl2, 0.0003 % MgSO4· 7H2O, and 2 %
10
(w/v) agar, pH 6.2), in which alliin was added at the concentration of 0.1 % as the sole nitrogen source.
11 12
2.3 Identification of Alliinase Producing Microorganism
13
Alliinase producing strain was identified by 16S rRNA sequence in addition to morphological and biochemical
14
analysis, examined according to Bergey’s Manual of Systemic Bacteriology (Yabuuchi et al. 2005).
15
For 16 S rRNA sequencing the DNA was extracted using the method described by Cheng and Jiang (2006). The
16
16S rRNA gene was amplified by polymerase chain reaction (PCR), using prokaryotic universal primers Bact8F
17
(Forward
18
GACGGGCGGTGTGCA-3’. The PCR conditions were as follows: Initial denaturation at 94o C for 2 min, 30
19
cycles: 94oC for 1 min, 55oC for 45 sec, 72 o C for 2 min and a final extension at 72 oC for 20 min. The amplified
20
sequence was then sequenced using Applied Biosystems 3130 Genetic analyser. The acquired sequence was
21
used for a gene homology search, with the 16S rRNA sequences accessible in the public databases from BLAST
22
(http://www.ncbi.nlm.nih.gov/BLAST/, NCBI, Bethesda, MD, USA) (Altschul et al. 1997), and were identified
23
to the generic level by the CLUSTAL-X Multiple Sequence Alignment Program (Strasburg, France), the 16S
24
rRNA sequences of the isolated strain was aligned with sequences of related organisms obtained from Gen Bank
25
(Thompson et al. 1997). A phylogenetic tree was constructed via the neighbour-joining method using the Tree
26
View program (Saitou and Nei 1987).
primer):
5’-AGATTTGATCCTGGCTCAG-3’and
Bact
1391R
(Reverse
primer):
5’-
27 28 29
5
1 2
2.4 Optimization of growth conditions for alliinase production
3
Alliinase production by the isolate was optimized by varying glucose concentration (0.05-1 %), alliin
4
concentration (0.05-1 %), temperature (25 oC-55 oC), pH (3-11), incubation period (24-96 h) and incubation
5
condition (shaking and static).
6 7
2.5 Purification of Intracellular Alliinase
8
2.5.1 Preparation of cell-free extract
9
Cells from 1,000 ml of the medium were collected by centrifugation at 7,000 g for 5 min. 0.2 g of harvested
10
cells were suspended in 1 ml of lysis buffer and incubated at 4 oC for 10 min. The cell suspension was
11
centrifuged at 12,000 g for 20 min at 4 oC and the supernatant obtained after centrifugation was used as a crude
12
enzyme.
13
2.5.2 Ammonium sulphate precipitation
14
A typical batch was carried out using crude enzyme obtained from one litre of the medium. Solid ammonium
15
sulphate was added to the supernatant at 4 oC, to reach a saturation of 40 %. The mixture was kept overnight and
16
then centrifuged at 10,000 g for 20 min at 4 oC. It was dialysed overnight with 3-4 changes of buffer and was
17
further used for purification. Alliinase activity and protein content of the fraction were determined.
18
2.5.3 DEAE Cellulose Chromatography
19
The 40% fraction obtained from ammonium sulphate precipitation was passed through DEAE Cellulose Column
20
(2 X 6 cm) eluted using a linear discontinuous gradient of 0.05-0.5 M KCl in 0.1 M phosphate buffer (pH 7).
21
The active fractions were pooled and loaded onto the second column of DEAE Cellulose Column (2 X 6 cm)
22
and eluted using a linear continuous gradient of 0.1-0.3 M KCl in 0.1 M phosphate buffer, pH 7. The flow rate
23
was maintained at 20 ml/h. Each fraction was analysed for protein content and enzyme activity.
24
2.5.4 Sephadex G-100 Chromatography
25
The fractions exhibiting highest alliinase activity obtained from DEAE Cellulose Continuous gradient column
26
chromatography were pooled and loaded on Sephadex G-100 column (40.0 cm x 1.2 cm, bed volume 45 ml)
27
equilibrated with 0.1 M phosphate buffer (pH 7). Flow rate maintained was 20 ml/h. Fractions of 2 ml were
28
collected. The active fractions obtained were pooled and were used as the purified enzyme. The molecular
29
weight markers used included Blue dextran (2000 kDa), Thyroglobulin (669 kDa), Ferritin (440 kDa), Alcohol
30
dehydrogenase (150 kDa), Serum albumin (67 kDa) and Chymotrypsinogen (25 kDa).
6
1 2 3
2.6 SDS-PAGE analysis
4
Alliinase during the course of purification, was analysed using 10 % SDS-PAGE. Subunits of alliinase were
5
determined by electrophoresing it, in the presence and absence of β-ME (Gam and Latiff 2005). Puregene broad
6
range protein ladder was used as molecular weight standard. After separation, the proteins were detected using
7
silver staining.
8 9
2.7 Effect of temperature and pH on alliinase activity and stability
10
The alliinase activity was determined at different temperatures (0–50 °C) and different pH (3-11) under standard
11
assay conditions using alliin as a substrate to determine the optimum temperature and pH. The stability of
12
alliinase enzyme was determined by pre-incubating the purified enzyme at different temperatures (0–50 °C) and
13
pH (3-11) for 1 h and the alliinase activity was determined.
14 15
2.8 Effect of Metal Ions and Chemicals on Alliinase Activity
16
The effect of different metals on alliinase activity was assessed by adding each metal ion to the reaction mixture
17
and assayed for its activity. The final concentrations of the metal salts and Ethylenediaminetetraacetic acid
18
(EDTA) in the reaction mixture were maintained at 1mM, and Sodium dodecyl sulphate (SDS) was 0.1 %. The
19
metals used were chloride salts of Mn+2, Mg+2, Zn+2, Fe+2, Ca+2, Ag2+, Hg+2, K+ and Na+ except for Cu2+
20
(sulphate). The enzyme was incubated in the presence of various metal ions and chemicals, for 1 hr and relative
21
enzyme activity (%) was determined
22 23
2.9 Determination of Kinetic parameters
24
The Michaelis-Menten kinetic constants; the maximum reaction rate (Vmax) and Michaelis-Menten Constant
25
(Km) of the purified alliinase were determined using the different concentrations of alliin (0.5-5 mM). The
26
kinetic parameters were determined using Lineweaver-Burk plot. Km and Vmax were calculated using Graph Pad
27
PRISM software version 5.0
28 29
2.10 Alliinase assay
7
1
Alliinase was synthesized intracellularly by the isolate, hence cells were lysed using Lysis Buffer (25 mM Tris,
2
0.15 M NaCl, 0.01 M Phosphate Buffered Saline, 0.1 % Triton-X and 1 mM Phenyl methyl Sulphonyl Fluoride
3
(PMSF) for 10 mins at 4 oC and then after centrifugation the supernatant was assessed for alliinase activity. The
4
standard alliinase assay mixture contained 40 mM alliin, 20 mM PLP, 50 mM sodium phosphate (pH 7.0), and
5
100µl enzyme in a total volume of 1.0 ml. Alliinase activity was determined at 515 nm in UV-Visible Perkin
6
Elmer Lambda 25 spectrometer, by measuring the pyruvic acid produced in the reaction as described by Anthon
7
and Barrett (2003). One unit of the enzyme activity was defined as the enzyme amount that catalysed the
8
formation of 1 µmole of pyruvic acid per min.
9 10
2.11 Determination of Allicin, Ammonia, Pyruvate and Protein Content
11
Ammonia and pyruvate are by-products of the reaction catalysed by alliinase on alliin (Figure 1), hence it was
12
important to determine the concentration of allicin, ammonia and pyruvate generated, during the reaction.
13
Alliinase (10 Units) and 200 µM of alliin were incubated in PBS in the presence of 20 mM PLP at 37 oC, which
14
resulted in the generation of allicin, ammonia and pyruvate. Allicin, ammonia and pyruvate generated by 200
15
µM of alliin were determined by adopting 2-nitro-5-thiobenzoate (Miron et al. 2002), Nesslers reagent (Yuen
16
and Pollard 1954) and DNPH method (Anthon and Barrett 2003) respectively. Protein concentration was
17
determined according to the method of Lowry (1951), using bovine serum albumin fraction V as a standard.
18 19
2.12 Procurement and Maintenance of Cell Lines
20
Pancreatic carcinoma MIA PaCa-2 cell line was procured from National Centre for Cell Science (NCCS), Pune,
21
India and Human dermal Fibroblast HDF cell line was procured from V.G. Vaze College, Mumbai, India. Both
22
the cell lines were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, St.
23
Louis, MO, USA) with heat inactivated 10% fetal bovine serum (FBS) (Cell Clone, Genetix, India) and 1%
24
antibiotic solution of penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and amphotericin
25
(Himedia, Mumbai, India). Cells were maintained by passaging 1:3 at regular interval of 48 h using 0.5%
26
Trypsin–EDTA (Gibco, Paisley, UK) without phenol red. All reagents, media, buffers, chemicals and plastic-
27
ware used were of cell culture grade.
28 29
2.13 Cytotoxic activity of in-situ generated allicin
8
1
Cytotoxicity of in-situ generated allicin was determined by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-
2
Diphenyltetrazolium Bromide (MTT) assay on pancreatic cancer (MIA PaCa-2) cell line and human dermal
3
fibroblast (HDF) cell line. For each cell line, 1 x 105 cells were seeded in each well of 96 well plate and
4
incubated for 24 h. Growth medium was replaced with fresh medium containing 20 mM PLP. Alliinase (10
5
Units) was added to the cells along with increasing concentrations of alliin (20 µM -200 µM) for 24 h.
6
Cytotoxicity of reactants [alliin 200 µM, alliinase 10 U] and by-products of the reaction [ammonia (130 µM)
7
and pyruvate (149 µM)] were also assessed on both the cell lines. After 24 h incubation, 0.5 mg/ml of MTT
8
(Himedia, Mumbai, India) was added to each well and incubated for 4 h. The purple MTT formazan precipitate
9
was dissolved in 100 µl of DMSO. The absorbance was measured on a microplate reader (Bio-Rad, Hercules,
10
CA, USA). Data were represented as mean ± S.D. of three replicates from three independent experiments. The
11
values are represented as the percentage cell viability wherein viability of untreated cells was regarded as 100%.
12
Data was analyzed by one-way ANOVA followed by Dunnett’s post hoc test.
13
2.14 Acridine orange-ethidium bromide staining
14
MIA PaCa-2 cells were cultured at a density of 1 x 106 cells per well in 6 well flat-bottomed plate and incubated
15
for 24 h. Cells were then incubated with alliinase enzyme (10 U) in the presence of alliin (50 µM, 100 µM &
16
200 µM) for 24 h in the presence of 20mM PLP. Reactants [alliin (200 µM), alliinase (10 U)] and by-products
17
of the reaction [ammonia (130 µM) and pyruvate (149 µM)] were also assessed after 24 h incubation.
18
Thereafter, cells were stained with 100 µl of Acridine orange-ethidium bromide mixture (10 µg/ml) (MP
19
Biomedicals, USA) and cells were observed in an inverted fluorescent phase contrast microscope (Carl Zeiss,
20
Jena, Germany).
21 22
2.15 Statistical analysis
23
All experiments were performed in triplicates and the results were represented with standard deviation
24
calculated by Graph pad PRISM software version 5.
9
1
3.
Results
2
3.1 Isolation & identification of alliinase-producing microorganism
3
After 4-days of incubation at 37 °C, a colony formed was isolated as alliin-utilizing strain and was
4
independently inoculated onto the synthetic agar plate for evaluation of odorous compound production i.e.
5
allicin. Identification was carried out by morphological, biochemical and 16 S rRNA sequence analysis and the
6
strain was identified to be Cupriavidus necator. The 16 S rRNA sequence is available in the NCBI Gen Bank
7
repository, under the accession no. KM464555.
8 9
3.2 Optimization of Growth conditions for Alliinase production
10
Alliinase production was optimised by monitoring various nutritional and physiological parameters. Alliinase
11
production increased with an increase in the glucose concentration up to 0.2 % and then it remained stagnant up
12
to 0.4 % whereas a sharp decline was observed at 0.5 % and 1 % (Fig. 2a). Increase in alliin concentration
13
resulted in an increase in enzyme production up to 0.1 % whereas higher concentrations of alliin did not enhance
14
enzyme production by the organism (Fig. 2b).
15
Maximum alliinase production was observed at 37 oC (Fig. 2c) and at pH 7 (Fig. 2d). Organism when cultured
16
under shaking condition increased the enzyme production by 300 % as compared to when cultured under static
17
condition (Fig. 2e). Maximum enzyme production was observed at 48 h after its inoculation in the growth
18
medium after which it started to decline (Fig. 2f).
19 20
3.3 Purification of Alliinase and Molecular weight determination
21
The alliinase isolated from C.necator was purified using Ammonium sulphate precipitation, DEAE Cellulose
22
chromatography and Sephadex G-100 chromatography. The purification of alliinase enzyme, its SDS-PAGE
23
analysis and its elution profile using chromatography techniques during the course of enzyme purification is
24
summarized in Table 1, Fig. 3a and Fig. 4 respectively. The yield of alliinase enzyme was 1,660 U per gram of
25
the cell weight.
10
1
Alliinase in the presence of β-ME showed the presence of a single band at 55 kDa and in the absence of β- ME
2
showed the presence of a single band at 110 kDa (Fig. 3b); indicating alliinase from C.necator is a homodimer
3
consisting of two subunits of 55 kDa each.
4 5 6
3.4 Effect of temperature and pH on alliinase activity and stability
7
The enzyme was most active at 35 o C and it retained 80 % of its activity at 30 oC and 40 o C (Fig. 5a). Alliinase
8
exhibited maximum enzyme activity at pH 7 whereas more than 80 % activity was retained at pH 6 and pH 8
9
(Fig. 5b). Optimum temperature and pH for enzyme activity was found to be 35 oC and 7 respectively. The
10
alliinase enzyme from the isolate was stable at pH 7 and was more than 80 % stable over a pH range of 6–8
11
(Fig. 5b). Alliinase was more than 80% stable at temperatures lower than 40 oC, whereas a further increase in
12
temperature resulted in a sharp decrease in the enzyme activity (Fig 5a).
13 14
3.5 Effect of Metal Ions and chemicals on Alliinase activity
15
The enzyme activity was enhanced in the presence of 1 mM of Mn+2, Mg+2, Zn+2, Fe+2 and Ca2+. The highest
16
activity was seen with Zn+2 and Fe+2. Cu2+, Cs+, Li+, Ag2+, SDS and Hg+2 inhibited alliinase activity to 28%,
17
77%, 61%, 52%, 59%, and 27% respectively, whereas K+, Na+ and EDTA had no significant effect on alliinase
18
activity. (Fig. 6).
19 20
3.6 Determination of Kinetic parameters
21
Michaelis-Menten kinetic parameters, Km and Vmax of the purified alliinase were calculated from Lineweaver-
22
Burk double reciprocal plot (Fig. 7). The Km and Vmax values for the alliinase isolated from C.necator were
23
found to be 74.65 U/protein mg and 0.83mM respectively.
24 25
3.7 Ammonia, Pyruvate and Allicin determination
26
Alliin (200 µM) on reaction with alliinase (10 U) resulted in the generation of 89.43 (± 4.32) µM of allicin,
27
129.46 (± 7.28) µM of ammonia and 148.45 (± 8.98) µM of pyruvate.
28 29
3.8 Cytotoxicity of in-situ Generated Allicin
11
1
The cytotoxic effect of the in-situ generated allicin on MIA PaCa-2 and HDF cells was determined using MTT
2
assay for 24 h. A concentration-dependent decrease in cell viability was observed for both the cell lines. IC50 of
3
alliin was found to be 74.054 (± 4.545) µM for MIA PaCa-2 cells and 223.05 (± 16.83) µM for HDF cell line.
4
140 µM of alliin in the presence of alliinase resulted in an 87.58 % decrease in MIA PaCa-2 cell viability
5
(Figure 8) whereas the same concentration resulted in a 35.88 % decrease in HDF cell viability.
6
Treatment of both the cell lines with reactants (alliin, alliinase) and by-products (ammonia and pyruvate) did not
7
resulted in any statistically significant difference in the cell viability when compared to untreated control.
8 9
3.9 Assessment of cytotoxicity by Acridine orange-ethidium bromide staining
10
Acridine orange is a cell permeable dye and stains live cell green while ethidium bromide stains the cell that
11
have lost their membrane integrity as red. As shown in Figure 9 f-h, cells treated with alliinase + 50 µM, 100
12
µM and 200 µM alliin, exhibited orange-red colour signifying loss of membrane integrity, cell shrinkage,
13
nuclear fragmentation, membrane blebbing and condensed nuclei demonstrating cellular damage; whereas the
14
untreated, alliin, alliinase, ammonia and pyruvate treated groups did not show any evident morphological
15
changes (Figure 9 a-e).
12
1
4.
Discussion
2
Recent years have seen significant progress in the study of the biological activity and application of sulphur
3
containing compounds from garlic. Earlier, alliinases that have been purified to homogeneity and characterised
4
include those from garlic cloves (Rabinkov et al. 1994), Onion bulbs (Schwimmer and Mazelis 1963), leek
5
(Lohmuller et al. 1994), Chinese chive (Manabe et al. 1998), and wild garlic/ramson (Landshuter et al. 1994). In
6
this study, we have isolated alliinase producing bacterium from the soil, on the medium containing alliin as the
7
sole nitrogen source. The alliinase producing organism was identified to be C. necator and soil is one of its
8
natural habitats. C.necator is often used in bioremediation and is able to degrade a great number of chlorinated
9
aromatic (Chloroaromatic) compounds and chemically related pollutants (Schenzle et al. 1999). This is the first
10
report of the production of alliinase from C. necator. The synthesis of alliinase and its application for generation
11
of allicin on demand, is a plant associated activity for defense purpose and the acquisition of this activity by a
12
bacterium in the garlic grown soil is an important ecological development by plant-microorganism association
13
and permits the organism to use alliin as a source of nitrogen.
14
Alliinase production from C. necator was lowest at 1% w/v glucose. A higher concentration of glucose inhibited
15
alliinase production. Alliinase production from C. necator was highest during incubation under shaking
16
condition which indicated that the organism requires aeration and it showed maximum production at 37 oC
17
which was the average temperature of the area from where soil sample was collected. Alliinase isolated from C.
18
necator is a homodimeric 110 kDa enzyme, consisting of two subunits of 55 kDa each, which is similar to the
19
alliinase reported in bacterium Ensifer adhaerens, which is a 96 kDa homodimeric enzyme consisting of two
20
subunits of 48 kDa each (Yutani et al. 2011). Molecular weights of isolated alliinase vary depending on the
21
source of the enzyme, ranging from 67 kDa in Chinese chive (Manabe et al. 1998) to 580 kDa in leek
22
(Lohmuller et al. 1994). The number of enzyme subunits has been shown to differ depending on the source of
23
the alliinase, ranging from 1 for Chinese chive (Manabe et al. 1998) to 12 for leek (Lohmuller et al. 1994).
24
Musah et al. (2009) reported the presence of a heteropentameric alliinase from Petiveria alliacea.
13
1
PLP is a cofactor essential for the C-S lyase activity of alliinase, so far reported (Lohmuller et al. 1994).
2
Although each subunit of alliinase contains one tightly bound PLP, its exogenous addition enhances the enzyme
3
activity as the purification proceeds (Schwimmer and Mazelis 1963; Mazelis and Crews 1968). Most metal ions
4
had an effect on alliinase activity, amongst of which, enzyme activity was enhanced by Mn+2, Mg+2, Zn+2, Fe+2
5
and Ca2+, whereas Cu2+, Cs+, Li+, Ag2+, Hg2+ and SDS inhibited alliinase activity and K+, Na+ and EDTA had no
6
significant effect. Stimulation of alliinase activity with Mg2+ and Mn2+ is in accordance with the findings by
7
Kuettner et al. (2002) with other Allium species. The increase in activity of alliinase due to Mn +2, Mg+2, Zn+2,
8
Fe+2 and Ca2+ ions can be attributed to the vital role played by them in building active enzyme conformation.
9
Inhibition of alliinase activity with Cu2+ has also been reported for alliinase from garlic (Kuettner et al. 2002). In
10
comparison to the results of Mazelis and Crews (1968), EDTA did not enhance enzyme activity of alliinase
11
from C.necator, whereas EDTA appeared to enhance the activity of alliinase from garlic. The isolated alliinase
12
preparation from C. necator may not have the presence of heavy metal ions, which is one of the factors
13
responsible for EDTA activation of alliinase preparation of garlic.
14
The optimum reaction temperature and pH for alliinase from C.necator was found to be 35 °C and 7
15
respectively. These conditions are similar to the study reported by others, wherein the optimum temperature of
16
alliinase from garlic is 35 °C (Mazelis and Crews 1968). On the other hand, it is higher than that established by
17
Yutani et al. (2011) wherein the optimum reaction temperature for alliinase from Ensifer adhaerens is 30 °C. A
18
similar pattern was followed in pH, wherein alliinase from garlic was reported to be 6.5 (Mazelis and Crews
19
1968) and alliinase from Ensifer adhaerens was reported at pH 7 (Yutani et al. 2011). The alliinase enzymes
20
from A. sativum, A. odorum and A. chinense exhibited pH optima of pH 5.6-6.5 whereas alliinase from A.
21
tuberosum, A. cepa, A. porrum and A. fistulosum exhibited pH optima of pH 8.0 - 8.5 (Manabe et al. 1998). The
22
enzyme activity is stable over a narrow range of pH and temperature, a similar phenomenon is seen in most of
23
the alliinases reported so far. Extreme temperature and pH might be having an effect on the structure of
24
enzyme, rendering it inactive. This temperature and pH sensitivity of alliinase can be encountered during oral
25
administration, by a method reported by Lianga et al. (2013) wherein an enteric coated double-layer tablet of
26
alliin/alliinase was used, for optimum activity of alliinase on alliin under gastrointestinal condition.
27
The specific activity of alliinase isolated from C.necator was found to be 209 U/mg, which is the highest
28
reported specific activity from microbial origin till date, whereas specific activity of alliinase reported from
29
garlic varies from 218 to 660 U/mg depending on the source of garlic (Kuettner et al. 2002). Alliinase from
30
C.necator appears to exhibit the highest affinity towards alliin as compared to alliinases reported from different
14
1
sources. The Km of alliinase from C.necator was found to be 0.83 mM which is lower than the Km value of
2
alliinase reported from garlic (1.1 mM) (Tobkin and Mazelis 1979) and that from Ensifer adhaerens (2.3 mM)
3
(Yutani et al. 2011). A lower Km corresponds to the higher affinity of the enzyme towards the substrate. Purified
4
alliinase from Cupriavidus necator exhibited lowest Km reported so far, which indicates that the enzyme has the
5
highest affinity towards alliin, hence it is an ideal candidate for immobilisation on a suitable matrix and can be
6
exploited further for the generation of allicin
7
MTT assay is a well-recognized in vitro technique, practiced to investigate cytotoxicity of compounds against
8
cancer cell lines. It has been used as one of the conventional techniques for screening of compounds with
9
potential cytotoxic properties (Kondo et al. 2000). In-situ generated allicin induced concentration-dependent
10
decrease in cellular viability in MIA PaCa-2 cells and IC50 was determined to be 74.054 (± 4.545) µM of alliin
11
for MIA PaCa-2 cells and 223.05 (± 16.83) µM of alliin for HDF cell line which indicates that allicin is
12
selectively more cytotoxic to MIA PaCa-2 cells as compared to HDF cells Allicin-induced cytotoxicity was
13
accompanied by morphological changes, including nuclear condensation, membrane blebbing and cell shrinkage
14
which was confirmed using Acridine orange-ethidium bromide staining wherein a concentration dependent
15
change in cell morphology was observed.
16 17 Acknowledgement
18
This study was supported in part by Grant from Mumbai University Research Project (Grant no. 097). We are
19
grateful to Dr. Vibha Verma for her technical assistance and express our deep gratitude to Dr. Purvi Bhatt,
20
Assistant Professor, School of Science for her kind assistance in 16 S rRNA sequencing
21 22
Conflict of Interest
23
Authors declare no conflict of interest
24 25 26 27 28 29
15
1 2 3 4 5 6 7 8 9 10 11
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Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped
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BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25,
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killing of B-chronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-
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alliinase conjugate. Mol. Cancer Ther. 4, 325–331.
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Cheng, H.R., Jiang, N., 2006. Extremely rapid extraction of DNA from bacteria and yeasts. Biotech. Lett. 28, 55–59.
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Chhabria, S.V., Akbarsha, M.A., Li, A., Kharkar, P.S., Desai, K.B., 2015. In situ allicin generation using
21
targeted alliinase delivery for inhibition of MIA PaCa-2 cells via epigenetic changes, oxidative stress
22
and cyclin-dependent kinase inhibitor (CDKI) expression. Apoptosis. 20(10), 1388–1409.
23 24 25 26 27 28 29 30
Durbin, R.D., Uchytil, T.F., 1971. Purification and properties of alliin lyase from the fungus Penicillium corymbiferum. Biochim. Biophys. Acta. 235, 518–520. Fry, F.H., Okarter, N., Smith, C.B., Kershaw, M.J., Talbot, N.J., Jacob, C., 2005. Use of substrate/alliinase combination to generate antifungal activity in situ. J. Agri. Food Chem. 53, 574–580. Gam, L.H., Latiff, A., 2005. SDS-PAGE Electrophoretic property of Human Chorionic Gonadotropin (hCG) and its β-subunit. Int. J. Biol. Sci. 1, 103–109. Ilić, D.P., Nikolić, V.D., Nikolić, L.B., Stanković, M.Z., Stanojević, L.P., Cakić, M.D., 2011. Allicin and related compounds: biosynthesis, synthesis and pharmacological activity. Facta Universitatis 9, 9–20.
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Jones, M.G., Hughes, J., Tregova, A., Milne, J., Tomsett, A.B., Collin, H.A., 2004. Biosynthesis of the flavour precursors of onion and garlic. J. Exp. Bot. 55, 1903–1918. Ko, J.A., Lee, Y.L., Jeong, H.J., Park, H.J. 2012. Preparation of encapsulated alliinase in alginate microparticles. Biotech. Lett. 34, 515–518. Kondo, T., Yamauchi, M., Tominaga, S., 2000. Evaluation of usefulness of in-vitro drug sensitivity testing for adjuvant chemotherapy of stomach cancer. Int. J. Clin. Oncol. 5, 174–182. Kuettner, E.B., Hilgenfeld, R., Weiss, M.S., 2002. Purification, Characterization and Crystallization of Alliinase from Garlic. Arch. Biochem. Biophys. 402, 192–200. Landshuter, J., Lohmüller, E.M., Knobloch, K. 1994. Purification and Characterisation of a C-S Lyase from Ramson, the Wild Garlic, Allium ursinum. Planta Med. 60, 343–347.
11
Lianga, Y., Zhanga, J.J., Zhanga, Q., Wanga, Z.X., Yina, Z.N., Li, X.X., Chen, J., Ye, L.M. 2013. Release test
12
of alliin/alliinase double-layer tablet by HPLC-Allicin determination. J. Pharma Anal. 3, 187–192.
13
Lohmuller, E.M., Landshuter, J., Knobloch, K., 1994. On the Isolation and Characterisation of a C-S Lyase
14 15 16
Preparation from Leek, Allium porrum. Planta Med. 60, 337–342. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275.
17
Manabe, T., Hasumi, A., Sugiyama, M., Yamazaki, M., Saito, K. 1998. Alliinase [S-alk(en)yl-L-cysteine
18
sulfoxide lyase] from Allium tuberosum (Chinese chive) Purification, localization, cDNA cloning and
19
heterologous functional expression. Eur. J. Biochem. 257, 21–30.
20 21 22 23
Mazelis, M., Crews, L. 1968. Purification of the Alliin Lyase of Garlic, Allium sativum L. Biochem. J. 108, 725–730. Milka, P., Krest, I., Keusgen, M., 2000. Immobilisation of alliinase on porous aluminium oxide. Biotechnol. Bioeng. 69, 344–348.
24
Miron, T., Mironchik, M., Mirelman, D., Wilchek, M., Rabiknov, A., 2003. Inhibition of tumor growth by a
25
novel approach: in situ allicin generation using targeted alliinase delivery. Molecular Cancer
26
Therapeutics 2(12), 1295–1302.
27
Miron, T., Shin, I., Feigenblat, G., Weiner, L., Mirelman, D., Wilchek, M., Rabinkov,
A., 2002. A
28
spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction
29
of 4-mercaptopyridine with thiosulfinates. Anal. Biochem. 307, 76–83.
17
1 2
Musah, R.A., He, Q., Kubec, R., Jadhav, A., 2009. Studies of a novel cysteine Sulfoxide Lyase from Petiveria Alliacea: The first Heteropentameric Alliinase. Plant Physiology 151, 1304-1316.
3
Nock, L.P., Mazelis, M., 1987. The C-S Lyases of higher plants. Arch. Biochem. Biophys. 249, 27–33.
4
Oommen, S., Anto, R.J., Srinivas, G., Karunagaran, D., 2004. Allicin (from garlic) induces caspase-mediated
5 6 7 8 9
apoptosis in cancer cells. Eur. J. Pharmacol. 485, 97–103 Rabinkov, A., Zhu, X., Grafi, G., Galili, G., Mirelman, D., 1994. Alliin lyase (Alliinase) from garlic (Allium sativum), Biochemical characterization and cDNA cloning. Appl. Biochem. Biotechnol. 48, 149–171. Saitou, N., Nei, M., 1987. The Neighbour-joining Method: A New Method for Reconstructing Phylogenetic Trees. Mol. Bio. Evol. 4, 406-425.
10
Schenzle, A., Lenke, H., Spain, J., Knackmuss, H., 1999. Chemoselective nitro group reduction and reductive
11
dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134. Appl.
12
Enviro. Micro. 65, 2317–2323.
13 14 15 16
Schwimmer, S., Mazelis, M., 1963. Characterisation of alliinase of Allium cepa (onion). Arch. Biochem. Biophys. 100, 66–73. Slusarenko, A.J., Patel, A., Portz, D., 2008. Control of plant diseases by natural products: Allicin from garlic as a case study. Eur. J. Plant Pathol. 121, 313–322.
17
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X
18
windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
19
Nuc. Acids Res. 25, 4876-4882.
20 21 22 23 24 25 26 27
Tobkin, H., Mazelis, M., 1979. Alliin lyase: Preparation and characterisation of the homogeneous enzyme from garlic. Arch. Biochem. Biophys. 193, 150–157. Yabuuchi, E., Kawamura, Y., and Ezaki, T., 2005. in Bergey’s Manual of Systemic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore (MD), pp. 609-618. Yamazaki, M., Sugiyama, M., Siato, K., 2002. Intracellular Localisation of Cysteine Synthase and Alliinase in Bundle sheaths of Allium Plants. Plant Biotech. 19, 7–10. Yuen, S.H., Pollard, G.A., 1954. Determination of Nitrogen In Agricultural Materials by the Nessler Reagent. II Micro-deteremintaions in Plant Tissue and in Soil Extracts. J. Sci. Food. Agric. 5, 364–369.
28
Yutani, M., Taniguchi, H., Borjihan, H., Ogita, A., Fujita, K., Tanaka, T., 2011. Alliinase from Ensifer
29
adhaerens and Its Use for Generation of Fungicidal Activity. A.M.B. Express doi: 10.1186/2191-0855-
30
1-2.
18
1
Zhou, J.Q., Wang, J.W., 2009. Immobilisation of alliinase with a water soluble-insoluble reversible N-
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
succinyl-chitosan for allicin production. Enzyme and Microbial Tech. 45, 299–304.
Tables
Table 1: Summary of data on the course of purification of intracellular alliinase from C.necator
Purification step
Protein
Enzyme
Specific
activity
activity
(mg)
(Units/mg)
Yield (%)
Purification (Fold)
(units)
Crude extract
8907
17963
2.0167
100
1
Ammonium sulphate
1124
14954
13.3042
83.24
5.51
124
8490
68.467
47.28
33.2056
59
7861
133.237
43.762
79
24
5016
209
27.92
103.63
precipitation DEAE Cellulose Discontinuous Chromatography DEAE Cellulose Continuous Chromatography Sephadex G-100 Chromatography
20 21 22 23 24 25 19
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Figure legends Figure 1 Reaction of Alliinase on Alliin
15
Figure 2 Effect of various growth parameters on alliinase production from C.necator
16
(a) Effect of varying concentrations of glucose on alliinase production
17
(b) Effect of varying concentrations of alliin on alliinase production
18
(c) Effect of temperature on alliinase production
19
(d) Effect of pH on alliinase production
20
(e) Effect of incubation condition on alliinase production
21
(f) Effect of incubation time on alliinase production
22
Figure 3 SDS-PAGE Analysis
23
(a) The proteins at each step of the purification procedure were separated by 10% SDS-PAGE under non-
24
reducing conditions (in the absence of β-ME), followed by Silver staining. Lane 1- crude enzyme; Lane
25
2-Ammonium sulphate precipitation pooled fractions; Lane 3-DEAE Dis. Chromatography pooled
26
fractions; Lane 4 - DEAE Continuous Chromatography pooled fractions; Lane 5-Sephadex G 100
27
chromatography pooled fractions; Lane 6, Puregene protein molecular weight marker
28
(b) 10 % SDS- PAGE analysis for molecular weight determination of isolated alliinase. Lane 1 - Puregene
29
protein molecular weight marker; Lane 2 - Alliinase in absence of β ME; Lane 3 -Alliinase in presence
30
of β ME
31
Figure 4 Elution profile of alliinase from C.necator
32
(a) Elution profile of alliinase in DEAE Cellulose Discontinuous gradient chromatography
33
(b) Elution profile of alliinase in DEAE Cellulose Continuous chromatography
34
(c) Elution profile of alliinase in Sephadex G-100 Chromatography
20
1
Figure 5 (a) Effects of temperature on alliinase activity and stability (b) Effect of pH on alliinase activity and
2
stability. Relative activities were calculated by using the highest activity of free and immobilized alliinase as
3
100%, respectively.
4
Figure 6 Alliinase was incubated with different metals and chemicals under standard assay conditions. The
5
100% enzyme activity was obtained in the absence of metal ions or chemicals.
6
Figure 7 Lineweaver-Burk double reciprocal plot for Kinetic analysis of alliinase extracted from C.necator
7
using different concentrations of alliin.
8
Figure 8 Cytotoxic effect of in-situ generated allicin on MIA PaCa-2 cells. Cells were treated with increasing
9
concentrations of alliin in the presence of alliinase extracted from (C.necator) for 24 h. Data are represented as
10
mean ± SD of three replicates from three independent experiments. The values are represented as the percentage
11
cell viability where the viability of untreated cells was regarded as 100%. Data were analyzed by one-way
12
ANOVA followed by Dunnett’s post hoc test. Asterisk above bars indicate statistically significant difference
13
compared to untreated control (*** = p < 0.001, ** = p < 0.01, * = p < 0.05).
14
Figure 9 Morphological study of MiaPaCa-2 cells stained with Acridine orange ethidium bromide after 24 h
15
incubation with (a) untreated control, (b) alliin 200 µM, (c) alliinase (d) ammonia, (e) pyruvate, (f) alliinase +
16
alliin 50 µM, (g) alliinase + alliin 100 µM (h) alliinase + alliin 200 µM.
17
21
Figures Figure 1
Figure 2
21 22 23
1600
24
1400
25 26
1200
27
1600 1500 1400 1300
1500
1000
500 20
Concentration of Alliin (%)
30
40
50
b
c Enzyme activity (Units)
2000
Enzyme activity (Units)
36 37 38 39
1500 1000 500 0 0
1
2
3
4
5
6
pH
7
8
40
9 10 11 12
E n z ym e a ctiv it y (U n its)
2000 2000
1500 1000 500
1600 1400 1200 1000
0 0
Shaking
Incubation condition
f
1800
e
Stagnant
0
24
48
72
96
Time (hours)
f
48 49 50 51 52 53 54 55
60
Temperature (oC)
a 35
41 42 43 44 45 46 47
1700
1200 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
28 29 Concentration of Glucose (%)
1000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
30 31 32 33 34
2000
1800
Enzyme activity (Units)
Enzyme activity (Units)
201800
Enzyme activity (Units)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 3
22
a
b
Figure 4
1500 10 1000 5 500 0 0 40 41 42 43 44 45 46 47 48 49 50 51 52
Protein concentration
30
2000
20
1000
10
0
0 10
150
6000 100 4000 50
2000 0 0.0
0.1
0.2
0.3
0.4
0.5
0 0.6
Alliinase activity
Protein concentration
b
3000
5
8000
15
20
Protein Concentration (mg)
Alliinase activity (Units)
a
0
200
KCL (Molar)
Fractions Alliinase activity
10000
Alliinase Activity (Units)
Alliinase activity (U nits)
15
25
Fractions Allinase activity
Protein concentration
c
23
Protein Concentration (mg)
2000
Protein C oncentr ation (m g)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 5
120
120
Relative activity (%)
Relative activity (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
100 80 60 40 20 0 0
100 80 60 40 20
5 10 15 20 25 30 35 40 45 50 55 60
0 0
Temperature ( oC) Alliinase stability
1
2
3
4
5
6
7
8
9 10 11 12
pH
Alliinase activity
Alliinase activity
a
Alliinase stability
b
Figure 6
Relative Enzyme activity (%)
140 120 100 80 60 40 20 0 0 None Cu
Cs
Fe
Li
Mg
K
Ag
Na
Mn
Zn
Hg SDS EDTA
Metal ions (1mM)
Figure 7
0.04 0.03
1/V
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Ca
-1 /K m = - 1 .2 0 4
0.02 0.01
1 /V m a x = 0 .1 3 3 9
43 44 45
-2.0 -1.6 -1.2 -0.8 -0.4 -0.0 0.4 0.8 1.2 1.6 2.0 2.4
- 1/K m
1 /S
24
re
at e Al d C liin o Al (2 ntr o liin 0 0 l as µ e M) Al ( liin Am 10 ( Al 20 m U) liin µ P o n Al (40 M) yru ia liin µ + va A Al (60 M) lliin te liin µ + a A l ( 8 M A ll s e liin 0 ) + iin a A l ( 1 0 µ M A ll s e liin 0 ) + iin a A l ( 1 2 µ M A lli s e liin 0 ) + in a A l ( 1 4 µ M A ll s e liin 0 ) + iin a A l ( 1 6 µ M A lli s e liin 0 ) + in a A l ( 1 8 µ M A lli s e liin 0 ) + in (2 µ M A l a s e 0 0 ) liin µ M + A as ) + lliin e Al as e liin as e
U nt
Cell viability (%)
1
2
3 Figure 8
4
120
80
60
HDF cells MIA PaCa 2 cells
100
** *** * **
40
20
*** ***
*** *** *** *** *** ***
*** *** *** *** *** ***
0
Groups
5 6
25
Figure 9
a
d
b
c
e
g 100 µg/mL As-Chitosan NPs
f
h
26
S1319-562X(16)00005-X http://dx.doi.org/10.1016/j.sjbs.2016.01.003 SJBS 620
To appear in:
Saudi Journal of Biological Sciences
Received Date: Revised Date: Accepted Date:
12 September 2015 1 December 2015 2 January 2016
Please cite this article as: S. Chhabria, K. Desai, Purification and Characterization of Alliinase produced by Cupriavidus necator and its Application for Generation of Cytotoxic Agent: Allicin, Saudi Journal of Biological Sciences (2016), doi: http://dx.doi.org/10.1016/j.sjbs.2016.01.003
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1
Purification and Characterization of Alliinase produced by Cupriavidus necator and its Application for
2
Generation of Cytotoxic Agent: Allicin
3
Sagar Chhabria1, Krutika Desai2*
4 5
1
6
(W), Mumbai 400056, India.
7
2
8
College of Commerce & Economics, Vile Parle (W), Mumbai 400056, India
Dept. of Biological Sciences, Sunandan Divatia School of Science, SVKM’s NMIMS University, Vile Parle
Dept. Of Microbiology, SVKM’s Mithibai College of Arts, Chauhan Institute of Science &Amrutben Jivanlal
9 10
*Address all correspondence to:
11 12 13 14 15 16 17 18 19
Krutika B. Desai, Ph.D. Dept. of Microbiology, SVKM’s Mithibai College of Arts, Chauhan Institute of Science & Amrutben Jivanlal College of Commerce & Economics, Vile Parle (W), Mumbai 400056, India Tel: +91-22-42339000 Fax: +91-22-26130441 E-mail: [email protected]
20 21 22 23 24 25 26 27 28
29 30 1
1
Abstract
2
Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the
3
enzyme alliinase (EC 4.4.1.4). A bacterium Cupriavidus necator with the ability of alliinase production was
4
isolated from a soil sample and was identified by morphological, biochemical and 16 S rRNA sequence.
5
Alliinase production was optimized and further it was purified to apparent homogeneity with 103 fold
6
purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100
7
chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular
8
weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum
9
temperature was 35 oC. The enzyme exhibited maximum reaction rate (Vmax) at 74.65 U/mg and Michaelis-
10
Menten constant (Km) was determined to be 0.83 mM when alliin was used as a substrate. The cytotoxic activity
11
of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT
12
assay and Acridine orange-ethidium bromide staining. This approach of in-situ allicin generation suggests a
13
novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent
14
allicin.
15 16
Keywords: Alliinase; Characterisation, Cupriavidus necator; Purification, MIA Pa Ca-2 cells
17 18
Abbreviations: Diethylaminoethyl (DEAE), β-mercaptoethanol (β-ME), Pyridoxal 5’ Phosphate (PLP)
2
1
1.
Introduction
2
Allicin (Diallyl thiosulphate) is the best-known active compound of freshly crushed garlic extract and is known
3
to possess a wide range of pharmacological activities which include antimicrobial, anti-inflammatory,
4
antithrombotic, antifungal, antiviral, antioxidant, anticancer, and antiatherosclerotic (Ilić et al. 2011; Oommen et
5
al. 2004). This allyl-sulphur compound is synthesized as a result of C-S lyase activity of alliinase (Alliin Lyase;
6
EC 4.4.1.4) on alliin (S-allyl-L-cysteine sulphoxide), a naturally-occurring diastereomer molecule (Jones et al.
7
2004) (Fig. 1). Alliinase catalyses the release of volatile sulphur compounds which is responsible for the
8
characteristic flavour and odour of Allium species.
9
Alliinase has been purified from garlic, onion, and other plants of the genus Allium (Schwimmer and Mazelis
10
1963; Mazelis and Crews 1968; Tobkin and Mazelis 1979; Nock and Mazelis 1987; Landshuter et al. 1994;
11
Lohmuller et al. 1994; Rabinkov et al. 1994; Manabe et al. 1998). The enzymatic production of allicin occurs in
12
garlic, because of the injury of the plant tissue that enables interaction of the enzyme in vacuoles with alliin
13
gathered in the cytosol (Yamazaki et al. 2002); hence allicin production has been discussed as a defense
14
mechanism of the plant against microbial infection or insect attack (Slusarenko et al. 2008). Allicin generated
15
from alliinase which was purified from garlic, has been studied for fungicidal activity against Magnaporthe
16
grisea (Fry et al. 2005).
17
In the past, some microorganisms have also been studied for their alliinase producing ability, viz. Durbin and
18
Uchytil (1971) reported the presence of enzyme in Penicillium corymbiferum and Yutani et al. (2011) reported
19
the presence of an intracellular alliinase in Ensifer adhaerens; further allicin generated from this enzyme
20
exhibited potent fungicidal activity against Saccharomyces cerevisiae.
21
In the last few years, alliinase had found its application in agriculture, pharmaceutical and food industry.
22
Alliinase has been immobilised on N-succinyl chitosan polymer (Zhou and Wang 2009), calcium alginate beads
23
(Anifantaki et al. 2010), Monoclonal antibody (Chhabria et al. 2015), porous aluminium oxide (Milka et al.
24
2000) and alginate microparticles (Ko et al. 2012), further used to catalyse the conversion of alliin to allicin, an
25
active ingredient of pharmaceutical compositions and food additive. The immobilized alliinase in alginate beads
26
can be further used for the development of a biosensor for monitoring cysteine sulphoxides which could prove
27
valuable in several applications in the food industry.
28
Chemical instability and low miscibility of allicin in water obstruct its practical use; hence a binary system
29
consisting of purified alliinase and alliin, in which allicin was generated in-situ, had been demonstrated to be a
3
1
promising potential anticancer approach for inhibition of the gastric carcinoma cell line in vitro and in vivo by
2
Miron et al (2003). Arditti et al (2005) reported the application of purified alliinase, for in-situ generation of
3
allicin which further inhibited the growth of B-chronic lymphocytic leukemia tumour in vivo.
4
Currently, much attention is paid to the development of microbial enzyme technology, as these enzymes are
5
relatively more stable than the enzymes derived from plants. In addition, the microorganisms represent an
6
attractive source of enzymes because they are economically feasible, can be cultured in large quantities in a
7
short time by fermentation and enzyme yield is predictable and controllable.
8
In the present study, we report for the first time, purification and characterization of an intracellular alliinase
9
enzyme from Cupriavidus necator. The isolated enzyme was further used for on-site generation of cytotoxic
10
agent-allicin; which was assessed on pancreatic cancer (MIA PaCa-2) cell line using MTT assay and Acridine
11
orange-ethidium bromide staining.
4
1
2.
Material and methods
2
2.1 Chemicals and Reagents
3
Alliin & Allicin were purchased from LKT Laboratories, Inc (St. Paul, MN). DEAE Cellulose, Sephadex G-100
4
and Pyridoxal 5’ Phosphate (PLP) were purchased from Sigma-Aldrich. Molecular weight markers for
5
Sephadex G-100 chromatography were obtained from Bio-Rad laboratories, Inc, California. USA.
6 7
2.2 Isolation of Alliinase-Producing Microorganism
8
A 1:10 diluted suspension of soil sample collected from the garlic field was plated onto a synthetic medium (2
9
% Na2 HPO4, 0.3 % KH2PO4, 0.1 % D-glucose, 0.05 % NaCl, 0.002 % CaCl2, 0.0003 % MgSO4· 7H2O, and 2 %
10
(w/v) agar, pH 6.2), in which alliin was added at the concentration of 0.1 % as the sole nitrogen source.
11 12
2.3 Identification of Alliinase Producing Microorganism
13
Alliinase producing strain was identified by 16S rRNA sequence in addition to morphological and biochemical
14
analysis, examined according to Bergey’s Manual of Systemic Bacteriology (Yabuuchi et al. 2005).
15
For 16 S rRNA sequencing the DNA was extracted using the method described by Cheng and Jiang (2006). The
16
16S rRNA gene was amplified by polymerase chain reaction (PCR), using prokaryotic universal primers Bact8F
17
(Forward
18
GACGGGCGGTGTGCA-3’. The PCR conditions were as follows: Initial denaturation at 94o C for 2 min, 30
19
cycles: 94oC for 1 min, 55oC for 45 sec, 72 o C for 2 min and a final extension at 72 oC for 20 min. The amplified
20
sequence was then sequenced using Applied Biosystems 3130 Genetic analyser. The acquired sequence was
21
used for a gene homology search, with the 16S rRNA sequences accessible in the public databases from BLAST
22
(http://www.ncbi.nlm.nih.gov/BLAST/, NCBI, Bethesda, MD, USA) (Altschul et al. 1997), and were identified
23
to the generic level by the CLUSTAL-X Multiple Sequence Alignment Program (Strasburg, France), the 16S
24
rRNA sequences of the isolated strain was aligned with sequences of related organisms obtained from Gen Bank
25
(Thompson et al. 1997). A phylogenetic tree was constructed via the neighbour-joining method using the Tree
26
View program (Saitou and Nei 1987).
primer):
5’-AGATTTGATCCTGGCTCAG-3’and
Bact
1391R
(Reverse
primer):
5’-
27 28 29
5
1 2
2.4 Optimization of growth conditions for alliinase production
3
Alliinase production by the isolate was optimized by varying glucose concentration (0.05-1 %), alliin
4
concentration (0.05-1 %), temperature (25 oC-55 oC), pH (3-11), incubation period (24-96 h) and incubation
5
condition (shaking and static).
6 7
2.5 Purification of Intracellular Alliinase
8
2.5.1 Preparation of cell-free extract
9
Cells from 1,000 ml of the medium were collected by centrifugation at 7,000 g for 5 min. 0.2 g of harvested
10
cells were suspended in 1 ml of lysis buffer and incubated at 4 oC for 10 min. The cell suspension was
11
centrifuged at 12,000 g for 20 min at 4 oC and the supernatant obtained after centrifugation was used as a crude
12
enzyme.
13
2.5.2 Ammonium sulphate precipitation
14
A typical batch was carried out using crude enzyme obtained from one litre of the medium. Solid ammonium
15
sulphate was added to the supernatant at 4 oC, to reach a saturation of 40 %. The mixture was kept overnight and
16
then centrifuged at 10,000 g for 20 min at 4 oC. It was dialysed overnight with 3-4 changes of buffer and was
17
further used for purification. Alliinase activity and protein content of the fraction were determined.
18
2.5.3 DEAE Cellulose Chromatography
19
The 40% fraction obtained from ammonium sulphate precipitation was passed through DEAE Cellulose Column
20
(2 X 6 cm) eluted using a linear discontinuous gradient of 0.05-0.5 M KCl in 0.1 M phosphate buffer (pH 7).
21
The active fractions were pooled and loaded onto the second column of DEAE Cellulose Column (2 X 6 cm)
22
and eluted using a linear continuous gradient of 0.1-0.3 M KCl in 0.1 M phosphate buffer, pH 7. The flow rate
23
was maintained at 20 ml/h. Each fraction was analysed for protein content and enzyme activity.
24
2.5.4 Sephadex G-100 Chromatography
25
The fractions exhibiting highest alliinase activity obtained from DEAE Cellulose Continuous gradient column
26
chromatography were pooled and loaded on Sephadex G-100 column (40.0 cm x 1.2 cm, bed volume 45 ml)
27
equilibrated with 0.1 M phosphate buffer (pH 7). Flow rate maintained was 20 ml/h. Fractions of 2 ml were
28
collected. The active fractions obtained were pooled and were used as the purified enzyme. The molecular
29
weight markers used included Blue dextran (2000 kDa), Thyroglobulin (669 kDa), Ferritin (440 kDa), Alcohol
30
dehydrogenase (150 kDa), Serum albumin (67 kDa) and Chymotrypsinogen (25 kDa).
6
1 2 3
2.6 SDS-PAGE analysis
4
Alliinase during the course of purification, was analysed using 10 % SDS-PAGE. Subunits of alliinase were
5
determined by electrophoresing it, in the presence and absence of β-ME (Gam and Latiff 2005). Puregene broad
6
range protein ladder was used as molecular weight standard. After separation, the proteins were detected using
7
silver staining.
8 9
2.7 Effect of temperature and pH on alliinase activity and stability
10
The alliinase activity was determined at different temperatures (0–50 °C) and different pH (3-11) under standard
11
assay conditions using alliin as a substrate to determine the optimum temperature and pH. The stability of
12
alliinase enzyme was determined by pre-incubating the purified enzyme at different temperatures (0–50 °C) and
13
pH (3-11) for 1 h and the alliinase activity was determined.
14 15
2.8 Effect of Metal Ions and Chemicals on Alliinase Activity
16
The effect of different metals on alliinase activity was assessed by adding each metal ion to the reaction mixture
17
and assayed for its activity. The final concentrations of the metal salts and Ethylenediaminetetraacetic acid
18
(EDTA) in the reaction mixture were maintained at 1mM, and Sodium dodecyl sulphate (SDS) was 0.1 %. The
19
metals used were chloride salts of Mn+2, Mg+2, Zn+2, Fe+2, Ca+2, Ag2+, Hg+2, K+ and Na+ except for Cu2+
20
(sulphate). The enzyme was incubated in the presence of various metal ions and chemicals, for 1 hr and relative
21
enzyme activity (%) was determined
22 23
2.9 Determination of Kinetic parameters
24
The Michaelis-Menten kinetic constants; the maximum reaction rate (Vmax) and Michaelis-Menten Constant
25
(Km) of the purified alliinase were determined using the different concentrations of alliin (0.5-5 mM). The
26
kinetic parameters were determined using Lineweaver-Burk plot. Km and Vmax were calculated using Graph Pad
27
PRISM software version 5.0
28 29
2.10 Alliinase assay
7
1
Alliinase was synthesized intracellularly by the isolate, hence cells were lysed using Lysis Buffer (25 mM Tris,
2
0.15 M NaCl, 0.01 M Phosphate Buffered Saline, 0.1 % Triton-X and 1 mM Phenyl methyl Sulphonyl Fluoride
3
(PMSF) for 10 mins at 4 oC and then after centrifugation the supernatant was assessed for alliinase activity. The
4
standard alliinase assay mixture contained 40 mM alliin, 20 mM PLP, 50 mM sodium phosphate (pH 7.0), and
5
100µl enzyme in a total volume of 1.0 ml. Alliinase activity was determined at 515 nm in UV-Visible Perkin
6
Elmer Lambda 25 spectrometer, by measuring the pyruvic acid produced in the reaction as described by Anthon
7
and Barrett (2003). One unit of the enzyme activity was defined as the enzyme amount that catalysed the
8
formation of 1 µmole of pyruvic acid per min.
9 10
2.11 Determination of Allicin, Ammonia, Pyruvate and Protein Content
11
Ammonia and pyruvate are by-products of the reaction catalysed by alliinase on alliin (Figure 1), hence it was
12
important to determine the concentration of allicin, ammonia and pyruvate generated, during the reaction.
13
Alliinase (10 Units) and 200 µM of alliin were incubated in PBS in the presence of 20 mM PLP at 37 oC, which
14
resulted in the generation of allicin, ammonia and pyruvate. Allicin, ammonia and pyruvate generated by 200
15
µM of alliin were determined by adopting 2-nitro-5-thiobenzoate (Miron et al. 2002), Nesslers reagent (Yuen
16
and Pollard 1954) and DNPH method (Anthon and Barrett 2003) respectively. Protein concentration was
17
determined according to the method of Lowry (1951), using bovine serum albumin fraction V as a standard.
18 19
2.12 Procurement and Maintenance of Cell Lines
20
Pancreatic carcinoma MIA PaCa-2 cell line was procured from National Centre for Cell Science (NCCS), Pune,
21
India and Human dermal Fibroblast HDF cell line was procured from V.G. Vaze College, Mumbai, India. Both
22
the cell lines were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, St.
23
Louis, MO, USA) with heat inactivated 10% fetal bovine serum (FBS) (Cell Clone, Genetix, India) and 1%
24
antibiotic solution of penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and amphotericin
25
(Himedia, Mumbai, India). Cells were maintained by passaging 1:3 at regular interval of 48 h using 0.5%
26
Trypsin–EDTA (Gibco, Paisley, UK) without phenol red. All reagents, media, buffers, chemicals and plastic-
27
ware used were of cell culture grade.
28 29
2.13 Cytotoxic activity of in-situ generated allicin
8
1
Cytotoxicity of in-situ generated allicin was determined by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-
2
Diphenyltetrazolium Bromide (MTT) assay on pancreatic cancer (MIA PaCa-2) cell line and human dermal
3
fibroblast (HDF) cell line. For each cell line, 1 x 105 cells were seeded in each well of 96 well plate and
4
incubated for 24 h. Growth medium was replaced with fresh medium containing 20 mM PLP. Alliinase (10
5
Units) was added to the cells along with increasing concentrations of alliin (20 µM -200 µM) for 24 h.
6
Cytotoxicity of reactants [alliin 200 µM, alliinase 10 U] and by-products of the reaction [ammonia (130 µM)
7
and pyruvate (149 µM)] were also assessed on both the cell lines. After 24 h incubation, 0.5 mg/ml of MTT
8
(Himedia, Mumbai, India) was added to each well and incubated for 4 h. The purple MTT formazan precipitate
9
was dissolved in 100 µl of DMSO. The absorbance was measured on a microplate reader (Bio-Rad, Hercules,
10
CA, USA). Data were represented as mean ± S.D. of three replicates from three independent experiments. The
11
values are represented as the percentage cell viability wherein viability of untreated cells was regarded as 100%.
12
Data was analyzed by one-way ANOVA followed by Dunnett’s post hoc test.
13
2.14 Acridine orange-ethidium bromide staining
14
MIA PaCa-2 cells were cultured at a density of 1 x 106 cells per well in 6 well flat-bottomed plate and incubated
15
for 24 h. Cells were then incubated with alliinase enzyme (10 U) in the presence of alliin (50 µM, 100 µM &
16
200 µM) for 24 h in the presence of 20mM PLP. Reactants [alliin (200 µM), alliinase (10 U)] and by-products
17
of the reaction [ammonia (130 µM) and pyruvate (149 µM)] were also assessed after 24 h incubation.
18
Thereafter, cells were stained with 100 µl of Acridine orange-ethidium bromide mixture (10 µg/ml) (MP
19
Biomedicals, USA) and cells were observed in an inverted fluorescent phase contrast microscope (Carl Zeiss,
20
Jena, Germany).
21 22
2.15 Statistical analysis
23
All experiments were performed in triplicates and the results were represented with standard deviation
24
calculated by Graph pad PRISM software version 5.
9
1
3.
Results
2
3.1 Isolation & identification of alliinase-producing microorganism
3
After 4-days of incubation at 37 °C, a colony formed was isolated as alliin-utilizing strain and was
4
independently inoculated onto the synthetic agar plate for evaluation of odorous compound production i.e.
5
allicin. Identification was carried out by morphological, biochemical and 16 S rRNA sequence analysis and the
6
strain was identified to be Cupriavidus necator. The 16 S rRNA sequence is available in the NCBI Gen Bank
7
repository, under the accession no. KM464555.
8 9
3.2 Optimization of Growth conditions for Alliinase production
10
Alliinase production was optimised by monitoring various nutritional and physiological parameters. Alliinase
11
production increased with an increase in the glucose concentration up to 0.2 % and then it remained stagnant up
12
to 0.4 % whereas a sharp decline was observed at 0.5 % and 1 % (Fig. 2a). Increase in alliin concentration
13
resulted in an increase in enzyme production up to 0.1 % whereas higher concentrations of alliin did not enhance
14
enzyme production by the organism (Fig. 2b).
15
Maximum alliinase production was observed at 37 oC (Fig. 2c) and at pH 7 (Fig. 2d). Organism when cultured
16
under shaking condition increased the enzyme production by 300 % as compared to when cultured under static
17
condition (Fig. 2e). Maximum enzyme production was observed at 48 h after its inoculation in the growth
18
medium after which it started to decline (Fig. 2f).
19 20
3.3 Purification of Alliinase and Molecular weight determination
21
The alliinase isolated from C.necator was purified using Ammonium sulphate precipitation, DEAE Cellulose
22
chromatography and Sephadex G-100 chromatography. The purification of alliinase enzyme, its SDS-PAGE
23
analysis and its elution profile using chromatography techniques during the course of enzyme purification is
24
summarized in Table 1, Fig. 3a and Fig. 4 respectively. The yield of alliinase enzyme was 1,660 U per gram of
25
the cell weight.
10
1
Alliinase in the presence of β-ME showed the presence of a single band at 55 kDa and in the absence of β- ME
2
showed the presence of a single band at 110 kDa (Fig. 3b); indicating alliinase from C.necator is a homodimer
3
consisting of two subunits of 55 kDa each.
4 5 6
3.4 Effect of temperature and pH on alliinase activity and stability
7
The enzyme was most active at 35 o C and it retained 80 % of its activity at 30 oC and 40 o C (Fig. 5a). Alliinase
8
exhibited maximum enzyme activity at pH 7 whereas more than 80 % activity was retained at pH 6 and pH 8
9
(Fig. 5b). Optimum temperature and pH for enzyme activity was found to be 35 oC and 7 respectively. The
10
alliinase enzyme from the isolate was stable at pH 7 and was more than 80 % stable over a pH range of 6–8
11
(Fig. 5b). Alliinase was more than 80% stable at temperatures lower than 40 oC, whereas a further increase in
12
temperature resulted in a sharp decrease in the enzyme activity (Fig 5a).
13 14
3.5 Effect of Metal Ions and chemicals on Alliinase activity
15
The enzyme activity was enhanced in the presence of 1 mM of Mn+2, Mg+2, Zn+2, Fe+2 and Ca2+. The highest
16
activity was seen with Zn+2 and Fe+2. Cu2+, Cs+, Li+, Ag2+, SDS and Hg+2 inhibited alliinase activity to 28%,
17
77%, 61%, 52%, 59%, and 27% respectively, whereas K+, Na+ and EDTA had no significant effect on alliinase
18
activity. (Fig. 6).
19 20
3.6 Determination of Kinetic parameters
21
Michaelis-Menten kinetic parameters, Km and Vmax of the purified alliinase were calculated from Lineweaver-
22
Burk double reciprocal plot (Fig. 7). The Km and Vmax values for the alliinase isolated from C.necator were
23
found to be 74.65 U/protein mg and 0.83mM respectively.
24 25
3.7 Ammonia, Pyruvate and Allicin determination
26
Alliin (200 µM) on reaction with alliinase (10 U) resulted in the generation of 89.43 (± 4.32) µM of allicin,
27
129.46 (± 7.28) µM of ammonia and 148.45 (± 8.98) µM of pyruvate.
28 29
3.8 Cytotoxicity of in-situ Generated Allicin
11
1
The cytotoxic effect of the in-situ generated allicin on MIA PaCa-2 and HDF cells was determined using MTT
2
assay for 24 h. A concentration-dependent decrease in cell viability was observed for both the cell lines. IC50 of
3
alliin was found to be 74.054 (± 4.545) µM for MIA PaCa-2 cells and 223.05 (± 16.83) µM for HDF cell line.
4
140 µM of alliin in the presence of alliinase resulted in an 87.58 % decrease in MIA PaCa-2 cell viability
5
(Figure 8) whereas the same concentration resulted in a 35.88 % decrease in HDF cell viability.
6
Treatment of both the cell lines with reactants (alliin, alliinase) and by-products (ammonia and pyruvate) did not
7
resulted in any statistically significant difference in the cell viability when compared to untreated control.
8 9
3.9 Assessment of cytotoxicity by Acridine orange-ethidium bromide staining
10
Acridine orange is a cell permeable dye and stains live cell green while ethidium bromide stains the cell that
11
have lost their membrane integrity as red. As shown in Figure 9 f-h, cells treated with alliinase + 50 µM, 100
12
µM and 200 µM alliin, exhibited orange-red colour signifying loss of membrane integrity, cell shrinkage,
13
nuclear fragmentation, membrane blebbing and condensed nuclei demonstrating cellular damage; whereas the
14
untreated, alliin, alliinase, ammonia and pyruvate treated groups did not show any evident morphological
15
changes (Figure 9 a-e).
12
1
4.
Discussion
2
Recent years have seen significant progress in the study of the biological activity and application of sulphur
3
containing compounds from garlic. Earlier, alliinases that have been purified to homogeneity and characterised
4
include those from garlic cloves (Rabinkov et al. 1994), Onion bulbs (Schwimmer and Mazelis 1963), leek
5
(Lohmuller et al. 1994), Chinese chive (Manabe et al. 1998), and wild garlic/ramson (Landshuter et al. 1994). In
6
this study, we have isolated alliinase producing bacterium from the soil, on the medium containing alliin as the
7
sole nitrogen source. The alliinase producing organism was identified to be C. necator and soil is one of its
8
natural habitats. C.necator is often used in bioremediation and is able to degrade a great number of chlorinated
9
aromatic (Chloroaromatic) compounds and chemically related pollutants (Schenzle et al. 1999). This is the first
10
report of the production of alliinase from C. necator. The synthesis of alliinase and its application for generation
11
of allicin on demand, is a plant associated activity for defense purpose and the acquisition of this activity by a
12
bacterium in the garlic grown soil is an important ecological development by plant-microorganism association
13
and permits the organism to use alliin as a source of nitrogen.
14
Alliinase production from C. necator was lowest at 1% w/v glucose. A higher concentration of glucose inhibited
15
alliinase production. Alliinase production from C. necator was highest during incubation under shaking
16
condition which indicated that the organism requires aeration and it showed maximum production at 37 oC
17
which was the average temperature of the area from where soil sample was collected. Alliinase isolated from C.
18
necator is a homodimeric 110 kDa enzyme, consisting of two subunits of 55 kDa each, which is similar to the
19
alliinase reported in bacterium Ensifer adhaerens, which is a 96 kDa homodimeric enzyme consisting of two
20
subunits of 48 kDa each (Yutani et al. 2011). Molecular weights of isolated alliinase vary depending on the
21
source of the enzyme, ranging from 67 kDa in Chinese chive (Manabe et al. 1998) to 580 kDa in leek
22
(Lohmuller et al. 1994). The number of enzyme subunits has been shown to differ depending on the source of
23
the alliinase, ranging from 1 for Chinese chive (Manabe et al. 1998) to 12 for leek (Lohmuller et al. 1994).
24
Musah et al. (2009) reported the presence of a heteropentameric alliinase from Petiveria alliacea.
13
1
PLP is a cofactor essential for the C-S lyase activity of alliinase, so far reported (Lohmuller et al. 1994).
2
Although each subunit of alliinase contains one tightly bound PLP, its exogenous addition enhances the enzyme
3
activity as the purification proceeds (Schwimmer and Mazelis 1963; Mazelis and Crews 1968). Most metal ions
4
had an effect on alliinase activity, amongst of which, enzyme activity was enhanced by Mn+2, Mg+2, Zn+2, Fe+2
5
and Ca2+, whereas Cu2+, Cs+, Li+, Ag2+, Hg2+ and SDS inhibited alliinase activity and K+, Na+ and EDTA had no
6
significant effect. Stimulation of alliinase activity with Mg2+ and Mn2+ is in accordance with the findings by
7
Kuettner et al. (2002) with other Allium species. The increase in activity of alliinase due to Mn +2, Mg+2, Zn+2,
8
Fe+2 and Ca2+ ions can be attributed to the vital role played by them in building active enzyme conformation.
9
Inhibition of alliinase activity with Cu2+ has also been reported for alliinase from garlic (Kuettner et al. 2002). In
10
comparison to the results of Mazelis and Crews (1968), EDTA did not enhance enzyme activity of alliinase
11
from C.necator, whereas EDTA appeared to enhance the activity of alliinase from garlic. The isolated alliinase
12
preparation from C. necator may not have the presence of heavy metal ions, which is one of the factors
13
responsible for EDTA activation of alliinase preparation of garlic.
14
The optimum reaction temperature and pH for alliinase from C.necator was found to be 35 °C and 7
15
respectively. These conditions are similar to the study reported by others, wherein the optimum temperature of
16
alliinase from garlic is 35 °C (Mazelis and Crews 1968). On the other hand, it is higher than that established by
17
Yutani et al. (2011) wherein the optimum reaction temperature for alliinase from Ensifer adhaerens is 30 °C. A
18
similar pattern was followed in pH, wherein alliinase from garlic was reported to be 6.5 (Mazelis and Crews
19
1968) and alliinase from Ensifer adhaerens was reported at pH 7 (Yutani et al. 2011). The alliinase enzymes
20
from A. sativum, A. odorum and A. chinense exhibited pH optima of pH 5.6-6.5 whereas alliinase from A.
21
tuberosum, A. cepa, A. porrum and A. fistulosum exhibited pH optima of pH 8.0 - 8.5 (Manabe et al. 1998). The
22
enzyme activity is stable over a narrow range of pH and temperature, a similar phenomenon is seen in most of
23
the alliinases reported so far. Extreme temperature and pH might be having an effect on the structure of
24
enzyme, rendering it inactive. This temperature and pH sensitivity of alliinase can be encountered during oral
25
administration, by a method reported by Lianga et al. (2013) wherein an enteric coated double-layer tablet of
26
alliin/alliinase was used, for optimum activity of alliinase on alliin under gastrointestinal condition.
27
The specific activity of alliinase isolated from C.necator was found to be 209 U/mg, which is the highest
28
reported specific activity from microbial origin till date, whereas specific activity of alliinase reported from
29
garlic varies from 218 to 660 U/mg depending on the source of garlic (Kuettner et al. 2002). Alliinase from
30
C.necator appears to exhibit the highest affinity towards alliin as compared to alliinases reported from different
14
1
sources. The Km of alliinase from C.necator was found to be 0.83 mM which is lower than the Km value of
2
alliinase reported from garlic (1.1 mM) (Tobkin and Mazelis 1979) and that from Ensifer adhaerens (2.3 mM)
3
(Yutani et al. 2011). A lower Km corresponds to the higher affinity of the enzyme towards the substrate. Purified
4
alliinase from Cupriavidus necator exhibited lowest Km reported so far, which indicates that the enzyme has the
5
highest affinity towards alliin, hence it is an ideal candidate for immobilisation on a suitable matrix and can be
6
exploited further for the generation of allicin
7
MTT assay is a well-recognized in vitro technique, practiced to investigate cytotoxicity of compounds against
8
cancer cell lines. It has been used as one of the conventional techniques for screening of compounds with
9
potential cytotoxic properties (Kondo et al. 2000). In-situ generated allicin induced concentration-dependent
10
decrease in cellular viability in MIA PaCa-2 cells and IC50 was determined to be 74.054 (± 4.545) µM of alliin
11
for MIA PaCa-2 cells and 223.05 (± 16.83) µM of alliin for HDF cell line which indicates that allicin is
12
selectively more cytotoxic to MIA PaCa-2 cells as compared to HDF cells Allicin-induced cytotoxicity was
13
accompanied by morphological changes, including nuclear condensation, membrane blebbing and cell shrinkage
14
which was confirmed using Acridine orange-ethidium bromide staining wherein a concentration dependent
15
change in cell morphology was observed.
16 17 Acknowledgement
18
This study was supported in part by Grant from Mumbai University Research Project (Grant no. 097). We are
19
grateful to Dr. Vibha Verma for her technical assistance and express our deep gratitude to Dr. Purvi Bhatt,
20
Assistant Professor, School of Science for her kind assistance in 16 S rRNA sequencing
21 22
Conflict of Interest
23
Authors declare no conflict of interest
24 25 26 27 28 29
15
1 2 3 4 5 6 7 8 9 10 11
References Anifantaki, E., Touloupakis, E., Ghanotakis, D.F., 2010. Alliinase immobilisation in calcium alginate beads and layered double hydroxides matrices. J. Food Biochem. 36, 12–20. Anthon, G.E., Barrett, D.M., 2003. Modified method for the determination of pyruvic acid with dinitrophenylhydrazine in the assessment of onion pungency. J. Sci. Food Agri. 83, 1210–1213.
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Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped
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Tables
Table 1: Summary of data on the course of purification of intracellular alliinase from C.necator
Purification step
Protein
Enzyme
Specific
activity
activity
(mg)
(Units/mg)
Yield (%)
Purification (Fold)
(units)
Crude extract
8907
17963
2.0167
100
1
Ammonium sulphate
1124
14954
13.3042
83.24
5.51
124
8490
68.467
47.28
33.2056
59
7861
133.237
43.762
79
24
5016
209
27.92
103.63
precipitation DEAE Cellulose Discontinuous Chromatography DEAE Cellulose Continuous Chromatography Sephadex G-100 Chromatography
20 21 22 23 24 25 19
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Figure legends Figure 1 Reaction of Alliinase on Alliin
15
Figure 2 Effect of various growth parameters on alliinase production from C.necator
16
(a) Effect of varying concentrations of glucose on alliinase production
17
(b) Effect of varying concentrations of alliin on alliinase production
18
(c) Effect of temperature on alliinase production
19
(d) Effect of pH on alliinase production
20
(e) Effect of incubation condition on alliinase production
21
(f) Effect of incubation time on alliinase production
22
Figure 3 SDS-PAGE Analysis
23
(a) The proteins at each step of the purification procedure were separated by 10% SDS-PAGE under non-
24
reducing conditions (in the absence of β-ME), followed by Silver staining. Lane 1- crude enzyme; Lane
25
2-Ammonium sulphate precipitation pooled fractions; Lane 3-DEAE Dis. Chromatography pooled
26
fractions; Lane 4 - DEAE Continuous Chromatography pooled fractions; Lane 5-Sephadex G 100
27
chromatography pooled fractions; Lane 6, Puregene protein molecular weight marker
28
(b) 10 % SDS- PAGE analysis for molecular weight determination of isolated alliinase. Lane 1 - Puregene
29
protein molecular weight marker; Lane 2 - Alliinase in absence of β ME; Lane 3 -Alliinase in presence
30
of β ME
31
Figure 4 Elution profile of alliinase from C.necator
32
(a) Elution profile of alliinase in DEAE Cellulose Discontinuous gradient chromatography
33
(b) Elution profile of alliinase in DEAE Cellulose Continuous chromatography
34
(c) Elution profile of alliinase in Sephadex G-100 Chromatography
20
1
Figure 5 (a) Effects of temperature on alliinase activity and stability (b) Effect of pH on alliinase activity and
2
stability. Relative activities were calculated by using the highest activity of free and immobilized alliinase as
3
100%, respectively.
4
Figure 6 Alliinase was incubated with different metals and chemicals under standard assay conditions. The
5
100% enzyme activity was obtained in the absence of metal ions or chemicals.
6
Figure 7 Lineweaver-Burk double reciprocal plot for Kinetic analysis of alliinase extracted from C.necator
7
using different concentrations of alliin.
8
Figure 8 Cytotoxic effect of in-situ generated allicin on MIA PaCa-2 cells. Cells were treated with increasing
9
concentrations of alliin in the presence of alliinase extracted from (C.necator) for 24 h. Data are represented as
10
mean ± SD of three replicates from three independent experiments. The values are represented as the percentage
11
cell viability where the viability of untreated cells was regarded as 100%. Data were analyzed by one-way
12
ANOVA followed by Dunnett’s post hoc test. Asterisk above bars indicate statistically significant difference
13
compared to untreated control (*** = p < 0.001, ** = p < 0.01, * = p < 0.05).
14
Figure 9 Morphological study of MiaPaCa-2 cells stained with Acridine orange ethidium bromide after 24 h
15
incubation with (a) untreated control, (b) alliin 200 µM, (c) alliinase (d) ammonia, (e) pyruvate, (f) alliinase +
16
alliin 50 µM, (g) alliinase + alliin 100 µM (h) alliinase + alliin 200 µM.
17
21
Figures Figure 1
Figure 2
21 22 23
1600
24
1400
25 26
1200
27
1600 1500 1400 1300
1500
1000
500 20
Concentration of Alliin (%)
30
40
50
b
c Enzyme activity (Units)
2000
Enzyme activity (Units)
36 37 38 39
1500 1000 500 0 0
1
2
3
4
5
6
pH
7
8
40
9 10 11 12
E n z ym e a ctiv it y (U n its)
2000 2000
1500 1000 500
1600 1400 1200 1000
0 0
Shaking
Incubation condition
f
1800
e
Stagnant
0
24
48
72
96
Time (hours)
f
48 49 50 51 52 53 54 55
60
Temperature (oC)
a 35
41 42 43 44 45 46 47
1700
1200 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
28 29 Concentration of Glucose (%)
1000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
30 31 32 33 34
2000
1800
Enzyme activity (Units)
Enzyme activity (Units)
201800
Enzyme activity (Units)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 3
22
a
b
Figure 4
1500 10 1000 5 500 0 0 40 41 42 43 44 45 46 47 48 49 50 51 52
Protein concentration
30
2000
20
1000
10
0
0 10
150
6000 100 4000 50
2000 0 0.0
0.1
0.2
0.3
0.4
0.5
0 0.6
Alliinase activity
Protein concentration
b
3000
5
8000
15
20
Protein Concentration (mg)
Alliinase activity (Units)
a
0
200
KCL (Molar)
Fractions Alliinase activity
10000
Alliinase Activity (Units)
Alliinase activity (U nits)
15
25
Fractions Allinase activity
Protein concentration
c
23
Protein Concentration (mg)
2000
Protein C oncentr ation (m g)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 5
120
120
Relative activity (%)
Relative activity (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
100 80 60 40 20 0 0
100 80 60 40 20
5 10 15 20 25 30 35 40 45 50 55 60
0 0
Temperature ( oC) Alliinase stability
1
2
3
4
5
6
7
8
9 10 11 12
pH
Alliinase activity
Alliinase activity
a
Alliinase stability
b
Figure 6
Relative Enzyme activity (%)
140 120 100 80 60 40 20 0 0 None Cu
Cs
Fe
Li
Mg
K
Ag
Na
Mn
Zn
Hg SDS EDTA
Metal ions (1mM)
Figure 7
0.04 0.03
1/V
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Ca
-1 /K m = - 1 .2 0 4
0.02 0.01
1 /V m a x = 0 .1 3 3 9
43 44 45
-2.0 -1.6 -1.2 -0.8 -0.4 -0.0 0.4 0.8 1.2 1.6 2.0 2.4
- 1/K m
1 /S
24
re
at e Al d C liin o Al (2 ntr o liin 0 0 l as µ e M) Al ( liin Am 10 ( Al 20 m U) liin µ P o n Al (40 M) yru ia liin µ + va A Al (60 M) lliin te liin µ + a A l ( 8 M A ll s e liin 0 ) + iin a A l ( 1 0 µ M A ll s e liin 0 ) + iin a A l ( 1 2 µ M A lli s e liin 0 ) + in a A l ( 1 4 µ M A ll s e liin 0 ) + iin a A l ( 1 6 µ M A lli s e liin 0 ) + in a A l ( 1 8 µ M A lli s e liin 0 ) + in (2 µ M A l a s e 0 0 ) liin µ M + A as ) + lliin e Al as e liin as e
U nt
Cell viability (%)
1
2
3 Figure 8
4
120
80
60
HDF cells MIA PaCa 2 cells
100
** *** * **
40
20
*** ***
*** *** *** *** *** ***
*** *** *** *** *** ***
0
Groups
5 6
25
Figure 9
a
d
b
c
e
g 100 µg/mL As-Chitosan NPs
f
h
26