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ANALYTICAL

BIOCHEMISTRY
Analytical Biochemistry 351 (2006) 152154
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Notes & Tips

A method for continuous production of allicin


using immobilized alliinase
T. Miron, H. SivaRaman, A. Rabinkov, D. Mirelman, M. Wilchek
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
Received 3 November 2005
Available online 9 February 2006

Pure allicin was shown to possess strong antimicrobial,


antiparasitic, and antiviral activities [1]. Other studies
showed that allicin prevents the development of the atherosclerotic process, reduces serum cholesterol, normalizes
lipoprotein balance, decreases blood pressure, and has antithrombotic and antiinXammatory activities [25].
Lately, bacteria have been developing resistance to many
drugs and bacterial diseases that were long kept under control, such as tuberculosis and pneumonia, are reemerging.
The urgent need for new and unconventional antimicrobial
agents led us to propose the use of allicin as an alternative
drug. Allicin is formed in crushed garlic by the interaction of
the nonprotein amino acid alliin (+S-allyl-L-cysteine sulfoxide) and the PLP1-dependent enzyme alliinase (cysteine sulfoxide lyase; alliin lyase) (EC 4.4.1.4) [6] (Scheme 1):

production of allicin. The immobilized alliinase was stable


for at least 1 year at 4 C when used periodically. Alternatively, it could be used exhaustively for 2 weeks at room temperature. The previously described immobilization of
alliinase on membranes for the preparation of biosensors
enables allicin generation at only analytical quantities [7,8].
Setting our goal to use allicin therapeutically, and being particularly attracted by the idea of easy oral administration, we
tried to avoid the use of toxic carriers and developed a
method that would supply gram quantities of allicin [9]. Carrier activation with N,N-disuccinimidyl carbonate (DSC)
[10] or with p-nitrophenyl chloroformate [11] yielded the
most stable, the least leaky, and the most inexpensive (spacer
redundant) immobilized alliinase, maintaining the highest
speciWc activity. The immobilized alliinase was very stable

The production of allicin from alliin and alliinase is


hampered by the instability of both enzyme and allicin and
their tendency to aggregate. Furthermore, the reaction
stops after a limited number of cycles.
In this paper, we describe the stabilization of alliinase by
its immobilization through lysine residues on the surface of
various matrices including Sepharose and beaded cellulose,
or its entrapment within others, and their use for sustained

and there was no need to add any stabilizing agent such as


sodium chloride [12], thus eliminating additional components
in the allicin solution. The immobilization provides also an
eYcient solution to the allicin instability problem by its fresh
production when the need arises.
Garlic was obtained from the local market. Sepharose
CL-4B was from Amersham Biosciences AB (Uppsala, Sweden). Beaded cellulose (5080 m) and pyridoxal-5-phosphate were from Sigma Chemical Co. (St. Louis, MO, USA).
DSC was from CalbiochemNovabiochem (Basel, Switzerland). p-Nitrophenyl chloroformate, 4-(dimethylamino)-pyridine (DMAP), and -amino caproic acid were purchased
from Fluka (Buchs, Switzerland). Alliin was synthesized
from L-cysteine and allyl bromide following H2O2 oxidation

Corresponding author. Fax: +972 8 9468256.


E-mail address: meir.wilchek@weizmann.ac.il (M. Wilchek).
1
Abbreviations used: DMAP, 4-(dimethylamino)-pyridine; DSC,
N,N-disuccinimidyl carbonate; NTB, 2-nitro 5-thio-benzoic acid; PLP,
pyridoxal-5-phosphate.
0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2006.01.030

Notes & Tips / Anal. Biochem. 351 (2006) 152154

by using the procedure of Stoll and Seebeck [13]. 2-Nitro 5thio-benzoic acid (NTB) was prepared according to Degani
and Patchornik [14]. Alliinase was isolated from garlic cloves
as described [12,15]. Protein concentration was determined
by the Lowry method [16]. Protein bound to the carrier was
calculated by subtracting the amount of unbound protein
from the amount used initially for the coupling.
Quantitative analysis of alliin and allicin was performed
using the LKB HPLC system equipped with a chromatography station for Windows (DataApex Ltd. 1998, Czech
Republic). The substances were separated on a LiChrosorb
RP-18 (7 m) column, using methanol (60%) in water containing 0.01% triXuoroacetic acid, at a Xow rate of 0.55 ml/
min, and their absorbance was detected at 210 nm [17]. The
concentration of HPLC-puriWed allicin was determined
with NTB [18] and used thereafter as a standard.
Sepharose CL-4B and beaded cellulose with or without
spacer were activated with hydroxyl or carboxyl reactive
reagents to bind the alliinase through amino groups [19].
The immobilization was performed in the presence of pyridoxal phosphate to protect lys251 from reacting with the
activated carrier [20]. In a typical coupling procedure, 10 g
of CNBr or p-nitrophenyl-activated gel (1035 mol/g wet
gel) was washed on a sintered glass funnel with cold water,
resuspended in 10 ml of puriWed or partially puriWed alliinase solution (25 mg/ml, 520 U/mg) in 0.2 M NaHCO3,
and shaken at 4 C for 16 and 48 h, respectively. In the case
of coupling alliinase to N-hydroxysuccinimide-activated
gels, the coupling was performed in 0.02 M phosphate
buVer, pH 7.4, for 416 h at 4 C. Unbound protein was
Wltered and its amount and activity were determined.
Activity of alliinase was determined either by using the
NTB assay [18,21] or by assessing pyruvic acid formation
[22]. Immobilized alliinase activity was performed in a
batchwise mode (50 mg wet gel/ml assay buVer) in 0.05 M
phosphate buVer, pH 6.5, containing 0.02 mM pyridoxal-5phosphate. Aliquots (2050 l) were further diluted with
assay buVer to Wnal volume 0.95 ml. The enzymatic reaction
started upon adding alliin (50 l) of 25 mg/ml water. The
reaction proceeded for 5 min while shaking. Supernatants
were collected by either centrifugation or Wltration; 50- to
100-l samples of the supernatant were taken for determination of pyruvic acid or allicin concentration.
The speciWc activity of the immobilized enzyme depends
on the coupling method and its degree of purity. There was
no loss of activity as a result of immobilization. In some cases
immobilized alliinase showed higher speciWc activity than the
soluble enzyme. A putative explanation for this might be that
the free enzyme undergoes inactivation due to aggregation.

153

Since the free enzyme shows a time-dependent decrease of


activity even at 4 C, its immobilization stabilizes the active
enzyme conformation. Another explanation may be that alliinase is preferentially immobilized in relation to other proteins in the preparation. It is noteworthy that there was not
much of a diVerence between the various carriers and the
method of their activation. Therefore, preference was given
to carriers that maintain stable bonds (Table 1).
Samples of immobilized, entrapped, and soluble enzyme
were incubated with alliin at room temperature in various
buVers (0.05 M) ranging between pH 4.0 and 9.5 for a
period of 5 min to assess the enzyme pH dependence:
0.05 M Na acetate was used for pH 4.05.5, 0.05 M Na
phosphate for pH 6.08.0, and 0.05 M Na tetraborate for
pH 8.59.7.
The optimal pH for soluble and entrapped alliinase activity was 6.5, whereas that of covalently immobilized alliinase
ranged between pH 7.0 and 7.5. Despite the optimum shift of
the covalently immobilized alliinase, we chose to work at a
lower pH (6.5) due to the instability of allicin at higher pH.
The eVect of temperature on enzyme activity was examined. Samples of the immobilized and soluble enzyme were
incubated with alliin in assay buVer (Wnal volume 0.9 ml), pH
6.5, at temperatures ranging between 4 and 70 C for a period
of 5 min. The reaction was terminated by acidiWcation with
0.1 ml 50% trichloroacetic acid. There was no signiWcant
diVerence (10%) between the activities of the soluble enzyme
or the entrapped enzyme within the temperature range examined, however, the covalently immobilized enzyme was found
to be much more active at temperatures above 40 C. The
activity at room temperature (25 C) was about 50% of that
observed at 70 C. At temperatures higher than 70 C, all allinase preparations, soluble or immobilized, started to lose activity. We performed the enzymatic assay and the subsequent
production steps at room temperature, although the enzyme
was more active at a higher temperature, due to the high reaction rate, thus simplifying the system and saving energy. To
obtain continuous production of allicin, immobilized alliinase
was packed in columns (1 10 cm) at room temperature.
Alliin was continually loaded on the columns at a Xow rate of
7 ml/h. The eYciency of allicin production was in direct proportion to the speciWc activity of the immobilized alliinase
packed in the column. For example, a column (1 10 cm)
containing only 12 units activity/ml carrier converted continuously 7082% of alliin (4 mg/ml) to allicin within 48 hours,
whereas a column containing high speciWc activity, 4080
units activity/ml carrier, converted 100% of alliin (4 mg/ml)
within the same time period. We found a linear dependence of
allicin production on substrate concentration in high-speciWc-

Table 1
Yield of immobilization and residual speciWc activity of immobilized alliinase preparations
Carrier

Cellulose-6-aminocaproyl

Sepharose-6-aminocaproyl

Sepharose(p-nitrophenyl)

Sepharose(CNBr)

Sepharose(DSC)

Agar
entrapment

Protein bound mg/g wet gel


Residual speciWc activity (%)

21.7
105

5.6
114

5.6
119

5.3
119

5.5
119

1.9
97

The activity assay of the various gels was done after 1 week storage at 4 C.

154

Notes & Tips / Anal. Biochem. 351 (2006) 152154

[3]

[4]

[5]

[6]
[7]
[8]
Fig. 1. Allicin production by immobilized alliinase as a function of alliin
concentration. Alliinase covalently bound to Sepharose Cl 4B was packed
in columns (1 10 cm). Alliin in 0.05 M phosphate buVer, pH 6.5, containing 0.02 mM pyridoxal-5-phosphate was loaded at increasing concentrations on four columns at a rate of 7 ml/h at room temperature. The
concentration of allicin in the eluate was determined by HPLC after dilution (1:100) with the HPLC running buVer as described in the text.

activity preparations. This linear behavior is maintained up to


6 mg/ml substrate, since the product tends to form white
aggregates upon saturation at a concentration higher than
2 mg/ml (Fig. 1). We therefore used only 4 mg/ml substrate for
allicin production.
Immobilized alliinase packed in a column was used to
continuously produce allicin at room temperature for at least
2 weeks. The column showed no loss of activity over this
period, and it can therefore be used in health facilities for the
immediate production of allicin in cases such as bacterial or
fungal antibiotic resistance, where other drugs fail.
As opposed to all other methods of allicin synthesis,
whether chemical or enzymatic, immobilized enzyme yields
the highest degree of product purity. Covalently immobilized alliinase is the best up-to-date tool for large-scale production of allicin. In all our recent publications, allicin was
produced in the same manner as described in this communication [4,23,24]. The availability of pure allicin will enable
further studies of its biological eVects, its mechanism of
action, and its potential pharmaceutical applications.

[9]

[10]
[11]

[12]

[13]
[14]

[15]

[16]
[17]
[18]

[19]
[20]
[21]

Acknowledgments
This publication is dedicated in memory of Dr. Hephziba SivaRaman, our collaborator and friend.

[22]

[23]

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