Comparative studies on isolation and characterization of allinase from garlic and onion using PEGylation-A novel method

Article abstract of DOI:10.14233/ajchem.2014.17064

Allium plants including garlic (Allium sativum) and onion (Allium cepa) contain an enzyme allinase (E.C-4.4.1.4), which is an enzyme responsible for the production of thio sulphates, a promising therapeutically potential compound. PEG (polyethylene glycol) is used in this study for the first time, to precipitate a protein. Four fold purification was done for both garlic and onion. The protein yield of the garlic was greater than onion shoots. 6.2 mg protein was the yield at the step in garlic, whereas it was 0.6 mg in onion shoots. The total protein content in the garlic is 6.1 g in the 100 g of garlic where as it is 1.6 g in the onion shoots.

Full text of DOI:10.14233/ajchem.2014.17064

Asian Journal of Chemistry; Vol. 26, No. 12 (2014), 3733-3735
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.17064
Comparative Studies on Isolation and Characterization of Allinase
from Garlic and Onion using PEGylation-A Novel Method†
*
SENTHILKUMAR RATHNASAMY , L. RUFUS AUXILIA and PURUSOTHAMAN
School of Chemical and Biotechnology, SASTRA University, Thanjavur-613401, India
*Corresponding author: E-mail: rsenthilkumar@biotech.sastra.edu
Published online: 5 June 2014;
AJC-15335
Allium plants including garlic (Allium sativum) and onion (Allium cepa) contain an enzyme allinase (E.C-4.4.1.4), which is an enzyme
responsible for the production of thio sulphates, a promising therapeutically potential compound. PEG (polyethylene glycol) is used in
this study for the first time, to precipitate a protein. Four fold purification was done for both garlic and onion. The protein yield of the
garlic was greater than onion shoots. 6.2 mg protein was the yield at the step in garlic, whereas it was 0.6 mg in onion shoots. The total
protein content in the garlic is 6.1 g in the 100 g of garlic where as it is 1.6 g in the onion shoots.
Keywords: Allinase, Garlic, Onion, Liquid chromatography, Polyethylene glycol.
Allicin kills Helicobacter pylori, a kind of bacteria which
is implicated in the cause of some stomach cancer and ulcers.
National Cancer Institute is sponsoring a huge clinical trial on
garlic’s ability to prevent stomach cancer. In the Indian
traditional cooking the garlic and onion are used as the basic
ingredient in all types of food. It reduces the activity of many
of the chemical reactions and also reduces the adverse effects
of the chemical reaction.
INTRODUCTION
The garlic (Allium sativum) and the onion (Allium cepa)
are the important plants used for culinary and medicinal
purposes. The characteristic flavour of onion (Allium cepa L.)
and garlic (Allium sativum L.), occurs when the enzyme alliinase
(EC 4.4.1.4) hydrolyzes the S-alk(en)yl-L-Cys sulfoxides
(ACSOs) to form pyruvate, ammonia and odorous sulfur-
containing volatile metablites. The sulfoxide substrates are
located in the cytoplasm, but alliinase is present in the vacuole1.
Rupturing makes these compounds to communicate with. The
enzyme is a homo-dimeric glycoprotein from the fold-type I
family of PLP-dependent enzymes.
Allicin is a very labile and volatile compound when
exposed to air and the methods known today for its preparation
are not satisfactory. The chemical synthesis involves many
steps and is complicated, laborious, expensive and very ineffi-
cient. The enzymatic method seems to be more attractive,
however alliinase is a so-called “suicidal enzyme” that is rapidly
and irreversibly inactivated by its own reaction product, allicin.
Therefore a few minutes incubation of alliinase with the
substrate alliin or its product, allicin, leads to a biologically
inactive enzyme after few cycles.
It is very important to isolate and characterize the allinase
enzyme for the production of allicin and other associated
bioactive compounds. In this study allinase enzyme is isolated
from both garlic and onion and characterized.
Allin is the precursor which comes to a contact with allinase
enzyme in the presence of co-factor pyridoxal phosphate which
initially forms allicin, pyruvate and ammonia as products.
Allicin is the bioactive compound present in the garlic. It
possess antimicrobial, anticancer and cholesterol lowering
property2-4.
EXPERIMENTAL
Sample preparation: Garlic bulbs and onion shoots were
purchased from the local market and washed many times and
same size of shoots and bulbs were sorted out. They were
†Presented at PHYTOCONGRESS-2013, held on 7-8 March 2013, SASTRA University, Thanjavur, India

3734 Rathnasamy et al.
Asian J. Chem.
1.0
0.8
0.6
0.4
0.2
0
washed twice with water to remove soil. 100 g of shoots and
bulbs were weighed and extracted with 100 mL of extraction
buffer (Na-phosphate buffer 0.02 M pH 7.8, containing
glycerol 10 %, pyridoxal phosphate 0.02 mM and 0.2 % 2-
mercaptoethanol) and magnetically stirred for 30-45 min at
4 ºC5. The supernatant was collected and stored.
Slope = 0.825 mL/mg
0.2
0.4
0.6
0.8
1.0
PEG precipitation: Concentrations varying from 25-
55 % of PEG 6000 were used for both garlic and onion. They
were stirred using magnetic stirrer under 4 ºC for 4 h and
centrifuged for 10 min at 15000 rpm. The protein estimation
was carried out using Lowry et al.4 method with reference to
the standard BSA.
Concentration (mg/mL)
Fig. 1. Lowry’s calibration chart
mAU
55.98
Gel filtration chromatography:PEG precipitated sample
was desalted using Akta Prime Plus with the buffer system
Tris-Hcl buffer 0.02 mM pH-7.8. The matrix of the column is
made up of Sephadex G-25. Column volume- 5 mL, void
volume 1.5 mL, flow rate 5 mL/min and bead size 15- 70 µm.
Every time the columns were washed with alcohol and water
after a successful run. The optical density value and peak were
noted for further reference. The protein content was estimated
by Lowry’s et al.4.
1500
1000
500
0
56.43
Fraction Off
58.92
Fraction size 1.0 mL
54
57.38
52
56
min
58
60
Assay of Allinase: There is a simple and rapid spectropho-
tometric procedure for determination of allicin and allinase
activity, based on the reaction between 2-nitro-5-thiobenzoate
(NTB). The quantitative reaction of alliinase is assessed by
the reaction at 412 nm due to the consumption of 2-nitro-5-
thiobenzoate. 2-Nitro-5-thiobenzoate is not commercially
available, it should be synthesized from DTNB (Ellman’s
reagent)5,6.
Fig. 2. UV absorption spectrum of sample purified using HiTrap column
chromatography
liquid chromatography in an AKTA prime plus sephadex
column. The results obtained were presented in Fig. 3. There
was a peak at 36 min with 1938 mAU which was collected
and stored at 4 ºC for further use. Table-1 gives the total protein
yield of garlic and onion using fourfold purification steps. This
involved precipitation, desalting and liquid chromatographic
analysis. Results were tabulated in Table-1. The results exhibited
that garlic possessed greater protein yield than the onion. The
resultant solution from liquid chromatography was analyzed
using BRUKER NMR. DMSO was used as solvent. The proton
near the carboxylic group showed a triplet signal around 4.30
ppm and there were a triplet signal around 3.15 ppm for the
proton near NH group. There might be triplets of triplet peak
for the remaining protons which were slightly merged and
messed up Fig. 4. Allinase assay was done according to Miron
et al.7,8. The spectrophotometric results were diagrammatically
represented in Fig. 5. The results showed 5 % PEG garlic at
0.5 h and showed high peak allinase activity.
Preparation of 2-nitro-5-thiobenzoic acid: 2-Nitro-5-
thiobenzoate was prepared according to Degani and Patchornik
et al.6. 2-Mercaptoethanol (5 mL) was added to the solution
of DTNB (1 g) in 50 mL of 0.5 M Tris-HCl buffer, pH 8. After
5 min, the solution was acidified to pH 1.5 by the addition of
6 N HCl and kept overnight at 4 ºC. Orange colour crystals
were formed and filtered washed with dilute HCl.
Allinase assay: The assay of allinase activity is based on
the reaction of allicin produced during the enzymatic reaction
and 2-nitro-5-thiobenzoate. The standard reaction mixture
contained NTB (0.001 M) in 50 mM Na-phosphate, pH 6.5
containing EDTA (1 mM), pyridoxal phosphate (2 × 10 M)
and 2.5- 10 units allinase and alliin 10 mM in total volume of
1 mL, enzymatic activity at 23 ºC started by adding the alliin
and the initial rate was monitored spectrophotometrically by
recording the decrease in absorbance at 412 nm.
1.93
mAU
RESULTS AND DISCUSSION
20
Allinase assay: Lowrys et al.4 method was used to find
out the protein concentration in each and every step of purifi-
cation. Calibration chart was drawn and the slope was calcu-
lated and represented in Fig. 1. Desalting, process is used to
remove the salts in the precipitated sample. AKTA prime plus
is used for the desalting process along with HiTrap Desalting
column matrixes with sephadex G-25, cross linked dextran.
Desalting graph showed two peaks in Blue colour which
indicated the UV of the sample elution Shown in Fig. 2. The
sample was collected in a fresh, clean tube and the peak elutes
was stored at 10 ºC. This sample was later injected into the
14.02
23.93
0
-20
-40
-60
0
5
10
15
20
min
Fig. 3. Liquid chromatography elution profile through Sephadex G-25

Vol. 26, No. 12 (2014)
Comparative Studies on Isolation and Characterization of Allinase from Garlic and Onion using PEGylation 3735
TABLE-1
TOTAL PROTEIN YIELD
Protein
Units
Specific activity
Recovery
Purification
S.No.
Steps
Garlic
Onion
1.6 g
Garlic
365
91
Onion
123
47
Garlic
0.14
0.11
0.11
6.9
Onion
0.11
0.9
Garlic
Onion
98
Garlic
Onion
1
1
2
3
4
Raw sample
6.2 g
2.2 g
100
25
1.0
0.8
0.8
4.9
PEG precipitation
Desalting
0.9 g
25
0.8
0.6
2.2
1.9 g
0.8 g
81
28
0.9
20
22
Liquid chromatography
1.6 mg
0.6 mg
53
13
1.1
14
9
2.0
1.5
1.0
0.5
0
30min
80min
140min
200min
260min
PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG
6000 6000 6000 8000 8000 8000 6000 6000 6000 8000 8000 8000
onion onion onion onion onion onion garlic garlic garlic garlic garlic garlic
5% 10% 15% 5% 10% 15% 5% 10% 15% 5% 10% 15%
Fig. 5. Allinase assay
Conclusion
Since there are many secondary metabolites in allium
plants, the isolation of a single metabolite is a challenging
task, leading to the isolation of allinase catalysing reaction
that can help significantly to isolate a single desired metabolite.
Method discussed in this paper can help researchers in getting
a greater yield of enzyme and a product for a better drug design
and development.
ppm
4.40 4.35 4.30 4.25 4.20 4.15
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ppm
3.40 3.35 3.30 3.25 3.20 3.15 3.10 3.05
ppm
2.4
2.3
2.2
2.1
2.0
Fig. 4. Alliin NMR spectra